Mechano-bioconjugation Strategy Empowering Fusion Protein Therapeutics with Aggregation Resistance, Prolonged Circulation, and Enhanced Antitumor Efficacy.

Bioconjugation is a powerful protein modification strategy to improve protein properties. Herein, we report mechano-bioconjugation as a novel approach to empower fusion protein therapeutics and demonstrate its utility by a protein heterocatenane (cat-IFN-ABD) containing interferon-α2b (IFN) mechanically interlocked with a consensus albumin-binding domain (ABD). The conjugate was selectively synthesized in cellulo following a cascade of post-translational events using a pair of heterodimerizing p53dim variants and two orthogonal split-intein reactions. The catenane topology was proven by combined techniques of LC-MS, SDS-PAGE, SEC, and controlled proteolytic digestion. Not only did cat-IFN-ABD retain activities comparable to those of the wild-type IFN and ABD, the conjugate also exhibited enhanced aggregation resistance and prolonged circulation time over the simple linear and cyclic fusions. Consequently, cat-IFN-ABD potently inhibited tumor growth in the mouse xenograft model. Therefore, mechano-bioconjugation by catenation accomplishes function integration with additional benefits, providing an alternative pathway for developing advanced protein therapeutics.

Clinical implications of exosome-derived noncoding RNAs in liver.

Exosomes, one of three main types of extracellular vesicles, are ~30-100 nm in diameter and have a lipid bilayer membrane. They are widely distributed in almost all body fluids. Exosomes have the potential to regulate unknown cellular and molecular mechanisms in intercellular communication, organ homeostasis, and diseases. They are critical signal carriers that transfer nucleic acids, proteins, lipids, and other substances into recipient cells, participating in cellular signal transduction and material exchange. ncRNAs are non-protein-coding genes that account for over 90% of the genome and include microRNAs (miRNAs), long ncRNAs (lncRNAs), and circular RNAs (circRNAs). ncRNAs are crucial for physiological and pathological activities in the liver by participating in gene transcription, posttranscriptional epigenetic regulation, and cellular processes through interacting with DNA, RNA, or proteins. Recent evidence from both clinical and preclinical studies indicates that exosome-derived noncoding RNAs (ncRNAs) are highly involved in the progression of acute and chronic liver diseases by regulating hepatic lipid metabolism, innate immunity, viral infection, fibrosis, and cancer. Therefore, exosome-derived ncRNAs have promising potential and clinical implications for the early diagnosis, targeted therapy, and prognosis of liver diseases.

Constraining the Symmetron Model with the HUST-2020 Torsion Pendulum Experiment.

The search for dynamically screening the coupling between the scalar field and matter in high-density environment is achievable with the symmetron model. The high-accuracy and short-range gravity experiment is proposed to test the symmetron model. In this Letter, the data of the HUST-2020 torsion pendulum experiment testing the inverse-square law at submillimeter range is analyzed to constrain the symmetron model. The results show that the HUST-2020 experiment is uniquely sensitive to probe the symmetron model with a mass scale of µ=7.2×10^{-3} eV, and the self-coupling parameter λ≲105 is excluded at mass scale M=0.3 TeV. Especially, at the dark energy scale µ=2.4×10^{-3} eV, the constraint at M=1.3 TeV is improved by about 10 times the previous constraints on the torsion pendulum experiment.

Higher Order Protein Catenation Leads to an Artificial Antibody with Enhanced Affinity and In Vivo Stability.

The chemical topology is a unique dimension for protein engineering, yet the topological diversity and architectural complexity of proteins remain largely untapped. Herein, we report the biosynthesis of complex topological proteins using a rationally engineered, cross-entwining peptide heterodimer motif derived from p53dim (an entangled homodimeric mutant of the tetramerization domain of the tumor suppressor protein p53). The incorporation of an electrostatic interaction at specific sites converts the p53dim homodimer motif into a pair of heterodimer motifs with high specificity for directing chain entanglement upon folding. Its combination with split-intein-mediated ligation and/or SpyTag/SpyCatcher chemistry facilitates the programmed synthesis of protein heterocatenane or [n]catenanes in cells, leading to a general and modular approach to complex protein catenanes containing various proteins of interest. Concatenation enhances not only the target protein's affinity but also the in vivo stability as shown by its prolonged circulation time in blood. As a proof of concept, artificial antibodies have been developed by embedding a human epidermal growth factor receptor 2-specific affibody onto the [n]catenane scaffolds and shown to exhibit a higher affinity and a better pharmacokinetic profile than the wild-type affibody. These results suggest that topology engineering holds great promise in the development of therapeutic proteins.

