Mycophenolate mofetil as a successful therapy for idiopathic granulomatous mastitis.

Idiopathic granulomatous mastitis (IGM) is a benign chronic inflammatory breast disease of unknown etiology. No consensus exists as to the best therapeutic approach, though treatment choices include antibiotics, drainage, surgical excision, steroids, methotrexate, and observation. Herein we report a case of idiopathic granulomatous mastitis that was refractory to methotrexate and intralesional and systemic steroids but responded well to mycophenolate mofetil 1500mg twice daily. To our knowledge, this is the first report of the effective use of mycophenolate mofetil in idiopathic granulomatous mastitis.

Podoimbricatin A, a cytotoxic diterpenoid with an unprecedented 6/6/5/6-fused tetracyclic ring system from the twigs and leaves of Podocarpus imbricatus.

Podoimbricatin A (1), a diterpenoid possessing an unprecedented 6/6/5/6-fused tetracyclic ring system, and podoimbricatin B (2), a new abietane-type diterpenoid, together with four previously reported diterpenoids and a cyclic peptide were isolated from the twigs and leaves of Podocarpus imbricatus. Their structures were established on the basis of extensive spectroscopic analyses and the relative configuration of 1 was confirmed by single-crystal X-ray diffraction analysis. A plausible biogenetic pathway involving hetero-Diels-Alder cycloaddition as a key reaction is proposed for 1. Compounds 1, 5, and 7 showed inhibitory effects against the A549 and NCI-H292 cancer cell lines.

Individualised monitoring programme for pulmonary rehabilitation of patients with chronic obstructive pulmonary disease-study protocol for a randomised controlled trial.

Background: Chronic obstructive pulmonary disease (COPD) is characterised mainly by exertional dyspnoea, which may lead to activity reduction. Pulmonary rehabilitation (PR) is considered capable of mitigating these impairments. However, access to PR is limited to specialised centres, especially during the coronavirus disease 2019 pandemic. Moreover, low-cost home rehabilitation programmes have non-individualised prescriptions, which might lead to inconsistent clinical effects in patients with COPD. Therefore, it is important to develop new, low-cost protocols involving individualised prescriptions and staff supervision. Methods: This is a descriptive protocol for a randomised controlled study at the Grade III A Hospital in Tianjin. The sample size was calculated according to a described formula. Fifty-six participants will be selected and randomly allocated into two groups: (I) control (traditional PR training, medication, and nursing interventions); and (II) intervention [PR training in the hospital and at home by the Cardiopulmonary Rehabilitation System Management Platform (CSM)]. The protocol will be performed twice a week for 8 consecutive weeks in the outpatient clinic, and the training will be performed by the application of the CSM system in the final 6 months of the trial. The study will assess lung function and physical fitness and analyse the scores of the modified Medical Research Council Dyspnoea Scale, the COPD assessment test, the International Classification of Functioning, Disability, and Health, and the 6-minute walk test before and after the training protocol. Comparison of differences will be performed using repeated measures analysis of variance, a linear mixed effects analysis, or a non-parametric test, which will include only participants who completed all outcome measures and followed the intervention protocol. The study results will be disseminated through presentations at scientific conferences and publications in peer-reviewed journals. Discussion: The new, low-cost supervised rehabilitation programmes are expected to present positive results, making PR programmes more accessible and effective for patients with COPD. Trial Registration: The study was registered in the Chinese Clinical Trial Registry: ChiCTR2000040723.

Clinical characteristics, susceptibility profiles, and treatment of nocardiosis: a multicenter retrospective study in 2015-2021.

OBJECTIVES: This study aimed to investigate the microbiological characteristics, antimicrobial resistance profiles, antibiotic choice, and outcomes of Nocardia infection in various centers over a 7-year period (from 2015 to 2021). METHODS: We retrospectively analyzed the medical records of all hospitalized patients diagnosed with Nocardia between 2015 and 2021. The isolates were identified to the species level through the sequencing of 16S ribosomal RNA or secA1 or ropB genes. The susceptibility profiles were determined using the broth microdilution method. RESULTS: Of the 130 nocardiosis cases, 99 (76.2%) were established as pulmonary infection, of which the most common underlying disease was chronic lung disease (40.4%, 40/99), including bronchiectasis, chronic obstructive pulmonary disease, and chronic bronchitis. Among 130 isolates, 12 species were identified, with the most common species being Nocardia cyriacigeorgica (37.7%) and Nocardia farcinica (20.8%). All Nocardia strains were susceptible to linezolid and amikacin, and the susceptibility rate of trimethoprim-sulfamethoxazole (TMP-SMX) was 97.7%. Of the 130 patients, 86 (66.2%) received TMP-SMX monotherapy or multidrug regimen. Furthermore, 92.3% patients who were treated achieved clinical improvement. CONCLUSION: TMP-SMX was the treatment of choice for nocardiosis, and other combination drugs with TMP-SMX therapy yielded even better results.

