Surface Engineering of Natural Killer Cells with CD44-targeting Ligands for Augmented Cancer Immunotherapy.

Adoptive immunotherapy utilizing natural killer (NK) cells has demonstrated remarkable efficacy in treating hematologic malignancies. However, its clinical intervention for solid tumors is hindered by the limited expression of tumor-specific antigens. Herein, lipid-PEG conjugated hyaluronic acid (HA) materials (HA-PEG-Lipid) for the simple ex-vivo surface coating of NK cells is developed for 1) lipid-mediated cellular membrane anchoring via hydrophobic interaction and thereby 2) sufficient presentation of the CD44 ligand (i.e., HA) onto NK cells for cancer targeting, without the need for genetic manipulation. Membrane-engineered NK cells can selectively recognize CD44-overexpressing cancer cells through HA-CD44 affinity and subsequently induce in situ activation of NK cells for cancer elimination. Therefore, the surface-engineered NK cells using HA-PEG-Lipid (HANK cells) establish an immune synapse with CD44-overexpressing MIA PaCa-2 pancreatic cancer cells, triggering the "recognition-activation" mechanism, and ultimately eliminating cancer cells. Moreover, in mouse xenograft tumor models, administrated HANK cells demonstrate significant infiltration into solid tumors, resulting in tumor apoptosis/necrosis and effective suppression of tumor progression and metastasis, as compared to NK cells and gemcitabine. Taken together, the HA-PEG-Lipid biomaterials expedite the treatment of solid tumors by facilitating a sequential recognition-activation mechanism of surface-engineered HANK cells, suggesting a promising approach for NK cell-mediated immunotherapy.

Ameloblastoma modifies tumor microenvironment for enhancing invasiveness by altering collagen alignment.

Tumor progression is profoundly affected by crosstalk between cancer cells and their stroma. In the past decades, the development of bioinformatics and the establishment of organoid model systems have allowed extensive investigation of the relationship between tumor cells and the tumor microenvironment (TME). However, the interaction between tumor cells and the extracellular matrix (ECM) in odontogenic epithelial neoplasms and the ECM remodeling mechanism remain unclear. In the present study, transcriptomic comparison and histopathologic analysis revealed that TME-related genes were upregulated in ameloblastoma compared to in odontogenic keratocysts. Tumoroid analysis indicated that type I collagen is required for ameloblastoma progression. Furthermore, ameloblastoma shows the capacity to remodel the ECM independently of cancer-associated fibroblasts. In conclusion, ameloblastoma-mediated ECM remodeling contributes to the formation of an invasive collagen architecture during tumor progression.

Strategies for differentiation of hiPSCs into dental epithelial cell lineage.

Different stem cell-based strategies, especially induced pluripotent stem cells (iPSCs), have been exploited to regenerate teeth or restore biological and physiological functions after tooth loss. Further research is needed to establish an optimized protocol to effectively differentiate human iPSCs (hiPSCs) into dental epithelial cells (DECs). In this study, various factors were precisely modulated to facilitate differentiation of hiPSCs into DECs, which are essential for the regeneration of functional teeth. Embryoid bodies (EBs) were formed from hiPSCs as embryo-like aggregates, retinoic acid (RA) was used as an early ectodermal inducer, and bone morphogenic protein 4 (BMP4) activity was manipulated. The characteristics of DECs were enhanced and preserved after culture in keratinocyte serum-free medium (K-SFM). The yielded cell population exhibited noticeable DEC characteristics, consistent with the expression of epithelial cell and ameloblast markers. DECs demonstrated odontogenic abilities by exerting an inductive effect on human dental pulp stem cells (hDPSCs) and forming a tooth-like structure with the mouse tooth mesenchyme. Overall, our differentiation protocol provides a practical approach for applying hiPSCs for tooth regeneration.

Transfer of a healthy microbiota reduces amyloid and tau pathology in an Alzheimer's disease animal model.

