Establishment and validation of a multigene model to predict the risk of relapse in hormone receptor-positive early-stage Chinese breast cancer patients / 中华医学杂志(英文版)

BACKGROUND@#Breast cancer patients who are positive for hormone receptor typically exhibit a favorable prognosis. It is controversial whether chemotherapy is necessary for them after surgery. Our study aimed to establish a multigene model to predict the relapse of hormone receptor-positive early-stage Chinese breast cancer after surgery and direct individualized application of chemotherapy in breast cancer patients after surgery.@*METHODS@#In this study, differentially expressed genes (DEGs) were identified between relapse and nonrelapse breast cancer groups based on RNA sequencing. Gene set enrichment analysis (GSEA) was performed to identify potential relapse-relevant pathways. CIBERSORT and Microenvironment Cell Populations-counter algorithms were used to analyze immune infiltration. The least absolute shrinkage and selection operator (LASSO) regression, log-rank tests, and multiple Cox regression were performed to identify prognostic signatures. A predictive model was developed and validated based on Kaplan-Meier analysis, receiver operating characteristic curve (ROC).@*RESULTS@#A total of 234 out of 487 patients were enrolled in this study, and 1588 DEGs were identified between the relapse and nonrelapse groups. GSEA results showed that immune-related pathways were enriched in the nonrelapse group, whereas cell cycle- and metabolism-relevant pathways were enriched in the relapse group. A predictive model was developed using three genes ( CKMT1B , SMR3B , and OR11M1P ) generated from the LASSO regression. The model stratified breast cancer patients into high- and low-risk subgroups with significantly different prognostic statuses, and our model was independent of other clinical factors. Time-dependent ROC showed high predictive performance of the model.@*CONCLUSIONS@#A multigene model was established from RNA-sequencing data to direct risk classification and predict relapse of hormone receptor-positive breast cancer in Chinese patients. Utilization of the model could provide individualized evaluation of chemotherapy after surgery for breast cancer patients.

International and domestic researches about neurogenic bladder: a visualized analysis / 中国康复理论与实践

@#Objective To evaluate the development, hot spots and trends of the fields of neurogenic bladder.Methods The relevant articles of neurogenic bladder from January, 2000 to June, 2021 in CNKI and Web of Science were retrieved.The countries, authors, institutions, cited reference and keywords were extracted with CiteSpace to draw knowledge mapping. Results and Conclusion A total of 5 064 articles were enrolled. At present, the research on the field of neurogenic bladder is in a stable period of development, and this field has been widely concerned by scholars at home and abroad. The cooperation between domestic authors and institutions is not close enough compared with foreign countries, and domestic cooperation is more between medical schools and their respective affiliated hospitals. In the future, China can further strengthen cross-regional and cross-agency cooperation. Low-frequency electrical stimulation and sacral nerve regulation are seem to be research hotspots, and children's neurogenic bladder and robot-assisted technologies are also needed more attention.

Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay

The papain-like protease (PLpro) of SARS-CoV-2 is a validated antiviral drug target. PLpro is involved in the cleavage of viral polyproteins and antagonizing host innate immune response through its deubiquitinating and deISG15ylating activities, rendering it a high profile antiviral drug target. Through a FRET-based high-throughput screening, several hits were identified as PLpro inhibitors with IC50 values at the single-digit micromolar range. Subsequent lead optimization led to potent inhibitors with IC50 values ranging from 0.56 to 0.90 {micro}M. To help prioritize lead compounds for the cellular antiviral assay against SARS-CoV-2, we developed the cell-based FlipGFP assay that is suitable for quantifying the intracellular enzymatic inhibition potency of PLpro inhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PLpro assay, Jun9-53-2 and Jun9-72-2, inhibited SARS-CoV-2 replication in Caco-2 hACE2 cells with EC50 values of 8.89 and 8.32 {micro}M, respectively, which were 3-fold more potent than GRL0617 (EC50 = 25.1 {micro}M). The X-ray crystal structures of PLpro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to the more closed conformation. Overall, the PLpro inhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PLpro assay might be a suitable surrogate for screening PLpro inhibitors in the BSL-2 setting.

