Dysregulated Ribosome Biogenesis Is a Targetable Vulnerability in Triple-Negative Breast Cancer: MRPS27 as a Key Mediator of the Stemness-inhibitory Effect of Lovastatin.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited effective therapeutic options readily available. We have previously demonstrated that lovastatin, an FDA-approved lipid-lowering drug, selectively inhibits the stemness properties of TNBC. However, the intracellular targets of lovastatin in TNBC remain largely unknown. Here, we unexpectedly uncovered ribosome biogenesis as the predominant pathway targeted by lovastatin in TNBC. Lovastatin induced the translocation of ribosome biogenesis-related proteins including nucleophosmin (NPM), nucleolar and coiled-body phosphoprotein 1 (NOLC1), and the ribosomal protein RPL3. Lovastatin also suppressed the transcript levels of rRNAs and increased the nuclear protein level and transcriptional activity of p53, a master mediator of nucleolar stress. A prognostic model generated from 10 ribosome biogenesis-related genes showed outstanding performance in predicting the survival of TNBC patients. Mitochondrial ribosomal protein S27 (MRPS27), the top-ranked risky model gene, was highly expressed and correlated with tumor stage and lymph node involvement in TNBC. Mechanistically, MRPS27 knockdown inhibited the stemness properties and the malignant phenotypes of TNBC. Overexpression of MRPS27 attenuated the stemness-inhibitory effect of lovastatin in TNBC cells. Our findings reveal that dysregulated ribosome biogenesis is a targetable vulnerability and targeting MRPS27 could be a novel therapeutic strategy for TNBC patients.

Effect of Tai Chi vs Aerobic Exercise on Blood Pressure in Patients With Prehypertension: A Randomized Clinical Trial.

Importance: Prehypertension increases the risk of developing hypertension and other cardiovascular diseases. Early and effective intervention for patients with prehypertension is highly important. Objective: To assess the efficacy of Tai Chi vs aerobic exercise in patients with prehypertension. Design, Setting, and Participants: This prospective, single-blinded randomized clinical trial was conducted between July 25, 2019, and January 24, 2022, at 2 tertiary public hospitals in China. Participants included 342 adults aged 18 to 65 years with prehypertension, defined as systolic blood pressure (SBP) of 120 to 139 mm Hg and/or diastolic BP (DBP) of 80 to 89 mm Hg. Interventions: Participants were randomized in a 1:1 ratio to a Tai Chi group (n = 173) or an aerobic exercise group (n = 169). Both groups performed four 60-minute supervised sessions per week for 12 months. Main Outcomes and Measures: The primary outcome was SBP at 12 months obtained in the office setting. Secondary outcomes included SBP at 6 months and DBP at 6 and 12 months obtained in the office setting and 24-hour ambulatory BP at 12 months. Results: Of the 1189 patients screened, 342 (mean [SD] age, 49.3 [11.9] years; 166 men [48.5%] and 176 women [51.5%]) were randomized to 1 of 2 intervention groups: 173 to Tai Chi and 169 to aerobic exercise. At 12 months, the change in office SBP was significantly different between groups by -2.40 (95% CI, -4.39 to -0.41) mm Hg (P = .02), with a mean (SD) change of -7.01 (10.12) mm Hg in the Tai Chi group vs -4.61 (8.47) mm Hg in the aerobic exercise group. The analysis of office SBP at 6 months yielded similar results (-2.31 [95% CI, -3.94 to -0.67] mm Hg; P = .006). Additionally, 24-hour ambulatory SBP (-2.16 [95% CI, -3.84 to -0.47] mm Hg; P = .01) and nighttime ambulatory SBP (-4.08 [95% CI, -6.59 to -1.57] mm Hg; P = .002) were significantly reduced in the Tai Chi group compared with the aerobic exercise group. Conclusions and Relevance: In this study including patients with prehypertension, a 12-month Tai Chi intervention was more effective than aerobic exercise in reducing SBP. These findings suggest that Tai Chi may help promote the prevention of cardiovascular disease in populations with prehypertension. Trial Registration: Chinese Clinical Trial Registry Identifier: ChiCTR1900024368.