OBJECTIVE: Cerebral amyloidosis and severe tauopathy in the brain are key pathological features of Alzheimer's disease (AD). Despite a strong influence of the intestinal microbiota on AD, the causal relationship between the gut microbiota and AD pathophysiology is still elusive. DESIGN: Using a recently developed AD-like pathology with amyloid and neurofibrillary tangles (ADLPAPT) transgenic mouse model of AD, which shows amyloid plaques, neurofibrillary tangles and reactive gliosis in their brains along with memory deficits, we examined the impact of the gut microbiota on AD pathogenesis. RESULTS: Composition of the gut microbiota in ADLPAPT mice differed from that of healthy wild-type (WT) mice. Besides, ADLPAPT mice showed a loss of epithelial barrier integrity and chronic intestinal and systemic inflammation. Both frequent transfer and transplantation of the faecal microbiota from WT mice into ADLPAPT mice ameliorated the formation of amyloid ß plaques and neurofibrillary tangles, glial reactivity and cognitive impairment. Additionally, the faecal microbiota transfer reversed abnormalities in the colonic expression of genes related to intestinal macrophage activity and the circulating blood inflammatory monocytes in the ADLPAPT recipient mice. CONCLUSION: These results indicate that microbiota-mediated intestinal and systemic immune aberrations contribute to the pathogenesis of AD in ADLPAPT mice, providing new insights into the relationship between the gut (colonic gene expression, gut permeability), blood (blood immune cell population) and brain (pathology) axis and AD (memory deficits). Thus, restoring gut microbial homeostasis may have beneficial effects on AD treatment.

CDDO-Me, Sulforaphane and tBHQ attenuate the RANKL-induced osteoclast differentiation via activating the NRF2-mediated antioxidant response.

Metabolic bone diseases are global public health concerns and are primarily caused by uncontrolled osteoclast (OC) formation and activation. During OC differentiation, intracellular reactive oxygen species (ROS) stimulated by receptor activator of nuclear factor kappa-B ligand (RANKL) can serve as the signaling molecules to promote osteoclastic genes expression. Nuclear factor erythroid-2 related factor 2 (NRF2), a master mediator of cellular antioxidant response, also plays a critical role in OC differentiation through the regulation of redox homeostasis. In this study, we investigated the effects of three NRF2 inducers on osteoclastogenesis, including Bardoxolone methyl (CDDO-Me), Sulforaphane (SFN), and tert-butylhydroquinone (tBHQ). By treating RAW cells with three compounds, we found that NRF2 was activated and its downstream antioxidant genes were upregulated, and the RANKL-induced intracellular ROS production and osteoclastogenesis were impaired. Additionally, the expression of nuclear factor of activated T cells c1 (NFATC1), C-FOS and tumor necrosis factor alpha (TNFα) were inhibited after acute exposures (6 h) to the three compounds. Furthermore, suppressed the expression of osteoclast differentiation-associated genes, tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), matrix metalloproteinase-9 (MMP-9) and dendritic cell-specific transmembrane protein (DC-STAMP) were observed after prolonged exposures (5 days) to the compounds. Taken together, these results suggest that CDDO-Me, SFN and tBHQ attenuate RANKL-induced osteoclastogenesis via activation of NRF2-mediated antioxidant response. Among these compounds, relatively low concentrations of CDDO-Me showed stronger active and inhibitory effects on antioxidant response and osteoclastogenesis, respectively.

Wound healing mechanism in Mongolian gerbil skin.

The skin wound healing ability of animals differs depending on the environment. The gerbil wound model showed a different wound healing mechanism than was known thus far. Many other wound healing mechanisms have been found to involve transforming growth factor-beta 1 (TGF-ß1). However, in the wound healing of gerbil skin, the expression of TGF-ß1 seems to be not enough compared to mouse. In this study, we compared the wound healing process of gerbil and mouse back skin. At 3 days after wounding, the TGF-ß1 level was downregulated in gerbil skin wound healing compared mouse. In addition, gerbils have fewer integrin signals related to the regulation of TGF-ß activation and signaling. Despite lacking these factors, the wound healing results in the gerbil are similar to those for skin wound healing in mice. In contrast, in gerbil skin wound healing, the basal skin layer showed hyperplasia in re-epithelialization, more production of hair follicles, and low probability of collagen infiltration at the late stages of wound healing. These data suggest that different wound healing mechanisms are present in the mammals.

Micrometer Scale Resolution Limit of a Fiber-Coupled Electro-Optic Probe.

