Supporting her as the situation changes: A qualitative study of spousal support strategies for patients with breast cancer in China.

OBJECTIVE: Spouses who are the major source of social support for married breast cancer patients sometimes do not know how to support the patient effectively. This study aimed to investigate the experiences and strategies of spouses identified as supportive for patients throughout the disease. METHODS: A qualitative study using semi-structured in-depth interviews was conducted with 22 husbands of Chinese women with breast cancer, who had effectively supported their wives. Thematic analysis was used for data analysis. RESULTS: Three themes emerged from the data: (a) following the diagnosis, the spouse focused on "problem solving under stress" by preparing the patient for the physician's disclosure of the diagnosis, helping her to cope with the shock, and aiding her in dealing with the treatment recommendations; (b) during treatment, the spouse focused on "functional compensation" to offset the patient's reduced self-care and family care abilities; and (c) following treatment, the spouse focused on "role return" by adapting to changes in the patient and assisting her return to the family and society. CONCLUSION: Chinese spouses of women with breast cancer exhibited support strategies that varied with disease progress. Healthcare providers should aid spouses in providing support according to the changing needs of patients with breast cancer.

Monitoring the Response of PD-L1 Expression to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Nonsmall-Cell Lung Cancer Xenografts by Immuno-PET Imaging.

Accumulating evidence has suggested that the tumor microenvironment of nonsmall-cell lung cancer (NSCLC) may be impacted by chemotherapy, radiotherapy, or epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). PD-L1 is an important biomarker in the tumor microenvironment that can predict patient response to immunotherapies. Therefore, it is highly desirable to achieve a real-time, noninvasive assessment of PD-L1 expression, which can provide critical information for recruiting patients as well as monitoring therapeutic efficacy. We herein studied the EGFR-TKI-induced effects on PD-L1 levels in NSCLC tumor models using immuno-PET imaging with 89Zr-Df-KN035, an imaging tracer previously established by our group. A549 human NSCLC xenografts were established in BALB/c nude mice and treated with different doses of an EGFR-TKI gefitinib. PET imaging with 89Zr-Df-KN035 was performed before and after the treatment to evaluate PD-L1 expression, which was further verified by immunohistochemical staining. Our results demonstrate that 89Zr-Df-KN035 can specifically evaluate PD-L1 levels in NSCLC tumor models. Compared to the untreated control, the high dose of gefitinib inhibited tumor growth and lowered the tumor uptake of 89Zr-Df-KN035. In comparison, the low dose of gefitinib did not affect tumor growth, although the extensive tumor necrosis also led to the lower uptake of 89Zr-Df-KN035. In conclusion, our results demonstrate that immuno-PET imaging with 89Zr-Df-KN035 is a promising tool to noninvasively monitor PD-L1 expression in NSCLC treated with EGFR-TKIs and can be used to optimize treatment plans for immunotherapy.

Immuno-PET Imaging of 89Zr Labeled Anti-PD-L1 Domain Antibody.

Recently, various immuno-PET tracers based on monoclonal antibodies (mAbs), engineered scaffold proteins, and peptides were developed to target either programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 (PD-L1), showing promise in assessment of immune checkpoints. We sought to develop an immunotherapeutic agent based PET probe that enables real-time assessment of PD-L1 expression and evaluation of antibody drug biodistribution to select eligible candidates for anti-PD-1/PD-L1 immunotherapies. KN035, a 79.6 kDa size anti-PD-L1 domain antibody under analysis in clinical trials, was used to develop the immuno-PET probe, 89Zr-Df-KN035. Immuno-PET studies were performed to monitor PD-L1 levels in nude mice bearing LN229 xenografts with positive expression for PD-L1, and to evaluate the whole-body biodistribution in healthy non-human primates (NHPs). LN229 xenografts were markedly visualized from 24 h after injection of 89Zr-Df-KN035, with elevated accumulation persisting for up to 120 h. Tumor radioactivity was notably reduced in the presence of excess KN035. Mouse ex vivo biodistribution studies performed at 24 and 120 h revealed tumor-to-muscle ratios as high as 5.64 ± 0.65 and 7.70 ± 1.37, respectively. In the NHP model, PET imaging demonstrated low background. The liver and kidney showed moderate accumulation with the highest SUVmean value of 1.15 ± 0.15 and 2.13 ± 0.10 at 72 h, respectively. The spleen, lymph nodes, and salivary glands were also slightly visualized. In conclusion, 89Zr-Df-KN035, a novel anti-PD-L1 domain antibody-based probe, shows the feasibility of noninvasive in vivo evaluation of PD-L1 expression. This work further provides a template for immunotherapeutic agent based imaging to evaluate human PD-L1 expression and to augment our understanding of therapeutic agent biodistribution, leading to better therapeutic strategies in the future.

