Septins provide microenvironment sensing and cortical actomyosin partitioning in motile amoeboid T lymphocytes.

The all-terrain motility of lymphocytes in tissues and tissue-like gels is best described as amoeboid motility. For amoeboid motility, lymphocytes do not require specific biochemical or structural modifications to the surrounding extracellular matrix. Instead, they rely on changing shape and steric interactions with the microenvironment. However, the exact mechanism of amoeboid motility remains elusive. Here, we report that septins participate in amoeboid motility of T cells, enabling the formation of F-actin and α-actinin-rich cortical rings at the sites of cell cortex-indenting collisions with the extracellular matrix. Cortical rings compartmentalize cells into chains of spherical segments that are spatially conformed to the available lumens, forming transient "hourglass"-shaped steric locks onto the surrounding collagen fibers. The steric lock facilitates pressure-driven peristaltic propulsion of cytosolic content by individually contracting cell segments. Our results suggest that septins provide microenvironment-guided partitioning of actomyosin contractility and steric pivots required for amoeboid motility of T cells in tissue-like microenvironments.

Dynamic neuroinflammatory profiles predict Alzheimer's disease pathology in microglia-containing cerebral organoids.

Neuroinflammation and the underlying dysregulated immune responses of microglia actively contribute to the progression and, likely, the initiation of Alzheimer's disease (AD). Fine-tuned therapeutic modulation of immune dysfunction to ameliorate disease cannot be achieved without the characterization of diverse microglial states that initiate unique, and sometimes contradictory, immune responses that evolve over time in chronic inflammatory environments. Because of the functional differences between human and murine microglia, untangling distinct, disease-relevant reactive states and their corresponding effects on pathology or neuronal health may not be possible without the use of human cells. In order to profile shifting microglial states in early AD and identify microglia-specific drivers of disease, we differentiated human induced pluripotent stem cells (iPSCs) carrying a familial AD PSEN2 mutation or its isogenic control into cerebral organoids and quantified the changes in cytokine concentrations over time with Luminex XMAP technology. We used partial least squares (PLS) modeling to build cytokine signatures predictive of disease and age to identify key differential patterns of cytokine expression that inform the overall organoid immune milieu and quantified the corresponding changes in protein pathology. AD organoids exhibited an overall reduction in cytokine secretion after an initial amplified immune response. We demonstrate that reduced synapse density observed in the AD organoids is prevented with microglial depletion. Crucially, these differential effects of dysregulated immune signaling occurred without the accumulation of pathological proteins. In this study, we used microglia-containing AD organoids to quantitatively characterize an evolving immune milieu, made up of a diverse of collection of activation patterns and immune responses, to identify how a dynamic, overall neuroinflammatory state negatively impacts neuronal health and the cell-specific contribution of microglia.

Septins Enable T Cell Contact Guidance via Amoeboid-Mesenchymal Switch.

Lymphocytes exit circulation and enter in-tissue guided migration toward sites of tissue pathologies, damage, infection, or inflammation. By continuously sensing and adapting to the guiding chemo-mechano-structural properties of the tissues, lymphocytes dynamically alternate and combine their amoeboid (non-adhesive) and mesenchymal (adhesive) migration modes. However, which mechanisms guide and balance different migration modes are largely unclear. Here we report that suppression of septins GTPase activity induces an abrupt amoeboid-to-mesenchymal transition of T cell migration mode, characterized by a distinct, highly deformable integrin-dependent immune cell contact guidance. Surprisingly, the T cell actomyosin cortex contractility becomes diminished, dispensable and antagonistic to mesenchymal-like migration mode. Instead, mesenchymal-like T cells rely on microtubule stabilization and their non-canonical dynein motor activity for high fidelity contact guidance. Our results establish septin's GTPase activity as an important on/off switch for integrin-dependent migration of T lymphocytes, enabling their dynein-driven fluid-like mesenchymal propulsion along the complex adhesion cues.

Dynein-Powered Cell Locomotion Guides Metastasis of Breast Cancer.

The principal cause of death in cancer patients is metastasis, which remains an unresolved problem. Conventionally, metastatic dissemination is linked to actomyosin-driven cell locomotion. However, the locomotion of cancer cells often does not strictly line up with the measured actomyosin forces. Here, a complementary mechanism of metastatic locomotion powered by dynein-generated forces is identified. These forces arise within a non-stretchable microtubule network and drive persistent contact guidance of migrating cancer cells along the biomimetic collagen fibers. It is also shown that the dynein-powered locomotion becomes indispensable during invasive 3D migration within a tissue-like luminal network formed by spatially confining granular hydrogel scaffolds (GHS) made up of microscale hydrogel particles (microgels). These results indicate that the complementary motricity mediated by dynein is always necessary and, in certain instances, sufficient for disseminating metastatic breast cancer cells. These findings advance the fundamental understanding of cell locomotion mechanisms and expand the spectrum of clinical targets against metastasis.

