BACKGROUND: To evaluate the efficacy and safety of the first cohort of people in China treated with a responsive neurostimulation system (Epilcure TM , GenLight MedTech, Hangzhou, China) for focal drug-resistant epilepsy in this study. METHODS: This multicenter, before-and-after self-controlled study was conducted across 8 centers from March 2022 to June 2023, involving patients with drug-resistant epilepsy who were undergoing responsive neurostimulation (RNS). The study was based on an ongoing multi-center, single-blind, randomized controlled study. Efficacy was assessed through metrics including median seizure count, seizure frequency reduction (SFR), and response rate. Multivariable linear regression analysis was conducted to explore the relationships of basic clinical factors and intracranial electrophysiological characteristics with SFR. The postoperative quality of life, cognitive function, depression, and anxiety were evaluated as well. RESULTS: The follow-up period for the 19 participants was 10.7 ± 3.4 months. Seizure counts decreased significantly 6 months after device activation, with median SFR of 48% at the 6th month (M6) and 58% at M12 ( P  <0.05). The average response rate after 13 months of treatment was 42%, with 21% ( n  = 4) of the participants achieving seizure freedom. Patients who have previously undergone resective surgery appear to achieve better therapeutic outcomes at M11, M12 and M13 ( β  <0, P  <0.05). No statistically significant differences were observed in patients' scores of quality of life, cognition, depression and anxiety following stimulation when compared to baseline measurements. No serious adverse events related to the devices were observed. CONCLUSIONS: The preliminary findings suggest that Epilcure TM exhibits promising therapeutic potential in reducing the frequency of epileptic seizures. However, to further validate its efficacy, larger-scale randomized controlled trials are required. REGISTRATION Chinese Clinical Trial Registry (No. ChiCTR2200055247).
Humans ; Female ; Male ; Drug Resistant Epilepsy/therapy* ; Adult ; Young Adult ; Middle Aged ; China ; Adolescent ; Treatment Outcome ; Quality of Life ; Single-Blind Method ; Seizures ; Electric Stimulation Therapy/methods*

Traditional development of small protein scaffolds has relied on display technologies and mutation-based engineering, which limit sequence and functional diversity, thereby constraining their therapeutic and application potential. Protein design tools have significantly advanced the creation of novel protein sequences, structures, and functions. However, further improvements in design strategies are still needed to more efficiently optimize the functional performance of protein-based drugs and enhance their druggability. Here, we extended an evolution-based design protocol to create a novel minibinder, BindHer, against the human epidermal growth factor receptor 2 (HER2). It not only exhibits super stability and binding selectivity but also demonstrates remarkable properties in tissue specificity. Radiolabeling experiments with ^99mTc, ⁶⁸Ga, and ¹⁸F revealed that BindHer efficiently targets tumors in HER2-positive breast cancer mouse models, with minimal nonspecific liver absorption, outperforming scaffolds designed through traditional engineering. These findings highlight a new rational approach to automated protein design, offering significant potential for large-scale applications in therapeutic mini-protein development.

Objective:We genetically modified our non-replicating vaccinia virus NTV to improve its immunogenicity.Methods:We constructed NTV-modified strain NTV-ΔF1L-C7L by homologous recombination of vaccinia virus based on CRISPR-Cas9 technology by inserting the C7L gene while deleting the F1L gene. The recombinant virus NTV-ΔF1L-C7L was then immunized with 10 7 PFU in BALB/c mice, and the levels of humoral and cellular immunity induced by NTV-ΔF1L-C7L were detected by ELISA and ELISpot method, respectively, and the levels of neutralizing antibodies were determined by the phage-reduced neutralization assay. Results:The PCR and western- blot identification proved that the F1L gene of the constructed NTV-modified strain NTV-ΔF1L-C7L was missing, while the C7L gene was inserted back in the region, and the C7L gene could be expressed normally, indicating that the recombinant virus was constructed correctly. After immunization of mice with NTV-ΔF1L-C7L, ELISA result showed that the recombinant virus NTV-ΔF1L-C7L induced a higher level of IgG antibody than NTV; ELISpot result also showed that the recombinant virus was able to induce a higher level of IFN-γ; and the result of plaque reduction neutralization test showed that the recombinant virus was able to induce a higher level of IFN-γ antibody than that of NTV.Conclusions:We correctly constructed the NTV gene-modified strain NTV-ΔF1L-C7L, which induced stronger humoral and cellular immunity compared with NTV, and provided reference data for the research and development of replacement products for smallpox or monkeypox vaccines.