Development and characterization of a novel long-acting recombinant follicle stimulating hormone agonist by fusing Fc to an FSH-ß subunit.

STUDY QUESTION: Does a novel long-acting recombinant human FSH, KN015, a heterodimer composed of FSHα and FSHß-Fc/Fc, offer a potential FSH alternative? SUMMARY ANSWER: KN015 had in vitro activity and superior in vivo bioactivity than recombinant human FSH (rhFSH), suggesting KN015 could serve as a potential FSH agonist for clinical therapy. WHAT IS KNOWN ALREADY: rhFSH has very short half-life so that repeat injections are needed, resulting in discomfort and inconvenience for patients. The longest-acting rhFSH available in clinics is corifollitropin alpha (FSH-CTP), but its half-life is not long enough to sustain the whole therapy period, and additional injections of rhFSH are needed. STUDY DESIGN, SIZE, DURATION: Plasmids containing FSHα, FSHß-Fc and Fc cDNA were transfected into Chinese hamster ovary (CHO) cells for KN015 production. The pharmacokinetics of KN015 was investigated in 6-week-old SD rats (n = 6/group) and healthy Cynomolgus monkeys in two different dose groups (n = 2/group). A series of experiments were designed for in vitro and in vivo characterization of the bioactivity of KN015 relative to rhFSH. PARTICIPANTS/MATERIALS, SETTING, METHODS: The purity and molecular weight of KN015 were determined by reducing and non-reducing SDS-PAGE. To measure KN015 half-life, sera were collected at increasing time points and the remaining FSH concentration was measured by enzyme-linked immunosorbent assay. To assess the bioactivity of KN015 versus rhFSH in vitro, firstly cAMP production was assessed in CHO cells expressing FSH receptor (FSHR) with the treatment of Fc/Fc, rhFSH or KN015 at eight different doses (0.03, 0.09, 0.28, 0.83, 2.5, 7.5, 22.5, 67.5 nM), and secondly cumulus oocyte complexes (COCs; n = 20/group) of ICR mice (primed-PMSG 44 h before sacrificed) were collected and cultured in medium containing 1.25 pM Fc/Fc, rhFSH or KN015 at 37°C and then germinal vesicle breakdown (GVBD) and COC expansion were observed at 4 and 16 h, respectively. The in vivo activity of KN015 was compared with rhFSH by ovary weight gain and ovulation assays. In the former, ovary weight gains in 21-day-old female SD rats, after a single subcutaneous injection of KN015, were compared with those after several injections of rhFSH over a range of doses (n = 8/group). Sera were harvested for estradiol (E2) analysis, and the ovaries were processed for hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL), RT-PCR and western blot. In the latter, 26-day-old female SD rats (n = 8/group) were injected with different doses of KN015 or rhFSH, and were sacrificed at 24 h after an injection of hCG (20 IU/rat). Moreover, the molecular responses stimulated by KN015 or rhFSH in the ovary were also analyzed through detecting expression of the FSH target genes (Cyp19a1, Fshr and Lhcgr) and phosphatidylinositide 3-kinase (PI3K) pathway activation. MAIN RESULTS AND THE ROLE OF CHANCE: KN015 has a molecular weight of 82 kD and its half-life is 84 h in SD rats (10-fold longer than that of rhFSH) and 215 h in Cynomolgus monkeys. The EC50 value of the cAMP induction in CHO cells (KN015 versus rhFSH, 1.84 versus 0.87 nM), COC expansion and oocyte maturation assays showed KN015 had approximately half of rhFSH's activity in vitro. A single dose of KN015 (1.5 pmol/rat, 166.1 ± 19.7 mg, P < 0.01) stimulated significantly larger ovary weight gain than several injections of rhFSH (1.5 pmol/rat, 59.3 ± 28.1 mg, P < 0.01). The serum E2 level in the KN015 group was significantly higher than that in rhFSH group. The number of oocytes obtained by ovulation induction was comparable with or higher in the KN015 group than in the rhFSH group. KN015 was more effective than rhFSH in inducing FSH target genes (Cyp19a1, Fshr, Lhcgr) or activating the PI3K pathway in vivo. Moreover, a single injection of KN015 promoted granulosa cell proliferation and prevented follicle atresia to the same extent as several injections of rhFSH. LIMITATIONS, REASONS FOR CAUTION: All assays in this study were operated only in animals and clinical trials are needed to confirm they can be extrapolated to humans. WIDER IMPLICATIONS OF THE FINDINGS: KN015 is a valuable alternative to FSH and may have great potential for therapeutic applications. STUDY FUNDING/COMPETING INTERESTS: This study was supported by National Basic Research Program of China (2011|CB944504, 2012CB944403) and National Natural Science Foundation of China (81172473, 31371449). The authors have no conflicts of interest to declare.

