The liquid-to-solid transition of FUS is promoted by the condensate surface.

A wide range of macromolecules can undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed reversibly, and carry out essential functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics, and mechanics in time and space, that single-component FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases coexist simultaneously within the same condensate, resulting in highly inhomogeneous structures. Furthermore, our results show that this transition originates at the interface between the condensate and the dilute continuous phase, and once initiated, the gelation process propagates toward the center of the condensate. To probe such spatially inhomogeneous rheology during condensate aging, we use a combination of established micropipette aspiration experiments together with two optical techniques, spatial dynamic mapping and reflective confocal dynamic speckle microscopy. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a critical element in driving pathological protein aggregation.

Combination of novel oncolytic herpesvirus with paclitaxel as an efficient strategy for breast cancer therapy.

BACKGROUND: New strategies are needed to improve the treatment of patients with breast cancer (BC). Oncolytic virotherapy is a promising new tool for cancer treatment but still has a limited overall durable antitumor response. A novel replicable recombinant oncolytic herpes simplex virus type 1 called VG161 has been developed and has demonstrated antitumor effects in several cancers. Here, we explored the efficacy and the antitumor immune response of VG161 cotreatment with paclitaxel (PTX) which as a novel oncolytic viral immunotherapy for BC. METHODS: The antitumor effect of VG161 and PTX was confirmed in a BC xenograft mouse model. The immunostimulatory pathways were tested by RNA-seq and the remodeling of tumor microenvironment was detected by Flow cytometry analysis or Immunohistochemistry. Pulmonary lesions were analyzed by the EMT6-Luc BC model. RESULTS: In this report, we demonstrate that VG161 can significantly represses BC growth and elicit a robust antitumor immune response in a mouse model. The effect is amplified when combined with PTX treatment. The antitumor effect is associated with the infiltration of lymphoid cells, including CD4+ T cells, CD8+ T cells, and NK cells (expressing TNF and IFN-γ), and myeloid cells, including macrophages, myeloid-derived suppressor cells, and dendritic cell cells. Additionally, VG161 cotreatment with PTX showed a significant reduction in BC lung metastasis, which may result from the enhanced CD4+ and CD8+ T cell-mediated responses. CONCLUSIONS: The combination of PTX and VG161 is effective for repressing BC growth by inducing proinflammatory changes in the tumor microenvironment and reducing BC pulmonary metastasis. These data will provide a new strategy and valuable insight for oncolytic virus therapy applications in primary solid or metastatic BC tumors.

Oncolytic virotherapy: basic principles, recent advances and future directions.

Oncolytic viruses (OVs) have attracted growing awareness in the twenty-first century, as they are generally considered to have direct oncolysis and cancer immune effects. With the progress in genetic engineering technology, OVs have been adopted as versatile platforms for developing novel antitumor strategies, used alone or in combination with other therapies. Recent studies have yielded eye-catching results that delineate the promising clinical outcomes that OVs would bring about in the future. In this review, we summarized the basic principles of OVs in terms of their classifications, as well as the recent advances in OV-modification strategies based on their characteristics, biofunctions, and cancer hallmarks. Candidate OVs are expected to be designed as "qualified soldiers" first by improving target fidelity and safety, and then equipped with "cold weapons" for a proper cytocidal effect, "hot weapons" capable of activating cancer immunotherapy, or "auxiliary weapons" by harnessing tactics such as anti-angiogenesis, reversed metabolic reprogramming and decomposing extracellular matrix around tumors. Combinations with other cancer therapeutic agents have also been elaborated to show encouraging antitumor effects. Robust results from clinical trials using OV as a treatment congruously suggested its significance in future application directions and challenges in developing OVs as novel weapons for tactical decisions in cancer treatment.

VG161 activates systemic antitumor immunity in pancreatic cancer models as a novel oncolytic herpesvirus expressing multiple immunomodulatory transgenes.