Applications of Isolated-Check Visual Evoked Potential in Early Stage of Open-Angle Glaucoma Patients.

BACKGROUND: Standard automated perimetry does not sufficiently detect early open-angle glaucoma (OAG) in the clinic. New visual function tests for early glaucoma damage are therefore needed. The present study evaluated whether an isolated-check visual evoked potential (icVEP) could be used to detect visual function abnormalities in early-stage OAG and to explore potential related factors. METHODS: This was a cross-sectional study. Thirty-seven OAG patients with early-stage visual field loss (mean deviation ≥ -6.00 dB) detected by the Humphrey Field Analyzer (30-2 SITA program) and 26 controls were included in this study. Optical coherence tomography (OCT) was used to detect retinal nerve fiber layer (RNFL) defects. The icVEP preferentially evaluates the magnocellular-ON pathway. VEPs were recorded and signal-to-noise ratios (SNRs) were derived based on multivariate analysis. Eyes that yielded an SNR ≤1 were considered abnormal. Receiver operating characteristic (ROC) curve analysis was used to estimate the accuracy of group classification. Correlations between SNRs and related factors were analyzed. RESULTS: Based on an SNR criterion of 1, the icVEP had a sensitivity of 62.2% and a specificity of 92.3% for diagnosing early-stage OAG with 74.6% classification accuracy. The ROC curve analysis, however, suggested that an SNR criterion of 0.93 would produce the highest classification accuracy (77.3%). Both RNFL thinning in the temporal superior quadrant on OCT and number of abnormal test points in the central 11° visual field (pattern deviation, P < 0.5%) significantly correlated with the SNR (P < 0.05). CONCLUSIONS: The icVEP detected visual function abnormalities in approximately 3/5 of eyes with early-stage OAG with greater than 90% specificity. SNR correlated with both a decrease in RNFL thickness and severity of central visual field loss.

Cost of Routine Herpes Simplex Virus Infection Visits to U.S. Emergency Departments 2006-2013.

INTRODUCTION: Little is known about emergency department (ED) utilization for herpes simplex viruses (HSV) types 1 and 2 in the United States. Our goal was to determine the utilization and cost burden associated with HSV infection visits to U.S. EDs in recent years from 2006-2013. METHODS: We analyzed the Nationwide Emergency Department Sample (NEDS) database, the largest national database of hospital-based ED visits in the U.S., to determine the number of visits and the cost associated with HSV visits from 2006-2013. We also analyzed trends across years. RESULTS: From 2006-2013, there were 704,728 ED visits with a primary diagnosis of HSV infection. Of these, 658,805 (93.5%) resulted in routine discharges without inpatient admission, amounting to a total ED charge of $543.0 million. After adjusting for inflation, there was a doubling of total ED spending for HSV from 2006 to 2013 ($45.0 million to $90.7 million) and a 24% increase in number of visits (73,227 visits in 2006, vs. 90,627 visits in 2013). ED visits for genital herpes have increased while visits for herpes gingivostomatitis have decreased. CONCLUSION: HSV-associated ED use and associated costs have increased between 2006-2013. Most of these cases could likely be managed in non-emergent outpatient settings as 93.5% of visits resulted in routine discharges without admission. Our findings add to knowledge regarding HSV utilization and epidemiology in the U.S. and highlight the need for continued prevention, patient education, and emphasis of care in non-emergency settings to prevent unnecessary ED utilization.