Applying Noncontact Sensing Technology in the Customized Product Design of Smart Clothes Based on Anthropometry.

Electrocardiograms (ECGs) provide important information for diagnosing cardiovascular diseases. In clinical practice, the conventional Ag/AgCl electrode is generally used; however, it is not suitable for long-term ECG measurement because of the risk of allergic reactions on the skin and the dying issue of electrolytic gels. In previous studies, several dry electrodes have been proposed to address these issues. However, most dry electrodes, which are the mode of conductive materials, have to contact the skin well and are easily affected by motion artifacts in daily life. In the smart clothes developed in this study, a noncontact electrode was used to assess the biopotential across the clothes to prevent skin irritation and discomfort. Moreover, a three-dimensional parametric model based on anthropometric data was built, and the technique of customized product design was introduced into the smart clothes development process to reduce the influence of motion artifacts. The experimental results show that the proposed smart clothes can maintain a good ECG signal quality stably under motion from different activities.

NMR Spectroscopic Studies Reveal the Critical Role of the Isopeptide Bond in Forming the Otherwise Unstable SpyTag-SpyCatcher Mutant Complexes.

The interplay between protein folding and chemical reaction has been an intriguing subject. In this contribution, we report the study of SpyTag and SpyCatcher reactive mutants using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, liquid chromatography and mass spectrometry, circular dichroism, and NMR spectroscopy. It was found that the wild-type SpyCatcher is well-folded in solution and docks with SpyTag to form an intermediate that promotes isopeptide bond formation. By contrast, the double mutant SpyCatcherVA is disordered in solution yet remains reactive toward SpyTag, forming a well-folded covalent complex. Control experiments using the catalytically inactive mutants further reveal the critical role of the isopeptide bond in stabilizing the otherwise loose SpyTag-SpyCatcherVA complex, amplifying the effect of the minute sequence disparity. We believe that the synergy between protein folding and isopeptide bonding is an effective way to enhance protein stability and engineer protein-protein interactions.

Lasso Proteins: Modular Design, Cellular Synthesis, and Topological Transformation.

Entangled proteins have attracted significant research interest. Herein, we report the first rationally designed lasso proteins, or protein [1]rotaxanes, by using a p53dim-entwined dimer for intramolecular entanglement and a SpyTag-SpyCatcher reaction for side-chain ring closure. The lasso structures were confirmed by proteolytic digestion, mutation, NMR spectrometry, and controlled ligation. Their dynamic properties were probed by experiments such as end-capping, proteolytic digestion, and heating/cooling. As a versatile topological intermediate, a lasso protein could be converted to a rotaxane, a heterocatenane, and a "slide-ring" network. Being entirely genetically encoded, this robust and modular lasso-protein motif is a valuable addition to the topological protein repertoire and a promising candidate for protein-based biomaterials.

Galectin-1 from conditioned medium of three-dimensional culture of adipose-derived stem cells accelerates migration and proliferation of human keratinocytes and fibroblasts.