Posttraumatic growth and psychological distress in Chinese early-stage breast cancer survivors: a longitudinal study.

OBJECTIVE: To describe the dynamic changes in posttraumatic growth (PTG) and psychological distress in hospitalized early-stage breast cancer (BC) survivors over a 6-month period. METHODS: A longitudinal study design was adopted. The PTG inventory (PTGI) and distress management screening measure were used 3 months after diagnosis, then again at 6 and 9 months after diagnosis. For baseline data, 155 BC patients who were receiving chemotherapy were selected from four first-class tertiary hospitals in Beijing from April 2010 to March 2011 using a purposive sampling method. Of these, 120 BC patients completed the follow-up investigation. A repeated measures analysis of variance, followed by least significant difference post-hoc analysis, was used to compare PTG and psychological distress. RESULTS: The total score of the PTGI was 62.72 ± 14.66 in BC survivors at 3 months after diagnosis.There was a weak negative relationship between PTG and psychological distress (r = ­0.282, p<0.001).PTG increased and psychological distress decreased from 3 to 9 months after diagnosis. The PTGI scores were 63.24 ± 14.21, 68.26 ± 15.29, and 70.29 ± 16.07 at 3, 6, and 9 months after diagnosis, respectively, with distress thermometer scores of 3.62 ± 1.98, 2.59 ± 2.00, and 2.51 ± 1.00, respectively. CONCLUSIONS: At 3 months after diagnosis, BC survivors develop PTG at a low level while they are receiving chemotherapy. PTG showed a weak negative association with psychological distress. The level of PTG shows an increasing tendency, whereas the degree of psychological distress exhibits a downward trend in the 9 months after diagnosis.

Ki-67 Change in Anthracyline-containing Neoadjuvant Chemotherapy Response in Breast Cancer.

OBJECTIVE: Anthracycline-containing regimens are irreplaceable in neoadjuvant chemotherapy (NAC) for breast cancer (BC) at present. However, 30% of early breast cancer (EBC) patients are resistant to anthracycline-containing chemotherapy, leading to poor prognosis and higher mortality. Ki-67 is associated with the prognosis and response to therapy, and it changes after NAC. METHODS: A total of 105 BC patients who received anthracycline-containing NAC were enrolled. Then, the optimal model of Ki-67 was selected, and its predictive efficacy was analyzed. Immunohistochemistry (IHC) was used to determine the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) status and Ki-67 level. Fluorescent in situ hybridization (FISH) was used to verify the HER-2 when the IHC score was 2+. RESULTS: The post-NAC Ki67 level after treatment with anthracycline drugs was lower than pre-NAC Ki-67 (19.6%±23.3% vs. 45.6%±23.1%, P<0.001). Furthermore, patients with the Ki-67 decrease had a border line higher pathological complete response (pCR) rate (17.2% vs. 0.0%, P=0.068), and a higher overall response rate (ORR) (73.6% vs. 27.8%, P<0.001), when compared to patients without the Ki-67 decrease. The ΔKi-67 and ΔKi-67% were valuable markers for the prediction of both the pCR rate and ORR. The area under the curve (AUC) for ΔKi-67 on pCR and ORR was 0.809 (0.698-0.921) and 0.755 (0.655-0.855), respectively, while the AUC for ΔKi-67% on pCR and ORR was 0.857 (0.742-0.972) and 0.720 (0.618-0.822), respectively. Multivariate logistic regression model 1 revealed that ΔKi-67 was an independent predictor for both pCR [odds ratio (OR)=61.030, 95% confidence interval (CI)=4.709-790.965; P=0.002] and ORR (OR=10.001, 95% CI: 3.044-32.858; P<0.001). Multivariate logistic regression model 2 revealed that ΔKi-67% was also an independent predictor for both pCR (OR=408.922, 95% CI=8.908-18771.224; P=0.002) and ORR (OR=5.419, 95% CI=1.842-15.943; P=0.002). CONCLUSIONS: The present study results suggest that ΔKi67 and ΔKi67% are candidate predictors for anthracycline-containing NAC response, and that they may provide various information for further systematic therapy after surgery in clinical practice.