The VG161 represents the first recombinant oncolytic herpes simplex virus type 1 carrying multiple synergistic antitumor immuno-modulating factors. Here, we report its antitumor mechanisms and thus provide firm theoretical foundation for the upcoming clinical application in pancreatic cancer. Generally, the VG161-mediated antitumor outcomes were analyzed by a collaboration of techniques, namely the single-cell sequencing, airflow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADSI-MSI) and nanostring techniques. In vitro, the efficacy of VG161 together with immune checkpoint inhibitors (ICIs) has been successfully shown to grant a long-term antitumor effect by altering tumor immunity and remodeling tumor microenvironment (TME) metabolisms. Cellular functional pathways and cell subtypes detected from patient samples before and after the treatment had undergone distinctive changes including upregulated CD8+ T and natural killer cells. More importantly, significant antitumor signals have emerged since the administration of VG161 injection. In conclusion, VG161 can systematically activate acquired and innate immunity in pancreatic models, as well as improve the tumor immune microenvironment, indicative of strong antitumor potential. The more robusting antitumor outcome for VG161 monotherapy or in combination with other therapies on pancreatic cancer is worth of being explored in further clinical trials.

Dynamic Assembly of Viscoelastic Networks by Aqueous Liquid-Liquid Phase Separation and Liquid-Solid Phase Separation (AqLL-LS PS2 ).

Living cells comprise diverse subcellular structures, such as cytoskeletal networks, which can regulate essential cellular activities through dynamic assembly and synergistic interactions with biomolecular condensates. Despite extensive efforts, reproducing viscoelastic networks for modulating biomolecular condensates in synthetic systems remains challenging. Here, a new aqueous two-phase system (ATPS) is proposed, which consists of poly(N-isopropylacrylamide) (PNIPAM) and dextran (DEX), to construct viscoelastic networks capable of being assembled and dissociated dynamically to regulate the self-assembly of condensates on-demand. Viscoelastic networks are generated using liquid-liquid phase-separated DEX droplets as templates and the following liquid-to-solid transition of the PNIPAM-rich phase. The resulting networks can dissolve liquid fused in sarcoma (FUS) condensates within 5 min. This work demonstrates rich phase-separation behaviors in a single ATPS through incorporating stimuli-responsive polymers. The concept can potentially be applied to other macromolecules through other stimuli to develop materials with rich phase behaviors and hierarchical structures.

Aquabots.

Soft robots, made from elastomers, easily bend and flex, but deformability constraints severely limit navigation through and within narrow, confined spaces. Using aqueous two-phase systems we print water-in-water constructs that, by aqueous phase-separation-induced self-assembly, produce ultrasoft liquid robots, termed aquabots, comprised of hierarchical structures that span in length scale from the nanoscopic to microsciopic, that are beyond the resolution limits of printing and overcome the deformability barrier. The exterior of the compartmentalized membranes is easily functionalized, for example, by binding enzymes, catalytic nanoparticles, and magnetic nanoparticles that impart sensitive magnetic responsiveness. These ultrasoft aquabots can adapt their shape for gripping and transporting objects and can be used for targeted photocatalysis, delivery, and release in confined and tortuous spaces. These biocompatible, multicompartmental, and multifunctional aquabots can be readily applied to medical micromanipulation, targeted cargo delivery, tissue engineering, and biomimetics.

Line optical tweezers as controllable micromachines: techniques and emerging trends.

In the past three decades, the technology of optical tweezers has made significant contributions in various scientific areas, including optics, photonics, and nanosciences. Breakthroughs include manipulating particles in both static and dynamic ways, particle sorting, and constructing controllable micromachines. Advances in shaping and controlling the laser beam profile enable control over the position and location of the trap, which has many possible applications. A line optical tweezer (LOT) can be created by rapidly moving a spot optical tweezer using a tool such as a galvanometer mirror or an acousto-optic modulator. By manipulating the intensity profile along the beam line to be asymmetric or non-uniform, the technique can be adapted to various specific applications. Among the many exciting applications of line optical tweezers, in this work, we discuss in detail applications of LOT, including probing colloidal interactions, transporting and sorting of colloidal microspheres, self-propelled motions, trapping anisotropic particles, exploring colloidal interactions at fluid-fluid interfaces, and building optical thermal ratchets. We further discuss prospective applications in each of these areas of soft matter, including polymeric and biological soft materials.