Keratinocytes and fibroblasts cells play important roles in the skin-wound healing process and are the cell types activated by trauma. Activated cells participate in epithelialization, granulation, scar tissue formation, wound remodeling, and angiogenesis via a series of cellular activities including migration and proliferation. Previous studies reported that the conditioned medium (CM) of adipose-derived stem cells (ADSCs) stimulated the migration and proliferation of cell types involved in the skin wound healing process; however, these studies only show ADSC-CM effects that were obtained using 2-dimensional (2D) culture. Recently, 3-dimensional (3D) culture has been considered as a more physiologically appropriate system than 2D culture for ADSC cultures; therefore, ADSC-CM was collected from 3D culture (ADSC-CM-3D) and compared with ADSC-CM from 2D culture (ADSC-CM-2D) to investigate the effects on the migration and proliferation of human keratinocytes (HaCaTs) and fibroblasts. The migrations of the HaCaT cells and fibroblasts were significantly higher with ADSC-CM-3D compared with the 2D culture; similarly, the proliferation of HaCaT cells was also highly stimulated by ADSC-CM-3D. Proteomic analyses of the ADSC-CM revealed that collagens and actins were highly expressed in the 3D-culture system. Chitinase 3-like 1 (CHI3L1), tissue inhibitor of metalloproteinases (TIMP), and galectin-1 were specifically expressed only in ADSC-CM-3D. Especially, through antibody neutralization, galectin-1 in ADSC-CM-3D was found to be an important factor for the migration of human keratinocytes. Therefore, these results suggest that ADSC-CM-3D was more effective in the wound healing than ADSC-CM-2D, and galectin-1 in ADSC-CM-3D was could be a promising option for skin-wound healing. Furthermore, the differential expressions of several ADSC-CM proteins between the 2D- and 3D-culture systems may be used as basic information for the development of efficient wound-healing strategies.

Brainstem gangliogliomas: prognostic factors, surgical indications and functional outcomes.

To explore the prognostic factors and discuss the surgical indications of brainstem gangliogliomas. Twenty-one patients with brainstem ganglioglioma were surgically treated at our hospital between 2006 and 2014. The clinical, radiological, operative, and pathological findings of these patients were retrospectively reviewed. The 3-years overall survival and event-free survival (EFS) rates were 90.5 % and 68.4 %, respectively. Four patients (4/18, 22 %) experienced a recurrence with a mean recurrence-free survival of 5.5 months and a mean follow-up of 37 months. Three patients died of surgery-related complications. Three growth patterns were identified: exophytic (6/21), intrinsic (2/21), and endo-exophytic (13/21). Eight patients (8/15, 53 %) harbored a BRAF V600E mutation. All recurrent tumors were endo-exophytic, and except the one without molecular information, were BRAF V600E mutants. A Cox hazard proportion ratio model was used to identify factors influencing EFS, including sex, age, location, growth patterns, extent of resection (EOR), and BRAF V600E mutation status. On univariate analysis, none of these factors reached statistical significance. Among them, EOR and growth patterns were strongly associated with each other (Fisher's exact test, P < 0.01). A multivariate analysis demonstrated that growth patterns were the only factor associated with EFS (P = 0.02; HR 49.05; 95 % CI 1.76-1365.13). Growth patterns may be useful to select surgery candidates and predict prognosis for patients with brainstem gangliogliomas. BRAF V600E was frequently present and appeared to be associated with shorter recurrence-free survival. Studies on BRAF V600E-targeted therapy for patients with high surgical risks are needed.

Fibrocyte trafficking in patients with chronic obstructive asthma and during an acute asthma exacerbation.

BACKGROUND: Fibrocytes express several chemokine receptors (CCR7 and CXCR4) that regulate their recruitment and trafficking into tissue-damage sites in response to specific chemokine gradients (CCL19 and CXCL12). OBJECTIVE: We investigated whether these chemoattractants and S100A9, through the receptor for advanced glycation end-products (RAGE; ie, its receptor), are involved in fibrocyte trafficking in patients with chronic obstructive asthma (COA) and during an acute exacerbation (AE) in patients without airflow obstruction (Asthma AE group). METHODS: We collected peripheral blood from 14 asthmatic patients with normal pulmonary function, 14 patients with COA, 11 patients in the Asthma AE group, and 14 healthy subjects. Isolated circulating fibrocytes were used for migration assay. Expression of CCR7, CXCR4, S100A9, and RAGE in fibrocytes was measured by using flow cytometry. CCL19 and CXCL12 expression in bronchial tissues was determined by using immunohistochemistry and RT-PCR. RESULTS: There were higher numbers of circulating fibrocytes in patients in the Asthma AE group and patients with COA. The expression of CXCL12 in bronchial tissues and CXCR4 in circulating fibrocytes was higher in the Asthma AE group and, to a lesser extent, in patients with COA. The expression of CCL19 in bronchial tissues and CCR7 in fibrocytes was higher in patients with COA. CXCL12/CXCR4 and CCL19/CCR7 enhanced fibrocyte transmigration in the Asthma AE group and in patients with COA, respectively. The upregulated expression of S100A9 and RAGE in fibrocytes of patients in the Asthma AE group and those with COA contributes to the enhanced basal migratory motility of fibrocytes. CONCLUSION: The CXCR4/CXCL12 axis contributes to chemotaxis of fibrocytes in patients in the Asthma AE group, whereas the CCR7/CCL19 axis plays an important role in patients with COA. S100A9 enhances the basal migratory motility of fibrocytes from patients in the Asthma AE group and patients with COA.