CG001, a C3b-targeted complement inhibitor blocks three complement pathways: development and preclinical evaluation.

Excessively activated or dysregulated complement activation may contribute to the pathogenesis of a wide range of human diseases, thus leading to a surge in complement inhibitors. Herein, we developed a human-derived and antibody-like C3b-targeted fusion protein (CRIg-FH-Fc) *2, termed CG001, that could potently block all three complement pathways. CRIg and FH bind to distinct sites in C3b and synergistically inhibit complement activation. CRIg occupancy in C3b prevents the recruitment of C3 and C5 substrates, while FH occupancy in C3b accelerates the decay of C3/C5 convertases and promotes the Factor I-mediated degradation and inactivation of C3b. CG001 also showed therapeutic effects in AP-induced hemolytic mouse and CP-induced MsPGN rat models. In the pharmacological/toxicological evaluation in rats and cynomolgus monkeys, CG001 displayed an antibody-like pharmacokinetic profile, a convincing complement inhibitory effect, and no observable toxic effects. Therefore, CG001 holds substantial potential for human clinical studies.

Factors associated with perceived cognitive function in breast cancer patients treated with chemotherapy: A multicenter cross-sectional study.

PURPOSE: This study aimed to investigate the factors associated with perceived cognitive function among breast cancer patients treated with chemotherapy in China. METHODS: The study was a multicenter cross-sectional design. Data were collected from 10 public hospitals in China between April 2022 and February 2023. A total of 741 participants completed questionnaires assessing sociodemographic and medical characteristics, perceived cognitive function, sleep quality, fatigue, anxiety, and depression. Hierarchical multiple regression analysis was used to assess the determinants of cognitive function. RESULTS: The hierarchical multiple regression model accounted for 31.5% of variation in perceived cognitive function (sociodemographic 4.5%; medical 6.6%; exercise frequency 6.6%; sleep quality 2.1%; fatigue 2.8%; anxiety combined with depression 9.0%). Education level, chemotherapy type, number of chemotherapy cycles, and cyclophosphamide drug use were significant predisposing factors of perceived cognitive function (p < 0.001). Exercising ≥3 times/week (p < 0.001) was a significant factor positively influencing perceived cognitive function, meanwhile, anxiety (p < 0.001) and depression (p < 0 0.001) were negative factors. CONCLUSION: Our findings suggest that patients with low education levels, postoperative chemotherapy, cyclophosphamide treatment, and a greater number of chemotherapy cycles need more assessment. Sedentary patients, those who have never exercised, and those with anxiety or depression all showed greater cognitive decline. By identifying susceptible populations, encouraging regular exercise, and addressing anxiety and depression, healthcare professionals can contribute significantly to prevent patients' cognitive decline throughout chemotherapy.

Identification of key genes in HER2-positive breast cancer with brain metastasis via bioinformatics methods.