99mTc-methoxyisobutylisonitrile single-photon emission computed tomography/computed tomography parathyroid imaging in the diagnosis of functional parathyroid cysts: an initial experience.

Background: Functional parathyroid cysts (FPCs) are rare and difficult to diagnose with noninvasive methods. The aim of this study was to evaluate the diagnostic value of 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) single-photon emission computed tomography/computerized tomography (SPECT/CT) parathyroid imaging in the diagnosis of FPCs and to account for its performance. Methods: The data from 10 patients with suspected parathyroid cysts (PCs) who underwent 99mTc-MIBI SPECT/CT parathyroid imaging between 2012 and 2022 were retrospectively evaluated. The diagnostic value of 99mTc-MIBI SPECT/CT parathyroid imaging for FPCs was analyzed. Results: Surgical resection was performed in six cases and parathyroid puncture was performed in four cases. The sensitivity of 99mTc-MIBI SPECT/CT for FPCs was 100.0% (3/3), with a specificity of 100.0% (7/7) and an accuracy of 100.0% (10/10). The postoperative pathological findings in three cases of FPCs were parathyroid adenoma, parathyroid adenoma with hemorrhage, and parathyroid adenoma with cystic degeneration, respectively. The diagnostic accuracy of ultrasound and CT for PCs was only 22.22% (2/9) and 25.0% (1/4), respectively, and neither modality could indicate whether the cysts were functional or not. Conclusions: 99mTc-MIBI parathyroid SPECT/CT imaging has a high value in the diagnosis of FPCs in patients with suspected PCs, and an intense ring-shaped accumulation of radioactivity in the cyst wall on 99mTc-MIBI imaging suggests that the patient may have FPCs.

Characterization of chicken-derived antibody against Alpha-Enolase of Streptococcus pneumoniae.

Streptococcus pneumoniae is a clinically relevant pathogen notorious for causing pneumonia, meningitis, and otitis media in immunocompromised patients. Currently, antibiotic therapy is the most efficient treatment for fighting pneumococcal infections. However, an arise in antimicrobial resistance in S. pneumoniae has become a serious health issue globally. To resolve the problem, alternative and cost-effective strategies, such as monoclonal antibody-based targeted therapy, are needed for combating bacterial infection. S. pneumoniae alpha-enolase (spEno1), which is thought to be a great target, is a surface protein that binds and converts human plasminogen to plasmin, leading to accelerated bacterial infections. We first purified recombinant spEno1 protein for chicken immunization to generate specific IgY antibodies. We next constructed two single-chain variable fragments (scFv) antibody libraries by phage display technology, containing 7.2 × 107 and 4.8 × 107 transformants. After bio-panning, ten scFv antibodies were obtained, and their binding activities to spEno1 were evaluated on ELISA, Western blot and IFA. The epitopes of spEno1 were identified by these scFv antibodies, which binding affinities were determined by competitive ELISA. Moreover, inhibition assay displayed that the scFv antibodies effectively inhibit the binding between spEno1 and human plasminogen. Overall, the results suggested that these scFv antibodies have the potential to serve as an immunotherapeutic drug against S. pneumoniae infections.

StAR-related lipid transfer domain protein 3 (STARD3) regulates HER2 and promotes HER2-positive breast cancer progression through interaction with HSP90 and SRC signaling.