We present the practical resolution limit of a fine electrical structure based on a fiber-coupled electro-optic probing system. The spatial resolution limit was experimentally evaluated on the sub-millimeter to micrometer scale of planar electrical transmission lines. The electrical lines were fabricated to have various potential differences depending on the dimensions and geometry. The electric field between the lines was measured through an electro-optic probe, which was miniaturized up to the optical bare fiber scale so as to investigate the spatial limit of electrical signals with minimal invasiveness. The experimental results show that the technical resolution limitation of a fiber-coupled probe can reasonably approach a fraction of the mode field diameter (~10 µm) of the fiber in use.

Field-calibrated magneto-optic sensor based on off-axis optical probing of intense magnetic fields.

We present a magneto-optic sensing system based on the "off-axis" optical probing technique to control the measurement sensitivity of magnets with various field strengths. The magnetic field is experimentally investigated in the absolute scale through a photonic calibration method with a standard electromagnet. Our all-dielectric magnetic field probe has a wide dynamic range (20 mT-3 T) with good responsivity and low probe invasiveness against the magnetic field being measured. Utilizing this magnetic-field-calibrated probing system, we obtain the magnetic field distribution of permanent magnets. Subsequently, we compare our results with numerical analyses to confirm the effectiveness of the probing system. Finally, we measure the intense magnetic field inside the bore of a 3.0-T clinical magnetic resonance imaging system with our probe.

Two-tier calibrated electro-optic sensing system for intense field characterization of high-power W-band gyrotron.

We present a field-calibrated electro-optic sensing system for measurement of the electric field radiating from a high-power vacuum oscillator at ~95 GHz. The intense electric field is measured in absolute scale via two probe-calibration steps, associated with a photonic heterodyne scheme. First, a micro-electro-optic probe, fabricated to less than one-tenth the oscillation wavelength scale to minimize field-perturbation due to the probe, is placed on the aperture of a field-calculable WR-10 waveguide to calibrate the probe in V/m scale. Then, using this arrangement as a calibrated reference probe at the first-tier position, another probe-bulkier, and thus more robust and sensitive but not accessible to the aperture-is calibrated at the second-tier position away from the waveguide aperture. This two-tier calibrated probe was utilized to diagnose the sub-MV/m scale of intense electric fields and emissions from a high-power W-band gyrotron. The experimental results obtained proved consistent with calculated analytical results-verifying the efficacy of the developed system.

Gastrin-releasing peptide expression and its effect on the calcification of developing mouse incisor.

Gastrin-releasing peptide (GRP) is considered to be one of the cancer growth factors. This peptide's receptor (GRPR) is known as a G protein-coupled receptor, regulating intracellular calcium storage and releasing signals. This study is the first to investigate the function of GRP during mouse incisor development. We hypothesized that GRP is one of the factors that affects the regulation of calcification during tooth development. To verify the expression pattern of GRP, in situ hybridization was processed during incisor development. GRP was expressed at the late bell stage and hard tissue formation stage in the epithelial tissue. To identify the genuine function of GRP during incisor development, a gain-of-function analysis was performed. After GRP overexpression in culture, the phenotype of ameloblasts, odontoblasts and predentin was altered compared to control group. Moreover, enamel and dentin thickness was increased after renal capsule transplantation of GRP-overexpressed incisors. With these results, we suggest that GRP plays a significant role in the formation of enamel and dentin by regulating ameloblasts and predentin formation, respectively. Thus, GRP signaling is strongly related to calcium acquisition and secretion during mouse incisor development.

In situ endoscopic observation of higher-order mode conversion in a microwave mode converter based on an electro-optic probe system.

Visualizing the electromagnetic field transformation inside a microwave mode conversion region has been considered to be only realizable by simulation studies. For the first time, we present a comprehensive experimental observation of the electric field transformation occurring inside a metallic waveguide TE(01)-to-TE(02) mode converter. An efficient electro-optic (EO) probe and its associated probing system were used for measuring the electric field pattern in the external near-field region as well as in the internal and penetrated region of the mode converter. Utilizing the optically measured field patterns at the aperture of the mode converter, the conversion performance from the TE(01) mode to the TE(02) mode can be also evaluated. Experimentally measured field patterns near the apertures show excellent agreement with simulation data. The mode conversion to the next higher-order mode (TE(01) to TE(02)) was experimentally demonstrated with phase-stabilized and field-animated post processing. The presented in situ endoscopic photonic measurement technique for the field evolution inside a semi-enclosed structure could be used for visually inspecting manufacturing errors in fabricated structures, and could be of great interest for research on higher-order mode formation and transmission.

