ObjectiveTo clarify whether METTL14 mediates the core role of acupuncture at Neiguan (PC6) in promoting myelination and improving behavior in young autistic rats through gene intervention technology. MethodsThe ASD model was established by intraperitoneal injection of valproic acid (VPA) in pregnant rats. Male offspring were intracerebroventricularly injected with adenovirus-packaged METTL14 shRNA (sh-METTL14) or its control (sh-NC) on postnatal day 1, with a model group set as well. Subsequently, the juvenile rats were divided into model group, acupuncture group, acupuncture+sh-NC group, and acupuncture+sh-METTL14 group. The acupuncture group received acupuncture at Neiguan (PC6) from postnatal day 7, once daily for 21 consecutive days. Neurobehavioral changes were evaluated by behavioral tests; METTL14 knockdown efficiency and the expression of METTL14, METTL3, and PTEN were detected by quantitative real-time PCR (qRT-PCR) and Western blot (WB); PTEN m⁶A levels were measured by RNA immunoprecipitation-qPCR (RIP-qPCR); myelin ultrastructure, expression of myelin basic protein (MBP) and neurofascin 155 (NF155), and dendritic spine density were observed using transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, qRT-PCR, and primary neuron culture. ResultsBehaviorally, knockdown of METTL14 significantly counteracted the beneficial effects of acupuncture in improving self-grooming, open field exploration, three-chamber social interaction, and Morris water maze learning and memory (P<0.05, P<0.01). Compared with the acupuncture+sh-NC group, the acupuncture+sh-METTL14 group showed significantly decreased mRNA and protein expression of hippocampal METTL14 (P<0.01), and the upregulating effects of acupuncture on METTL3 and PTEN expression were reversed (P<0.01). Meanwhile, knockdown of METTL14 significantly inhibited the acupuncture-induced increase in PTEN m⁶A levels (P<0.01). Morphologically, knockdown of METTL14 attenuated the improvement of myelin structure by acupuncture, reversed the downregulation of MBP and upregulation of NF155 induced by acupuncture, and blocked the increase in dendritic spine density (P<0.05, P<0.01). ConclusionMETTL14 is a key molecule mediating the therapeutic effect of acupuncture at Neiguan. Acupuncture at Neiguan upregulates METTL14, thereby enhancing m⁶A methylation modification of PTEN mRNA to stabilize its expression, ultimately promoting myelin development and improving behavioral symptoms in ASD juvenile rats. This preliminarily reveals the modern biological connotation of “opening Xuanfu and dredging myelin”.

ObjectiveTo clarify whether METTL14 mediates the core role of acupuncture at Neiguan (PC6) in promoting myelination and improving behavior in young autistic rats through gene intervention technology. MethodsThe ASD model was established by intraperitoneal injection of valproic acid (VPA) in pregnant rats. Male offspring were intracerebroventricularly injected with adenovirus-packaged METTL14 shRNA (sh-METTL14) or its control (sh-NC) on postnatal day 1, with a model group set as well. Subsequently, the juvenile rats were divided into model group, acupuncture group, acupuncture+sh-NC group, and acupuncture+sh-METTL14 group. The acupuncture group received acupuncture at Neiguan (PC6) from postnatal day 7, once daily for 21 consecutive days. Neurobehavioral changes were evaluated by behavioral tests; METTL14 knockdown efficiency and the expression of METTL14, METTL3, and PTEN were detected by quantitative real-time PCR (qRT-PCR) and Western blot (WB); PTEN m⁶A levels were measured by RNA immunoprecipitation-qPCR (RIP-qPCR); myelin ultrastructure, expression of myelin basic protein (MBP) and neurofascin 155 (NF155), and dendritic spine density were observed using transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, qRT-PCR, and primary neuron culture. ResultsBehaviorally, knockdown of METTL14 significantly counteracted the beneficial effects of acupuncture in improving self-grooming, open field exploration, three-chamber social interaction, and Morris water maze learning and memory (P<0.05, P<0.01). Compared with the acupuncture+sh-NC group, the acupuncture+sh-METTL14 group showed significantly decreased mRNA and protein expression of hippocampal METTL14 (P<0.01), and the upregulating effects of acupuncture on METTL3 and PTEN expression were reversed (P<0.01). Meanwhile, knockdown of METTL14 significantly inhibited the acupuncture-induced increase in PTEN m⁶A levels (P<0.01). Morphologically, knockdown of METTL14 attenuated the improvement of myelin structure by acupuncture, reversed the downregulation of MBP and upregulation of NF155 induced by acupuncture, and blocked the increase in dendritic spine density (P<0.05, P<0.01). ConclusionMETTL14 is a key molecule mediating the therapeutic effect of acupuncture at Neiguan. Acupuncture at Neiguan upregulates METTL14, thereby enhancing m⁶A methylation modification of PTEN mRNA to stabilize its expression, ultimately promoting myelin development and improving behavioral symptoms in ASD juvenile rats. This preliminarily reveals the modern biological connotation of “opening Xuanfu and dredging myelin”.