Although various HER2-targeted therapies have been approved clinically, drug resistance remains a considerable challenge. Studies have found that the cause of drug resistance is related to the expression of genes co-amplified with HER2 in breast cancer cells. Our study found that STARD3 was highly expressed in tumor tissues (n = 130, P < 0.001), especially in the HER2+ subtype (n = 35, P < 0.05), and correlated with poorer overall survival (HR = 1.47, P < 0.001). We discovered the interaction mechanism between STARD3 and HER2 proteins. We found that STARD3 overexpression increases HER2 levels by directly interacting with the HSP90 protein and inducing phosphorylated SRC, which may protect HER2 from degradation. Conversely, loss of STARD3 attenuates HER2 expression through lysosomal degradation. In addition, STARD3 overexpression induced cell cycle progression by inducing cyclin D1 and reducing p27. Therefore, the development of STARD3-specific targeted anti-cancer drugs would be helpful in the treatment of HER2+ patients. We further found that curcumin (15 µM) is a potent STARD3 inhibitor. STARD3-knockdown cells treated with curcumin (5 µM) showed a significant synergistic effect in inhibiting cancer cell growth and migration. The results suggest that targeting STARD3 would aid in treating HER2-positive breast cancer patients. This article uses curcumin as an example to prove that the targeted inhibition of STARD3 expression can be an option for the clinical treatment of HER2+ breast cancer patients.

Energy-Efficient Ingestible Drug Delivery System in the Dynamic Gastrointestinal Environment.

Ingestible electronics are promising platforms for on-demand health monitoring and drug delivery. However, these devices and their actuators must operate in the gastrointestinal (GI) environment, which has a pH range of 1 to 8. Drug delivery systems using electrochemical dissolution of metal films are particularly susceptible to pH changes. Optimal operation in this dynamic environment stands to transform our capacity to help patients across a range of conditions. Here we present an energy-efficient ingestible electronic electrochemical drug delivery system to support subjects through operation in this dynamic environment. The proposed system consists of a drug reservoir sealed with an electrochemically dissolvable gold membrane and an electronic subsystem. An electronic subsystem controls the rate of gold dissolution by sensing and adapting to the pH of the GI environment and provides an option for energy-efficient drug delivery, reducing energy consumption by up to 42.8 %. Integrating the electronics with electrochemical drug delivery enables the proposed system to adapt to the dynamic physiological environments which makes it suitable for drug and/or therapeutic delivery at different locations in the GI tract.

Analysis of false-positive and false-negative results in 99mTc-MIBI SPECT/CT parathyroid imaging.

Background: Exact preoperative localization is desirable to perform minimally invasive parathyroidectomy for hyperparathyroidism (HPT). This study aimed to evaluate the diagnostic values of 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) single photon emission computed tomography/computed tomography (SPECT/CT) of parathyroid glands by analyzing the relationship between lesion weight and false-negative (FN) results, as well as to explain the possible reason. Methods: The data from 314 patients with suspected HPT who underwent 99mTc-MIBI SPECT/CT parathyroid imaging between 2011 and 2022 were retrospectively evaluated. The sensitivity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of parathyroid 99mTc-MIBI SPECT/CT were calculated, and the false-positive (FP) and FN findings were analyzed. Results: Accurate localization by 99mTc-MIBI SPECT/CT was significantly associated with the parathyroid hormone (PTH) level. The 99mTc-MIBI SPECT/CT for diagnosis/lesion location reached a sensitivity of 84.6%/56.8%, a PPV of 97.3%/98.4%, an NPV of only 23.7%/4.18%, and an accuracy of 83.4%/57.1%, respectively. The largest diameter, shortest diameter, and lesion volume were lower in the FN group than in the TP group. A total of 7 FP cases were found, including 2 cases of thyroid nodules, 4 cases of thyroid tissue, and 1 case of hibernoma. A total of 45 FN patients, including 321 FN lesions, were confirmed, of which parathyroid hyperplasia accounted for 97.8%. Lesion weights greater than 20 µg were able to be detected, but lightweight lesions less than 100 mg were the principal source of FN results, accounting for approximately 39.3%. With lesion weights 0-100, 101-300, 301-1,000, and >1,000 mg, the FN rate was 70.8% (126/178), 51.8% (103/199), 34.6% (81/234), and 8.33% (11/132), respectively. Conclusions: 99mTc-MIBI SPECT/CT parathyroid imaging provides good sensitivity and high specificity in HPT location. Correct localization by 99mTc-MIBI SPECT/CT correlates positively with lesion weight and PTH levels. The smaller the lesion, the higher the FN rate in 99mTc-MIBI SPECT/CT parathyroid imaging, and lesions weighing less than 100 mg are the main source of FN results in 99mTc-MIBI SPECT/CT parathyroid imaging.

