Mechano-chemostats to study the effects of compressive stress on yeast.

Cells need to act upon the elastic extracellular matrix and against steric constraints when proliferating in a confined environment, leading to the build-up, at the population level, of a compressive, growth-induced, mechanical stress. Compressive mechanical stresses are ubiquitous to any cell population growing in a spatially-constrained environment, such as microbes or most solid tumors. They remain understudied, in particular in microbial populations, due to the lack of tools available to researchers. Here, we present various mechano-chemostats: microfluidic devices developed to study microbes under pressure. A mechano-chemostat permits researchers to control the intensity of growth-induced pressure through the control of cell confinement, while keeping cells in a defined chemical environment. These versatile devices enable the interrogation of physiological parameters influenced by mechanical compression at the single cell level and set a standard for the study of growth-induced compressive stress.

mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding.

Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed genetically encoded multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein that self-assemble into bright, stable particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can modulate the effective diffusion coefficient of particles ≥20 nm in diameter more than 2-fold by tuning ribosome concentration, without any discernable effect on the motion of molecules ≤5 nm. This change in ribosome concentration affected phase separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation.

Protein profiling comes of age.

Ever since DNA microarrays were first applied to the quantitation of RNA levels, there has been considerable interest in generating a protein homolog that can be used to assay cellular protein expression. A recent paper describes the first microarray that can be used for such protein profiling.

Bilateral occipital lobe stroke with inferior altitudinal defects.

BACKGROUND: Cerebrovascular disease is the most common cause of neurological disability in Western countries. Patients who survive cerebrovascular accidents exclusive to the occipital lobe often have no significant neurological deficits other than visual-field loss. Visual-field defects from occipital lobe stroke typically include congruous homonymous hemianopsias or quadranopsias, with or without macular sparing. CASE REPORT: A 61-year-old white man came to us with symptoms of sudden loss of vision and difficulty reading. Visual-field testing revealed a bilateral inferior altitudinal defect with normal optic nerve and fundus appearance in both eyes. On radiological examination, he was found to have had a bioccipital lobe cerebrovascular accident secondary to complete occlusion of the left vertebral artery. An embolic event causing the artery occlusion, in combination with bilaterally compromised cerebellar and posterior cerebral arteries, presumably caused the bilateral stroke. After appropriate medical and neurological consultation, optometric management consisted of maximizing the patient's remaining vision with a prismatic spectacle correction. DISCUSSION/CONCLUSION: Patients with infarction exclusive to the occipital lobe typically have no other neurological deficits except visual-field loss and are often easier to manage than patients with infarctions to other areas of the cerebral cortex or multiple infarctions. Visual-field loss from occipital lobe damage can be successfully managed with optical systems and/or visual rehabilitation. Factors related to management include location and extent of visual-field damage, functional visual needs, and both personal and health concerns of the patient. A discussion is presented on cerebrovascular disease, occipital lobe infarction, imaging techniques, and visual rehabilitation.

The use of recombinant antibodies in proteomics.

Recombinant antibodies are becoming increasingly important in the field of proteomics. Recent advances include the development of large phage-antibody libraries that contain high-affinity binders to almost any target protein, and new methods for high-throughput selection of antibody-antigen interactions. Coupled with a range of new screening technologies that use high-density antibody arrays to identify differentially expressed proteins, these antibody libraries can be applied to whole proteome analysis.

By-passing selection: direct screening for antibody-antigen interactions using protein arrays.

We have developed a system to identify highly specific antibody-antigen interactions by protein array screening. This removes the need for selection using animal immunisation or in vitro techniques such as phage or ribosome display. We screened an array of 27 648 human foetal brain proteins with 12 well-expressed antibody fragments that had not previously been exposed to any antigen. Four highly specific antibody-antigen pairs were identified, including three antibodies that bind proteins of unknown function. The target proteins were expressed at a very low copy number on the array, emphasising the unbiased nature of the screen. The specificity and sensitivity of binding demonstrates that this 'naive' screening approach could be applied to the high throughput isolation of specific antibodies against many different targets in the human proteome.

Stability of trypsin immobilized on inorganic orthopedic biomaterials.

Biochemical surface modification of biomaterials utilizes immobilized biomolecules to induce preferred tissue responses. Although several techniques are available for immobilizing biomolecules on organic substrates, comparatively few are available for use with inorganic materials, such as those used in many orthopedic applications. The present study investigated the stability/elutability of a model enzyme immobilized on Co-Cr-Mo and Ti-6Al-4V alloys using p-nitrophenyl chloroformate (p-NPC). Trypsin-conjugated biomaterials were incubated in cell culture medium at 37 degrees C for up to 96 hr, and the residual immobilized activity was measured. Although all samples initially bound enzymatically active trypsin, significant decreases were observed within the first 2 hr of incubation. Immobilization of trypsin on Co-Cr-Mo treated with 0.65 mg p-NPC/cm2 of nominal surface area gave significantly higher residual activity than on untreated samples at 24-96 hr of incubation and prevented the nearly complete loss of enzymatic activity that was observed with free (not immobilized) enzyme. Derivatization of Ti-6Al-4V with p-NPC was not beneficial to the level of immobilized enzymatic activity after incubation in medium for longer than 6 hr.