The glymphatic system is a network of perivascular spaces that promotes movement of cerebrospinal fluid (CSF) into the brain and clearance of metabolic waste. This fluid transport system is supported by the water channel aquaporin-4 (AQP4) localized to vascular endfeet of astrocytes. The glymphatic system is more effective during sleep, but whether sleep timing promotes glymphatic function remains unknown. We here show glymphatic influx and clearance exhibit endogenous, circadian rhythms peaking during the mid-rest phase of mice. Drainage of CSF from the cisterna magna to the lymph nodes exhibits daily variation opposite to glymphatic influx, suggesting distribution of CSF throughout the animal depends on timeof-day. The perivascular polarization of AQP4 is highest during the rest phase and loss of AQP4 eliminates the day-night difference in both glymphatic influx and drainage to the lymph nodes. We conclude that CSF distribution is under circadian control and that AQP4 supports this rhythm.

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.

N2 fixation should be a critical process in the nitrogen-poor surface water of the eastern Mediterranean Sea. Despite favorable conditions, diazotroph abundance and N2 fixation rates remains low for reasons yet explained. The main goal of this study was to investigate the limiting nutrients for diazotrophy in this oligotrophic environment. Hence, we conducted dedicated bottle-microcosms with eastern Mediterranean Sea water that were supplemented with mono and polysaccharides as well as inorganic nitrogen and phosphorous. Our results indicate that the diazotrophic community expressing nifH was primarily represented by heterotrophic Proteobacteria. N2 fixation and heterotrophic bacterial activity increased up-to tenfold following two days of dark incubations, once seawater was supplemented with organic carbon substrate in the form of glucose (monosaccharides) or gum-xanthan (polysaccharide surrogate). Furthermore, our results point that carbon-rich polysaccharides, such as transparent exopolymer particles, enhance heterotrophic N2 fixation, by forming microenvironments of intense metabolic activity, high carbon: nitrogen ratio, and possibly low O2 levels. The conclusions of this study indicate that diazotrophs in the eastern Mediterranean coast are primarily limited by organic carbon substrates, as possibly in many other marine regions.

Membrane distillation (MD) is an emerging desalination technology that uses low-grade heat to drive water vapor across a microporous hydrophobic membrane. Currently, little is known about the biofilms that grow on MD membranes. In this study, we use estuarine water collected from Long Island Sound in a bench-scale direct contact MD system to investigate the initial stages of biofilm formation. For comparison, we studied biofilm formation in a bench-scale reverse osmosis (RO) system using the same feedwater. These two membrane desalination systems expose the natural microbial community to vastly different environmental conditions: high temperatures with no hydraulic pressure in MD and low temperature with hydraulic pressure in RO. Over the course of 4 days, we observed a steady decline in bacteria concentration (nearly 2 orders of magnitude) in the MD feed reservoir. Even with this drop in planktonic bacteria, significant biofilm formation was observed. Biofilm morphologies on MD and RO membranes were markedly different. MD membrane biofilms were heterogeneous and contained several colonies, while RO membrane biofilms, although thicker, were a homogeneous mat. Phylogenetic analysis using next-generation sequencing of 16S rDNA showed significant shifts in the microbial communities. Bacteria representing the orders Burkholderiales, Rhodobacterales, and Flavobacteriales were most abundant in the MD biofilms. On the basis of the results, we propose two different regimes for microbial community shifts and biofilm development in RO and MD systems.

The glymphatic system is a fluid transport network of cerebrospinal fluid (CSF) entering the brain along arterial perivascular spaces, exchanging with interstitial fluid (ISF), ultimately establishing directional clearance of interstitial solutes. CSF transport is facilitated by the expression of aquaporin-4 (AQP4) water channels on the perivascular endfeet of astrocytes. Mice with genetic deletion of AQP4 (AQP4 KO) exhibit abnormalities in the brain structure and molecular water transport. Yet, no studies have systematically examined how these abnormalities in structure and water transport correlate with glymphatic function. Here we used high-resolution 3D magnetic resonance (MR) non-contrast cisternography, diffusion-weighted MR imaging (MR-DWI) along with intravoxel-incoherent motion (IVIM) DWI, while evaluating glymphatic function using a standard dynamic contrast-enhanced MR imaging to better understand how water transport and glymphatic function is disrupted after genetic deletion of AQP4. AQP4 KO mice had larger interstitial spaces and total brain volumes resulting in higher water content and reduced CSF space volumes, despite similar CSF production rates and vascular density compared to wildtype mice. The larger interstitial fluid volume likely resulted in increased slow but not fast MR diffusion measures and coincided with reduced glymphatic influx. This markedly altered brain fluid transport in AQP4 KO mice may result from a reduction in glymphatic clearance, leading to enlargement and stagnation of fluid in the interstitial space. Overall, diffusion MR is a useful tool to evaluate glymphatic function and may serve as valuable translational biomarker to study glymphatics in human disease.

In dynamic optical coherence elastography (OCE), surface acoustic waves are the predominant perturbations. They constrain the quantification of elastic modulus to the direction of wave propagation only along the surface of tissues, and disregard elasticity gradients along depth. Longitudinal shear waves (LSW), on the other hand, can be generated at the surface of the tissue and propagate through depth with desirable properties for OCE: (1) LSW travel at the shear wave speed and can discriminate elasticity gradients along depth, and (2) the displacement of LSW is longitudinally polarized along the direction of propagation; therefore, it can be measured by a phase-sensitive optical coherence tomography system. In this study, we explore the capabilities of LSW generated by a circular glass plate in contact with a sample using numerical simulations and tissue-mimicking phantom experiments. Results demonstrate the potential of LSW in detecting an elasticity gradient along axial and lateral directions simultaneously. Finally, LSW are used for the elastography of ex vivo mouse brain and demonstrate important implications in in vivo and in situ measurements of local elasticity changes in brain and how they might correlate with the onset and progression of degenerative brain diseases.

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The tight coupling between cerebral blood flow and neural activity is a key feature of normal brain function and forms the basis of functional hyperemia. The mechanisms coupling neural activity to vascular responses, however, remain elusive despite decades of research. Recent studies have shown that cerebral functional hyperemia begins in capillaries, and red blood cells (RBCs) act as autonomous regulators of brain capillary perfusion. RBCs then respond to local changes of oxygen tension (PO2) and regulate their capillary velocity. Using ex vivo microfluidics and in vivo two-photon microscopy, we examined RBC capillary velocity as a function ofPO2and showed that deoxygenated hemoglobin and band 3 interactions on RBC membrane are the molecular switch that responds to localPO2changes and controls RBC capillary velocity. Capillary hyperemia can be controlled by manipulating RBC properties independent of the neurovascular unit, providing an effective strategy to treat or prevent impaired functional hyperemia.