OBJECTIVE To screen a single matrix， prepare dissolving microneedles of Pterocarya hupehensis total flavonoids（PHTF-MN）， and characterize it while evaluating its analgesic effects.METHODS Using microneedle morphology and puncture rate as evaluation indicators， the matrix was screened from polyvinyl alcohol （PVA）， polyvinylpyrrolidone K30 （PVP K30）， PVP K90， and sodium hyaluronate with relative molecular weights of 15 000 and 60 000 （HA 1.5w and HA 6w ）. PHTF-MN was prepared by the layer-by-layer casting method with the selected matrix， and the appearance and morphology， total flavonoid content， mechanical properties， puncture performance， dissolution properties， and drug release behavior of the obtained PHTF-MN were characterized. Using male BALB/c mice as test subjects and aspirin （ASA） as a positive control， the analgesic effects of the microneedles were evaluated using a formalin-induced pain model.RESULTS Blank microneedles prepared with 30% HA 1.5w exhibited optimal surface flatness and the membrane puncture rate （through 4 layers of parafilm membrane） reaching 78%. The PHTF-MNs （with PHTF contents of 10， 20， 30， 40， and 50 mg/g in the matrix） exhibited uniform arrays and intact tips， with RSDs for total flavonoid contents all below 7% （ n ＝8）. The single-needle puncture forces were 1.46 and 1.07 N （with PHTF contents of 30 and 50 mg/g in the matrix， respectively）. The skin penetration rate approached 100%， with visible puncture marks in the stratum corneum and drug residue （with a PHTF content of 50 mg/g in the matrix）. After 30 min of fixation， the needles had almost completely dissolved， and the skin retention rates all exceeded 90% （with PHTF contents of 10， 30， and 50 mg/g in the matrix， respectively）. The cumulative release within 8 h was （134.64±2.03） μg/cm 2 ， and the fit to the first-order kinetic equation （ R 2 ＝0.950 1） was highest （with a PHTF content of 50 mg/g in the matrix）. Low- and high-dose PHTF-MN （containing 85.26 and 145.03 μg of total flavonoids per patch， respectively； 1 patch/day） significantly shortened the cumulative time of phase Ⅱ pain response in mice （ P ＜0.05）， with pain inhibition rates （43.67% and 59.78%） comparable to those of ASA （53.70%）.CONCLUSIONS PHTF-MN using HA 1.5w as a single matrix was successfully prepared. This formulation features a simplified preparation process， uniform drug loading， favorable mechanical properties， resistance to fracture， and rapid dissolution. Its drug release behavior conforms to a first-order kinetic model， and its analgesic effect is comparable to that of ASA.

AIM: To delineate the specific mutation responsible for retinitis pigmentosa(RP)in a Yi pedigree, and to analyze the correlation of RHO gene mutation with clinical phenotype.METHODS:A comprehensive clinical evaluation was conducted on the proband diagnosed with RP and other familial members, complemented by a thorough ophthalmic examination. Peripheral blood samples were obtained from the proband and familial members, from which genomic DNA was extracte. Subsequent whole exome sequencing(WES)was employed to identify the variant genes in the proband. The identified variant gene was validated through Sanger sequencing, then an in-depth analysis of the mutation genes was carried out using genetic databases to ascertain the pathogenic mutation sites. Furthermore, an exhaustive analysis was performed to delineate the genotype and phenotype characteristics.RESULTS:The RP pedigree encompasses 5 generations with 42 members, including 19 males and 23 females. A total of 13 cases of RP were identified, consisting of 4 males and 9 females, which conforms to the autosomal dominant inheritance pattern. The clinical features of this family include an early onset age, rapid progression, and a more severe condition. The patients were found to have night blindness around 6 years old, representing the earliest reported case of night blindness in RP families. The retina was manifested by progressive osteocytoid pigmentation of the fundus, a reduced visual field, and significantly decreased or even vanished a and b amplitudes of ERG. The combined results of WES and Sanger sequencing indicated that the proband had a heterozygous missense mutation of the RHO gene c.1040C>T:p.P347L, where the 1 040 base C of cDNA was replaced by T, causing codon 347 to encode leucine instead of proline. Interestingly, this mutation has not been reported in the Chinese population.CONCLUSION:This study confirmed that the mutant gene of RP in a Yi nationality pedigree was RHO(c.1040C>T). This variant leads to the change of codon 347 from encoding proline to encoding leucine, resulting in a severe clinical phenotype among family members. This study provides a certain molecular, clinical, and genetic basis for genetic counseling and gene diagnosis of RHO.