Hybrid spin-microcantilever sensor for environmental, chemical, and biological detection.

Nowadays hybrid spin-micro/nanomechanical systems are being actively explored for potential quantum sensing applications. In combination with the pump-probe technique or the spin resonance spectrum, we theoretically propose a realistic, feasible, and an exact way to measure the cantilever frequency in a hybrid spin-micromechanical cantilever system which has a strong coherent coupling of a single nitrogen vacancy center in the single-crystal diamond cantilever with the microcantilever. The probe absorption spectrum which exhibits new features such as mechanically induced three-photon resonance and ac Stark effect is obtained. Simultaneously, we further develop this hybrid spin-micromechanical system to be an ultrasensitive mass sensor, which can be operated at 300 K with a mass responsivity 0.137 Hz ag(-1), for accurate sensing of gaseous or aqueous environments, chemical vapors, and biomolecules. And the best performance on the minimum detectable mass can be [Formula: see text] in vacuum. Finally, we illustrate an in situ measurement to detect Angiopoietin-1, a marker of tumor angiogenesis, accurately with this hybrid microcantilever at room temperature.

Sensitive detection of Majorana fermions based on a hybrid spin-microcantilever via enhanced spin resonance spectrum.

Motivated by recent experimental progress towards the detection and manipulation of Majorana fermions in ferromagnetic atomic chains on a superconductor, we present a novel proposal based on a single-crystal diamond (SCD) microcantilever with a single nitrogen-vacancy (NV) center spin embedded in ultrapure diamond substrate to probe Majorana fermions in an all-optical domain. With this scheme, a possible distinct Majorana signature is investigated via the electron spin resonance spectrum. In the proposal, the SCD microcantilever behaves as a phonon cavity and is robust for detecting of Majorana fermions, while the NV center spin can be considered as a sensitive probe. Further, the vibration of the microcantilever will enhance the coupling effect, which makes the Majorana fermions more sensitive to detection and the well-established optical NV spin readout technology will certainly promote the detection. This proposed method may provide a potential supplement for the detection of Majorana fermions.

Proposition of a Silica Nanoparticle-Enhanced Hybrid Spin-Microcantilever Sensor Using Nonlinear Optics for Detection of DNA in Liquid.

We theoretically propose a method based on the combination of a nonlinear optical mass sensor using a hybrid spin-microcantilever and the nanoparticle-enhanced technique, to detect and monitor DNA mutations. The technique theoretically allows the mass of external particles (ssDNA) landing on the surface of a hybrid spin-microcantilever to be detected directly and accurately at 300 K with a mass responsivity 0.137 Hz/ag in situ in liquid. Moreover, combined with the nanoparticle-enhanced technique, even only one base pair mutation in the target DNA sequence can be identified in real time accurately, and the DNA hybridization reactions can be monitored quantitatively. Furthermore, in situ detection in liquid and measurement of the proposed nonlinear optical spin resonance spectra will minimize the experimental errors.

Omental fibroma combined with right indirect inguinal hernia masquerades as a scrotal tumor: A case report.

