Cryo-shocked tumor cells deliver CRISPR-Cas9 for lung cancer regression by synthetic lethality.

Although CRISPR-mediated genome editing holds promise for cancer therapy, inadequate tumor targeting and potential off-target side effects hamper its outcomes. In this study, we present a strategy using cryo-shocked lung tumor cells as a CRISPR-Cas9 delivery system for cyclin-dependent kinase 4 (CDK4) gene editing, which initiates synthetic lethal in KRAS-mutant non-small cell lung cancer (NSCLC). By rapidly liquid nitrogen shocking, we effectively eliminate the pathogenicity of tumor cells while preserving their structure and surface receptor activity. This delivery system enables the loaded CRISPR-Cas9 to efficiently target to lung through the capture in pulmonary capillaries and interactions with endothelial cells. In a NSCLC-bearing mouse model, the drug accumulation is increased nearly fourfold in lung, and intratumoral CDK4 expression is substantially down-regulated compared to CRISPR-Cas9 lipofectamine nanoparticles administration. Furthermore, CRISPR-Cas9 editing-mediated CDK4 ablation triggers synthetic lethal in KRAS-mutant NSCLC and prolongs the survival of mice.

In situ formed depot of elastin-like polypeptide-hirudin fusion protein for long-acting antithrombotic therapy.

Thrombosis, induced by abnormal coagulation or fibrinolytic systems, is the most common pathology associated with many life-threatening cardio-cerebrovascular diseases. However, first-line anticoagulant drugs suffer from rapid drug elimination and risk of hemorrhagic complications. Here, we developed an in situ formed depot of elastin-like polypeptide (ELP)-hirudin fusion protein with a prodrug-like feature for long-term antithrombotic therapy. Highly secretory expression of the fusion protein was achieved with the assistance of the Ffu312 tag. Integration of hirudin, ELP, and responsive moiety can customize fusion proteins with properties of adjustable in vivo retention and controllable recovery of drug bioactivity. After subcutaneous injection, the fusion protein can form a reservoir through temperature-induced coacervation of ELP and slowly diffuse into the blood circulation. The biological activity of hirudin is shielded due to the N-terminal modification, while the activated key proteases upon thrombus occurrence trigger the cleavage of fusion protein together with the release of hirudin, which has antithrombotic activity to counteract thrombosis. We substantiated that the optimized fusion protein produced long-term antithrombotic effects without the risk of bleeding in multiple animal thrombosis models.

Amino acid formula induces microbiota dysbiosis and depressive-like behavior in mice.

Amino acid formula (AAF) is increasingly consumed in infants with cow's milk protein allergy; however, the long-term influences on health are less described. In this study, we established a mouse model by subjecting neonatal mice to an amino acid diet (AAD) to mimic the feeding regimen of infants on AAF. Surprisingly, AAD-fed mice exhibited dysbiotic microbiota and increased neuronal activity in both the intestine and brain, as well as gastrointestinal peristalsis disorders and depressive-like behavior. Furthermore, fecal microbiota transplantation from AAD-fed mice or AAF-fed infants to recipient mice led to elevated neuronal activations and exacerbated depressive-like behaviors compared to that from normal chow-fed mice or cow's-milk-formula-fed infants, respectively. Our findings highlight the necessity to avoid the excessive use of AAF, which may influence the neuronal development and mental health of children.

The Structure, Biology, and Mechanical Function of Tendon/Ligament-Bone Interfaces.

After tendon or ligament reconstruction, the interface between the hard bone and soft connective tissue is considerably weakened and is difficult to restore through healing. The tendon/ligament-bone interface is mechanically the weakest point under tensile loading and is often the source of various postoperative complications, such as bone resorption and graft laxity. A comprehensive understanding of the macro- and microfeatures of the native tendon/ligament-bone interface would be beneficial for developing strategies for regenerating the tissue. This article discusses the structural, biological, and mechanical features of the tendon/ligament-bone interfaces and how these can be affected by aging and loading conditions.

Rationally designed approaches to augment CAR-T therapy for solid tumor treatment.