The causality between obstructive sleep apnea and ventricular structure and function: a bidirectional Mendelian randomization study.

Background: Multiple observational studies have discovered a substantial link between obstructive sleep apnea (OSA) and ventricular dysfunction. However, conventional observational studies are vulnerable to causal reversal and confounding, making it challenging to infer the causes of effects and their direction. Methods: With the help of a bidirectional, two-sample Mendelian randomization (MR) study, we assessed the potential causality between OSA and left and right ventricular (LV, RV) structure and function. We conducted our analysis utilizing summary data from genome-wide association studies of OSA (16,761 cases and 201,194 controls) in the FinnGen Study, as well as LV (36,041 participants) and RV (29,506 participants) in the UK Biobank cardiovascular magnetic resonance research. The inverse variance weighted (IVW) was selected as the main strategy, with the MR-Egger and weighted median methods serving as supplements. Other methods were employed as sensitivity analysis tools to look at heterogeneity and pleiotropy, including MR-Egger intercept, Cochran Q statistic, MR-PRESSO, and leave-one-out analysis. Results: In the primary IVW analysis, genetically predicted OSA was strongly causative on LV end-diastolic volume (ß = 0.114, 95% CI = 0.034-0.194, p = 0.006) and LV stroke volume (ß = 0.111, 95% CI = 0.031-0.191, p = 0.007), and genetically predicted LV ejection fraction was linked to an increased risk of OSA (OR = 1.161, 95% CI = 1.029-1.309, p = 0.015). However, there was no connection found between OSA and any RV parameters. Conclusion: Our genetic analysis raises a potential causative link between OSA and ventricular structure and function, which may improve the knowledge of OSA as a risk factor for cardiovascular disease by demonstrating a direct impact on cardiac structure and function.

Successful replantation of amputated facial tissues by supermicrosurgery.

BACKGROUND: Although replantation of amputated facial segments remains challenging in reconstructive surgery, it offers excellent aesthetic and functional outcomes. METHODS: From May 2004 to October 2019, 12 patients underwent replantation of amputated facial tissues by supermicrosurgery. The case details, such as the rationale for replantation, the operation method, and postoperative therapy, are described. Four cases are discussed to demonstrate the replantation of different facial parts. RESULTS: Facial tissue replantation was successful in all 12 patients without secondary surgery. The cases included the nose (1 patient), ears (8 patients), lips (2 patients), and one of the soft tissue segments surrounding the lower jaw. Venous congestion occurred in three patients who received a solitary arterial repair and were treated with bloodletting. All patients expressed satisfaction with the cosmetic and functional results at the final follow-up. CONCLUSIONS: Supermicrosurgical facial tissue replantation is a promising and effective procedure for providing patients with the best aesthetic and functional outcomes.

Defect Engineering of a High-Entropy Metallic Glass Surface for High-Performance Overall Water Splitting at Ampere-Level Current Densities.

Platinum-based electrocatalysts possess high water electrolysis activity and are essential components for hydrogen evolution reaction (HER). A major challenge, however, is how to break the cost-efficiency trade-off. Here, a novel defect engineering strategy is presented to construct a nanoporous (FeCoNiB0.75 )97 Pt3 (atomic %) high-entropy metallic glass (HEMG) with a nanocrystalline surface structure that contains large amounts of lattice distortion and stacking faults to achieve excellent electrocatalytic performance using only 3 at% of Pt. The defect-rich HEMG achieves ultralow overpotentials at ampere-level current density of 1000 mA cm-2 for HER (104 mV) and oxygen evolution reaction (301 mV) under alkaline conditions, while retains a long-term durability exceeding 200 h at 100 mA cm-2 . Moreover, it only requires 81 and 122 mV to drive the current densities of 1000 and 100 mA cm-2 for HER under acidic and neutral conditions, respectively. Modelling results reveal that lattice distortion and stacking fault defects help to optimize atomic configuration and modulate electronic interaction, while the surface nanoporous architecture provides abundant active sites, thus synergistically contributing to the reduced energy barrier for water electrolysis. This defect engineering approach combined with a HEMG design strategy is expected to be widely applicable for development of high-performance alloy catalysts.