Vimentin intermediate filaments and filamentous actin form unexpected interpenetrating networks that redefine the cell cortex.

The cytoskeleton of eukaryotic cells is primarily composed of networks of filamentous proteins, F-actin, microtubules, and intermediate filaments. Interactions among the cytoskeletal components are important in determining cell structure and in regulating cell functions. For example, F-actin and microtubules work together to control cell shape and polarity, while the subcellular organization and transport of vimentin intermediate filament (VIF) networks depend on their interactions with microtubules. However, it is generally thought that F-actin and VIFs form two coexisting but separate networks that are independent due to observed differences in their spatial distribution and functions. In this paper, we present a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks. We characterize the structure of VIFs and F-actin networks within the cell cortex using structured illumination microscopy and cryo-electron tomography. We find that VIFs and F-actin form an interpenetrating network (IPN) with interactions at multiple length scales, and VIFs are integral components of F-actin stress fibers. From measurements of recovery of cell contractility after transient stretching, we find that the IPN structure results in enhanced contractile forces and contributes to cell resilience. Studies of reconstituted networks and dynamic measurements in cells suggest direct and specific associations between VIFs and F-actin. From these results, we conclude that VIFs and F-actin work synergistically, both in their structure and in their function. These results profoundly alter our understanding of the contributions of the components of the cytoskeleton, particularly the interactions between intermediate filaments and F-actin.

Topological analysis of hepatocellular carcinoma tumour microenvironment based on imaging mass cytometry reveals cellular neighbourhood regulated reversely by macrophages with different ontogeny.

OBJECTIVE: Hepatocellular carcinoma (HCC) tumour microenvironment (TME) is highly complex with diverse cellular components organising into various functional units, cellular neighbourhoods (CNs). And we wanted to define CN of HCC while preserving the TME architecture, based on which, potential targets for novel immunotherapy could be identified. DESIGN: A highly multiplexed imaging mass cytometry (IMC) panel was designed to simultaneously quantify 36 biomarkers of tissues from 134 patients with HCC and 7 healthy donors to generate 562 highly multiplexed histology images at single-cell resolution. Different function units were defined by topological analysis of TME. CN relevant to the patients' prognosis was identified as specific target for HCC therapy. Transgenic mouse models were used to validate the novel immunotherapy target for HCC. RESULTS: Three major types of intratumour areas with distinct distribution patterns of tumorous, stromal and immune cells were identified. 22 cellular metaclusters and 16 CN were defined. CN composed of various types of cells formed regional function units and the regional immunity was regulated reversely by resident Kupffer cells and infiltrating macrophages with protumour and antitumour function, respectively. Depletion of Kupffer cells in mouse liver largely enhances the T cell response, reduces liver tumour growth and sensitises the tumour response to antiprogrammed cell death protein-1 treatment. CONCLUSION: Our findings reveal for the first time the various topological function units of HCC TME, which also presents the largest depository of pathological landscape for HCC. This work highlights the potential of Kupffer cell-specific targeting rather than overall myeloid cell blocking as a novel immunotherapy for HCC treatment.

Combined targeted therapy and immunotherapy for cancer treatment.

Although targeted therapies and immunotherapies have been effective against several malignancies, the respective monotherapies are limited by low and/or short-term responses. Specific inhibitors of oncogenic signaling pathways and tumor-associated angiogenesis can activate the anti-tumor immune responses by increasing tumor antigen presentation or intratumor T cell infiltration. Additional insights into the effects and mechanisms of targeted therapies on the induction of anti-tumor immunity will facilitate development of rational and effective combination strategies that synergize rapid tumor regression and durable response. In this review, we have summarized the recent combinations of targeted therapies and immunotherapies, along with the associated clinical challenges.

Effects of Vimentin Intermediate Filaments on the Structure and Dynamics of In Vitro Multicomponent Interpenetrating Cytoskeletal Networks.

