ObjectiveBased on the conjoint analysis of transcriptome and metabolome， the key genes in the phenylpropanoid biosynthesis pathway of Lonicera macranthoides were explored， which provided a basis for further exploring the synthesis and regulation mechanism of phenylpropanoid compounds in "Xianglei" L. macranthoides. MethodsThe stem， leaves， and three flowering flowers of "Xianglei" L. macranthoides were selected as experimental materials to construct transcriptome and metabolome. The transcriptome and metabolomics were conjointly analyzed by the Kyoto Encyclopedia of Genes and Genomes （KEGG） database and weighted correlation network analysis （WGCNA）， and the key genes in the phenylpropanoid biosynthesis pathway of L. macranthoides were explored. ResultsIn this study， 77 differential phenylpropanoids and 315 differential genes were found. Through the joint analysis of transcription and metabolism， nine key differential metabolites and four key genes related to them were finally discovered. Among them， cinnamic acid， 5-O-caffeoylshikimic acid，sinapyl alcohol， and chlorogenic acid were higher in flowers， and the content of the iconic effective component， namely chlorogenic acid，decreased sharply during the withering period. Caffeic acid，ferulic acid， 5-hydroxyconiferaldehyde，p-coumaryl alcohol， and syringin were higher in leaves. These four key genes belong to the cinnamic alcohol dehydrogenase （CAD） family， 4-coumaric acid： Coenzyme A （4CL） family， hydroxycinnamyl transferase （HCT） family， and L-phenylalanine ammonlyase （PAL） family genes. ConclusionAmong the four key genes excavated from L. macranthoides， TRINITY_DN42767_c0_g6 is related to the synthesis of p-coumaryl alcohol and sinapyl alcohol. TRINITY_DN43525_c4_g1 uses caffeic acid，ferulic acid，and cinnamic acid as substrates to catalyze the next reaction. TRINITY_DN47958_c3_g4 correlates with the synthesis of 3-p-coumaroyl quinic acid and caffeoyl-CoA， and TRINITY_DN52595_c1_g2 correlates with cinnamic acid synthesis. These findings provide a basis for further exploring the synthesis and regulation mechanism of phenylpropanoids in "Xianglei" L. macranthoides.

