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Microscale Fluid Behavior during Cryo-EM Sample Blotting
Blotting has been the standard technique for preparing aqueous samples for single-particle electron cryo-microscopy for over three decades. This technique removes the excess solution from a transmission electron microscope grid by pressing absorbent filter paper against the specimen before vitrification. However, this standard technique produces vitreous ice with inconsistent thickness from specimen to specimen and from region to region within the same specimen, the reasons for which are not understood. Here, high-speed interference contrast microscopy is used to demonstrate that the irregular pattern of fibers in the filter paper imposes tortuous, highly variable boundaries during the removal of excess liquid from a flat, hydrophilic surface. As a result, aqueous films of nonuniform thickness are formed while the filter paper is pressed against the substrate. This pattern of nonuniform liquid thickness changes again after the filter paper is pulled away, but the thickness still does not become completely uniform. We suggest that similar topographical features of the liquid film are produced during the standard technique used to blot EM grids and that these manifest in nonuniform ice after vitrification. These observations suggest that alternative thinning techniques, which do not rely on direct contact between the filter paper and the grid, may result in more repeatable and uniform sample thicknesses
Isolation of a Collagenase cDNA Clone and Measurement of Changing Collagenase mRNA Levels during Induction in Rabbit Synovial Fibroblasts.
To facilitate our studies on the mechanisms controlling collagenase production at a molecular level in rabbit synovial fibroblasts, we have constructed a cDNA library using mRNAs isolated from cells induced with crystals of monosodium urate monohydrate. We have screened this library with cDNA probes made from induced and control mRNA populations. From among 30 clones that hybridized preferentially to the induced-cell probe, 4 contained collagenase sequences. The largest, a clone of 650 base pairs, was identified by its ability to hybrid select a mRNA that could be translated in a cell-free system into a product that was precipitable with monospecific antibody to collagenase. Using this clone to probe blots of RNA from induced cells, we detected the appearance of a collagenase mRNA of 2.7 kilobases within 5 hr of addition of urate. The level of collagenase mRNA continued to increase for 35-40 hr, when it was 60 to 90 times more abundant in induced cells than in control cells. The increase in mRNA levels correlated with an increase in immunoreactive collagenase protein that was detectable in culture medium by 10 hr
The Rostral Migratory Stream Plays a Key Role in Intranasal Delivery of Drugs into the CNS
Background: The blood brain barrier (BBB) is impermeable to most drugs, impeding the establishment of novel neuroprotective therapies and strategies for many neurological diseases. Intranasal administration offers an alternative path for efficient drug delivery into the CNS. So far, the anatomical structures discussed to be involved in the transport of intranasally administered drugs into the CNS include the trigeminal nerve, olfactory nerve and the rostral migratory stream (RMS), but the relative contributions are debated. Methods and Findings: In the present study we demonstrate that surgical transection, and the resulting structural disruption of the RMS, in mice effectively obstructs the uptake of intranasally administered radioligands into the CNS. Furthermore, using a fluorescent cell tracer, we demonstrate that intranasal administration in mice allows agents to be distributed throughout the entire brain, including olfactory bulb, hippocampus, cortex and cerebellum. Conclusions: This study provides evidence of the vital role the RMS has in the CNS delivery of intranasally administered agents. The identification of the RMS as the major access path for intranasally administered drugs into the CNS may contribute to the development of treatments that are tailored for efficient transport within this structure. Research into the RMS needs to continue to elucidate its limitations, capabilities, mechanisms of transport and potential hazards before w
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