Engineering nanomedicines for immunogenic eradication of cancer cells: Recent trends and synergistic approaches.

Resistance to cancer immunotherapy is mainly attributed to poor tumor immunogenicity as well as the immunosuppressive tumor microenvironment (TME) leading to failure of immune response. Numerous therapeutic strategies including chemotherapy, radiotherapy, photodynamic, photothermal, magnetic, chemodynamic, sonodynamic and oncolytic therapy, have been developed to induce immunogenic cell death (ICD) of cancer cells and thereby elicit immunogenicity and boost the antitumor immune response. However, many challenges hamper the clinical application of ICD inducers resulting in modest immunogenic response. Here, we outline the current state of using nanomedicines for boosting ICD of cancer cells. Moreover, synergistic approaches used in combination with ICD inducing nanomedicines for remodeling the TME via targeting immune checkpoints, phagocytosis, macrophage polarization, tumor hypoxia, autophagy and stromal modulation to enhance immunogenicity of dying cancer cells were analyzed. We further highlight the emerging trends of using nanomaterials for triggering amplified ICD-mediated antitumor immune responses. Endoplasmic reticulum localized ICD, focused ultrasound hyperthermia, cell membrane camouflaged nanomedicines, amplified reactive oxygen species (ROS) generation, metallo-immunotherapy, ion modulators and engineered bacteria are among the most innovative approaches. Various challenges, merits and demerits of ICD inducer nanomedicines were also discussed with shedding light on the future role of this technology in improving the outcomes of cancer immunotherapy.

Thermal-Induced Microstructure Deterioration of Egyptian Granodiorite and Associated Physico-Mechanical Responses.

Mineral transformations often induce microstructural deteriorations during temperature variations. Hence, it is crucial to understand why and how this microstructure weakens due to mineral alteration with temperature and the correlated physical and mechanical responses. Therefore, in this study, physical, chemical, thermal, petrographic, and mechanical analyses were carried out to comprehend better the thermal behaviors of Egyptian granodiorite exposed to temperatures as high as 800 °C. The experimental results indicate that the examined attributes change in three distinct temperature phases. Strength zone (up to 200 °C): During this phase, the temperature only slightly impacts the granodiorite mass loss and porosity, and the P-wave velocity and E slightly decrease. However, the rock structure was densified, which resulted in a minor increase in strength. After that, the transition zone (200-400 °C) was distinguished by the stability of most studied parameters. For instance, mass and porosity did not significantly alter, and the uniaxial compressive strength steadily increased with an axial failure mode. When the temperature rises, transgranular cracks cause the P-wave velocity and elastic modulus to decrease moderately. The decay zone started after 400 °C and continued to 800 °C. This zone is characterized by complicated factors that worsen the granodiorite properties, lead to color shift, and produce a shear failure mode. The properties of granodiorite became worse because of chemical reactions, structural and crystal water evaporation, rising thermal expansion coefficient variation, and quartz inversion at 575 °C (α to ß, according to the differential thermal analysis). Thermal damage greatly affected granodiorite's physical and mechanical properties and microstructure at 800 °C. As a result, UCS measurements were extremely small with a complex failure pattern, making Vp and E unattainable.

Circularized Nanodiscs for Multivalent Mosaic Display of SARS-CoV-2 Spike Protein Antigens.