Potent in vitro Neutralization of SARS-CoV-2 by Hetero-bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain

Cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, while RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VHH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity KD ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (KD ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, fusion of two Nbs with non-overlapping epitopes resulted in hetero-bivalent Nbs, namely aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (KD of 59.2 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND50) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL ([~]0.043 nM) and 3.18 ng/mL ([~]0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnosis reagents for COVID-19. ImportanceTo date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics including SARS-CoV-2 targeting antibodies remains critical. Due to their small size (13-15 kDa), highly solubility and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multi-valent formats, compared to the conventional antibody. Here, we report a serial of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.

An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity

The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II and XII, each containing a reactive warhead that covalently modifies the catalytic Cys145. In this study, we report an expedited drug discovery approach by coupling structure-based design and Ugi four-component (Ugi-4CR) reaction methodology to the design of non-covalent Mpro inhibitors. The most potent compound 23R had cellular antiviral activity similar to covalent inhibitors such as GC376. Our designs were guided by overlaying the structure of SARS-CoV Mpro + ML188 (R), a non-covalent inhibitor derived from Ug-4CR, with the X-ray crystal structures of SARS-CoV-2 Mpro + calpain inhibitor XII/GC376/UAWJ247. Binding site analysis suggests a strategy of extending the P2 and P3 substitutions in ML188 (R) to achieve optimal shape complementary with SARS-CoV-2 Mpro. Lead optimization led to the discovery of 23R, which inhibits SARS-CoV-2 Mpro and SARS-CoV-2 viral replication with an IC50 of 0.31 M and EC50 of 1.27 M, respectively. The binding and specificity of 23R to SARS-CoV-2 Mpro were confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 Mpro with 23R revealed the P2 biphenyl fits snuggly into the S2 pocket and the benzyl group in the -methylbenzyl faces towards the core of the enzyme, occupying a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study revealed the most potent non-covalent SARS-CoV-2 Mpro inhibitors reported to date and a novel binding pocket that can be explored for Mpro inhibitor design.

Signatures and mechanisms of efficacious therapeutic ribonucleotides against SARS-CoV-2 revealed by analysis of its replicase using magnetic tweezers

The nucleotide analog Remdesivir (RDV) is the only FDA-approved antiviral therapy to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The physical basis for efficient utilization of RDV by SARS-CoV-2 polymerase is unknown. Here, we characterize the impact of RDV and other nucleotide analogs on RNA synthesis by the polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. The location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We reveal that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into deep backtrack, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this nucleotide analog well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases. TeaserWe revise Remdesivirs mechanism of action and reveal SARS-CoV-2 ability to evade interferon-induced antiviral ddhCTP

Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L

The main protease (Mpro) of SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, is a key antiviral drug target. While most SARS-CoV-2 Mpro inhibitors have a {gamma}-lactam glutamine surrogate at the P1 position, we recently discovered several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II/XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. To determine the binding mode of these calpain inhibitors and establish a structure-activity relationship, we solved X-ray crystal structures of Mpro in complex with calpain inhibitors II and XII, and three analogues of GC-376, one of the most potent Mpro inhibitors in vitro. The structure of Mpro with calpain inhibitor II confirmed the S1 pocket of Mpro can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. Interestingly, the structure of calpain inhibitor XII revealed an unexpected, inverted binding pose where the P1 pyridine inserts in the S1 pocket and the P1 norvaline is positioned in the S1 pocket. The overall conformation is semi-helical, wrapping around the catalytic core, in contrast to the extended conformation of other peptidomimetic inhibitors. Additionally, the structures of three GC-376 analogues UAWJ246, UAWJ247, and UAWJ248 provide insight to the sidechain preference of the S1, S2, S3 and S4 pockets, and the superior cell-based activity of the aldehyde warhead compared with the -ketoamide. Taken together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of Mpro inhibitors as SARS-CoV-2 antivirals.