OBJECTIVE: To explore the improvement effect of electroacupuncture (EA) based on Xingnao Kaiqiao acupuncture (acupuncture for regaining consciousness and opening orifices) on cognitive impairment in mice with Parkinson's disease (PD), and to explore its regulatory mechanisms on the kinesin family member 5A (KIF5A)/mitochondrial Rho GTPase 1 (Miro1) pathway and mitophagy in prefrontal cortical neurons. METHODS: A total of 70 male C57BL/6J mice of clean grade were randomly divided into a normal group (12 mice), a sham operation group (12 mice), and a model pre-screening group (46 mice). Unilateral stereotaxic injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle was adopted to establish the PD model in the model pre-screening group. Twenty-four mice after successful modeling were randomly selected and divided into a model group and an EA group, 12 mice in each one. In the EA group, acupuncture was applied at "Shuigou" (GV26) and bilateral "Sanyinjiao" (SP6) and "Neiguan" (PC6), ipsilateral "Sanyinjiao" (SP6) and "Neiguan" (PC6) were connected to EA respectively, with disperse-dense wave, 5 Hz/20 Hz in frequency, 0.5 mA in current intensity, 20 min a time, 6 times a week for 30 days. Cognitive function was assessed by Y-maze and Morris water maze tests; morphology of prefrontal cortex was observed by H.E. staining; reactive oxygen species (ROS) level in prefrontal cortex was detected by fluorescence probe method; mitochondrial morphology and autophagosome ultrastructure were observed by transmission electron microscopy; the mRNA expression of tyrosine hydroxylase (TH) was detected by quantitative real-time PCR; the protein expression of TH, KIF5A, Miro1, p62, Parkin and PTEN induced kinase 1 (PINK1) was detected by Western blot. RESULTS: Compared with the sham operation group, both the model group and the EA group exhibited increased rotation number of per minute (P<0.001). Compared with the sham operation group, in the model group, the novel arm exploration time of Y-maze test was shortened (P<0.001), the escape latency of Morris water maze test was prolonged (P<0.05) and the platform crossing number of Morris water maze test was reduced (P<0.01); in the prefrontal cortex, the number of cellular vacuole and neurons with karyopyknosis was increased (P<0.001), and mitochondrial autophagosomes could be observed; in the prefrontal cortex, the relative expression of ROS was increased (P<0.001), the protein and mRNA expression of TH was decreased (P<0.001), the protein expression of Miro1, PINK1, Parkin was increased (P<0.001, P<0.01), the protein expression of KIF5A and p62 was decreased (P<0.001). Compared with the model group, in the EA group, the novel arm exploration time of Y-maze test was prolonged (P<0.01), the escape latency of Morris water maze test was shortened (P<0.05) and the platform crossing number of Morris water maze test was increased (P<0.05); in the prefrontal cortex, the number of cellular vacuole and neurons with karyopyknosis was decreased (P<0.001), and the number of mitochondrial autophagosomes reduced and the mitochondrial morphology was improved; in the prefrontal cortex, the relative expression of ROS was decreased (P<0.01), the protein and mRNA expression of TH was increased (P<0.001, P<0.01), the protein expression of Miro1, PINK1, Parkin was decreased (P<0.001, P<0.01, P<0.05), the protein expression of KIF5A and p62 was increased (P<0.01, P<0.05). CONCLUSION Xingnao Kaiqiao electroacupuncture effectively alleviates cognitive impairment and damage of neuronal function in PD mice, its mechanism may be related to the regulation of KIF5A/Miro1 pathway, hence reducing the mitophagy in prefrontal cortical neurons.
Animals ; Electroacupuncture ; Male ; Mice ; Parkinson Disease/physiopathology* ; Cognitive Dysfunction/psychology* ; Kinesins/genetics* ; Humans ; Mitophagy ; Mice, Inbred C57BL ; rho GTP-Binding Proteins/genetics* ; Mitochondria/genetics* ; Prefrontal Cortex/metabolism*

Objective To investigate prokaryotic expression of the antigen sequence (amino acids 59-145) of mouse leukemia-related protein 16 (LRP16) protein and preparation of rabbit anti-mouse LRP16 polyclonal antibody. Methods The prokaryotic expression plasmid pLS962-LRP16 was constructed by the molecular cloning method and transferred into E.coli Rosetta to express LRP16 protein induced by IPTG. The recombinant protein was purified using Ni-NTA affinity columns followed by gel filtration chromatography. New Zealand white rabbits were immunized with the purified antigen to generate polyclonal antiserum, with antibody titer quantified by ELISA. Antigen-specific IgG was affinity-purified using Sepharose-coupled LRP16 and validated through Western blot and immunofluorescence assays. Results SDS-PAGE analysis confirmed insoluble expression of the LRP16 fusion protein as inclusion bodies. ELISA demonstrated exceptional antiserum titer (1:256 000). Western blot and immunofluorescence verified that the polyclonal antibody could specifically recognize endogenous LRP16 in murine tissues. Conclusion The prokaryotic expression of the LRP16 gene is successfully achieved, and the rabbit anti-mouse LRP16 polyclonal antibody exhibiting high specificity is prepared. This lays the foundation for further studies on the function of the LRP16 gene.
Animals ; Rabbits ; Mice ; Antibodies/immunology* ; Escherichia coli/metabolism* ; Enzyme-Linked Immunosorbent Assay ; Blotting, Western ; Antibody Specificity