This study aimed to investigate the influence of anti-cross-reactive carbohydrate determinant IgE antibodies (anti-CCD IgE) on the detection of allergen-specific IgE (sIgE) antibodies, as well as the application value of anti-CCD IgE adsorbents in detecting allergen sIgE. In this cross-sectional study, a total of 2 636 test samples from patients who received treatment in West China Hospital of Sichuan University and tested allergen sIgE using the western blot method from October 2020 to May 2021 were analyzed. In these samples, 709 samples tested postive of allergen sIgE. 46 stochastic venous serum samples that tested positive in both sIgE and anti-CCD IgE and 1 serum sample that tested positive in sIgE but negative in anti-CCD IgE were collected. These samples were processed by anti-CCD IgE adsorbents, followed by allergen sIgE detection. The difference between the two detection results before and after adsorption was analyzed. The allergen test results showed that the positive rate of anti-CCD IgE in samples was 2.6% (69/2 636) during the period of sample collection. After treatment with anti-CCD IgE adsorbents, the top three allergen-sIgE of the positive rate changed from tree combination 2 (willow/poplar/elm), common ragweed and peanut to dust mite combination, cockroach and crab. The positive anti-CCD IgE results of 46 samples all turned negative and the total positive sIgE antibody dropped by 62.8%; the positive rate of sIgE antibodies with the class result ≥2 significantly decreased after treatment with anti-CCD IgE adsorbents, especially the positive rate of common ragweed dropped by 96.2%. The results of positive samples showed that multiple sIgE antibodies declined by different ranges, involving up to 11 antibodies with a maximum decline of 4 classes. Strongly positive sIgE antibodies (the class result ≥4) also had a high conversion rate of negative (25.0%-100%). The positive sIgE antibodies in about 60% of the samples decreased by more than 2, and the sIgE antibodies in 17.4% of the samples turned completely negative. There was no change in the allergen sIgE detection results of the sample with negative anti-CCD IgE after treatment. In conclusion, sIgE antibodies including targeting common ragweed, humulus, tree combination 2 (willow/poplar/elm), etc. are susceptible to false positives caused by anti-CCD IgE. Treatment of samples with anti-CCD IgE adsorbents can significantly reduce the risk of false positives caused by anti-CCD IgE. It is necessary to pretreat samples that were anti-CCD IgE positive with anti-CCD IgE adsorbents, which can make laboratory results more accurate and provide a reference for diagnosis and prevention of allergic diseases.

This study aimed to investigate the influence of anti-cross-reactive carbohydrate determinant IgE antibodies (anti-CCD IgE) on the detection of allergen-specific IgE (sIgE) antibodies, as well as the application value of anti-CCD IgE adsorbents in detecting allergen sIgE. In this cross-sectional study, a total of 2 636 test samples from patients who received treatment in West China Hospital of Sichuan University and tested allergen sIgE using the western blot method from October 2020 to May 2021 were analyzed. In these samples, 709 samples tested postive of allergen sIgE. 46 stochastic venous serum samples that tested positive in both sIgE and anti-CCD IgE and 1 serum sample that tested positive in sIgE but negative in anti-CCD IgE were collected. These samples were processed by anti-CCD IgE adsorbents, followed by allergen sIgE detection. The difference between the two detection results before and after adsorption was analyzed. The allergen test results showed that the positive rate of anti-CCD IgE in samples was 2.6% (69/2 636) during the period of sample collection. After treatment with anti-CCD IgE adsorbents, the top three allergen-sIgE of the positive rate changed from tree combination 2 (willow/poplar/elm), common ragweed and peanut to dust mite combination, cockroach and crab. The positive anti-CCD IgE results of 46 samples all turned negative and the total positive sIgE antibody dropped by 62.8%; the positive rate of sIgE antibodies with the class result ≥2 significantly decreased after treatment with anti-CCD IgE adsorbents, especially the positive rate of common ragweed dropped by 96.2%. The results of positive samples showed that multiple sIgE antibodies declined by different ranges, involving up to 11 antibodies with a maximum decline of 4 classes. Strongly positive sIgE antibodies (the class result ≥4) also had a high conversion rate of negative (25.0%-100%). The positive sIgE antibodies in about 60% of the samples decreased by more than 2, and the sIgE antibodies in 17.4% of the samples turned completely negative. There was no change in the allergen sIgE detection results of the sample with negative anti-CCD IgE after treatment. In conclusion, sIgE antibodies including targeting common ragweed, humulus, tree combination 2 (willow/poplar/elm), etc. are susceptible to false positives caused by anti-CCD IgE. Treatment of samples with anti-CCD IgE adsorbents can significantly reduce the risk of false positives caused by anti-CCD IgE. It is necessary to pretreat samples that were anti-CCD IgE positive with anti-CCD IgE adsorbents, which can make laboratory results more accurate and provide a reference for diagnosis and prevention of allergic diseases.