Field analysis of electro-optic probes for minimally invasive microwave sampling.

We numerically and experimentally investigate the field invasiveness of microwave signals using an electro-optic technique. The distortion of the standing wave voltage and pulse waveform probed by the electro-optic technique is explored through both minimally invasive external and non-invasive internal sensing configurations. First, we analyzed the continuous wave microwave field imaging on a millimeter- scale coaxial transmission line using a highly accurate and stable electro- optic scanning system. The electric field images from the microwave device are attained virtually non-invasively using a miniaturized fiber-coupled electro-optic probe. The accuracy of the field imaging associated with various probe styles is investigated by numerical analysis and experiment. Then, we analyzed the waveform of the coaxial transmission line up to 50 GHz using a pulsed electro-optic system with an external probe set. Finally, the invasive analysis was extended to the sub-millimeter-scale on-wafer coplanar waveguides, where the voltage waveforms are measured using a minimally invasive external probe as well as an internal wafer probe for non-invasive sampling.

MAST4 regulates stem cell maintenance with DLX3 for epithelial development and amelogenesis.

The asymmetric division of stem cells permits the maintenance of the cell population and differentiation for harmonious progress. Developing mouse incisors allows inspection of the role of the stem cell niche to provide specific insights into essential developmental phases. Microtubule-associated serine/threonine kinase family member 4 (Mast4) knockout (KO) mice showed abnormal incisor development with low hardness, as the size of the apical bud was decreased and preameloblasts were shifted to the apical side, resulting in amelogenesis imperfecta. In addition, Mast4 KO incisors showed abnormal enamel maturation, and stem cell maintenance was inhibited as amelogenesis was accelerated with Wnt signal downregulation. Distal-Less Homeobox 3 (DLX3), a critical factor in tooth amelogenesis, is considered to be responsible for the development of amelogenesis imperfecta in humans. MAST4 directly binds to DLX3 and induces phosphorylation at three residues within the nuclear localization site (NLS) that promotes the nuclear translocation of DLX3. MAST4-mediated phosphorylation of DLX3 ultimately controls the transcription of DLX3 target genes, which are carbonic anhydrase and ion transporter genes involved in the pH regulation process during ameloblast maturation. Taken together, our data reveal a novel role for MAST4 as a critical regulator of the entire amelogenesis process through its control of Wnt signaling and DLX3 transcriptional activity.

Cyclic stretch induced epigenetic activation of periodontal ligament cells.

Periodontal ligament (PDL) cells play a crucial role in maintaining periodontal integrity and function by providing cell sources for ligament regeneration. While biophysical stimulation is known to regulate cell behaviors and functions, its impact on epigenetics of PDL cells has not yet been elucidated. Here, we aimed to investigate the cytoskeletal changes, epigenetic modifications, and lineage commitment of PDL cells following the application of stretch stimuli to PDL. PDL cells were subjected to stretching (0.1 Hz, 10 %). Subsequently, changes in focal adhesion, tubulin, and histone modification were observed. The survival ability in inflammatory conditions was also evaluated. Furthermore, using a rat hypo-occlusion model, we verified whether these phenomena are observed in vivo. Stretched PDL cells showed maximal histone 3 acetylation (H3Ace) at 2 h, aligning perpendicularly to the stretch direction. RNA sequencing revealed stretching altered gene sets related to mechanotransduction, histone modification, reactive oxygen species (ROS) metabolism, and differentiation. We further found that anchorage, cell elongation, and actin/microtubule acetylation were highly upregulated with mechanosensitive chromatin remodelers such as H3Ace and histone H3 trimethyl lysine 9 (H3K9me3) adopting euchromatin status. Inhibitor studies showed mechanotransduction-mediated chromatin modification alters PDL cells behaviors. Stretched PDL cells displayed enhanced survival against bacterial toxin (C12-HSL) or ROS (H2O2) attack. Furthermore, cyclic stretch priming enhanced the osteoclast and osteoblast differentiation potential of PDL cells, as evidenced by upregulation of lineage-specific genes. In vivo, PDL cells from normally loaded teeth displayed an elongated morphology and higher levels of H3Ace compared to PDL cells with hypo-occlusion, where mechanical stimulus is removed. Overall, these data strongly link external physical forces to subsequent mechanotransduction and epigenetic changes, impacting gene expression and multiple cellular behaviors, providing important implications in cell biology and tissue regeneration.