The emergence of vaccine-evading SARS-CoV-2 variants urges the need for vaccines that elicit broadly neutralizing antibodies (bnAbs). Here, we assess covalently circularized nanodiscs decorated with recombinant SARS-CoV-2 spike glycoproteins from several variants for eliciting bnAbs with vaccination. Cobalt porphyrin-phospholipid (CoPoP) was incorporated into the nanodisc to allow for anchoring and functional orientation of spike trimers on the nanodisc surface through their His-tag. Monophosphoryl-lipid (MPLA) and QS-21 were incorporated as immunostimulatory adjuvants to enhance vaccine responses. Following optimization of nanodisc assembly, spike proteins were effectively displayed on the surface of the nanodiscs and maintained their conformational capacity for binding with human angiotensin-converting enzyme 2 (hACE2) as verified using electron microscopy and slot blot assay, respectively. Six different formulations were prepared where they contained mono antigens; four from the year 2020 (WT, Beta, Lambda, and Delta) and two from the year 2021 (Omicron BA.1 and BA.2). Additionally, we prepared a mosaic nanodisc displaying the four spike proteins from year 2020. Intramuscular vaccination of CD-1 female mice with the mosaic nanodisc induced antibody responses that not only neutralized matched pseudo-typed viruses, but also neutralized mismatched pseudo-typed viruses corresponding to later variants from year 2021 (Omicron BA.1 and BA.2). Interestingly, sera from mosaic-immunized mice did not effectively inhibit Omicron spike binding to human ACE-2, suggesting that some of the elicited antibodies were directed towards conserved neutralizing epitopes outside the receptor binding domain. Our results show that mosaic nanodisc vaccine displaying spike proteins from 2020 can elicit broadly neutralizing antibodies that can neutralize mismatched viruses from a following year, thus decreasing immune evasion of new emerging variants and enhancing healthcare preparedness.

Comparing the effectiveness of L-carnitine and paraffin oil in acute aluminum phosphide poisoning using predictive biomarkers and scores: A randomized controlled clinical trial.

Aluminum phosphide (AlP) poisoning is a serious medical emergency with a high mortality rate. The absence of an exact antidote for AlP poisoning necessitates the quest for alternative treatment options. The study sought to assess the efficacy of adding L-carnitine or medicated paraffin oil to the conventional approach of treatment employed in cases of acute AlP poisoning. We conducted a 1 year, randomized, controlled, parallel-group, single-blind clinical study. 96 individuals with acute AlP poisoning were randomly assigned to one of three groups. The standard AlP therapy was administered to all groups according to the Poison Control Center guidelines at the Ain-Shams University hospitals. All patients underwent a medical history review, clinical examination, and laboratory tests. The outcomes were assessed. The participants in the study groups had mean ages ranging from 25.6 to 26.3 years. The cases analyzed were evenly distributed between genders, with the majority originating from rural areas. The average delay time varied from 2.9 to 4.2 h. All patients in the study reported ingesting AlP during suicide attempts. 12 hours after admission, many clinical and biochemical data improved in both intervention groups including cytochrome c oxidase, caspase-3, caspase-9, catalase, and superoxide dismutase. The intervention groups required significantly less mechanical ventilation and had a lower mortality rate than the control group. Decontamination with paraffin oil could be advantageous for reducing the severity of AlP poisoning, improving prognosis, and lowering the mortality rate.

Engineered nanomedicines to overcome resistance of pancreatic cancer to immunotherapy.

Pancreatic cancer (PC) is a highly aggressive malignant type of cancer. Although immunotherapy has been successfully used for treatment of many cancer types, many challenges limit its success in PC. Therefore, nanomedicines were engineered to enhance the responsiveness of PC cells to immune checkpoint inhibitors (ICIs). In this review, we highlight recent advances in engineering nanomedicines to overcome PC immune resistance. Nanomedicines were used to increase the immunogenicity of PC cells, inactivate stromal cancer-associated fibroblasts (CAFs), enhance the antigen-presenting capacity of dendritic cells (DCs), reverse the highly immunosuppressive nature of the tumor microenvironment (TME), and, hence, improve the infiltration of cytotoxic T lymphocytes (CTLs), resulting in efficient antitumor immune responses.

DNA-nanostructure-templated assembly of planar and curved lipid-bilayer membranes.

Lipid-bilayer nanodiscs and liposomes have been developed to stabilize membrane proteins in order to study their structures and functions. Nanodiscs are detergent-free, water-soluble, and size-controlled planar phospholipid-bilayer platforms. On the other hand, liposomes are curved phospholipid-bilayer spheres with an aqueous core used as drug delivery systems and model membrane platforms for studying cellular activities. A long-standing challenge is the generation of a homogenous and monodispersed lipid-bilayer system with a very wide range of dimensions and curvatures (elongation, bending, and twisting). A DNA-origami template provides a way to control the shapes, sizes, and arrangements of lipid bilayers via enforcing the assembly of lipid bilayers within the cavities created by DNA nanostructures. Here, we provide a concise overview and discuss how to design planar and curved lipid-bilayer membranes by using DNA-origami nanostructures as templates. Finally, we will discuss the potential applications of DNA-origami nanostructures in the structural and functional studies of large membrane proteins and their complexes.

KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney.

SARS-CoV-2 precipitates respiratory distress by infection of airway epithelial cells and is often accompanied by acute kidney injury. We report that Kidney Injury Molecule-1/T cell immunoglobulin mucin domain 1 (KIM-1/TIM-1) is expressed in lung and kidney epithelial cells in COVID-19 patients and is a receptor for SARS-CoV-2. Human and mouse lung and kidney epithelial cells express KIM-1 and endocytose nanoparticles displaying the SARS-CoV-2 spike protein (virosomes). Uptake was inhibited by anti-KIM-1 antibodies and TW-37, a newly discovered inhibitor of KIM-1-mediated endocytosis. Enhanced KIM-1 expression by human kidney tubuloids increased uptake of virosomes. KIM-1 binds to the SARS-CoV-2 Spike protein in vitro . KIM-1 expressing cells, not expressing angiotensin-converting enzyme 2 (ACE2), are permissive to SARS-CoV-2 infection. Thus, KIM-1 is an alternative receptor to ACE2 for SARS-CoV-2. KIM-1 targeted therapeutics may prevent and/or treat COVID-19.

Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs.

G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1ß1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.

Antiretroviral Drugs Promote Amyloidogenesis by De-Acidifying Endolysosomes.

Antiretroviral therapeutics (ART) have effectively increased the long-term survival of HIV-1 infected individuals. However, the prevalence of HIV-1 associated neurocognitive disorders (HAND) has increased and so too have clinical manifestations and pathological features of Alzheimer's disease (AD) in people living with HIV-1/AIDS. Although underlying mechanisms are not clear, chronic exposure to ART drugs has been implicated in the development of AD-like symptoms and pathology. ART drugs are categorized according to their mechanism of action in controlling HIV-1 levels. All ART drugs are organic compounds that can be classified as being either weak acids or weak bases, and these physicochemical properties may be of central importance to ART drug-induced AD-like pathology because weak bases accumulate in endolysosomes, weak bases can de-acidify endolysosomes where amyloidogenesis occurs, and endolysosome de-acidification increases amyloid beta (Aß) protein production and decreases Aß degradation. Here, we investigated the effects of ART drugs on endolysosome pH and Aß levels in rat primary cultured neurons. ART drugs that de-acidified endolysosomes increased Aß levels, whereas those that acidified endolysosomes decreased Aß levels. Acidification of endolysosomes with the mucolipin transient receptor potential (TRPML) channel agonist ML-SA1 blocked ART drug-induced increases in Aß levels. Further, ART drug-induced endolysosome de-acidification increased endolysosome sizes; effects that were blocked by ML-SA1-induced endolysosome acidification. These results suggest that ART drug-induced endolysosome de-acidification plays an important role in ART drug-induced amyloidogenesis and that endolysosome acidification might attenuate AD-like pathology in HIV-1 positive people taking ART drugs that de-acidify endolysosomes. Graphical Abstract.

Conformational gating, dynamics and allostery in human monoacylglycerol lipase.

Inhibition of human Monoacylglycerol Lipase (hMGL) offers a novel approach for treating neurological diseases. The design of inhibitors, targeting active-inactive conformational transitions of the enzyme, can be aided by understanding the interplay between structure and dynamics. Here, we report the effects of mutations within the catalytic triad on structure, conformational gating and dynamics of hMGL by combining kinetics, NMR, and HDX-MS data with metadynamics simulations. We found that point mutations alter delicate conformational equilibria between active and inactive states. HDX-MS reveals regions of the hMGL that become substantially more dynamic upon substitution of catalytic acid Asp-239 by alanine. These regions, located far from the catalytic triad, include not only loops but also rigid α-helixes and ß-strands, suggesting their involvement in allosteric regulation as channels for long-range signal transmission. The results identify the existence of a preorganized global communication network comprising of tertiary (residue-residue contacts) and quaternary (rigid-body contacts) networks that mediate robust, rapid intraprotein signal transmission. Catalytic Asp-239 controls hMGL allosteric communications and may be considered as an essential residue for the integration and transmission of information to enzymes' remote regions, in addition to its well-known role to facilitate Ser-122 activation. Our findings may assist in the identification of new druggable sites in hMGL.

KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney

SARS-CoV-2 precipitates respiratory distress by infection of airway epithelial cells and is often accompanied by acute kidney injury. We report that Kidney Injury Molecule-1/T cell immunoglobulin mucin domain 1 (KIM-1/TIM-1) is expressed in lung and kidney epithelial cells in COVID-19 patients and is a receptor for SARS-CoV-2. Human and mouse lung and kidney epithelial cells express KIM-1 and endocytose nanoparticles displaying the SARS-CoV-2 spike protein (virosomes). Uptake was inhibited by anti-KIM-1 antibodies and TW-37, a newly discovered inhibitor of KIM-1-mediated endocytosis. Enhanced KIM-1 expression by human kidney tubuloids increased uptake of virosomes. KIM-1 binds to the SARS-CoV-2 Spike protein in vitro. KIM-1 expressing cells, not expressing angiotensin-converting enzyme 2 (ACE2), are permissive to SARS-CoV-2 infection. Thus, KIM-1 is an alternative receptor to ACE2 for SARS-CoV-2. KIM-1 targeted therapeutics may prevent and/or treat COVID-19.

Large Nanodiscs: A Potential Game Changer in Structural Biology of Membrane Protein Complexes and Virus Entry.

Phospho-lipid bilayer nanodiscs have gathered much scientific interest as a stable and tunable membrane mimetic for the study of membrane proteins. Until recently the size of the nanodiscs that could be produced was limited to ~ 16 nm. Recent advances in nanodisc engineering such as covalently circularized nanodiscs (cND) and DNA corralled nanodiscs (DCND) have opened up the possibility of engineering nanodiscs of size up to 90 nm. This enables widening the application of nanodiscs from single membrane proteins to investigating large protein complexes and biological processes such as virus-membrane fusion and synaptic vesicle fusion. Another aspect of exploiting the large available surface area of these novel nanodiscs could be to engineer more realistic membrane mimetic systems with features such as membrane asymmetry and curvature. In this review, we discuss the recent technical developments in nanodisc technology leading to construction of large nanodiscs and examine some of the implicit applications.

Large nanodiscs going viral.

Covalently circularized and DNA-corralled nanodisc technologies have enabled engineering of large-sized bilayer nanodiscs up to 90nm. These large nanodiscs have the potential to extend the applicability of nanodisc technology from studying small and medium-sized membrane proteins to acting as surrogate membranes to investigate functional and structural aspects of viral entry. Here, we discuss the recent technical developments leading to construction of large circularized and DNA-corralled nanodiscs and examine their application in viral entry.

Topological analysis of the gp41 MPER on lipid bilayers relevant to the metastable HIV-1 envelope prefusion state.

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.

Calcifying Epithelial Odontogenic (Pindborg) Tumor in a Child: A Case Report and Literature Review.

Calcifying epithelial odontogenic tumor (CEOT) is a rare neoplasm, which accounts for < 1% of all odontogenic tumors. CEOT occurs more frequently in adults with a peak incidence in the 5th decade of life and is extremely rare in the pediatric population. We present a case of a 13-year-old girl who was found to have a mandibular CEOT. We summarize the radiological features, pathological findings, clinical management and literature review focusing on this entity in children.

Role of endolysosomes and pH in the pathogenesis and treatment of glioblastoma.