Chimeric antigen receptor T cell denoted as CAR-T therapy has realized incredible therapeutic advancements for B cell malignancy treatment. However, its therapeutic validity has yet to be successfully achieved in solid tumors. Different from hematological cancers, solid tumors are characterized by dysregulated blood vessels, dense extracellular matrix, and filled with immunosuppressive signals, which together result in CAR-T cells' insufficient infiltration and rapid dysfunction. The insufficient recognition of tumor cells and tumor heterogeneity eventually causes cancer reoccurrences. In addition, CAR-T therapy also raises safety concerns, including potential cytokine release storm, on-target/off-tumor toxicities, and neuro-system side effects. Here we comprehensively review various targeting aspects, including CAR-T cell design, tumor modulation, and delivery strategy. We believe it is essential to rationally design a combinatory CAR-T therapy via constructing optimized CAR-T cells, directly manipulating tumor tissue microenvironments, and selecting the most suitable delivery strategy to achieve the optimal outcome in both safety and efficacy.

Gasdermin D licenses MHCII induction to maintain food tolerance in small intestine.

The intestinal epithelial cells (IECs) constitute the primary barrier between host cells and numerous foreign antigens; it is unclear how IECs induce the protective immunity against pathogens while maintaining the immune tolerance to food. Here, we found IECs accumulate a less recognized 13-kD N-terminal fragment of GSDMD that is cleaved by caspase-3/7 in response to dietary antigens. Unlike the 30-kD GSDMD cleavage fragment that executes pyroptosis, the IEC-accumulated GSDMD cleavage fragment translocates to the nucleus and induces the transcription of CIITA and MHCII molecules, which in turn induces the Tr1 cells in upper small intestine. Mice treated with a caspase-3/7 inhibitor, mice with GSDMD mutation resistant to caspase-3/7 cleavage, mice with MHCII deficiency in IECs, and mice with Tr1 deficiency all displayed a disrupted food tolerance phenotype. Our study supports that differential cleavage of GSDMD can be understood as a regulatory hub controlling immunity versus tolerance in the small intestine.

Microbial sensing in the intestine.

The gut microbiota plays a key role in host health and disease, particularly through their interactions with the immune system. Intestinal homeostasis is dependent on the symbiotic relationships between the host and the diverse gut microbiota, which is influenced by the highly co-evolved immune-microbiota interactions. The first step of the interaction between the host and the gut microbiota is the sensing of the gut microbes by the host immune system. In this review, we describe the cells of the host immune system and the proteins that sense the components and metabolites of the gut microbes. We further highlight the essential roles of pattern recognition receptors (PRRs), the G protein-coupled receptors (GPCRs), aryl hydrocarbon receptor (AHR) and the nuclear receptors expressed in the intestinal epithelial cells (IECs) and the intestine-resident immune cells. We also discuss the mechanisms by which the disruption of microbial sensing because of genetic or environmental factors causes human diseases such as the inflammatory bowel disease (IBD).

Comparison of the morphology of the anterior cruciate ligament and related bony structures between pigs and humans.

Introduction: Pigs are widely used for clinical research on the anterior cruciate ligament (ACL) because of the similarity of the knee structure to the human knee. But evidence to support the suitability of using porcine samples to guide clinical practices is limited. This study aims to explore the qualitative and quantitative morphological features of the porcine knee and ACL, and to compare these with data on humans reported in literature. Methods: Nineteen porcine knees were used for this study. The bone structures were measured on coronal X-ray images. The length of the ACL was measured using a caliper. The ACL bone insertion sites were marked and measured on a digital photograph. The lengths of the long and short axis of the ACL isthmus were measured on the X-ray microscopy reconstructed images. The outcomes were compared with previously reported data on humans using an abstract independent-samples T test. Results: Qualitative observation indicated a similar location, orientation and general morphology of the porcine ACL to human ACLs. The major difference was the location of the ACL tibial insertion with respect to the anterior horn of the lateral meniscus (AHLM). The porcine ACL was split into AM and PL bundles by the AHLM, while the AHLM was adjacent to the anterolateral border of the ACL tibial insertion in human knees. The quantitative comparison showed no significant difference between the human and porcine ACL in terms of the length of the ACL, the width of the femoral condyle and tibial plateau, and the tibial interspinal width. However, the CSA, the lengths of the long and short axis of the ACL isthmus, and the femoral and tibial insertion areas of the porcine ACL were all significantly larger than the reported features in human knees. Conclusion: The location, orientation and basic morphology of the porcine ACL and knee are similar to humans. However, the two-bundle structure is more distinct in a porcine ACL, and the dimensions of the porcine ACL are generally larger. This study may provide useful information to researchers when assessing the feasibility and limitations of using porcine samples for research on the human ACL and knee.