AIM: To delineate the specific mutation responsible for retinitis pigmentosa(RP)in a Yi pedigree, and to analyze the correlation of RHO gene mutation with clinical phenotype.METHODS:A comprehensive clinical evaluation was conducted on the proband diagnosed with RP and other familial members, complemented by a thorough ophthalmic examination. Peripheral blood samples were obtained from the proband and familial members, from which genomic DNA was extracte. Subsequent whole exome sequencing(WES)was employed to identify the variant genes in the proband. The identified variant gene was validated through Sanger sequencing, then an in-depth analysis of the mutation genes was carried out using genetic databases to ascertain the pathogenic mutation sites. Furthermore, an exhaustive analysis was performed to delineate the genotype and phenotype characteristics.RESULTS:The RP pedigree encompasses 5 generations with 42 members, including 19 males and 23 females. A total of 13 cases of RP were identified, consisting of 4 males and 9 females, which conforms to the autosomal dominant inheritance pattern. The clinical features of this family include an early onset age, rapid progression, and a more severe condition. The patients were found to have night blindness around 6 years old, representing the earliest reported case of night blindness in RP families. The retina was manifested by progressive osteocytoid pigmentation of the fundus, a reduced visual field, and significantly decreased or even vanished a and b amplitudes of ERG. The combined results of WES and Sanger sequencing indicated that the proband had a heterozygous missense mutation of the RHO gene c.1040C>T:p.P347L, where the 1 040 base C of cDNA was replaced by T, causing codon 347 to encode leucine instead of proline. Interestingly, this mutation has not been reported in the Chinese population.CONCLUSION:This study confirmed that the mutant gene of RP in a Yi nationality pedigree was RHO(c.1040C>T). This variant leads to the change of codon 347 from encoding proline to encoding leucine, resulting in a severe clinical phenotype among family members. This study provides a certain molecular, clinical, and genetic basis for genetic counseling and gene diagnosis of RHO.

ObjectiveMagnetoencephalography (MEG), a non-invasive neuroimaging technique, meticulously captures the magnetic fields emanating from brain electrical activity. Compared with MEG based on superconducting quantum interference devices (SQUID), MEG based on optically pump magnetometer (OPM) has the advantages of higher sensitivity, better spatial resolution and lower cost. However, most of the current studies are clinical studies, and there is a lack of animal studies on MEG based on OPM technology. Pain, a multifaceted sensory and emotional phenomenon, induces intricate alterations in brain activity, exhibiting notable sex differences. Despite clinical revelations of pain-related neuronal activity through MEG, specific properties remain elusive, and comprehensive laboratory studies on pain-associated brain activity alterations are lacking. The aim of this study was to investigate the effects of inflammatory pain (induced by Complete Freund’s Adjuvant (CFA)) on brain activity in a rat model using the MEG technique, to analysis changes in brain activity during pain perception, and to explore sex differences in pain-related MEG signaling. MethodsThis study utilized adult male and female Sprague-Dawley rats. Inflammatory pain was induced via intraplantar injection of CFA (100 μl, 50% in saline) in the left hind paw, with control groups receiving saline. Pain behavior was assessed using von Frey filaments at baseline and 1 h post-injection. For MEG recording, anesthetized rats had an OPM positioned on their head within a magnetic shield, undergoing two 15-minute sessions: a 5-minute baseline followed by a 10-minute mechanical stimulation phase. Data analysis included artifact removal and time-frequency analysis of spontaneous brain activity using accumulated spectrograms, generating spectrograms focused on the 4-30 Hz frequency range. ResultsMEG recordings in anesthetized rats during resting states and hind paw mechanical stimulation were compared, before and after saline/CFA injections. Mechanical stimulation elevated alpha activity in both male and female rats pre- and post-saline/CFA injections. Saline/CFA injections augmented average power in both sexes compared to pre-injection states. Remarkably, female rats exhibited higher average spectral power 1 h after CFA injection than after saline injection during resting states. Furthermore, despite comparable pain thresholds measured by classical pain behavioral tests post-CFA treatment, female rats displayed higher average power than males in the resting state after CFA injection. ConclusionThese results imply an enhanced perception of inflammatory pain in female rats compared to their male counterparts. Our study exhibits sex differences in alpha activities following CFA injection, highlighting heightened brain alpha activity in female rats during acute inflammatory pain in the resting state. Our study provides a method for OPM-based MEG recordings to be used to study brain activity in anaesthetized animals. In addition, the findings of this study contribute to a deeper understanding of pain-related neural activity and pain sex differences.