BACKGROUND: Glioblastoma multiforme (GBM) is a Grade IV astrocytoma with an aggressive disease course and a uniformly poor prognosis. Pathologically, GBM is characterized by rapid development of primary tumors, diffuse infiltration into the brain parenchyma, and robust angiogenesis. The treatment options that are limited and largely ineffective include a combination of surgical resection, radiotherapy, and chemotherapy with the alkylating agent temozolomide. RECENT FINDINGS: Similar to many other forms of cancer, the extracellular environment near GBM tumors is acidified. Extracellular acidosis is particularly relevant to tumorgenesis and the concept of tumor cell dormancy because of findings that decreased pH reduces proliferation, increases resistance to apoptosis and autophagy, promotes tumor cell invasion, increases angiogenesis, obscures immune surveillance, and promotes resistance to drug and radio-treatment. Factors known to participate in the acidification process are nutrient starvation, oxidative stress, hypoxia and high levels of anaerobic glycolysis that lead to increases in lactate. Also involved are endosomes and lysosomes (hereafter termed endolysosomes), acidic organelles with highly regulated stores of hydrogen (H+) ions. Endolysosomes contain more than 60 hydrolases as well as about 50 proteins that are known to affect the number, sizes and distribution patterns of these organelles within cells. Recently, vacuolar ATPase (v-ATPase), the main proton pump that is responsible for maintaining the acidic environment in endolysosomes, was identified as a novel therapeutic target for glioblastoma. CONCLUSIONS: Thus, a greater understanding of the role of endolysosomes in regulating cellular and extracellular acidity could result in a better elucidation of GBM pathogenesis and new therapeutic strategies.

Acidifying Endolysosomes Prevented Low-Density Lipoprotein-Induced Amyloidogenesis.

Cholesterol dyshomeostasis has been linked to the pathogenesis of sporadic Alzheimer's disease (AD). In furthering the understanding of mechanisms by which increased levels of circulating cholesterol augments the risk of developing sporadic AD, others and we have reported that low-density lipoprotein (LDL) enters brain parenchyma by disrupting the blood-brain barrier and that endolysosome de-acidification plays a role in LDL-induced amyloidogenesis in neurons. Here, we tested the hypothesis that endolysosome de-acidification was central to amyloid-ß (Aß) generation and that acidifying endolysosomes protects against LDL-induced increases in Aß levels in neurons. We demonstrated that LDL, but not HDL, de-acidified endolysosomes and increased intraneuronal and secreted levels of Aß. ML-SA1, an agonist of endolysosome-resident TRPML1 channels, acidified endolysosomes, and TRPML1 knockdown attenuated ML-SA1-induced endolysosome acidification. ML-SA1 blocked LDL-induced increases in intraneuronal and secreted levels of Aß as well as Aß accumulation in endolysosomes, prevented BACE1 accumulation in endolysosomes, and decreased BACE1 activity levels. LDL downregulated TRPML1 protein levels, and TRPML1 knockdown worsens LDL-induced increases in Aß. Our findings suggest that endolysosome acidification by activating TRPML1 may represent a protective strategy against sporadic AD.

DNA-Corralled Nanodiscs for the Structural and Functional Characterization of Membrane Proteins and Viral Entry.

Here we present a modular method for manufacturing large-sized nanodiscs using DNA-origami barrels as scaffolding corrals. Large-sized nanodiscs can be produced by first decorating the inside of DNA barrels with small lipid-bilayer nanodiscs, which open up when adding extra lipid to form large nanodiscs of diameters ∼45 or ∼70 nm as prescribed by the enclosing barrel dimension. Densely packed membrane protein arrays are then reconstituted within these large nanodiscs for potential structure determination. Furthermore, we demonstrate the potential of these nanodiscs as model membranes to study poliovirus entry.

Covalently circularized nanodiscs; challenges and applications.

Covalently circularized nanodiscs (cNDs) represent a significant advance in the durability and applicability of nanodisc technology. The new cNDs demonstrate higher size homogeneity and improved stability compared with that of non-circularized forms. Moreover, cNDs can be prepared at various defined sizes up to 80-nm diameter. The large cNDs can house much larger membrane proteins and their complexes than was previously possible with the conventional nanodiscs. In order to experience the full advantages of covalent circularization, high quality circularized scaffold protein and nanodisc samples are needed. Here, we give a concise overview and discuss the technical challenges that needed to be overcome in order to obtain high quality preparations. Furthermore, we review some potential new applications for the cNDs.