Graft Diameter Should Reflect the Size of the Native Anterior Cruciate Ligament (ACL) to Improve the Outcome of ACL Reconstruction: A Finite Element Analysis.

The size of the anterior cruciate ligament (ACL) often varies between individuals, but such variation is not typically considered during ACL reconstruction (ACLR). This study aimed to explore how the size of the ACL affects the selection of a suitable graft diameter. A finite element model of a human knee was implanted with intact ACLs of different dimensions (0.95, 1 and 1.05 times the size of the original ACL) and with grafts of different diameters, to simulate ACLR (diameter 7.5-12 mm in 0.5 mm increments). The knee models were flexed to 30° and loaded with an anterior tibial load of 103 N, internal tibial moment of 7.5 Nm, and valgus tibial moment of 6.9 Nm. Knee kinematics (anterior tibial translation (ATT), internal tibial rotation (ITR) and valgus tibial rotation (VTR)) and ligament forces were recorded and compared among the different groups. The results showed that, compared with the intact knee, a graft diameter of 7.5 mm was found to increase the ATT and VTR, but reduce the graft force. Increasing the graft diameter reduced knee laxity and increased the graft force. A 10% increase in the size of the ACL corresponded to a 3 mm larger graft diameter required to restore knee stability and graft force after ACLR. It was concluded that the graft diameter should be selected according to the dimensions of the native ACL, for better restoration of knee functionality. This study may help to improve the clinical treatment of ACL ruptures.

Hourglass-shaped grafts are superior to conventional grafts for restoring knee stability and graft force at knee flexion angle of 30° following anterior cruciate ligament reconstruction: A finite element analysis.

Background: Anterior cruciate ligament reconstruction (ACLR) using a generally columnar graft is considered the gold standard for treating anterior cruciate ligament ruptures, but such grafts cannot replicate the geometry and mechanical properties of the native anterior cruciate ligament. Purpose: To evaluate the effectiveness of an innovative hourglass-shaped graft versus a traditional columnar graft for restoring joint stability and graft force, while avoiding notch impingement following anterior cruciate ligament reconstruction. Methods: Finite element models of a human knee were developed to simulate ① An intact state, ② anterior cruciate ligament reconstruction using columnar grafts with different diameters (7.5-12 mm in 0.5 mm increments), ③ anterior cruciate ligament reconstruction using columnar grafts with different Young's moduli (129.4, 168.0 and 362.2 MPa) and ④ anterior cruciate ligament reconstruction using hourglass-shaped grafts with different Young's moduli. The knee model was flexed to 30° and loaded with an anterior tibial load of 103 N, internal tibial moment of 7.5 Nm, and valgus tibial moment of 6.9 Nm. The risk of notch impingement, knee stability and graft forces were compared among the different groups. Results: This study found that columnar grafts could not simultaneously restore knee stability in different degree of freedoms (DOFs) and graft force to a level similar to that of the intact knee. The anterior tibial translation and graft force were restored to a near-normal condition when the internal tibial rotation was over-restrained and valgus tibial rotation was lax. A graft diameter of at least 10 mm was needed to restore knee stability and graft force to physiological levels, but such large grafts were found to be at high risk of notch impingement. In contrast, the hourglass-shaped graft was able to simultaneously restore both knee stability and graft force at knee flexion of 30° while also having a much lower risk of impingement. Conclusion: Under knee flexion angle of 30°, an hourglass-shaped graft was better able to restore joint stability and graft force to a near-physiological level than columnar grafts, while also reducing the risk of notch impingement.

The comfort assessment in healthy adults during constant-flow mode in noninvasive ventilator.

BACKGROUND: From the point of view of machine construction and hydrodynamics, this paper innovatively proposes that the essence of high-flow nasal cannula (HFNC) is a constant-flow mode in noninvasive ventilator (NIVCFM). This study enrolled healthy adults as study subjects to assess the subjective comfort assessed by visual analog scoring scale of NIVCFM/HFNC and objective comfort measured by the noise level generated by NIVCFM/HFNC, aiming to provide a scientific basis for the rational clinical application of NIVCFM/HFNC. METHODS: Forty-four healthy adults participated in this study. The noise generated by NIVCFM/HFNC is measured, and the comfort is evaluated during NIVCFM delivery at flow rates of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 L/min. RESULTS: When the ventilator flow rate is 60 L/min, the maximum noise is 65.9 dB, increasing noise by 23.7 dB from a baseline of 42.2 dB at the flow rate of 0 L/min. There was a strong nonlinear positive correlation between the noise level and the flow rates. The median score for dry mouth, nose or throat, dysphagia, sore throat, and other discomfort was 0. The median score for dyspnea was 0 at 0-30 L/min, 1 at 35-55 L/min, and 2 at 60 L/min. CONCLUSIONS: The grater the flow rate, the greater the noise generated by NIVCFM/HFNC (<65.9 dB). The maximum flow rate that most healthy adults can able to tolerate is 30 L/min, and the main discomfort is dyspnea.