Objective:To accurately ascertain the whole genome sequencing and the composition of open reading frames (ORFs) of non-replicating Tiantan strain of vaccinia virus (NTV) using next-generation long-read sequencing technology.Methods:NTV, obtained from our laboratory stock, was amplified and purified on chicken embryo fibroblast cells(CEFs), and the full-length genomic nucleic acid of NTV was extracted. The PacBio HiFi sequencing platform was utilized for de novo assembly to obtain the complete genomic sequence of NTV. Using a homology annotation strategy, we identified its ORF composition and compared it with known non-replicating vaccinia virus strains. Results:The total length of NTV′s genome was 171 729 bp, with a GC content of 33%. Its unique inverted terminal repeat (ITR) region comprised hairpin structures, two tandem repeat regions, and three non-repeat regions. NTV contained 166 ORFs, with major differences observed in the ITR and its surrounding regions when compared to MVA-BN and NYVAC. These three strains shared a common set of 138 ORFs. NTV encoded six unique ORFs related to virus evasion of host antiviral response.Conclusions:This study accurately determines the whole genome sequencing and ORFs composition of NTV, and reveals its similarities and differences with other replication-deficient vaccinia virus strains, which pave a way for the development and application of the next generation of monkeypox vaccines and novel viral vectors.

OBJECTIVE: To verify the effect of Buyang Huanwu Decoction (BHD) in ameliorating erectile dysfunction (ED) after radical prostatectomy (RP). METHODS: The composition of BHD was verified by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) analysis. Bilateral cavernous nerve crush injury (BCNI) in rats was used to mimic the neurovascular injury occurring after RP. By the envelope method, forty rats were randomly divided into 4 groups as follows: sham (cavernous nerves exposed only), model (BCNI), low-dosage BHD [LBHD, 12.8 g/(kg·d)], and high-dosage BHD [HBHD, 51.2 g/(kg·d)] groups, 10 rats in each group, feeding for 3 weeks respectively. Erectile function was evaluated by measuring intracavernosal pressure (ICP). Changes in the histopathology of corpus cavernosum (CC) were examined by hematoxylin-eosin staining. Meanwhile, the fibrosis of CC was measured by Masson's trichrome staining and Western blot was used to detect the expressions of collagen I, transforming growth factor beta 1 (TGF- β 1) and α-smooth muscle actin (α-SMA). Apoptosis index was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) and Western blot for determining the expressions of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax). The oxidative stress in the CC were assessed by the superoxide dismutase (SOD), malondialdehyde (MDA) and reactive oxygen species (ROS) levels. The proteins expression of c-Jun N-terminal kinase (JNK) and c-Jun were detected by Western blot. In addition, the expression of α-SMA and p-c-Jun in the CC was observed by double immunofluorescence staining. RESULTS: The UPLC-QTOF-MS/MS analysis showed that BHD contained calycosin-7-O- β -D-glucoside, ononin, calycosin and formononetin. Compared with the model group, LBHD and HBHD treatment improved the ICP and the circumference, area, and weight of CC (P<0.05 or P<0.01). Furthermore, LBHD and HBHD treatments increased CC smooth muscle content and decreased apoptosis index (P<0.05 or P<0.01). LBHD and HBHD also elevated SOD and expression level of α -SMA and Bcl-2, and reduced MDA and ROS levels, as well as expression of TGF- β 1, collagen I, Bax, p-c-JNK, p-JNK in the CC compared with the model group (P<0.05 or P<0.01). The double immunofluorescence staining showed that the fluorescence degree of p-c-Jun in both LBHD and HBHD treatment groups was significantly reduced, whereas the α -SMA expression increased (P<0.05 or P<0.01). CONCLUSIONS BHD can improve ED of rats with BCNI, which is related to inhibiting fibrosis, apoptosis, and oxidative stress of CC. The ROS/JNK/c-Jun signaling pathway may play an important role in the process.
Male ; Humans ; Rats ; Animals ; Reactive Oxygen Species ; Tandem Mass Spectrometry ; bcl-2-Associated X Protein ; Rats, Sprague-Dawley ; Erectile Dysfunction/drug therapy* ; Collagen ; Fibrosis ; Disease Models, Animal

Objective:To develop a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a-based nucleic acid assay for monkeypox virus with high specificity and sensitivity.Methods:RAA primers and CRISPR RNA (crRNA) were designed based on the known conserved regions of the monkeypox virus gene and synthesized, and specific crRNAs were screened using fluorescence detection. The sensitivity and specificity of the detection system were evaluated.Results:An RAA-CRISPR/Cas12a-based nucleic acid assay for monkeypox virus was developed with a sensitivity of 2.5 copies/reaction and high specificity without cross-reactivity with ectromelia virus and vaccinia virus.Conclusions:An RAA-CRISPR/Cas12a-based nucleic acid assay for monkeypox virus was established, which would provide a powerful tool for efficient, rapid and specific detection of monkeypox virus.