Abnormal liver differentiation and excessive angiogenesis in mice lacking Runx3.

Runt-related transcription factor 3 (Runx3) is essential for normal mouse development, and Runx3 knock-out (KO) mice (FVB strain), which die within 24 h after birth, show various organ defects, such as lung hyperplasia. For proper early liver development, angiogenesis and liver cell differentiation mechanisms are necessary in mammals. Previous studies have reported that various signaling molecules, such as vascular endothelial growth factor (VEGF), von Willebrand factor (vWF) and cluster of differentiation 31 (CD31), are closely related to angiogenesis in the developing liver. Proper expression levels of molecules that induce liver cell differentiation, such as phosphorylated Smad2 (pSmad2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), Wilms tumor-1 (WT-1) and CD90 (Thy-1), are necessary for fetal liver development. To confirm the pathogenesis of liver defects caused by the loss of function of Runx3, the localization of proliferating cells was examined in wild-type and Runx3 KO mouse livers at postnatal day 1 (PN1). Specimens were also stained for various liver differentiation markers to confirm the function of Runx3. Moreover, gene expression level was examined by real-time quantitative polymerase chain reaction (RT-qPCR). Our results indicate that VEGF, vWF, CD31, pSmad2, NF-kB, WT-1 and Thy-1 were markedly up-regulated by the loss of Runx3. Therefore, our results indicate that liver development is controlled by Runx3. Clarifying the mechanisms of angiogenesis and liver differentiation might aid in the design of efficient and safe antiangiogenic therapy and gene therapy for liver disorders.

Harnessing the dental cells derived from human induced pluripotent stem cells for hard tissue engineering.

INTRODUCTION: Most mineralized tissues in our body are present in bones and teeth. Human induced pluripotent stem cells (hiPSCs) are promising candidates for cell therapy to help regenerate bone defects and teeth loss. The extracellular matrix (ECM) is a non-cellular structure secreted by cells. Studies on the dynamic microenvironment of ECM are necessary for stem cell-based therapies. OBJECTIVES: We aim to optimize an effective protocol for hiPSC differentiation into dental cells without utilizing animal-derived factors or cell feeders that can be applied to humans and to mineralize differentiated dental cells into hard tissues. METHODS: For the differentiation of both dental epithelial cells (DECs) and dental mesenchymal cells (DMCs) from hiPSCs, an embryoid body (EB) was formed from hiPSCs. hiPSC were differentiated into neural crest cells with an induction medium utilized in our previous study, and hiPSC-derived DECs were differentiated with a BMP-modulated customized medium. hiPSC-dental cells were then characterized, analyzed, and validated with transcriptomic analysis, western blotting, and RT-qPCR. To form mineralized tissues, hiPSC-derived DECs were recombined with hiPSC-derived DMCs encapsulated in various biomaterials, including gelatin methacryloyl (GelMA), collagen, and agar matrix. RESULTS: These hiPSC-derived dental cells are highly osteogenic and chondro-osteogenic in photocrosslinkable GelMA hydrogel and collagen type I microenvironments. Furthermore, hiPSC-derived dental cells in agar gel matrix induced the formation of a bioengineered tooth. CONCLUSION: Our study provides an approach for applying hiPSCs for hard tissue regeneration, including tooth and bone. This study has immense potential to provide a novel technology for bioengineering organs for various regenerative therapies.

Epithelial plasticity enhances regeneration of committed taste receptor cells following nerve injury.

Taste receptor cells are taste bud epithelial cells that are dependent upon the innervating nerve for continuous renewal and are maintained by resident tissue stem/progenitor cells. Transection of the innervating nerve causes degeneration of taste buds and taste receptor cells. However, a subset of the taste receptor cells is maintained without nerve contact after glossopharyngeal nerve transection in the circumvallate papilla in adult mice. Here, we revealed that injury caused by glossopharyngeal nerve transection triggers the remaining differentiated K8-positive taste receptor cells to dedifferentiate and acquire transient progenitor cell-like states during regeneration. Dedifferentiated taste receptor cells proliferate, express progenitor cell markers (K14, Sox2, PCNA) and form organoids in vitro. These data indicate that differentiated taste receptor cells can enter the cell cycle, acquire stemness, and participate in taste bud regeneration. We propose that dedifferentiated taste receptor cells in combination with stem/progenitor cells enhance the regeneration of taste buds following nerve injury.