We investigate the rheological properties of interpenetrating networks reconstituted from the main cytoskeletal components: filamentous actin, microtubules, and vimentin intermediate filaments. The elastic modulus is determined largely by actin, with little contribution from either microtubules or vimentin. However, vimentin dramatically impacts the relaxation, with even small amounts significantly increasing the relaxation time of the interpenetrating network. This highly unusual decoupling between dissipation and elasticity may reflect weak attractive interactions between vimentin and actin networks.

Biomaterial Cues Regulated Differentiation of Neural Stem Cells into GABAergic Neurons through Ca2+/c-Jun/TLX3 Signaling Promoted by Hydroxyapatite Nanorods.

Directed differentiation enables the production of a specific cell type by manipulating signals in development. However, there is a lack of effective means to accelerate the regeneration of neurons of particular subtypes for pathogenesis and clinical therapy. In this study, we find that hydroxyapatite (HAp) nanorods promote neural differentiation of neural stem cells due to their chemical compositions. Lysosome-mediated degradation of HAp nanorods elevates intracellular calcium concentrations and accelerates GABAergic neurogenesis. As a mechanism, the enhanced activity of a Ca2+ peak initiated by HAp nanorods leads to the activation of c-Jun and thus suppresses the expression of GABAergic/glutamatergic selection gene TLX3. We demonstrate the capability of HAp nanorods in promoting the differentiation into GABAergic neurons at both molecular and cellular function levels. Given that GABAergic neurons are responsible for various physiological and pathological processes, our findings open up enormous opportunities in efficient and precise stem cell therapy of neurodegenerative diseases.

Preoperative transarterial chemoembolization for barcelona clinic liver cancer stage A/B hepatocellular carcinoma beyond the milan criteria: a propensity score matching analysis.

BACKGROUND: Debate continues about the benefits of preoperative transarterial chemoembolization (TACE) for treatment of hepatocellular carcinoma (HCC). This study aimed to assess the impact of preoperative TACE on long-term outcomes after curative resection for HCC beyond the Milan criteria. METHODS: Patients who underwent HCC resection exceeding the Milan criteria without macrovascular invasion between 2015 and 2018 were identified (n = 393). Short- and long-term outcomes were compared between patients who underwent preoperative TACE and patients who did not before and after propensity score matching (PSM). Factors associated with recurrence after resection were analyzed. RESULTS: 100 patients (25.4%) underwent preoperative TACE. Recurrence-free survival (RFS) and overall survival (OS) were comparable with patients who underwent primary liver resection. 7 patients (7.0%) achieved total necrosis with better RFS compared with patients who had an incomplete response to TACE (P=0.041). PSM created 73 matched patient pairs. In the PSM cohort, preoperative TACE improved RFS (P=0.002) and OS (P=0.003). The maximum preoperatively diagnosed tumor diameter (HR 3.230, 95% CI: 1.116-9.353; P=0.031) and hepatitis B infection (HR 2.905, 95%CI: 1.281-6.589; P=0.011) were independently associated with favorable RFS after HCC resection. CONCLUSION: Preoperative TACE made no significant difference to perioperative complications and was correlated with an improved prognosis after surgical resection for patients with HCC beyond the Milan criteria.

A preoperative nomogram predicts prognosis of patients with hepatocellular carcinoma after liver transplantation: a multicenter retrospective study.

BACKGROUND: Although criteria for liver transplantation, such as the Milan criteria and Hangzhou experiences, have become popular, criteria to guide adjuvant therapy for patients with hepatocellular carcinoma after liver transplantation are lacking. METHODS: We collected data from all consecutive patients from 2012 to 2019 at three liver transplantation centers in China retrospectively. Univariate and multivariate analyses were used to analyze preoperative parameters, such as demographic and clinical data. Using data obtained in our center, calibration curves and the concordance Harrell's C-indices were used to establish the final model. The validation cohort comprised the patients from the other centers. RESULTS: Data from 233 patients were used to construct the nomogram. The validation cohort comprised 36 patients. Independent predictors of overall survival (OS) were identified as HbeAg positive (P = 0.044), blood-type compatibility unmatched (P = 0.034), liver transplantation criteria (P = 0.003), and high MELD score (P = 0.037). For the validation cohort, to predict OS, the C-index of the nomogram was 0.874. Based on the model, patients could be assigned into low-risk (≥ 50%), intermediate-risk (30-50%), and high-risk (≤ 30%) groups to guide adjuvant therapy after surgery and to facilitate personalized management. CONCLUSIONS: The OS in patients with hepatocellular carcinoma after liver transplantation could be accurately predicted using the developed nomogram.