Objective:Through the study of the cell biological characteristics and virulence in mice in vivo of three non-replicating vaccinia virus modified strains to provide reference for the development of smallpox/monkeypox vaccine replacement products.Methods:Replicating vaccinia virus Tiantan strain VTT and non-replicating Tiantan Vaccinia Strain NTV were studied in BHK-21/CEF and its modified strains NTV-C7L, NTV-△F1L-C7L, NTV-K1L were amplified, purified, and identified by Western blotting. The virulence and diffusivity of each strain in cells were evaluated by immune-plaque assay. The replication dynamics curves were used to compare the replication differences between the strains, and the weight loss was observed by intranasal route in the mouse model to compare the virulence levels of the viruses.Results:In this study, Western blotting result proved that the amplified and purified vaccinia virus strains were correct. Immunophagocytosis and replication kinetics showed that the replication capacity of the three NTV modified strains in CEF was similar to that of NTV. The diffusion ability and replication ability between Vero cells were improved, but the replication multiple was less than 100 times. The replication level of MRC-5 was significantly enhanced compared with that of NTV, and the replication ratio of NTV-C7L was more than 20000 times. The virulence in mice showed that the body weight of the three NTV modified strains had no statistical significance compared with that of NTV.Conclusions:The three NTV modified strains recovered their replication ability in human MRC-5 cells, but their virulence in mice was similar to that of NTV, which provided the preliminary conditions for being candidate strains of smallpox/monkeypox vaccine replacement products.

Objective:Based on targeted amplicon technology combined with high-throughput sequencing technology and bioinformatic analysis technology, to understand the characteristics of the whole genome of the monkeypox virus and its variation, and to construct a method for the analysis of monkeypox virus variation and molecular traceability of the case in Qinghai province, and to provide technical support for the prevention and control of monkeypox epidemic in the future.Methods:The extracted viral DNA was used as a template, and the genome of monkeypox virus was specifically amplified by Ion AmpliSeq Monkeypox Panel with the number of amplicons 1 609 and the length of 125 bp-275 bp, and the sequencing library was constructed by Ion AmpliSeq Library Kit Plus, and sequenced by Ion Torrent GeneStudio S5. The sequencing library was constructed by Ion AmpliSeq Library Kit Plus, and the monkeypox virus genome was sequenced using Ion Torrent GeneStudio S5 sequencer. Monkeypox virus was analyzed for genomic profiling and mutation site analysis using the online analysis tool Nextclade. The genomic sequence of the case virus in this study was compared with some sequences in the GIASID monkeypox virus database and a phylogenetic tree was constructed to analyze the potential origin of the case virus.Results:The Ct values of monkeypox virus genes in the rash swab and oropharyngeal swab samples were 32.13 and 36.91, respectively. The rash swab sample had a reads number match of 99.99% and a genome coverage of 99.45% after whole-genome sequencing of monkeypox virus, and the sequences belonged to the IIb (West African branch) B. 1.3 type. The analysis of nucleotide mutation sites and phylogenetic tree showed that the sequences were in the same branch with four monkeypox virus genome sequences recently submitted by China and Japan in the GISAID monkeypox virus database, and had the closest evolutionary relationship with the sequence EPI_ISL_18059184 (sampled on 2023-07-03) submitted by Yunnan, China, which shared 82 single-nucleotide mutation sites, among which the sequence from Yunnan was only present in all of the shared 82 single-nucleotide mutation sites. The sequence in this study has 2 additional nucleotide mutation sites on top of the shared 82 single nucleotide mutation sites. The sequence submitted by Japan, EPI_ISL_17692269 (sampled on 2023-04-28), is more closely related in evolution, sharing 78 single nucleotide mutation sites, with 7 single nucleotide mutation site differences, and the Japanese sequence shares 78 single nucleotide mutation sites. The Japanese sequence shared 78 mutation sites with one additional nucleotide mutation site (G57786A), while the present sequence had six additional nucleotide mutation sites (G13563A, C21062T, G101241A, C142797T, G152866A, T169721A).Conclusions:The whole genome sequence of monkeypox virus of 197 084 bp was successfully obtained from a sample with low viral load, and the average. We constructed a method for sequencing and analyzing the whole genome of monkeypox virus.