Clinical analysis of medical hemostatic materials for prevention and treatment of subcutaneous hydrops after breast cancer operation / 中国综合临床

Objective@#To observe the effect of medical instant hemostasis gauze combined with filament speed instant gauze on the drainage and flap healing after modified radical mastectomy.@*Methods@#From August 2015 to August 2016, a total of 80 patients with modified radical mastectomy for breast cancer admitted to Huanxing Tumor Hospital, Chaoyang District, Beijing were selected.According to the random number table method, 80 patients who were ready for modified radical mastectomy for breast cancer were randomly divided into study group (40 cases) and control group (40 cases). Two kinds of hemostatic materials (medical hemolytic hemostatic gauze combined with fibril quick hemostatic gauze) were applied to the surgical wounds in the study group during the operation, while no medical hemostatic materials were used in the control group during the operation, and the other treatment was the same as that in the study group.Total drainage volume and drainage tube removal time were compared between the two groups 1 to 5 days after operation.@*Results@#There were no statistically significant differences in the age, body mass index, and effusion production between the two groups (all P>0.05). The total drainage volume of the study group was (289.23±5.36) ml, and the total drainage volume of the control group was (492.15±8.56) ml.The difference between the two groups was statistically significant (t=8.543, P<0.05). The drainage time of the study group was (6.24±1.23) days, and the extraction time of the control group was (10.12±2.21) days.The difference between the two groups was statistically significant (t=6.203, P<0.05).@*Conclusion@#In addition to hemostatic function, using absorbable hemostatic gauze combined with surgicel fibrillar during the surgical process can significantly reduce postoperative subcutaneous fluid accumulation.

Clinical analysis of medical hemostatic materials for prevention and treatment of subcutaneous hydrops after breast cancer operation / 中国综合临床

Objective:To observe the effect of medical instant hemostasis gauze combined with filament speed instant gauze on the drainage and flap healing after modified radical mastectomy.Methods:From August 2015 to August 2016, a total of 80 patients with modified radical mastectomy for breast cancer admitted to Huanxing Tumor Hospital, Chaoyang District, Beijing were selected.According to the random number table method, 80 patients who were ready for modified radical mastectomy for breast cancer were randomly divided into study group (40 cases) and control group (40 cases). Two kinds of hemostatic materials (medical hemolytic hemostatic gauze combined with fibril quick hemostatic gauze) were applied to the surgical wounds in the study group during the operation, while no medical hemostatic materials were used in the control group during the operation, and the other treatment was the same as that in the study group.Total drainage volume and drainage tube removal time were compared between the two groups 1 to 5 days after operation.Results:There were no statistically significant differences in the age, body mass index, and effusion production between the two groups (all P>0.05). The total drainage volume of the study group was (289.23±5.36) ml, and the total drainage volume of the control group was (492.15±8.56) ml.The difference between the two groups was statistically significant ( t=8.543, P<0.05). The drainage time of the study group was (6.24±1.23) days, and the extraction time of the control group was (10.12±2.21) days.The difference between the two groups was statistically significant ( t=6.203, P<0.05). Conclusion:In addition to hemostatic function, using absorbable hemostatic gauze combined with surgicel fibrillar during the surgical process can significantly reduce postoperative subcutaneous fluid accumulation.

Sentinel lymph node biopsy after neoadjuvant chemotherapy for axillary lymph node-positive breast cancer / 中华普通外科杂志

Objective To investigate the feasibility and clinical significance of sentinel lymph node biopsy(SLNB) after neoadjuvant chemotherapy (NAC) for axillary lymph node-positive breast cancer.Methods Enrolled for a prospective cohort study were 167 patients from Jan 2016 to Jan 2018 with axillary lymph node-positive breast cancer admitted to the Cancer Hospital of Chinese Academy of Medical Sciences.SLNB was performed after NAC by lymphatic dual mapping,followed by axillary lymph node dissection.The primary end point was sentinel lymph node identification rate (IR) and false negative rate (FNR).Results 62 patients (37.1％) had complete pathological response of axillary lymph nodes.There was a significant difference of NAC response in patients with different subtypes (P ＜0.001).The IR of SLNB after NAC was 94.6％,the FNR was 6.7％,the sensitivity was 93.3％,the specificity was 100％,and the accuracy was 95.8％.Univariate analysis showed that there was no significant difference between tumor stage,hormone receptor status,HER2 expression,and pathological remission in SLN detection group and the SLN undetected group (P ＞ 0.05).The proportion of patients who received breast conserving surgery in the undetected group was significantly higher than that in the test group (P =0.006).Conclusions Sentinel lymph node biopsy after breast neoadjuvant chemotherapy by lymphatic dual mapping is highly accurate with a high identification rate and a low false negative rate.