Vitamin D Receptor Antagonist MeTC7 Inhibits PD-L1.

Small-molecule inhibitors of PD-L1 are postulated to control immune evasion in tumors similar to antibodies that target the PD-L1/PD-1 immune checkpoint axis. However, the identity of targetable PD-L1 inducers is required to develop small-molecule PD-L1 inhibitors. In this study, using chromatin immunoprecipitation (ChIP) assay and siRNA, we demonstrate that vitamin D/VDR regulates PD-L1 expression in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells. We have examined whether a VDR antagonist, MeTC7, can inhibit PD-L1. To ensure that MeTC7 inhibits VDR/PD-L1 without off-target effects, we examined competitive inhibition of VDR by MeTC7, utilizing ligand-dependent dimerization of VDR-RXR, RXR-RXR, and VDR-coactivators in a mammalian 2-hybrid (M2H) assay. MeTC7 inhibits VDR selectively, suppresses PD-L1 expression sparing PD-L2, and inhibits the cell viability, clonogenicity, and xenograft growth of AML cells. MeTC7 blocks AML/mesenchymal stem cells (MSCs) adhesion and increases the efferocytotic efficiency of THP-1 AML cells. Additionally, utilizing a syngeneic colorectal cancer model in which VDR/PD-L1 co-upregulation occurs in vivo under radiation therapy (RT), MeTC7 inhibits PD-L1 and enhances intra-tumoral CD8+T cells expressing lymphoid activation antigen-CD69. Taken together, MeTC7 is a promising small-molecule inhibitor of PD-L1 with clinical potential.

A noncommutative combinatorial protein logic circuit controls cell orientation in nanoenvironments.

Single-protein-based devices that integrate signal sensing with logical operations to generate functional outputs offer exceptional promise for monitoring and modulating biological systems. Engineering such intelligent nanoscale computing agents is challenging, as it requires the integration of sensor domains into a functional protein via intricate allosteric networks. We incorporate a rapamycin-sensitive sensor (uniRapR) and a blue light-responsive LOV2 domain into human Src kinase, creating a protein device that functions as a noncommutative combinatorial logic circuit. In our design, rapamycin activates Src kinase, causing protein localization to focal adhesions, whereas blue light exerts the reverse effect that inactivates Src translocation. Focal adhesion maturation induced by Src activation reduces cell migration dynamics and shifts cell orientation to align along collagen nanolane fibers. Using this protein device, we reversibly control cell orientation by applying the appropriate input signals, a framework that may be useful in tissue engineering and regenerative medicine.

Dynein-Powered Cell Locomotion Guides Metastasis of Breast Cancer.

Metastasis is a principal cause of death in cancer patients, which remains an unresolved fundamental and clinical problem. Conventionally, metastatic dissemination is linked to the actomyosin-driven cell locomotion. However, locomotion of cancer cells often does not strictly line up with the measured actomyosin forces. Here, we identify a complementary mechanism of metastatic locomotion powered by the dynein-generated forces. These forces that arise within a non-stretchable microtubule network drive persistent contact guidance of migrating cancer cells along the biomimetic collagen fibers. We also show that dynein-powered locomotion becomes indispensable during invasive 3D migration within a tissue-like luminal network between spatially confining hydrogel microspheres. Our results indicate that the complementary contractile system of dynein motors and microtubules is always necessary and in certain instances completely sufficient for dissemination of metastatic breast cancer cells. These findings advance fundamental understanding of cell locomotion mechanisms and expand the spectrum of clinical targets against metastasis.

Effect of omega-3 fatty acid use on sepsis and mortality in patients with Covid-19.