Background: Brain metastasis (BM) represents one of the most common advanced disease states in breast cancer (BC), especially in human epidermal growth factor receptor 2 (HER2)-positive BC, and is associated with poor survival outcomes. Methods: In this study, in-depth analysis of the microarray data from the GSE43837 dataset with 19 BM samples of HER2-positive BC patients and 19 HER2-positive nonmetastatic primary BC samples was conducted. The differentially expressed genes (DEGs) between BM and primary BC samples were identified and function enrichment analysis of the DEGs was conducted to identify potential biological functions. The hub genes were identified by constructing the protein-protein interaction (PPI) network using STRING and Cytoscape. UALCAN and Kaplan-Meier plotter online tools were used to verify the clinical roles of the hub DEGs in HER2-positive BC with BM (BCBM). Results: A total of 1,056 DEGs including 767 downregulated and 289 upregulated genes were identified by comparing the microarray data of the HER2-positive BM and primary BC samples. Functional enrichment analysis demonstrated that the DEGs were mainly enriched in pathways related to extracellular matrix (ECM) organization, cell adhesion, and collagen fibril organization. PPI network analysis identified 14 hub genes. Among these, CD44, COL1A2, MMP14, POSTN, and SOX9 were associated with the survival outcomes of HER2-positive patients. Conclusions: In summary, 5 BM-specific hub genes were identified in the study; those are potential prognostic biomarkers and therapeutic targets for HER2-positive BCBM patients. However, further investigations are necessary to unravel the mechanisms by which these 5 hub genes regulate BM in HER2-positive BC.

The Identification of Gene Expression Profiles Associated with Granulomatous Mastitis.

BACKGROUND: Granulomatous mastitis (GM) is a rare chronic inflammatory disease of the breast. The current therapeutic effects of the antibiotic therapy and surgical or immunomodulatory (steroid) treatment are normally poor due to the unclear etiology. METHOD: This study aimed to identify the differentially expressed mRNAs in GM tissues using RNA sequencing and further explored the functions of differentially expressed mRNAs resulting in GM. Moreover, we revealed the relationship between GM and breast cancer by shared highly expressed genes in GM tissues and breast cancer tissues. RESULTS: A total of 12,115 mRNAs were analyzed in the whole expression profile, and 207 mRNAs (136 upregulated and 71 downregulated mRNAs) were differently expressed between the GM tissues and normal tissues. The enrichment analysis showed that the differentially expressed mRNAs were enriched in the biological processes and played a significant role in the immune system. Besides, the genes expressed significantly highly in breast cancer tissues are found to be enriched with GM genes, which may explain the similar clinical features between breast cancer and GM. We also found that the HSD11B1 gene which was differentially expressed in GM was used as drug target of prednisone, which is a common treatment for GM. CONCLUSION: This study is the first to use sequencing technology to elucidate the genetic mechanisms of GM. The finding of this study may have potential value in GM diagnosis and also provides potential drug targets for GM treatment.

miR-27-3p Enhances the Sensitivity of Triple-Negative Breast Cancer Cells to the Antitumor Agent Olaparib by Targeting PSEN-1, the Catalytic Subunit of Γ-Secretase.

Olaparib has been used in the treatment of triple-negative breast cancer (TNBC) with BRCA mutations. In the present study, we demonstrated the effect of miR-27-3p on the γ-secretase pathway by regulating the sensitivity of TNBC cells to olaparib. miR-27-3p, a microRNA with the potential to target PSEN-1, the catalytic subunit of γ-secretase mediating the second step of the cleavage of the Notch protein, was identified by the online tool miRDB and found to inhibit the expression of PSEN-1 by directly targeting the 3'-untranslated region (3'-UTR) of PSEN-1. The overexpression of miR-27-3p inhibited the activation of the Notch pathway via the inhibition of the cleavage of the Notch protein, mediated by γ-secretase, and, in turn, enhanced the sensitivity of TNBC cells to the antitumor agent olaparib. Transfection with PSEN-1 containing mutated targeting sites for miR-27-3p or the expression vector of the Notch protein intracellular domain (NICD) almost completely blocked the effect of miR-27-3p on the Notch pathway or the sensitivity of TNBC cells to olaparib, respectively. Therefore, our results suggest that the miR-27-3p/γ-secretase axis participates in the regulation of TNBC and that the overexpression of miR-27-3p represents a potential approach to enhancing the sensitivity of TNBC to olaparib.

Nomogram for predicting axillary lymph node pathological response in node-positive breast cancer patients after neoadjuvant chemotherapy.