Deficiency of Follistatin-Like Protein 1 Accelerates the Growth of Breast Cancer Cells at Lung Metastatic Sites / 한국유방암학회지

PURPOSE: Follistatin-like protein 1 (FSTL1) is a secreted glycoprotein that has been shown to play a role in various types of cancer. However, the clinical significance and function of FSTL1 in breast cancer have not been reported. We investigated the role of FSTL1 in breast cancer in this study. METHODS: Enzyme-linked immunosorbent assays, western blot analysis, and reverse transcription polymerase chain reaction were used to monitor the expression of FSTL1 in breast cancer tissue and in serum samples from breast cancer patients. We employed a 4T1 breast cancer model and Fstl1(+/−) mice for in vivo studies. Hematoxylin and eosin staining, western blot analysis, and RNA sequencing were used to analyze the effect of FSTL1 on primary tumor growth and lung metastasis. RESULTS: We demonstrated that the expression of FSTL1 is reduced in both the breast cancer tissue and the serum of breast cancer patients. We showed that reduced levels of FSTL1 in serum correlate with elevated expression of Ki-67 and epidermal growth factor receptor (EGFR) in cancer tissues. Moreover, lowered expression of FSTL1 was associated with decreased survival in breast cancer patients. Experiments on the Fstl1(+/−) mouse model established that FSTL1 deficiency had no effect on primary tumor growth, but increased the lung metastases of breast cancer cells, resulting in reduced survival of tumor-bearing mice. RNA sequencing found significantly reduced expression of Egln3 and increased expression of EGFR in Fstl1(+/−) mice. Thus, our results suggest that FSTL1 may affect the expression of EGFR through Egln3, inhibiting the proliferation of breast cancer cells at lung metastatic sites. CONCLUSION: In conclusion, we suggest a suppressor role of FSTL1 in breast cancer lung metastasis. Furthermore, FSTL1 may represent a potential prognostic biomarker and a candidate therapeutic target in breast cancer patients.

Differential expression and localization of neuropeptide Y peptide in pancreatic islet of diabetic and high fat fed rats.

Neuropeptide Y (NPY) inhibits insulin secretion. Increased numbers of pancreatic islet cells expressing NPY have been observed in type 1 diabetic rats. To understand the functional significance of NPY expression in islet cells, we investigated the effects of high fat feeding and diabetic conditions on the expression and location of NPY expressing cells in normal and diabetic rats. Twenty rats were maintained on either normal chow (ND) or a high fat dietary regimen (HFD) for 4 weeks. In half of each group, type 1 or type 2 diabetes (groups T1DM and T2DM, respectively) was induced by injection of streptozotocin. At 8 weeks rats were euthanized and the pancreases were processed for immunofluorescence labeling (NPY/insulin, NPY/glucagon, NPY/somatostatin, and NPY/pancreatic polypeptide). Compared with the ND group, HFD rats had significantly fewer alpha cells, but beta cells were similar, while T1DM and T2DM rats showed significant increases in the proportions of alpha, delta, and PP cells. Robust increases in NPY-positive islet cells were found in the HFD, T1DM, and T2DM rats compared with ND controls. In ND rats, 99.7% of the NPY-positive cells were PP cells. However, high fat feeding and diabetes resulted in significant increases in NPY-positive delta cells, with concomitant decreases in NPY-positive PP cells. In summary, high-fat feeding and diabetes resulted in changes in the hormonal composition of pancreatic islet and increased number of NPY-expressing islet cells. Under diabetic conditions NPY expression switched from predominantly a characteristic of PP cells to predominantly that of delta cells. This may be a factor in reduced pancreatic hormone secretion during diabetes.