Background: This study aimed to investigate the effects of omega-3 fatty acid use on sepsis and mortality in patients treated for COVID-19 disease in the intensive care unit (ICU) based on clinical and laboratory results. Aim: To determine the effect of omega-3 fatty acid use on sepsis and mortality in patients with COVID-19. Patients and Methods: A total of 80 patients with confirmed COVID-19 infection who were hospitalized in the ICU of Ankara City Hospital, received (n = 40) or did not receive (n = 40) omega-3 fatty acid dietary supplementation, were included in this single-center, retrospective study. The clinical and laboratory data of eligible patients were extracted from the hospital records. Results: The mean age was 65.5 (13.6). The mean length of stay in the intensive care unit was 11.5 (6.3) days. Mortality and sepsis development rates were similar in the groups. The frequency of patients who received pulse steroid therapy was higher in the group of patients who did not receive omega-3 (P < 0.05). Hypertension was more common in the patient group receiving omega-3 supplements (P < 0.05). Mean procalcitonin and interleukin-6 (IL-6) levels were significantly lower in patients who received omega-3 supplements compared to those who did not receive supplements (P < 0.001 and P < 0.05). Mean prothrombin time (PT) was shorter in patients receiving omega-3 supplementation (P < 0.05). Conclusions: Study results obtained in this study indicate that providing omega-3 fatty acid supplements may be beneficial to patients with severe COVID-19, however further research with large-scale randomized controlled trials is necessary.

Effects of mastoid and middle-ear volume on graft success and hearing outcomes in paediatric tympanoplasty.

OBJECTIVE: To examine the effects of mastoid and middle-ear volume on the anatomical and functional success of type 1 tympanoplasty in paediatric patients. METHODS: This study included 45 paediatric patients who underwent type 1 cartilage tympanoplasty. Patients' demographic data, pre- and post-operative audiological evaluation results, and post-operative graft status were evaluated. Middle-ear and mastoid cavity volumes were calculated (in cubic centimetres) using temporal bone high-resolution computed tomography. Middle-ear and mastoid cavity volume values were compared between patients with and without post-operative anatomical and functional success. RESULTS: Anatomical success was achieved in 82.2 per cent of patients (n = 37), and functional success in 68.9 per cent (n = 31). When anatomical success and failure groups were compared, a statistically significant difference was found in mean mastoid volume (p = 0.037), while there was no significant difference in relation to mean middle-ear volume (p = 0.827). The comparison of functional success and failure groups revealed no significant difference in mean mastoid volume (p = 0.492) or middle-ear volume (p = 0.941). CONCLUSION: The study showed that mastoid pneumatisation volume affects surgical success in paediatric tympanoplasty.

Identification of a Vitamin-D Receptor Antagonist, MeTC7, which Inhibits the Growth of Xenograft and Transgenic Tumors In Vivo.

Vitamin-D receptor (VDR) mRNA is overexpressed in neuroblastoma and carcinomas of lung, pancreas, and ovaries and predicts poor prognoses. VDR antagonists may be able to inhibit tumors that overexpress VDR. However, the current antagonists are arduous to synthesize and are only partial antagonists, limiting their use. Here, we show that the VDR antagonist MeTC7 (5), which can be synthesized from 7-dehydrocholesterol (6) in two steps, inhibits VDR selectively, suppresses the viability of cancer cell-lines, and reduces the growth of the spontaneous transgenic TH-MYCN neuroblastoma and xenografts in vivo. The VDR selectivity of 5 against RXRα and PPAR-γ was confirmed, and docking studies using VDR-LBD indicated that 5 induces major changes in the binding motifs, which potentially result in VDR antagonistic effects. These data highlight the therapeutic benefits of targeting VDR for the treatment of malignancies and demonstrate the creation of selective VDR antagonists that are easy to synthesize.

Two-input protein logic gate for computation in living cells.

Advances in protein design have brought us within reach of developing a nanoscale programming language, in which molecules serve as operands and their conformational states function as logic gates with precise input and output behaviors. Combining these nanoscale computing agents into larger molecules and molecular complexes will allow us to write and execute "code". Here, in an important step toward this goal, we report an engineered, single protein design that is allosterically regulated to function as a 'two-input logic OR gate'. Our system is based on chemo- and optogenetic regulation of focal adhesion kinase. In the engineered FAK, all of FAK domain architecture is retained and key intramolecular interactions between the kinase and the FERM domains are externally controlled through a rapamycin-inducible uniRapR module in the kinase domain and a light-inducible LOV2 module in the FERM domain. Orthogonal regulation of protein function was possible using the chemo- and optogenetic switches. We demonstrate that dynamic FAK activation profoundly increased cell multiaxial complexity in the fibrous extracellular matrix microenvironment and decreased cell motility. This work provides proof-of-principle for fine multimodal control of protein function and paves the way for construction of complex nanoscale computing agents.

Mechanical Counterbalance of Kinesin and Dynein Motors in a Microtubular Network Regulates Cell Mechanics, 3D Architecture, and Mechanosensing.