Lymphoma is a highly heterogeneous malignancy characterized by complex molecular regulatory mechanisms that result in significant differences in aggressiveness and prognosis across its subtypes.Gene copy number alteration(CNA)analysis,an emerging technology,has become a pivotal tool in the precision re-search and management of lymphoma.By detecting DNA deletions,amplifications,and chromosomal copy number changes,CNA analysis addresses the limitations of traditional cytogenetic techniques,enhances the ac-curacy of subtype classification,and aids in evaluating tumor heterogeneity and disease progression.This re-view provides a comprehensive summary of CNA detection methods and their applications in lymphoma,with a focus on recent advancements in the field.It offers a comparative analysis of CNA detection techniques and discusses their role in precision diagnosis,subtype classification,monitoring disease progression,predicting therapeutic resistance,and assessing prognosis.Additionally,the review explores the potential applications of CNA analysis in uncovering molecular regulatory mechanisms,optimizing therapeutic strategies,and impro-ving patient survival outcomes.

Objective To investigate the therapeutic effects and underlying mechanisms of Ganluqingwen formula on lipopolysaccharide(LPS)-induced acute lung injury/acute respiratory distress syndrome(ALI/ARDS)in mice.Methods Fifty ICR mice were randomly divided into five groups:control,model,and Ganluqingwen formula(GLQW)low dose(7.10 g/kg),medium dose(15.21 g/kg),and high dose(30.42 g/kg)groups,with 10 mice per group.On days 1-3,mice in GLQW groups were daily gavaged with the corresponding dose of GLQW,while control and model groups received equal volumes of saline.On day 4,ALI/ARDS was induced in model and GLQW groups using intraperitoneal injection of LPS(20 mg/kg),while control group received an equal volume of PBS.At 24 h post-treatment,survival rate,wet-to-dry weight ratio(W/D)and lung histological changes(HE staining)were observed.Serum levels of tumor necrosis factor(TNF)-α,interferon gamma(IFN-γ),interleukin(IL)-4,IL-10,IL-12,as well as lung tissue levels of TNF-α,IFN-γ,IL-1β,IL-4,IL-6,IL-10 were measured by ELISA.Western blotting was used to determine the expression levels of NOD-like receptor thermal protein domain associated protein 3(NLRP3),cystatinase-1(Caspase-1),apoptosis-associated speck-like protein(ASC),and membrane perforating protein Gasdermin D(GSDMD)in lung tissue.Results No significant differences in survival rates were observed among the groups(P>0.05).Compared with control group,ELISA and Western blotting results showed that lung tissue W/D,IFN-γ,TNF-α,IL-4,IL-12,IL-1β,IL-6,NLRP3,ASC,and Caspase-1,GSDMD and serum IFN-γ,TNF-α,IL-4,IL-12 levels were significantly higher(P<0.05),and IL-10 levels in lung tissue and serum were significantly lower in mice of model group(P<0.05).Compared with model group,lung tissue W/D,IFN-γ,TNF-α,IL-1β,IL-4,IL-6,IL-12,NLRP3,ASC,Caspase-1,and GSDMD,and serum IFN-γ,TNF-α,IL-4,and IL-12 levels were significantly lower(P<0.05),and lung tissue IL-10 levels were significantly higher(P<0.05)in GLQW low,medium,and high dose groups,with high-dose group showing significantly higher level in serum IL-10(P<0.05).Compared with GLQW low-dose group,the lung tissue levels of IFN-γ,IL-6,NLRP3,ASC,Caspase-1,and GSDMD,and serum TNF-α were significantly lower(P<0.05),and lung and serum IL-10 levels were significantly higher in GLQW high-dose group(P<0.05).HE staining results showed that lung structure was clear and normal in control group;part of the lung interstitium was congested and hemorrhagic,and some of the fine bronchial periphery was infiltrated with inflammatory cells in model group;the phenomena of lung interstitial congestion and hemorrhage were reduced,and the degree of infiltration of inflammatory cells was alleviated in GLQW low-,medium-,and high-dose groups.Conclusion Ganluqingwen formula can delay the development of ALI/ARDS in mice by inhibiting NLRP3/Caspase-1/GSDMD pathway,thereby suppressing cellular pyroptosis.