Comparison of glucose and lipid metabolism in two types of Parkinson’ s disease rat models / 解剖学报

Objective To select an ideal Parkinson ’ s disease ( PD) animal model with metabolic abnormalities for subsequent experimental studies .Methods A total of 62 Sprague-Dawley male rats were randomly divided into four groups:damaged medial forebrain bundle ( MFB) model group, damaged medial forebrain bundle ( MFB) sham group, damaged Striatum model group and damaged Striatum sham group .After detecting the rotation experiment , successful model rats of two groups were selected to detect the changes of food intake , body weight , blood glucose and intra-abdominal adipose tissue.Results It was easier to produce a PD model by destroying MFB than striatum .Compared with sham-operated rats, MFB model rats showed significant abnormality both in reduction of body weight [(218.1 ±13.99) g vs (252.7 ±10.1)g, P<0.05] and high blood glucose appeared at 15min and 30min after introperitoneal glucose tolerance test ( IPGTT) .Their perirenal white adipose tissue was significantly reduced ( both left and right side ) .Striatum model rats only appeared decreased food intake [(13.95 ±0.25)g vs (20.23 ±0.86)g, P<0.001] and impaired glucose regulation at 15min, 30min and 60min after IPGTT.Their body weight and adipose tissue did not change significantly .Conclusion No matter in the success rate or metabolism-related indicators , MFB damaged rat model of PD is more suitable to study PD patients with abnormal lipid metabolism compared with Striatum rat model .

Study on the postmortem change of plasma and urine concentrations of H-FABP in rat / 医学研究生学报

Objective: To observe the changes of heart-type fatty acid binding-protein(H-FABP) in the femoral vein(periphe ral blood) and urine of rats with myocardial infarction in different postmortem intervals. Methods: Acute myocardial infarction (AMI) was duplicated by ligating the anterior branch of the left coronary artery following the method of Johns in rats. Concentrations of H-FABP were detected with enzyme-linked immuno-sorbent assay (ELISA). Results: The concentrations of H-FABP in experiment group were obviously higher than that in control group. Along with the time, the concentrations of H-FABP in experiment group rose consistently, the concentration of H-FABP in 12 h group rose markedly, and it was obviously higher than in 48h that in 24h. The H-FABP concentrations in urine of the experiment group were obviouslyhigher than the one in control group. The urine concentrations of H-FABP in experiment group increased by wavy in postmortem intervals, the concentration of H-FABP in (6h) group was obviouslyhigher than that in 0h group, 12h and 24h postmortem, the H-FABP concentrations decreased, but still higher than that of 0h group and 48h group, the concentration of H-FABP rose markedly and washigher than 6h group. Conclusion: Affected by autolysis, the H-FABP concentrations of plasma and urine appeared abnormal raise and can not be used in postmortem diagnosis of AMI.

Assessing and Subtypes of Writing in Chinese School Children / 中国心理卫生杂志

Objective: To develop tools for assessing writing skills in Chinese children and classifying its subtypes. Methods: Using tasks of character copy, free writing according to pictures, shape copy and writing to dictation, 65 children with writing difficulties reported by their parents were investigated.Results: the inter rater reliability of character copy, free writing, and shape copy were high. Children could be classified into four subtypes based on character copy and free writing.Conclusion:Character writing skill and written expression are two factors for assessing Chinese writing. Patterns between subtypes at these two tasks are different.

Molecular cloning,nucleotides sequence and transient expression of S and M genome segment of hantavirus strain 84Fli / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To sequence, analyze and express the nucleotide sequences of S and M segments of hantavirus strain 84Fli.</p><p><b>METHODS</b>S and M segments of hantavirus 84Fli strain were amplified by RT-PCR, the PCR products were cloned into plasmid pCR2.1-TOPOr. Three clones of each segment which have been sequenced were randomly selected. The coding region of S and M segments were amplified by PCR, and cloned into expressing vector pcDNA3.0. Transient expression of nucleocapsid protein, G1 and G2 glycoproteins in COS7 cells were performed by Lipofectin transfection. The expression of NP, G1 and G2 have been conformed by using immunofluorescence, Western blot and immuno-precipitation methods.</p><p><b>RESULTS</b>The full length S and M segments cDNA have been amplified and cloned. The S and M segments of hantavirus strain 84Fli contained 1 688 and 3 616 nucleotides respectively.</p><p><b>CONCLUSIONS</b>Deduced amino acid sequences of NP and glycoproteins revealed high homologue to other Hantaan viruses. NP, G1 and G2 proteins of 84Fli can be transiently expressed in COS7 cells.</p>