Cell-based and cell-free immunotherapies for glioblastoma: current status and future directions.

Glioblastoma (GBM) is among the most fatal and recurring malignant solid tumors. It arises from the GBM stem cell population. Conventional neurosurgical resection, temozolomide (TMZ)-dependent chemotherapy and radiotherapy have rendered the prognosis of patients unsatisfactory. Radiotherapy and chemotherapy can frequently induce non-specific damage to healthy brain and other tissues, which can be extremely hazardous. There is therefore a pressing need for a more effective treatment strategy for GBM to complement or replace existing treatment options. Cell-based and cell-free immunotherapies are currently being investigated to develop new treatment modalities against cancer. These treatments have the potential to be both selective and successful in minimizing off-target collateral harm in the normal brain. In this review, several aspects of cell-based and cell-free immunotherapies related to GBM will be discussed.

Predicting post-laminoplasty kyphosis in cervical spondylotic myelopathy patients without preoperative kyphosis: a retrospective study.

BACKGROUND: This study aimed to determine potential risk factors for post-laminoplasty kyphosis and the effect of postoperative kyphosis on neurologic function recovery. METHODS: A total of 266 patients with cervical spondylotic myelopathy (CSM) underwent traditional cervical laminoplasty with a minimum of a 12-month follow-up period. The patients were divided into non-kyphosis (NK group) and kyphosis (K group) groups based on the postoperative C2-7 Cobb angle. Clinical and radiological measurements were collected preoperatively and at the final follow-up. RESULTS: Of the 266 patients, 26 (9.77%) developed postoperative kyphosis at the final follow-up. The postoperative Japanese Orthopedic Association score did not differ significantly between the NK and K groups (P > 0.05). The postoperative numeric rating scale (NRS) also showed no significant difference between the NK and K groups; however, postoperative NRS improved better than the preoperative values in the NK group (P < 0.001). Multivariate analysis revealed that the preoperative C2-7 extension Cobb angle and C2-7 Cobb angle were independent predictors of post-laminoplasty kyphosis. Cut-off values for predicting postoperative kyphosis were a C2-7 extension Cobb angle of 18.00° and a C2-7 Cobb angle of 9.30°. CONCLUSIONS: Low preoperative C2-7 extension Cobb angle and C2-7 Cobb angle may be associated with post-laminoplasty kyphosis in CSM patients without preoperative kyphosis. The cut-off value of the C2-7 extension Cobb angle and C2-7 Cobb angle were 18.00° and 9.30°, respectively.

Generation and characterization of avian single chain variable fragment against human Alpha-Enolase.

Overexpression of human alpha-enolase (hEno1)has been reported in a wide range of cancers and is tightly associated with poor prognosis, making it a remarkable biomarker and therapeutic target. In this study, polyclonal yolk-immunoglobulin (IgY) antibodies purified from hEno1-immunized chickens showed a noticeable specific humoral response. Phage display technology was used to construct two antibody libraries of IgY gene-derived single-chain variable fragments (scFvs) containing 7.8 × 107 and 5.4 × 107 transformants, respectively. Phage-based ELISA indicated that specific anti-hEno1 clones were significantly enriched. The nucleotide sequences of scFv-expressing clones were determined and classified into seven groups either in the short linker or the long linker. Moreover, higher mutation rates were revealed in the CDR regions, especially in the CDR3. Three distinguish antigenic epitopes were identified on the hEno1 protein. The binding activities of selected anti-hEno1 scFv on hEno1-positive PE089 lung cancer cells were confirmed using Western blot, flow cytometry, and immunofluorescence assay. In particular, hEnS7 and hEnS8 scFv antibodies significantly suppressed the growth and migration of PE089 cells. Taken together, these chicken-derived anti-hEno1 IgY and scFv antibodies have great potential to develop diagnostic and therapeutic agents for the treatment of lung cancer patients with high expression levels of hEno1 protein.