Study on the mechanism of haitongpi prescription extract ointment in the treatment of knee osteoarthritis based on transcriptome / 中国骨伤

OBJECTIVE@#To explore the mechanism of Haitongpi Prescription extract in the treatment of knee osteoarthritis based on transcriptome.@*METHODS@#Total of 12 SPF grade rats were divided into control group(group C), model group(group M), and Haitongpi prescription group(group HP). The knee osteoarthritis rat model was established using the Panicker method for group M and group HP, and group HP was intervened by local topical application of Haitongpi Prescription extract for 4 weeks. Total RNA from mouse knee cartilage was extracted and three sets of differential genes were obtained through sequencing.Differential genes were prediction and analysis through GO function and KEGG pathway enrichment analysis.@*RESULTS@#A total of 109 differentially expressed genes were identified in Group C versus Group M, while 118 differentially expressed genes were identified in Group M versus Group HP, resulting in a total of 28 genes. GO functional enrichment analysis showed that the mechanism of HP extract in treating knee osteoarthritis mainly involved immunoglobulin mediated immune response, immunoglobulin complexes, and antigen binding; KEGG pathway enrichment analysis showed correlation with tumor necrosis factor (TNF) signaling pathway, interleukin 17(IL-17) signaling pathway, and estrogen signaling pathway.@*CONCLUSION@#HP extract can exert therapeutic effects on knee osteoarthritis through mechanisms such as immunoglobulin mediated immune response, immunoglobulin complexes, and antigen binding, as well as signaling pathways such as TNF signaling pathway, IL-17 signaling pathway, and estrogen signaling pathway.

Effects of omitting elective neck irradiation to nodal Level IB in nasopharyngeal carcinoma patients with negative Level IB lymph nodes treated by intensity-modulated radiotherapy: a Phase 2 study.

OBJECTIVE: To investigate the need for elective neck irradiation (ENI) to nodal Level IB in patients with nasopharyngeal carcinoma (NPC) with negative Level IB lymph nodes (IB-negative) treated by intensity-modulated radiotherapy (IMRT). METHODS: We conducted a Phase 2 prospective study in 123 newly diagnosed IB-negative patients with NPC treated by IMRT, who met at least 1 of the following criteria: (1) unilateral or bilateral Level II involvement with 1 of the following: Level IIA involvement or any Level II node ≥2 cm/with extracapsular spread; (2) ≥2 unilateral node-positive regions. Bilateral Level IB nodes were not contoured as part of the treatment target and treated electively. Level IB regional recurrence rate; pattern of treatment failure; 3-year overall survival (3y-OS), 3-year local control (3y-LC) and 3-year regional control (3y-RC) rates; toxicities; and dosimetric data for planning target volumes, organs at risk, Level IB and submandibular glands (SMGs) were evaluated. RESULTS: Two patients developed failures at Level IB (1.6%). The 3y-LC, 3y-RC and 3y-OS rates were 93.5%, 93.5% and 78.0%, respectively. Bilateral Level IB received unplanned high-dose irradiation with a mean dose (Dmean) ≥50 Gy in 60% of patients. The average Dmean of bilateral SMGs was approximately 53 Gy. CONCLUSION: ENI to Level IB may be unnecessary in IB-negative patients with NPC treated by IMRT. A further Phase 3 study is warranted. ADVANCES IN KNOWLEDGE: Based on the results of this first Phase 2 study, we suggest omitting ENI to Level IB in Ib-negative patients with NPC with extensive nodal disease treated by IMRT.

Identification of Bupleurum marginatum var. Wall. ex DC stenophyllum (Wolff) Shan et Li and its analogues based on ITS2 barcode / 中草药

Objective: DNA barcoding technology, a molecular identification method, is applied to distinguish Bupleurum marginatum var. stenophyllum from its analogues in order to ensure the quality and clinical curative effect. Methods: In this study, the internal transcribed spacer 2 (ITS2) regions of 50 samples were amplified by PCR and sequenced bi-directionally. Obtained sequences were assembled using CodonCode Aligner. The genetic distances were computed by MEGA 6.0 in accordance with the kimura 2-parameter (K2P) model and the phylogenetic tree was constructed by Neighbor-joining (NJ) method. Moreover, the secondary structure of ITS2 was predicted using ITS2 database websites. Results: The intra-specific genetic distances were smaller than inter-specific ones in ITS2 regions of B. marginatum var. stenophyllum. The NJ tree and secondary structure results could distinctly differentiate B. marginatum var. stenophyllum and its analogues. Conclusion: ITS2 sequence can scientifically and reliably identify the authenticity of B. marginatum var. stenophyllum and could provide more new and reliable techniques to ensure clinical safety of this traditional Chinese medicine.