MAST4 controls cell cycle in spermatogonial stem cells.

Spermatogonial stem cell (SSC) self-renewal is regulated by reciprocal interactions between Sertoli cells and SSCs in the testis. In a previous study, microtubule-associated serine/threonine kinase 4 (MAST4) has been studied in Sertoli cells as a regulator of SSC self-renewal. The present study focused on the mechanism by which MAST4 in Sertoli cells transmits the signal and regulates SSCs, especially cell cycle regulation. The expression of PLZF, CDK2 and PLZF target genes was examined in WT and Mast4 KO testes by Immunohistochemistry, RT-qPCR and western blot. In addition, IdU and BrdU were injected into WT and Mast4 KO mice and cell cycle of SSCs was analysed. Finally, the testis tissues were cultured in vitro to examine the regulation of cell cycle by MAST4 pathway. Mast4 KO mice showed infertility with Sertoli cell-only syndrome and reduced sperm count. Furthermore, Mast4 deletion led to decreased PLZF expression and cell cycle progression in the testes. MAST4 also induced cyclin-dependent kinase 2 (CDK2) to phosphorylate PLZF and activated PLZF suppressed the transcriptional levels of genes related to cell cycle arrest, leading SSCs to remain stem cell state. MAST4 is essential for maintaining cell cycle in SSCs via the CDK2-PLZF interaction. These results demonstrate the pivotal role of MAST4 regulating cell cycle of SSCs and the significance of spermatogenesis.

Enhanced Bone Formation by Rapidly Formed Bony Wall over the Bone Defect Using Dual Growth Factors.

BACKGROUND: In guided bone regeneration (GBR), there are various problems that occur in the bone defect after the wound healing period. This study aimed to investigate the enhancement of the osteogenic ability of the dual scaffold complex and identify the appropriate concentration of growth factors (GF) for new bone formation based on the novel GBR concept that is applying rapid bone forming GFs to the membrane outside of the bone defect. METHODS: Four bone defects with a diameter of 8 mm were formed in the calvaria of New Zealand white rabbits each to perform GBR. Collagen membrane and biphasic calcium phosphate (BCP) were applied to the bone defects with the four different concetration of BMP-2 or FGF-2. After 2, 4, and 8 weeks of healing, histological, histomorphometric, and immunohistochemical analyses were conducted. RESULTS: In the histological analysis, continuous forms of new bones were observed in the upper part of bone defect in the experimental groups, whereas no continuous forms were observed in the control group. In the histomorphometry, The group to which BMP-2 0.5 mg/ml and FGF-2 1.0 mg/ml was applied showed statistically significantly higher new bone formation. Also, the new bone formation according to the healing period was statistically significantly higher at 8 weeks than at 2, 4 weeks. CONCLUSION: The novel GBR method in which BMP-2, newly proposed in this study, is applied to the membrane is effective for bone regeneration. In addition, the dual scaffold complex is quantitatively and qualitatively advantageous for bone regeneration and bone maintenance over time.

Gut Microbiota as a Hidden Player in the Pathogenesis of Alzheimer's Disease.

Alzheimer's disease (AD), the most common neurodegenerative disorder, is accompanied by cognitive impairment and shows representative pathological features, including senile plaques and neurofibrillary tangles in the brain. Recent evidence suggests that several systemic changes outside the brain are associated with AD and may contribute to its pathogenesis. Among the factors that induce systemic changes in AD, the gut microbiota is increasingly drawing attention. Modulation of gut microbiome, along with continuous attempts to remove pathogenic proteins directly from the brain, is a viable strategy to cure AD. Seeking a holistic understanding of the pathways throughout the body that can affect the pathogenesis, rather than regarding AD solely as a brain disease, may be key to successful therapy. In this review, we focus on the role of the gut microbiota in causing systemic manifestations of AD. The review integrates recently emerging concepts and provides potential mechanisms about the involvement of the gut-brain axis in AD, ranging from gut permeability and inflammation to bacterial translocation and cross-seeding.