A wireless and battery-less implant for multimodal closed-loop neuromodulation in small animals.

Fully implantable wireless systems for the recording and modulation of neural circuits that do not require physical tethers or batteries allow for studies that demand the use of unconstrained and freely behaving animals in isolation or in social groups. Moreover, feedback-control algorithms that can be executed within such devices without the need for remote computing eliminate virtual tethers and any associated latencies. Here we report a wireless and battery-less technology of this type, implanted subdermally along the back of freely moving small animals, for the autonomous recording of electroencephalograms, electromyograms and body temperature, and for closed-loop neuromodulation via optogenetics and pharmacology. The device incorporates a system-on-a-chip with Bluetooth Low Energy for data transmission and a compressed deep-learning module for autonomous operation, that offers neurorecording capabilities matching those of gold-standard wired systems. We also show the use of the implant in studies of sleep-wake regulation and for the programmable closed-loop pharmacological suppression of epileptic seizures via feedback from electroencephalography. The technology can support a broader range of applications in neuroscience and in biomedical research with small animals.

Construction of endothelial cell-related and autophagy-related prognostic models for hepatocellular carcinoma based on single-cell data.

BACKGROUND: We aimed to develop an autophagy-related prognostic model with single-cell RNA sequencing (ScRNA-Seq) data for hepatocellular carcinoma (HCC) patients. METHODS: ScRNA-Seq datasets of HCC patients were analyzed by Seurat. The expression of genes involved in canonical and noncanonical autophagy pathways in scRNA-seq data was also compared. Cox regression was applied to construct an AutRG risk prediction model. Subsequently, we examined the characteristics of AutRG high-risk and low-risk group patients. RESULTS: Six major cell types (hepatocytes, myeloid cells, T/NK cells, B cells, fibroblast cells, and endothelial cells) were identified in the scRNA-Seq dataset. The results showed that most of the canonical and noncanonical autophagy genes were highly expressed in hepatocytes, with the exception of MAP 1LC3B, SQSTM1, MAP 1LC3A, CYBB, and ATG3. Six AutRG risk prediction models originating from different cell types were constructed and compared. The AutRG prognostic signature (GAPDH, HSP90AA1, and TUBA1C) in endothelial cells had the best overall performance for predicting the overall survival of HCC patients, with 1-year, 3-year, and 5-year AUCs equal to 0.758, 0.68, and 0.651 in the training cohort and 0.760, 0.796, and 0.840 in the validation cohort, respectively. The different tumor mutation burden, immune infiltration, and gene set enrichment characteristics of the AutRG high-risk and low-risk group patients were identified. CONCLUSION: We constructed an endothelial cell-related and autophagy-related prognostic model of HCC patients using the ScRNA-Seq dataset for the first time. This model demonstrated the good calibration ability of HCC patients and provided a new understanding of the evaluation of prognosis.

Potential roles of heparanase in cancer therapy: Current trends and future direction.

Heparanase (HPSE; heparanase-1) is an endo-ß-glucuronidase capable of degrading the carbohydrate moiety of heparan sulfate proteoglycans, thus modulating and facilitating the remodeling of the extracellular matrix and basement membrane. HPSE activity is strongly associated with major human pathological complications, including but not limited to tumor progress and angiogenesis. Several lines of literature have shown that overexpression of HPSE leads to enhanced tumor growth and metastatic transmission, as well as poor prognosis. Gene silencing of HPSE or treatment of tumor with compounds that block HPSE activity are shown to remarkably attenuate tumor progression. Therefore, targeting HPSE is considered as a potential therapeutical strategy for the treatment of cancer. Intriguingly, recent findings disclose that heparanase-2 (HPSE-2), a close homolog of HPSE but lacking enzymatic activity, can also regulate antitumor mechanisms. Given the pleiotropic roles of HPSE, further investigation is in demand to determine the precise mechanism of regulating action of HPSE in different cancer settings. In this review, we first summarize the current understanding of HPSE, such as its structure, subcellular localization, and tissue distribution. Furthermore, we systematically review the pro- and antitumorigenic roles and mechanisms of HPSE in cancer progress. In addition, we delineate HPSE inhibitors that have entered clinical trials and their therapeutic potential.