Correlation analysis of hepatic steatosis, blood lipids and uric acid metabolism in elderly patients with chronic hepatitis B / 公共卫生与预防医学

Objective To analyze the correlation of hepatic steatosis with blood lipids and uric acid metabolism in elderly patients with chronic hepatitis B (CHB). Methods The clinical data of 120 patients with CHB admitted to the hospital from January to December 2021 were retrospectively analyzed. According to the presence or absence of hepatic steatosis, the patients were divided into steatosis group (n=35) and non-steatosis group (n=85). The general clinical data, serological indicators of hepatitis B virus, blood lipid and uric acid levels were compared between the two groups. The correlation of hepatic steatosis grading with blood lipids and uric acid metabolism was analyzed. Results The inflammation and fibrosis degree of liver tissues were significantly different in the two groups (P0.05). Pearson correlation analysis found that the grade of hepatic steatosis in patients with CHB was negatively correlated with liver tissue inflammation, fibrosis degree and HDL-C level (P<0.05), and positively correlated with TG and TC levels (P<0.05). Conclusion Elderly patients with CHB and hepatic steatosis have abnormal blood lipid metabolism. Hepatic steatosis will exacerbate abnormal blood lipid metabolism but not liver tissue inflammation or fibrosis degree. Clinically, attention should be paid to blood lipid monitoring of elderly patients with CHB.

MicroRNA expression in the serum of dust exposure population / 中国职业医学

OBJECTIVE: To analyze the microRNA levels in the serum of dust exposure population. METHODS: By cluster random sampling method,479 dust exposure workers were selected as dust exposure group. Four hundred and thirty-four normal healthy people without occupational dust exposure history were selected as control group. Forty-three pneumoconiosis patients were selected as pneumoconiosis group. The peripheral venous blood of the objects of 3 groups were collected,and the plasma serum were separated for detecting the relative expression of miR-16,miR-21,miR-29 a,miR-155,miR-200 c,miR-204 and miR-206 in serum by real time quantitative polymerase chain reaction. The difference of microRNA expression in the above 3 groups was observed. RESULTS: From high to low,the relative expression levels of miR-16,miR-29 a and miR-200 c were pneumoconiosis group > dust exposure group > control group( P < 0. 05),while the relative expression levels of miR-204 were control group > dust exposure group > pneumoconiosis group( P < 0. 05).The relative expression levels of miR-21 in dust exposure and pneumoconiosis groups were higher than that of control group,respectively( P < 0. 05). The relative expression level of miR-155 in dust exposure group was lower than that of control group( P < 0. 05). The relative expression level of miR-206 in pneumoconiosis group was higher than those of dust exposure group and control group respectively( P < 0. 05). CONCLUSION: The miR-16,miR-29 a,miR-200 c and miR-204 could be used as biomarkers in early diagnosis of pneumoconiosis disease.

Impact of basic diseases on the clinical symptoms and prognosis of cryptococcosis / 医学研究生学报

Objective Cryptococcosis is a potential life-threatening systemic mycosis with a heterogeneous susceptible popu-lation which is classified into three groups according to the current guidelines, including AIDS patients, organ transplantation recipients ( OTR) , and non-HIV-infected and non-transplant hosts ( NHNT) .This study aimed to discuss the influence of basic diseases on the clinical features and prognosis of NHNT cryptococcosis patients. Methods Using a retrospective cohort study design, we retrieved the clinical data about 73 NHNT cryptococcosis patients treated in Jinling Hospital.Based on the presence or absence of immunodefi-ciency or infection-increasing complications, we divided the patients into a basic disease group ( n=35) and a non-basic disease group ( n=38) and analyzed their clinical characteristics, chest radiographic features, laboratory results, and clinical outcomes. Results Compared with the non-basic disease group, the basic disease group showed a significantly higher incidence rate of disseminated disea-ses (62.9%vs 21.1%, P<0.01), more cases of patchy consolidation (47.4%vs 16.7%, P<0.05) and mixed lesion (31.6%vs 3.3%, P<0.05) in chest radiography, and a higher mortality (30.0%vs 5.3%, P=0.016). Conclusion Basic diseases have a great impact on the clinical features and prognosis of NHNT cryptococcosis.NHNT patients with basic diseases are susceptible to dis-seminated diseases with severer clinical symptoms and a higher mortality.