BACKGROUND: The most common causes of scrotal enlargement in patients include primary tumor of the scrotum, inflammation, hydrocele of the tunica vaginalis, and indirect inguinal hernia; scrotal enlargement caused by external tumors of the scrotum is rare. The patient had both a greater omentum tumor and an inguinal hernia, and the tumor protruded into the scrotum through the hernia sac, which is even rarer. Moreover, omental tumors are mostly metastatic, and primary omental fibroma is rare. CASE SUMMARY: Here, we report a rare case of a 25-year-old young man with scrotal enlargement and pain for 3 months. Preoperative examination and multidisciplinary discussions considered intra-abdominal tumor displacement and inguinal hernia, and intraoperative exploration confirmed that the greater omentum tumor protruded into the scrotum. Therefore, tumor resection and tension-free inguinal hernia repair were performed. The final diagnosis was benign fibroma of the greater omentum accompanied by an indirect inguinal hernia. CONCLUSION: This unusual presentation of a common inguinal hernia disease illustrates the necessity of performing detailed history taking, physical examination, and imaging before surgery.

Corticotropin-releasing hormone downregulates IL-10 production by adaptive forkhead box protein 3-negative regulatory T cells in patients with atopic dermatitis.

BACKGROUND: Corticotropin-releasing hormone (CRH) is the central regulating hormone of the hypothalamic-pituitary-adrenal axis. CRH also has diverse functional effects in the periphery and is related to the aggravation of several cutaneous diseases; however, the effect of CRH on T cells in patients with atopic dermatitis (AD) has not been well evaluated. OBJECTIVE: We investigated whether CRH directly affects peripheral T(H)1, T(H)2, and regulatory T (Treg) cells in patients with AD. METHODS: We assessed whether T cells express the CRH receptor protein and mRNA by using flow cytometry, Western blotting, immunofluorescence, immunohistochemistry, and RT-PCR. We evaluated cytokine expression using ELISA after treating the T cells extracted from patients with AD and healthy control subjects (HCs) with CRH. Flow cytometry was then used to evaluate any direct effects of CRH on T(H)1, T(H)2, and Treg cells from patients with AD and HCs. RESULTS: T cells from patients with AD expressed significantly lower CRH receptor 1/2 mRNA levels than T cells from HCs. T cells from HCs reacted with different IL-4 and IFN-γ secretions to CRH treatment, whereas T cells from patients with AD did not. IL-10 production was significantly decreased in the supernatants from both the HCs and patients with AD after CRH treatment. CRH upregulated IL-4 production by T(H)2 cells and downregulated IFN-γ production by T(H)1 cells in HCs. CRH also suppressed the production of IL-10 by forkhead box protein 3-negative Treg cells in both groups, but the difference was only significant in patients with AD. CONCLUSIONS: CRH-mediated suppression of IL-10 secretion from Treg cells might explain stress-related exacerbations in patients with AD.

Orchestrating Chemical and Physical Cross-Linking in Protein Hydrogels to Regulate Embryonic Stem Cell Growth.

Protein hydrogels are ideal candidates for next-generation biomaterials due to their genetically programmable properties. Herein, we report an entirely protein-based hydrogel as an artificial extracellular matrix (ECM) for regulating the embryonic stem cell growth. A synergy between chemical and physical cross-linking was achieved in one step by SpyTag/SpyCatcher reaction and P zipper association at 37 °C. The hydrogels' stress relaxation behaviors can be tuned across a broad spectrum by single-point mutation on a P zipper. It has been found that faster relaxation can promote the growth of HeLa tumor spheroids and embryonic stem cells, and mechanical regulation of embryonic stem cells occurs via retention of the cells at the G1 phase. The results highlight the promise of genetically encoded protein materials as a platform of artificial ECM for understanding and controlling the complex cell-matrix interactions in a 3D cell culture.

Data-driven evolutionary game models for the spread of fairness and cooperation in heterogeneous networks.