Living-DNA Nanogel Appendant Enables In Situ Modulation and Quantification of Regulation Effects on Membrane Proteins.

Screening appendants on membrane proteins to understand their varied regulation effects is desirable for finding the potential candidates of the membrane-protein-targeted drugs. However, most artificial appendants can hardly support in situ condition screening because they cannot evolve in situ, neither can they send out signals to reflect the modulation. Here, we designed living-DNA appendants to enable such screening. First, the living-cell rolling-circle amplification (LCRCA) strategy was developed to elongate the DNA appendants for self-tangled physical nanogels. The nanogels unify both the functions of membrane-protein modulation and quantification: their sizes increase with the increased time length of LCRCA, which change the regulation effect on the membrane proteins; their large number of repeating short sequences allow quantification of their sizes in the presence of the complementary fluorophore-tagged short DNA. Then, the performance of the living-DNA appendants was examined taking α6ß4 integrins as the target, where effective regulation over the distribution of actin filaments, cell viability, and chances of anoikis are all validated. The screening also clearly elucidates the interesting nonlinear relationships between the regulations and the effects. We hope this screening strategy based on living-DNA appendants can stand for a prototype for deeper understanding of natural behaviors of membrane proteins and help in the accurate designing of the membrane-protein-targeted drugs.

Oncolytic virus combined with traditional treatment versus traditional treatment alone in patients with cancer: a meta-analysis.

BACKGROUND: Oncolytic virus therapy has shown benefits for multiple cancers, while limitations remain for traditional treatment. However, few studies have concentrated on comparing whether oncolytic virus combined with traditional treatment is better than traditional treatment alone in patients with cancer. We conducted a meta-analysis of the curative effect and safety of oncolytic virus combination therapy. METHODS: We searched the PubMed, Embase, Cochrane Library, and Web of Science databases comprehensively for articles comparing oncolytic virus combined with traditional treatment to traditional treatment alone in patients with cancer. A meta-analysis and trial sequential analysis were performed. RESULTS: A total of 12 studies involving 1494 patients (combination therapy group, 820 patients; traditional treatment group, 674 patients) were included in the study. Compared with traditional treatment alone, combination therapy was significantly associated with high objective response rate [odds ratio (OR) 1.35, 95% confidence interval (CI) 1.01-1.82, p = 0.04]. There were no significant differences for other outcomes such as 1- and 2-year survival rate, and 4- and 12-month progression-free survival rate. Combination therapy was significantly associated with high incidence of grade ≥ 3 adverse effects (OR 1.47, 95% CI 1.06-2.05, p = 0.02) and high incidence of grade ≥ 3 neutropenia (OR 1.65, 95% CI 1.13-2.43, p = 0.01). There were no significant differences for other grade ≥ 3 adverse effects, e.g., gastrointestinal adverse effects, influenza-like illness, fatigue, anemia, and thrombocytopenia. CONCLUSION: Despite partially increased toxicity, the combination therapy improves the effectiveness of cancer treatment. However, high-quality, large-scale studies are needed to evaluate its effectiveness and safety.

Effect of Divalent Cations on the Structure and Mechanics of Vimentin Intermediate Filaments.

Divalent cations behave as effective cross-linkers of intermediate filaments (IFs) such as vimentin IF (VIF). These interactions have been mostly attributed to their multivalency. However, ion-protein interactions often depend on the ion species, and these effects have not been widely studied in IFs. Here, we investigate the effects of two biologically important divalent cations, Zn2+ and Ca2+, on VIF network structure and mechanics in vitro. We find that the network structure is unperturbed at micromolar Zn2+ concentrations, but strong bundle formation is observed at a concentration of 100 µM. Microrheological measurements show that network stiffness increases with cation concentration. However, bundling of filaments softens the network. This trend also holds for VIF networks formed in the presence of Ca2+, but remarkably, a concentration of Ca2+ that is two orders higher is needed to achieve the same effect as with Zn2+, which suggests the importance of salt-protein interactions as described by the Hofmeister effect. Furthermore, we find evidence of competitive binding between the two divalent ion species. Hence, specific interactions between VIFs and divalent cations are likely to be an important mechanism by which cells can control their cytoplasmic mechanics.