Construction of microRNA Let-7b-mediated recombinant influenza A (H1N1) virus with mutated PB1 / 医学研究生学报

Objective Vaccination is a most effective method for the prevention of severe diseases caused by pandemic influenza and microRNA ( miRNA) mediated gene silencing has offered a novel approach to the construction of new vaccines.Our study aimed to construct a recombinant influenza A ( H1 N1 ) virus with the PB1 gene that carries the target fragment of miRNA Let-7b. Methods After comparing the sequence of the A/Nanjing/108/2009 H1N1 viral fragments with that of Let-7b, we selected PB1 as the optimal gene sequence, inserted the Let-7b binding target gene into PB1, ligated the modified fragments with pDP 2000, and named the recombinant plasmids pDP-mu-PB1 and pDP-sclb-PB1, respectively.We co-transfected the MDCK and 293T cells with the recombinant and other seven plasmids and injected the supernatant into the allantoic cavity of the chickenembryo for virus propagation, followed by detection of the virus by hemagglutination ( HA) assay and measurement of the viral titer by TCID50 .We amplified the viral cRNA by RT-PCR and identified the viruses by agarose gel electrophoresis and nucleotide sequence analysis. Results PB1 was the optimal sequence ( 83 bp -107bp) for the attenuation of viruses.The HA-titers of miRT-H1N1 and scbl-H1N1 were 1∶32 and 1∶64, and their viral loads were 4.68 ×105 and 7.94 ×104 TCID50/mL, respectively.Nucleotide sequence analysis showed the expected fragment in the rescued virus. Conclusion A recombinant strain vaccine was successfully constructed, which has laid the foundation for fur-ther assessment of virulence.

Experimental Study on Apoptosis of Kasumi-1 Cells Induced by Sertraline and Its Molecular Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the biological effects of sertraline, one of psychotropic drugs, on actue myeloid leukemia cell line Kasumi-1.</p><p><b>METHODS</b>Cells were treated by different concentrations of sertraline for different times. The effects of sertraline were evaluated by cell growth, cell morphology, cell cycle distribution and markers of cell apoptosis, respectively. Western blot was used to detect the expression change of related proteins.</p><p><b>RESULTS</b>Sertraline could inhibit cell proliferation and induce apoptosis. After treatment with 15 µmol/L and 20 µmol/L sertraline for 24 h, the inhibitory rate of Kasumi-1 cell proliferation was (19.00 ± 7.37)% and (47.90 ± 11.19)%, respectively. Meanwhile, compared with the control group, the percentage of Annexin V positive cells in Kasumi-1 cells treated with sertraline for 24 h raised obviously from (9.71 ± 2.12)% to (20.54 ± 2.52)% and (45.37 ± 7.88)% (P < 0.01), respectively. The cells were arrested in G0/G1 and G2/M phase. In addition, it was found that sertraline could also down-regulate the level of translationally controlled tumor protein (TCTP) in Kasumi-1 cells.</p><p><b>CONCLUSION</b>Sertraline can significantly induce the apoptosis of Kasumi-1 cells, that probably is associated with the down-regulation of TCTP protein expression.</p>

Construction and identification of a recombinant adenoviral vector expressing murine dendritic cell-associ-atedC-type lectin-1 / 医学研究生学报

Objective Dendritic cell-associatedC-type lectin-1 ( Dectin-1) is one of the most important receptors in antifungal innate immune response.This study was to construct a recombinant adenovirus vector expressing themurine Dectin-1gene and acquire a high-concentration adenovirus by amplification and purification. Methods The PCR amplification product CLEC7A-pIRES2-EGFP was cloned into the intermediate vector pDONR221, and then recom-bined with the backbone vector pAD/CMV/V5-DEST to produce a re-combinant plasmid pAD-CLEC7A-pIRE2S -EGFP.The recombinant plasmid was linearized with Pac I and transfected into human embryon-ic kidney ( HEK293) cells to produce recombinant adenovirus pAD-CLEC7Ap-IRES 2-EGFP. The adenovirus was propagated in the HEK293 cells and purified by filtering through the cellulose acetate membrane and concentrating column.Fluorescence microscopy and re-al-time PCR were used to determine the expression of the Dectin-1 gene. Results PCR identification, enzyme digestion, and sequen-cing results manifested theDectin-1 gene in the vector, with the final adenovirus titer of 5×1011 IU/mL.Fluorescence microscopy revealed green fluorescence and real-time PCR assay confirmed that the expression of Dectin-1 was improved by 8677.25 times. Conclusion A relatively high-titer adenovirus expressing Dectin-1 was acquired,which may help to further study the high expression of Dectin-1 in anti-fungal innate immunity in vitro and in vivo.