Objective To explore the correlation between the proximal angle of carotid atherosclerotic plaques and neovascularization scores,and their clinical application value in predicting recurrent cerebral infarction.Methods A total of 88 patients who underwent carotid plaque ultrasound examination in our hospital were selected.According to CT/MRI results,patients were divided into the non-cerebral infarction group(45 cases)and the cerebral infarction group(43 cases).Conventional ultrasound examination was performed followed by contrast-enhanced ultrasound.Plaque length,thickness and proximal angle were measured,and the neovascularization score of the proximal end was evaluated using contrast-enhanced ultrasound,and the results were compared and analyzed.Results In the cerebral infarction group,plaque thickness,proximal angle,and neovascularization score were significantly higher than those in the non-cerebral infarction group(P＜0.05),while there was no significant difference in plaque length.The proportion of plaques with a proximal neovascularization score of 2 or 3 was higher in the cerebral infarction group than those of the non-cerebral infarction group(79.1%vs.24.4%,P＜0.01).A positive correlation was found between the proximal angle and neovascularization score in all patients(rs=0.374,P＜0.01).There was no significant difference between the area under the ROC curve for neovascularization score in predicting recurrent cerebral infarction and the proximal angle(P＞0.05).The optimal cutoff value of the proximal angle was 18.8,and the sensitivity and the specificity for predicting recurrent cerebral infarction were 93.0%and 62.2%.In the cerebral infarction group,seven patients(16.3%)had recurrent infarction within one year,and these patients had higher proximal neovascularization scores,with angles greater than 18.8°.Conclusion There is a strong correlation between proximal angle of carotid plaques and neovascularization score,which has a positive predictive role in the recurrence of cerebral infarction,providing a reliable auxiliary diagnostic basis for clinical practice.

Objective:To investigate the effects of Roxadustat and recombinant human erythropoietin (rHuEPO) on novel inflammatory immune indices in anemic patients with non-dialysis type 2 diabetes mellitus combined with chronic kidney disease (CKD).Methods:A retrospective case-control study was performed in this study. Patients with non-dialysis type 2 diabetes mellitus combined with CKD admitted to Shanghai First People's Hospital from December 2015 to December 2023 were selected. Among those patients, 252 cases were treated with rHuEPO (rHuEPO group) and 103 cases were treated with Roxadustat (Roxadustat group). Both group had a course of treatment over three months. The baseline data and novel inflammatory immunity indices, systemic immuno-inflammation index (SII), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) were compared between the two groups of patients before and after 3 months of treatment.Results:The differences in gender, age, body mass index, systolic blood pressure, diastolic blood pressure, history of cardiovascular and cerebrovascular disease, history of hypertension, estimated glomerular filtration rate, and use of hypoglycemic, antihypertensive, and lipid-lowering medications were not statistically significant when compared between the two groups of patients (all P>0.05). Before treatment, the differences in NLR, PLR, SII, and LMR between the two groups were not statistically significant (all P>0.05); after 3 months of treatment, NLR, PLR, SII, and LMR were lower in both groups than before treatment [rHuEPO group: (2.3±0.8)% vs. (2.8±0.8)%, (83±33)% vs. (160±49)%, (2.3±0.8)% vs. (3.1±0.7)%, (476±227)% vs. (594±243)%, with t values of 9.25, 23. 20, 26.67, and 9.62, respectively, all P<0.001; Roxadustat group: (1.7±0.6)% versus (2.9±1.0)%, (72±30)% versus (162±47)%, (2.0±0.8)% versus (3.1±0.9)%, (408±151)% versus (605±267)%, with t values of 8. 38, 14.27, 8.62, and 5.97, respectively, all P<0.001], and NLR, PLR, and SII were lower in the Roxadustat group than in the rHuEPO group ( t=5.00, P<0.001, t=2.44, P=0.015, t=2.09, P=0.040). Conclusion:In patients with anemia in non-dialysis type 2 diabetes mellitus associated with CKD,Roxadustat had similar ability of reducing the level of novel inflammatory markers compared to rHuEPO.