Dissolvable Polyacrylamide Beads for High-Throughput Droplet DNA Barcoding.

Droplet-based single cell sequencing technologies, such as inDrop, Drop-seq, and 10X Genomics, are catalyzing a revolution in the understanding of biology. Barcoding beads are key components for these technologies. What is limiting today are barcoding beads that are easy to fabricate, can efficiently deliver primers into drops, and thus achieve high detection efficiency. Here, this work reports an approach to fabricate dissolvable polyacrylamide beads, by crosslinking acrylamide with disulfide bridges that can be cleaved with dithiothreitol. The beads can be rapidly dissolved in drops and release DNA barcode primers. The dissolvable beads are easy to synthesize, and the primer cost for the beads is significantly lower than that for the previous barcoding beads. Furthermore, the dissolvable beads can be loaded into drops with >95% loading efficiency of a single bead per drop and the dissolution of beads does not influence reverse transcription or the polymerase chain reaction (PCR) in drops. Based on this approach, the dissolvable beads are used for single cell RNA and protein analysis.

Oncolytic virotherapy in hepato-bilio-pancreatic cancer: The key to breaking the log jam?

Traditional therapies have limited efficacy in hepatocellular carcinoma, pancreatic cancer, and biliary tract cancer, especially for advanced and refractory cancers. Through a deeper understanding of antitumor immunity and the tumor microenvironment, novel immunotherapies are becoming available for cancer treatment. Oncolytic virus (OV) therapy is an emerging type of immunotherapy that has demonstrated effective antitumor efficacy in many preclinical studies and clinical studies. Thus, it may represent a potential feasible treatment for hard to treat gastrointestinal (GI) tumors. Here, we summarize the research progress of OV therapy for the treatment of hepato-bilio-pancreatic cancers. In general, most OV therapies exhibits potent, specific oncolysis both in cell lines in vitro and the animal models in vivo. Currently, several clinical trials have suggested that OV therapy may also be effective in patients with refractory hepato-bilio-pancreatic cancer. Multiple strategies such as introducing immunostimulatory genes, modifying virus capsid and combining various other therapeutic modalities have been shown enhanced specific oncolysis and synergistic anti-cancer immune stimulation. Combining OV with other antitumor therapies may become a more effective strategy than using virus alone. Nevertheless, more studies are needed to better understand the mechanisms underlying the therapeutic effects of OV, and to design appropriate dosing and combination strategies.

Prediction of postoperative pancreatic fistula using a nomogram based on the updated definition.

PURPOSE: The International Study Group on Pancreatic Fistula's definition of postoperative pancreatic fistula (POPF) has recently been updated. This study aimed to identify risk factors for POPF in patients having pancreaticoduodenectomy (PD) and to generate a nomogram to predict POPF. METHODS: Data on 298 patients who underwent PD from March 2012 to October 2017 was retrospectively reviewed and POPF statuses were redefined. A nomogram was constructed using data from 220 patients and validated using the remaining 78 patients. Independent risk factors for POPF were identified using univariate and multivariate analyses. A predictive nomogram was established based on the independent risk factors and was compared with existing models. RESULTS: Texture of the pancreas, size of the main pancreatic duct, portal vein invasion, and definitive pathology were the identified risk factors. The nomogram had a C-index of 0.793 and was internally validated. The nomogram performed better (C-index of 0.816) than the other most cited models (C-indexes of 0.728 and 0.735) in the validation cohort. In addition, the nomogram can assign patients into low- (less than 10%), intermediate- (10% to 30%), and high-risk (equal or higher than 30%) groups to facilitate personalized management. CONCLUSION: The nomogram accurately predicted POPF in patients having PD.