CT axial imaging of the iliolumbar ligament and its significance on locating lumbosacral vertebral segments / 中国骨伤

<p><b>OBJECTIVE</b>To study the CT axial manifestations of iliolumbar ligament(ILL) and discusses its clinical effects on locating lumbosacral vertebral segments.</p><p><b>METHODS</b>From May 2008 to March 2010, 706 adult patients diagnosed lumbar disc disease were performed with axial scans by single slice helical CT. Among the patients, 436 patients were male and 270 patients were female, ranging in age from 25 to 82 years, the median age was 44 years, 78 cases with lumbosacral transitional vertebrae (LSTV) were verified by X-radiography or fluoroscopy. The morphology, origin and insertion, courses of ILL and the relationship of ligament and spinal segments on axial plane images were used to study. The location method of spinal segments by ILL was compared with the other four location methods on CT.</p><p><b>RESULTS</b>Of the 628 cases with normal lumbosacral segmentations sides of ligament, the main part of ILL originated from L5 transverse processes and terminated at the iliac crest, the morphological characters were divided into two types: double band (71.8%, 451/628) and single band (28.2%, 177/628). The tiny branches from posterior and outside edge of L4, lumbar disc were seen simultaneity in 3 cases. The ILL of 78 cases with LSTV all also originated from L5 transverse processes. Using ILL as a marker of the L5 vertebral level, 78 cases with LSTV were correctly numbered, the accuracy rate was higher than the other location methods, there was statistical significance between the location method by ILL and the location method by iliac crest (P < 0.05).</p><p><b>CONCLUSION</b>The main part of ILL originates from L5 transverse processes, the anatomic location is relatively steady and can be clearly displayed on axial CT, which can be used as a measure in the idenlification of LSTV in clinical practice, it is worthy to be applied widely in basic-level hospitals.</p>

Quantitative analysis of synaptic vesicle release and readily releasable pool size in hippocampal neurons / 生理学报

In central nervous system only a limited number of vesicles exist in the presynaptic terminals. The size and fusion modes of the vesicles were particularly important because of their potential impact on neuronal communications. Efficient methods were needed to analyze the recycling kinetics of synaptic vesicle and the size of readily releasable pool (RRP). In this study, fluorescent dyes with different affinity for membranes (FM1-43 with high affinity and FM2-10 with low affinity) were used to stain the functional synaptic vesicles of cultured hippocampal neurons and the kinetics of vesicle recycling was measured. The results showed that the destaining proportion was larger for FM2-10 than that for FM1-43 during the first trial, while it was greater for FM1-43 than FM2-10 during the second and third trials (first round, 93.0%+/-5.9% versus 57.9%+/-3.5% for FM2-10 and FM1-43, respectively, P<0.0001; second round, 1.4%+/-3.8% versus 24.0%+/-2.3%, P<0.0001; third round, 2.3%+/-1.6% versus 8.6%+/-1.5%, P=0.005). The results indicated that rapid endocytosis existed not only in the first round but also occurred when the vesicles were reused. Moreover, Both high-frequency stimuli and hypertonic sucrose stimuli were used to estimate the RRP sizes in the mix cultured hippocampal inhibitory neurons at 13-14 days in vitro (DIV). We found that the RRP size estimated by hypertonic sucrose stimuli [(200+/-23.0) pC] was much larger than that estimated by high-frequency stimuli [(51.1+/-10.5) pC]. One possible reason for the discrepancies in RRP estimates is that in mix cultured conditions, one neuron may receive inputs from several neurons and hypertonic sucrose stimuli will cause RRP of all those neurons release, while using dual patch recording, only the connection between two neurons was analyzed. Thus, to exclude out the impacts of inputs numbers on RRP sizes, it is more reasonable to use high-frequency stimuli to estimate the RRP size in mix cultured neurons.