Microtubules (MTs) and MT motor proteins form active 3D networks made of unstretchable cables with rod-like bending mechanics that provide cells with a dynamically changing structural scaffold. In this study, we report an antagonistic mechanical balance within the dynein-kinesin microtubular motor system. Dynein activity drives the microtubular network inward compaction, while isolated activity of kinesins bundles and expands MTs into giant circular bands that deform the cell cortex into discoids. Furthermore, we show that dyneins recruit MTs to sites of cell adhesion, increasing the topographic contact guidance of cells, while kinesins antagonize it via retraction of MTs from sites of cell adhesion. Actin-to-microtubule translocation of septin-9 enhances kinesin-MT interactions, outbalances the activity of kinesins over that of dyneins, and induces the discoid architecture of cells. These orthogonal mechanisms of MT network reorganization highlight the existence of an intricate mechanical balance between motor activities of kinesins and dyneins that controls cell 3D architecture, mechanics, and cell-microenvironment interactions.

Programmable DNA-augmented hydrogels for controlled activation of human lymphocytes.

Contractile forces within the planar interface between T cell and antigen-presenting surface mechanically stimulate T cell receptors (TCR) in the mature immune synapses. However, the origin of mechanical stimulation during the initial, i.e., presynaptic, microvilli-based TCR activation in the course of immune surveillance remains unknown and new tools to help address this problem are needed. In this work, we develop nucleic acid nanoassembly (NAN)-based technology for functionalization of hydrogels using isothermal toehold-mediated reassociation of RNA/DNA heteroduplexes. Resulting platform allows for regulation with NAN linkers of 3D force momentum along the TCR mechanical axis, whereas hydrogels contribute to modulation of 2D shear modulus. By utilizing different lengths of NAN linkers conjugated to polyacrylamide gels of different shear moduli, we demonstrate an efficient capture of human T lymphocytes and tunable activation of TCR, as confirmed by T-cell spreading and pY foci.

Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments.

Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding escape from antitumor immunity and optimizing T cell-related therapeutic strategies. Here, we engineered nanotextured elastic platforms to study and enhance T cell migration through complex microenvironments and define how the balance between contractility localization-dependent T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues. Using these platforms, we characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration whereas clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. We show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. Thus, engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics.

Engineering Elastic Nano- and Micro-Patterns and Textures for Directed Cell Motility.

We present a reproducible protocol for fabrication of polyacrylamide (PAA) hydrogel-based nano-patterns and nano-textures with a wide range of elastic rigidities to study fundamental cell behaviors, such as mechanosensitivity and motility. We explore the benefits of this protocol by successfully testing the compatibility of the PAA platforms with super-resolution microscopy, which is largely unavailable with platforms of nano-scale textures made from different polymers. We also utilized soft and rigid nano-textures to study the mechanosensing basis of T cell behavior and phenotype. For complete information on the generation and use of this protocol, please refer to Tabdanov et al. (2018b).

The role of nonmuscle myosin 2A and 2B in the regulation of mesenchymal cell contact guidance.

Contact guidance refers to the ability of cells to sense the geometrical features of the microenvironment and respond by changing their shape and adopting the appropriate orientation. Inhibition and ablation of nonmuscle myosin 2 (NM2) paralogues have demonstrated their importance for contact guidance. However, the specific roles of the NM2 paralogues have not been systematically studied. In this work we use micropatterned substrates to examine the roles of NM2A and NM2B and to elucidate the relationship of the microenvironment, actomyosin, and microtubules in contact guidance. We show that contact guidance is preserved following loss of NM2B and that expression of NM2A alone is sufficient to establish an appropriate orientation of the cells. Loss of NM2B and overexpression of NM2A result in a prominent cell polarization that is found to be linked to the increased alignment of microtubules with the actomyosin scaffold. Suppression of actomyosin with blebbistatin reduces cell polarity on a flat surface, but not on a surface with contact guidance cues. This indicates that the lost microtubule-actomyosin interactions are compensated for by microtubule-microenvironment interactions, which are sufficient to establish cell polarity through contact guidance.

Bimodal sensing of guidance cues in mechanically distinct microenvironments.