BACKGROUND: Pathological complete response (pCR) of axillary lymph nodes (ALNs) is frequently achieved in patients with clinically node-positive breast cancer after neoadjuvant chemotherapy (NAC), and ALN status is an important prognostic factor for breast cancer patients. This study aims to develop a new predictive clinical model to assess the ALN pCR rate after NAC. METHODS: This was a retrospective series of 467 patients who had biopsy-proven positive ALNs at diagnosis and underwent ALN dissection from 2007 to 2014 at the National Cancer Center/Cancer Hospital of the Chinese Academy of Medical Sciences. We analyzed the clinicopathologic features of the patients and developed a nomogram to predict the probability of ALN pCR. A multivariable logistic regression stepwise model was used to construct a nomogram to predict ALN pCR in node-positive patients. The adjusted area under the receiver operating characteristic curve (AUC) was calculated to quantify the ability to rank patients by risk. Internal validation was performed using the 50/50 hold-out validation method. The nomogram was externally validated with prospective cohorts of 167 patients from 2016 to 2018 at the Cancer Hospital of the Chinese Academy of Medical Sciences and 114 patients from 2018 to 2020 at Beijing Tiantan Hospital. RESULTS: In this retrospective study, 115 (24.6%) patients achieved ALN pCR after NAC. Multivariate analysis showed that clinical tumor stage (Odds ratio [OR]: 0.321, 95% confidence interval [CI]: 0.121-0.856; P = 0.023); primary tumor response (OR: 0.189; 95% CI: 0.123-0.292; P < 0.001), and estrogen receptor status (OR: 0.530, 95% CI: 0.304-0.925; P = 0.025) were independent predictors of ALN pCR. The nomogram was constructed based on the result of multivariate analysis. In the internal validation of performance of nomogram, the AUCs for the training and test sets were 0.719 and 0.753, respectively. The nomogram was validated in external cohorts with AUCs of 0.720, which demonstrated good discriminatory power in these data sets. CONCLUSION: We developed a nomogram to predict the likelihood of axillary pCR in node-positive breast cancer patients after NAC. The predictive model performed well in multicenter prospective external validation. This practical tool could provide information to surgeons regarding whether to perform additional ALN dissection after NAC. TRIAL REGISTRATION: ChiCTR.org.cn, ChiCTR1800014968.

Non-fibrillar amyloid-beta peptide reduces NMDA-induced neurotoxicity, but not AMPA-induced neurotoxicity.

Amyloid-beta peptide (Abeta) is thought to be linked to the pathogenesis of Alzheimer's disease. Recent studies suggest that Abeta has important physiological roles in addition to its pathological roles. We recently demonstrated that Abeta42 protects hippocampal neurons from glutamate-induced neurotoxicity, but the relationship between Abeta42 assemblies and their neuroprotective effects remains largely unknown. In this study, we prepared non-fibrillar and fibrillar Abeta42 based on the results of the thioflavin T assay, Western blot analysis, and atomic force microscopy, and examined the effects of non-fibrillar and fibrillar Abeta42 on glutamate-induced neurotoxicity. Non-fibrillar Abeta42, but not fibrillar Abeta42, protected hippocampal neurons from glutamate-induced neurotoxicity. Furthermore, non-fibrillar Abeta42 decreased both neurotoxicity and increases in the intracellular Ca(2+) concentration induced by N-methyl-d-aspartate (NMDA), but not by alpha-amino-3-hydrozy-5-methyl-4-isoxazole propionic acid (AMPA). Our results suggest that non-fibrillar Abeta42 protects hippocampal neurons from glutamate-induced neurotoxicity through regulation of the NMDA receptor.

Rac1 inhibition negatively regulates transcriptional activity of the amyloid precursor protein gene.