Polycystic ovary syndrome (PCOS) is the predominant cause of subfertility in reproductive-aged women; however, its pathophysiology remains unknown. Neurotensin (NTS) is a member of the gut-brain peptide family and is involved in ovulation; its relationship with PCOS is unclear. Here, we found that NTS expression in ovarian granulosa cells and follicular fluids was markedly decreased in patients with PCOS. In the in vitro culture of cumulus-oocyte complexes, the neurotensin receptor 1 (NTSR1) antagonist SR48692 blocked cumulus expansion and oocyte meiotic maturation by inhibiting metabolic cooperation and damaging the mitochondrial structure in oocytes and surrounding cumulus cells. Furthermore, the ERK1/2-early growth response 1 pathway was found to be a key downstream mediator of NTS/NTSR1 in the ovulatory process. Animal studies showed that in vivo injection of SR48692 in mice reduced ovulation efficiency and contributed to irregular estrus cycles and polycystic ovary morphology. By contrast, NTS partially ameliorated the ovarian abnormalities in mice with dehydroepiandrosterone-induced PCOS. Our findings highlighted the critical role of NTS reduction and consequent abnormal NTSR1 signaling in the ovulatory dysfunction of PCOS, suggesting a potential strategy for PCOS treatment.
Polycystic Ovary Syndrome/physiopathology* ; Female ; Animals ; Neurotensin/metabolism* ; Receptors, Neurotensin/antagonists & inhibitors* ; Mice ; Ovulation/drug effects* ; Humans ; Granulosa Cells/metabolism* ; Adult ; Oocytes/metabolism* ; MAP Kinase Signaling System ; Signal Transduction ; Follicular Fluid/metabolism* ; Disease Models, Animal ; Gonadotropin-Releasing Hormone/analogs & derivatives*

Leukemia is a critical disease with a high incidence and extremely high fatality rate.Immune escape by leukemia stem cells(LSC)is the main factor for recurrence and progression after remission.Clinical diagnosis and treatment by Traditional Chinese Medicine(TCM)have distinct advantages of syndrome differentiation and treatment.Based on the purpose of diagnosis and treatment,leukemia treatment by TCM emphasizes the harmony of yin and yang to restore human functions,which is conducive to improve autoimmunity and conforms to the mechanism of intervention for tumor cell immune escape.This article discusses the mechanism and research progress of TCM interventions in LSC immune escape based on literature and TCM theory.

Objective To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2). Methods We designed,developed,and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection.The precision of the liquid transfer and temperature control was tested.A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction(RT-PCR).The entire process,from SARS-CoV-2 nucleic acid extraction to amplification,was evaluated. Results The precision of the syringe transfer volume was 19.2±1.9 μL(set value was 20),32.2±1.6(set value was 30),and 57.2±3.5(set value was 60).Temperature control in the amplification tube was measured at 60.0±0.0 ℃(set value was 60)and 95.1±0.2 ℃(set value was 95)respectively.SARS-Cov-2 nucleic acid extraction yield through the device was 7.10×106 copies/mL,while a commercial kit yielded 2.98×106 copies/mL.The mean time to complete the entire assay,from SARS-CoV-2 nucleic acid extraction to amplification detection,was 36 min and 45 s.The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL. Conclusion The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test(POCT).

Objective:This study aimed to develop a rapid and accurate integrated nucleic acid detection method tailored for the influenza virus.Methods:We designed primers and probes targeting the predominant influenza virus strains circulating in China in recent years. These were integrated with extraction and amplification reagents and a point of care testing (POCT) system to facilitate a seamless and expedited process involving nucleic acid extraction, reaction system preparation, amplification, and result interpretation for the influenza virus. The specificity of the POCT system was evaluated using cultured influenza viruses, while its cross-reactivity was assessed against common respiratory pathogens, including adenovirus and respiratory syncytial virus.Results:Our study successfully developed duplex amplification primers and probes for both influenza A and B viruses, achieving a detection threshold as low as 500 copies/ml. Specificity tests confirmed that the detection reagents did not show cross-reactivity with other respiratory pathogens such as adenovirus and respiratory syncytial virus. The POCT-based rapid nucleic acid detection method for influenza virus was established, it is capable of completing the entire process from nucleic acid extraction to amplification and result interpretation within 50 minutes, while enabling real-time data upload.Conclusions:The POCT-based rapid influenza virus detection kit developed in this study offers a " sample in, results out" convenience, making it suitable for rapid influenza virus detection in primary care settings. This innovation has significant potential for clinical application.