Contact guidance due to extracellular matrix architecture is a key regulator of carcinoma invasion and metastasis, yet our understanding of how cells sense guidance cues is limited. Here, using a platform with variable stiffness that facilitates uniaxial or biaxial matrix cues, or competing E-cadherin adhesions, we demonstrate distinct mechanoresponsive behavior. Through disruption of traction forces, we observe a profound phenotypic shift towards a mode of dendritic protrusion and identify bimodal processes that govern guidance sensing. In contractile cells, guidance sensing is strongly dependent on formins and FAK signaling and can be perturbed by disrupting microtubule dynamics, while low traction conditions initiate fluidic-like dendritic protrusions that are dependent on Arp2/3. Concomitant disruption of these bimodal mechanisms completely abrogates the contact guidance response. Thus, guidance sensing in carcinoma cells depends on both environment architecture and mechanical properties and targeting the bimodal responses may provide a rational strategy for disrupting metastatic behavior.

Microtubule-Actomyosin Mechanical Cooperation during Contact Guidance Sensing.

Cancer cell migration through and away from tumors is driven in part by migration along aligned extracellular matrix, a process known as contact guidance (CG). To concurrently study the influence of architectural and mechanical regulators of CG sensing, we developed a set of CG platforms. Using flat and nanotextured substrates with variable architectures and stiffness, we show that CG sensing is regulated by substrate stiffness and define a mechanical role for microtubules and actomyosin-microtubule interactions during CG sensing. Furthermore, we show that Arp2/3-dependent lamellipodia dynamics can compete with aligned protrusions to diminish the CG response and define Arp2/3- and Formins-dependent actin architectures that regulate microtubule-dependent protrusions, which promote the CG response. Thus, our work represents a comprehensive examination of the physical mechanisms influencing CG sensing.

The role of the nasal and paranasal sinus pathologies on the development of chronic otitis media and its subtypes: A computed tomography study.

OBJECTIVE: This objective of this study is to evaluate the presence and the coincidence of common nasal and paranasal sinus pathologies in adults suffering from chronic otitis media (COM) and its subtypes. MATERIALS AND METHODS: The study group comprised 354 ears of 177 patients who underwent tympanoplasty with or without mastoidectomy from January 2013 to February 2015 due to uni/bilateral COM. Chronic suppurative otitis media, intratympanic tympanosclerosis (ITTS), cholesteatoma, and tympanic membrane with retraction pockets constituted subtypes of COM. The control group consisted of 100 ears of 50 adult patients with aural diseases other than middle ear problems. All patients were evaluated for the evidence of mucosal disease on paranasal sinuses, the presence of concha bullosa (CB), and the angle of nasal septal deviation (NSD) and thickness of the medial mucosa of the inferior turbinate were measured by coronal computed tomography images. RESULTS: The incidence and the angle of NSD were found significantly higher in patients with COM (P = 0.028, P = 0.018; respectively). When ears with unilateral and bilateral COM compared in term of sinonasal pathologies, CB was found higher in patients with unilateral COM (P = 0.040). The presence of CB was significantly higher in ITTS when compared to other subtypes (P = 0.028). CONCLUSIONS: Our study suggests that obstructive nasal pathologies such as NSD and CB may play a role in the pathogenesis of especially unilateral COM. However, there was no correlation between COM and inflammatory pathologies such as sinusitis.

Reliability, validity and sensitivity to change of neurogenic bowel dysfunction score in patients with spinal cord injury.

STUDY DESIGN: Psychometrics study. OBJECTIVES: The aim of this study is to investigate the validity, reliability and sensitivity to change of neurogenic bowel dysfunction (NBD) score. SETTING: Dokuz Eylül University Faculty of Medicine, Turkey. METHODS: The study included 42 patients with spinal cord injury (SCI). The reliability of NBD score was assessed by test-retest reliability and internal consistency. Cronbach's alpha coefficient was calculated to determine internal consistency. The construct validity was evaluated by exploring correlations between the NBD score and SF-36 scales, patient assessment of impact of NBD on quality of life (QoL) and the physician global assessment (PGA). The Global Rating of Change (GRC) scale was used to assess the change of NBD to investigate the sensitivity of the score to change. RESULTS: Cronbach's alpha coefficient was 0.547. In test-retest reliability analysis, high correlations between total test-retest NBD score and answers of each question were found (r=1.000, P<0.001). NBD score had a strong and significant correlation with PGA (r=0.98, P<0.000) and the impact on QoL (r=0.92, P<0.001). There was a significant negative correlation between NBD score and subscales of SF-36 (P<0.05) except physical functioning, physical role functioning and physical component summary score. There was a significant improvement in NBD scores after treatment (P=0.011). A significant positive correlation was found between GRC scale and change in total NBD score (r=0.821, P=0.007). CONCLUSION: The Turkish version of the NBD score is a valid and reliable instrument and also sensitive to change in patients with SCI.