Phase separation drives RNA virus-induced activation of the NLRP6 inflammasome.

NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) in vitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.

The RNA helicase Dhx15 mediates Wnt-induced antimicrobial protein expression in Paneth cells.

RNA helicases play roles in various essential biological processes such as RNA splicing and editing. Recent in vitro studies show that RNA helicases are involved in immune responses toward viruses, serving as viral RNA sensors or immune signaling adaptors. However, there is still a lack of in vivo data to support the tissue- or cell-specific function of RNA helicases owing to the lethality of mice with complete knockout of RNA helicases; further, there is a lack of evidence about the antibacterial role of helicases. Here, we investigated the in vivo role of Dhx15 in intestinal antibacterial responses by generating mice that were intestinal epithelial cell (IEC)-specific deficient for Dhx15 (Dhx15 f/f Villin1-cre, Dhx15ΔIEC). These mice are susceptible to infection with enteric bacteria Citrobacter rodentium (C. rod), owing to impaired α-defensin production by Paneth cells. Moreover, mice with Paneth cell-specific depletion of Dhx15 (Dhx15 f/f Defensinα6-cre, Dhx15ΔPaneth) are more susceptible to DSS (dextran sodium sulfate)-induced colitis, which phenocopy Dhx15ΔIEC mice, due to the dysbiosis of the intestinal microbiota. In humans, reduced protein levels of Dhx15 are found in ulcerative colitis (UC) patients. Taken together, our findings identify a key regulator of Wnt-induced α-defensins in Paneth cells and offer insights into its role in the antimicrobial response as well as intestinal inflammation.

Impaired lipid biosynthesis hinders anti-tumor efficacy of intratumoral iNKT cells.

Dysfunction of invariant natural killer T (iNKT) cells in tumor microenvironment hinders their anti-tumor efficacy, and the underlying mechanisms remain unclear. Here we report that iNKT cells increase lipid biosynthesis after activation, and that is promoted by PPARγ and PLZF synergically through enhancing transcription of Srebf1. Among those lipids, cholesterol is required for the optimal IFN-γ production from iNKT cells. Lactic acid in tumor microenvironment reduces expression of PPARγ in intratumoral iNKT cells and consequently diminishes their cholesterol synthesis and IFN-γ production. Importantly, PPARγ agonist pioglitazone, a thiazolidinedione drug for type 2 diabetes, successfully restores IFN-γ production in tumor-infiltrating iNKT cells from both human patients and mouse models. Combination of pioglitazone and alpha-galactosylceramide treatments significantly enhances iNKT cell-mediated anti-tumor immune responses and prolongs survival of tumor-bearing mice. Our studies provide a strategy to augment the anti-tumor efficacy of iNKT cell-based immunotherapies via promoting their lipid biosynthesis.

The DNA Methylcytosine Dioxygenase Tet2 Sustains Immunosuppressive Function of Tumor-Infiltrating Myeloid Cells to Promote Melanoma Progression.

Ten-Eleven-Translocation-2 (Tet2) is a DNA methylcytosine dioxygenase that functions as a tumor suppressor in hematopoietic malignancies. We examined the role of Tet2 in tumor-tissue myeloid cells and found that Tet2 sustains the immunosuppressive function of these cells. We found that Tet2 expression is increased in intratumoral myeloid cells both in mouse models of melanoma and in melanoma patients and that this increased expression is dependent on an IL-1R-MyD88 pathway. Ablation of Tet2 in myeloid cells suppressed melanoma growth in vivo and shifted the immunosuppressive gene expression program in tumor-associated macrophages to a proinflammatory one, with a concomitant reduction of the immunosuppressive function. This resulted in increased numbers of effector T cells in the tumor, and T cell depletion abolished the reduced tumor growth observed upon myeloid-specific deletion of Tet2. Our findings reveal a non-cell-intrinsic, tumor-promoting function for Tet2 and suggest that Tet2 may present a therapeutic target for the treatment of non-hematologic malignancies.