Rac1, a member of the Rho family GTPases, participates in a variety of cellular functions including lamellipodia formation, actin cytoskeleton organization, cell growth, apoptosis, and neuronal development. Recent studies have implicated Rac1 in cytoskeletal abnormalities, production of reactive oxygen species, and generation of the amyloid beta-peptide (Abeta) observed in Alzheimer's disease. In this study, we examined the relationship between Rac1 and amyloid precursor protein (APP), because the abnormal proteolytic processing of APP is a pathologic feature of Alzheimer's disease. In primary hippocampal neurons, the Rac1-specific inhibitor NSC23766 decreased both Rac1 activity and APP protein levels in a concentration-dependent manner. To elucidate how NSC23766 decreases APP protein levels, we examined the effects of NSC23766 on APP processing, degradation, and biosynthesis. NSC23766 did not increase the levels of the proteolytic products of APP, sAPPalpha, Abeta40, and Abeta42. The proteasome inhibitor lactacystin did not reverse the NSC23766-induced decrease in APP protein levels. NSC23766 did, however, decrease the levels of both APP mRNA and APP protein. Decreased levels of APP mRNA and protein were also observed when HEK293 cells were transfected with an expression vector containing a dominant-negative Rac1 mutant or with siRNA targeting Rac1. By overexpressing progressively deleted fragments of the APP promoter in HEK293 cells, we identified a Rac1 response site at positions -233 to -41 bp in the APP promoter. Taken together, our results suggest that Rac1 regulates transcription of the APP gene in primary hippocampal neurons.

Structural basis of a novel heterodimeric Fc for bispecific antibody production.

Bispecific antibodies provide an efficient tool for combinational clinical therapy. Here we have engineered a heterodimeric Fc for bispecific antibodies production by combining the knob-into-hole and electrostatic steering strategies where a bulky hydrophobic residue Phe405 of the IgG CH3 interface is mutated to a charged residue Lys and Lys409 of the corresponding CH3 domain is mutated to Ala. The crystal structure of this Fc heterodimer solved here at 2.7Å resolution revealed how these two mutations resulted a complementary binding interface and explained why F405K mutation could effectively inhibit Fc homodimer formation during protein expression. An anti-HER2 bispecific antibody derived from trastuzumab and pertuzumab was generated by this heterodimeric Fc. It showed comparable or improved efficacy than the combination of trastuzumab and pertuzumab in inhibiting proliferation of cancer cells in vitro and in vivo. Overall this study shows that the heterodimeric Fc engineered here provides an efficient platform for generating active bispecific antibody for cancer treatment.

Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade.

The use of antibodies to target immune checkpoints, particularly PD-1/PD-L1, has made a profound impact in the field of cancer immunotherapy. Here, we identified KN035, an anti-PD-L1 nanobody that can strongly induce T-cell responses and inhibit tumor growth. The crystal structures of KN035 complexed with PD-L1 and free PD-L1, solved here at 1.7 and 2.7 Å resolution, respectively, show that KN035 competes with PD-1 (programmed death protein 1) for the same flat surface on PD-L1, mainly through a single surface loop of 21 amino acids. This loop forms two short helices and develops key hydrophobic and ionic interactions with PD-L1 residues, such as Ile54, Tyr56 and Arg113, which are also involved in PD-1 binding. The detailed mutagenesis study identified the hotspot residues of the PD-L1 surface and provides an explanation for the stronger (~1 000-fold) binding of KN035 to PD-L1 than PD-1 and its lack of binding to PD-L2. Overall, this study reveals how a single immunoglobulin-variable scaffold of KN035 or PD-1 can bind to a flat protein surface through either a single surface loop or beta-sheet strands; and provides a basis for designing new immune checkpoint blockers and generating bi-specific antibodies for combination therapy.

ADAM29 Expression in Human Breast Cancer and its Effects on Breast Cancer Cells In Vitro.

BACKGROUND: A Disintegrin and Metalloprotease Domain 29 (ADAM29) is involved in many important physiological processes. Recent studies have demonstrated that ADAM29 is a susceptibility locus showing traits as a risk factor for breast cancer under genome-wide significance, however, the clinical relevance and cellular function of ADAM29 in breast cancer have not been reported. MATERIALS AND METHODS: In this study, we assessed the expression levels of ADAM29 in a cohort of human breast cancer specimens. We also used MDA-MB-231 cells with differing ADAM29 expression and assessed the influence of ADAM29 and its mutations on the MDA-MB-231 cell line. RESULTS: Increased transcript expression of ADAM29 was observed in breast cancer tissues compared to normal ones. The expression of ADAM29 and its mutations in different domains significantly influenced proliferation, migration and invasion of breast cancer cells in vitro. CONCLUSION: ADAM29 may represent a prognostic factor in human breast cancer, as well as a novel molecular candidate to be used as a therapeutic target.