Remote control of muscle-driven miniature robots with battery-free wireless optoelectronics.

Bioengineering approaches that combine living cellular components with three-dimensional scaffolds to generate motion can be used to develop a new generation of miniature robots. Integrating on-board electronics and remote control in these biological machines will enable various applications across engineering, biology, and medicine. Here, we present hybrid bioelectronic robots equipped with battery-free and microinorganic light-emitting diodes for wireless control and real-time communication. Centimeter-scale walking robots were computationally designed and optimized to host on-board optoelectronics with independent stimulation of multiple optogenetic skeletal muscles, achieving remote command of walking, turning, plowing, and transport functions both at individual and collective levels. This work paves the way toward a class of biohybrid machines able to combine biological actuation and sensing with on-board computing.

Correction: The development of surgical risk score and evaluation of necrotizing soft tissue infection in 161 Naja atra envenomed patients.

[This corrects the article DOI: 10.1371/journal.pntd.0010066.].

Production and characterization of single-chain variable fragment antibodies targeting the breast cancer tumor marker nectin-4.

Background: Nectin-4 is a novel biomarker overexpressed in various types of cancer, including breast cancer, in which it has been associated with poor prognosis. Current literature suggests that nectin-4 has a role in cancer progression and may have prognostic and therapeutic implications. The present study aims to produce nectin-4-specific single-chain variable fragment (scFv) antibodies and evaluate their applications in breast cancer cell lines and clinical specimens. Methods: We generated recombinant nectin-4 ectodomain fragments as immunogens to immunize chickens and the chickens' immunoglobulin genes were amplified for construction of anti-nectin-4 scFv libraries using phage display. The binding capacities of the selected clones were evaluated with the recombinant nectin-4 fragments, breast cancer cell lines, and paraffin-embedded tissue sections using various laboratory approaches. The binding affinity and in silico docking profile were also characterized. Results: We have selected two clones (S21 and L4) from the libraries with superior binding capacity. S21 yielded higher signals when used as the primry antibody for western blot analysis and flow cytometry, whereas clone L4 generated cleaner and stronger signals in immunofluorescence and immunohistochemistry staining. In addition, both scFvs could diminish attachment-free cell aggregation of nectin-4-positive breast cancer cells. As results from ELISA indicated that L4 bound more efficiently to fixed nectin-4 ectodomain, molecular docking analysis was further performed and demonstrated that L4 possesses multiple polar contacts with nectin-4 and diversity in interacting residues. Conclusion: Overall, the nectin-4-specific scFvs could recognize nectin-4 expressed by breast cancer cells and have the merit of being further explored for potential diagnostic and therapeutic applications.

The Bottlenecks of Preparing Virus Particles by Size Exclusion for Antibody Generation.

Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient approach by preparing virus particles from size exclusion and immunization of chicken. Polyclonal yolk-immunoglobulin (IgY) was simply purified from egg yolk and monoclonal single-chain variable fragments (scFv) were selected via phage display technology with two scFv libraries containing 6.0 × 106 and 1.3 × 107 transformants. Specific clones were enriched after 5 rounds of bio-panning and four identical genes were classified after the sequence analysis. Moreover, the higher mutation rates were revealed in the CDR regions, especially in the CDR3. IgY showed specific binding activities to both EV71-infected and Coxsackievirus 16-infected cell lysates and high infectivity inhibitory activity of EV71. However, while IgY detected a 37 kDa protein, the selected scFv seemingly detected higher size proteins which could be cell protein instead of EV71 proteins. Despite the highly effective chicken antibody generation, the purity of virus particles prepared by size exclusion is the limitation of this study, and further characterization should be carried out rigorously.