【Objective】 To investigate the current state of infant responsive caregiving and to analyze its correlation with caregiver parenting confidence, in order to provide theoretical basis for improving the level of responsive care for infants and young children. 【Methods】 A cross-sectional survey was conducted from October 2022 to February 2023 to select 1 028 infants and young children under 3 years old who underwent health examinations in the Department of Child Healthcare. Participants completed a general data questionnaire, the Karitane Parenting Confidence Scale, and the Infant Responsive Caregiving Scale. Hierarchical multiple linear regression analysis was used to investigate the related influencing factors of infant responsive care and to analyze the relationship between caregiver parenting confidence and responsive caregiving. 【Results】 The level of responsive caregiving was found to be associated with various factors such as infant age(χ²=21.196), mode of pregnancy(Z=-2.072), history of pregnancy protection during pregnancy(Z=-4.713), history of pregnancy complications (Z=-4.504), gestational week at birth(χ²=41.358), small for term infants(Z=-3.497), neonatal intracranial hemorrhage(Z=-5.425), neonatal hyperbilirubinemia(Z=-2.184), maternal education level(χ²=9.419), family income(χ²=11.211) as well as type of family (χ²=15.360)(P < 0.05). Additionally, a significant correlation was observed between parenting confidence and responsive caregiving(r=0.421,P<0.001). Hierarchical multiple linear regression analysis revealed that caregiver parenting confidence had a significant positive effect on the level of responsive caregiving (B=0.623, P<0.05), even after controlling for the effect of demographic factors. 【Conclusion】 The level of infant responsive caregiving is influenced by caregiver parenting confidence, and increasing caregiver parenting confidence can effectively improve the quality of responsive caregiving for infants and young children.

【Objective】 To develop the Visual Cognitive Ability Assessment Scale for Preschool Children and to evaluate its reliability and validity, in order to provide reference for clinical evaluation of visual cognitive ability of preschool children. 【Methods】 1) From November 2021 to February 2022, construct the dimension framework was constructed and the pool of scale items was compiled according to the theory. 2) In March to June 2022, items were screened preliminarily through group discussion and Delphi method. In August 2022, the entries was revised by a pre-survey in a small sample (n=50). 3) Parents of children aged 4 - 7 from 8 kindergartens in 4 main urban areas of Nanjing were investigated by stratified cluster random sampling method in September to December 2022. The valid sample of the first survey (n=344) was analyzed to conduct item analysis and re-test reliability analysis, the valid sample of the second survey (n=695) was tested for reliability and confirmatory factor analysis, then the scale was finally compiled and evaluated. 【Results】 1) The scale contained 19 items in 4 dimensions:visual memory, discerning vision, spatial vision and visual integration. All items passed the project analysis test. 2) The Cronbach′s α coefficient of each dimension ranged from 0.604 to 0.886, and the Cronbach′s α coefficient of the whole scale was 0.917. During the two surveys, the scores of each item were correlated, and the average retest reliability coefficient was 0.601 (P< 0.05). 3) Content validity index (S-CVI) at scale level was 0.91, and item level content validity index (I-CVI) ranged from 0.8 to 1.0. After several rounds of model modification, the confirmatory factor model fit well. 【Conclusion】 The reliability and validity of the Visual Cognitive Ability Assessment Scale for Preschool Children are acceptable and meet the requirements of the scale, which provides a practical tool for clinical screening of visual and cognitive disorders.