Unique large-scale cooperation and fairness norms are essential to human society, but the emergence of prosocial behaviors is elusive. The fact that heterogeneous social networks prevail raised a hypothesis that heterogeneous networks facilitate fairness and cooperation. However, the hypothesis has not been validated experimentally, and little is known about the evolutionary psychological basis of cooperation and fairness in human networks. Fortunately, research about oxytocin, a neuropeptide, may provide novel ideas for confirming the hypothesis. Recent oxytocin-modulated network game experiments observed that intranasal administration of oxytocin to a few central individuals significantly increases global fairness and cooperation. Here, based on the experimental phenomena and data, we show a joint effect of social preference and network heterogeneity on promoting prosocial behaviors by building evolutionary game models. In the network ultimatum game and the prisoner's dilemma game with punishment, inequality aversion can lead to the spread of costly punishment for selfish and unfair behaviors. This effect is initiated by oxytocin, then amplified via influential nodes, and finally promotes global cooperation and fairness. In contrast, in the network trust game, oxytocin increases trust and altruism, but these effects are confined locally. These results uncover general oxytocin-initiated mechanisms underpinning fairness and cooperation in human networks.

PPAR-alpha/gamma agonists, glucagon-like peptide-1 receptor agonists and metformin for non-alcoholic fatty liver disease: A network meta-analysis.

OBJECTIVE: To undertake a network meta-analysis to compare the relative efficacy of a dual peroxisome proliferator-activated receptor (PPAR)α and PPARγ agonist, glucagon-like peptide-1 receptor agonists (GLP-1RAs) and metformin in patients with non-alcoholic fatty liver disease (NAFLD). METHODS: Electronic databases, including Embase®, PubMed® and The Cochrane Library, were searched systematically for eligible studies from inception to 20 July 2022. Randomized controlled trials (RCTs) that investigated aspartate aminotransferase, alanine aminotransferase (ALT) and triglyceride levels were considered for inclusion. Data were extracted using a standardized data collection table. A network meta-analysis was performed. Relative risk and 95% confidence interval were calculated for continuous data and I2 was used to assess the heterogeneity of studies. RESULTS: A total of 22 RCTs involving 1698 patients were eligible for inclusion in the analysis. Both direct analysis and indirect analysis showed that saroglitazar was significantly superior to GLP-1RAs in improving ALT levels. Metformin improved ALT levels, but the effect was not as good as saroglitazar. CONCLUSION: Saroglizatar was the most effective drug for improving NAFLD.INPLASY registration number: INPLASY202340066.

A single-domain green fluorescent protein catenane.

Natural proteins exhibit rich structural diversity based on the folds of an invariably linear chain. Macromolecular catenanes that cooperatively fold into a single domain do not belong to the current protein universe, and their design and synthesis open new territories in chemistry. Here, we report the design, synthesis, and properties of a single-domain green fluorescent protein catenane via rewiring the connectivity of GFP's secondary motifs. The synthesis could be achieved in two steps via a pseudorotaxane intermediate or directly via expression in cellulo. Various proteins-of-interest may be inserted at the loop regions to give fusion protein catenanes where the two subunits exhibit enhanced thermal resilience, thermal stability, and mechanical stability due to strong conformational coupling. The strategy can be applied to other proteins with similar fold, giving rise to a family of single-domain fluorescent proteins. The results imply that there may be multiple protein topological variants with desirable functional traits beyond their corresponding linear protein counterparts, which are now made accessible and fully open for exploration.

Ibrutinib facilitates the sensitivity of colorectal cancer cells to ferroptosis through BTK/NRF2 pathway.

Ibrutinib is a drug that inhibits the protein Burton's tyrosine kinase and thereby the nuclear translocation of Nrf2, which played a key role in mediating the activation of antioxidants during stress conditions and ferroptosis resistance. This study aimed to identify the effect of Ibrutinib and ferroptosis inducer on colorectal cancer (CRC) treatment and its underlying mechanism. In our study, we found the upregulation of Nrf2 was correlated with CRC progression and antioxidant proteins. Ibrutinib sensitized CRC to ferroptosis inducers, suggested by further reduced CRC cell viability, proliferation and decreased antioxidant protein levels in CRC cells after combination treatment of Ibrutinib and RSL3 or Ibrutinib and Erastin both in vivo and in vitro. Knockout of Nrf2 diminished the regulatory effect of Ibrutinib on CRC sensitivity to ferroptosis inducers. Altogether, this study demonstrated that Ibrutinib increases the sensitivity of CRC cell to ferroptosis inducers by inhibiting Nrf2.