Structural analysis of cDNAs coding for 4SNc-Tudor domain protein from fish and their expression in yellowtail organs.

We cloned complementary DNAs for 4SNc-Tudor protein (SN4TDR) from yellowtail (Seriola quinqueradiata), torafugu (Takifugu rubripes), and zebrafish (Danio rerio). This protein contains 4 staphylococcal nuclease domains at the N terminus followed by a Tudor domain. We also identified the 4SNc-Tudor proteins highly homologous to that in yellowtail from the Takifugu genomic database. According to the smart database, these fish proteins had an overlapping Tudor domain (smart00333) with a complete 5 SNc domain (smart00318). In addition, 2 possible translation start sites were observed at the 5' sequences in all 3 fish species. Northern blot analysis of different yellowtail organs showed that the full SN4TDR messenger RNA was approximately 4000 nucleotides long and that its expression was highest in liver and gallbladder, being about 2 to 5 times higher than in kidney, brain, ovary, and gills, and exceedingly low in spleen, heart, and muscle. A minor 2000-nucleotide transcript observed in kidney, spleen, and gallbladder, was attributable to an alternatively spliced variant of this gene. Total proteins extracted from yellowtail liver were fractionated by heparin affinity column chromatography and separated by sodium dodecylsulfate polyacrylamide gel electrophoresis. Analyses by SDS-PAGE and liquid chromatography with tandem mass spectroscopy identified the polypeptide encoded by SN4TDR as a single molecule of 102 kDa.

Ileal interposition reduces blood glucose levels and decreases insulin resistance in a type 2 diabetes mellitus animal model by up-regulating glucagon-like peptide-1 and its receptor.

This study is to explore the possible mechanism of ileal interposition (IT) treatment of glycemic control of the type 2 diabetes mellitus (T2DM) by establishing an IT animal model. Twelve T2DM rats (GK rats) of 8-week old were divided into GK IT surgery group (GK-IT) and GK sham group (GK-Sham). Six Wistar rats were used as the non-T2DM sham group (WS-Sham). Enzyme-linked immunosorbent assay was used to detect plasma insulin concentration and fasting pancreas glucagon-like peptide-1 (GLP-1) concentration changes. Homeostasis model assessment of insulin resistance was used to quantitatively measure insulin resistance. Glucagon-like peptide-1 receptor (GLP-1R) expression was detected by Western blotting. IT significantly decreased fasting blood glucose level and the oral glucose tolerance, and reduced insulin resistance of GK rats by increasing GLP-1 concentration and GLP-1R levels. The postoperative pancreatic ß-cell apoptosis rate of GK-Sham group was significantly higher than those in the GK-IT group and the WS-Sham group. IT significantly reduces blood glucose and decreases insulin resistance by up-regulating GLP-1 concentrations and GLP-1R levels, which may contribute to insulin secretion of pancreatic ß-cells and decreases apoptosis of pancreatic ß-cell.

Ileal interposition surgery-induced improvement of hyperglycemia and insulin resistance in Goto-Kakizaki rats by upregulation of TCF7L2 expression.

The aim of this study was to investigate the effects of ileal interposition (IT) on glucose and insulin resistance (IR) in type 2 diabetic mellitus (T2DM), and the role of T-cell factor 7-like 2 (TCF7L2), formerly known as TCF4, in the downregulation of hyperglycemia following IT. Goto-Kakizaki (GK) rats subjected to IT surgery (GK-IT group), GK rats subjected to sham surgery (GK-Sham group) and Wistar (WS) rats subjected to sham surgery (WS-Sham group) were investigated in this study. Fasting plasma glucose, body weight, food intake per 1 kg body weight, insulin and a homeostasis model assessment of insulin resistance (HOMA-IR) were measured pre- and post-surgery. The rats were euthanized 28 days post-surgery and the pancreas of each rat was dissected. The expression levels of TCF7L2 mRNA and protein were analyzed by quantitative RT-PCR and western blotting, respectively. Our results revealed that IT improved both fasting plasma glucose levels and IR in GK rats by upregulating the expression of the TCF7L2 protein. IT provides a valuable therapeutic option for patients with T2DM. Upregulation of TCF7L2 protein expression may be a possible mechanism underlying the improvement of T2DM following IT.