Optical control of endogenous proteins with a photoswitchable conditional subunit reveals a role for TREK1 in GABA(B) signaling

Selective ligands are lacking for many neuronal signaling proteins. Photoswitched tethered ligands (PTLs) have enabled fast and reversible control of specific proteins containing a PTL anchoring site and have been used to remote control overexpressed proteins. We report here a scheme for optical remote control of native proteins using?a "photoswitchable conditional subunit" (PCS), which contains the PTL anchoring site as well as a mutation that prevents it from reaching the plasma membrane. In cells lacking native subunits for the protein, the PCS remains nonfunctional internally. However, in cells expressing native subunits, the native subunit and PCS coassemble, traffic to the plasma membrane, and place the native protein under optical control provided by the coassembled PCS. We apply this approach to the TREK1 potassium channel, which lacks selective, reversible blockers. We find that TREK1, typically considered to be a leak channel, contributes to the hippocampal GABA(B) response.     

Quality control compartments coming of age

Maintenance of proteome integrity (proteostasis) is essential for cellular and organismal survival. Various cellular mechanisms work to preserve proteostasis by ensuring correct protein maturation and efficient degradation of misfolded and damaged proteins. Despite this cellular effort, under certain circumstances subsets of aggregation-prone proteins escape the quality control surveillance, accumulate within the cell and form insoluble aggregates that can lead to the development of disorders including late-onset neurodegenerative diseases. Cells respond to the appearance of insoluble aggregates by actively transporting them to designated deposition sites where they often undergo degradation. Although several protein aggregate deposition sites have been described and extensively studied, key questions regarding their biological roles and how they are affected by aging remained unanswered. Here we review the recent advances in the field, describe the different subtypes of these cellular compartments and outline the evidence that these structures change their properties over time. Finally, we propose models to explain the possible mechanistic links between aggregate deposition sites, neurodegenerative disorders and the aging process.     

Cell lineage analysis of the mammalian female germline

Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote). We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.     

Recursive construction of perfect DNA molecules from imperfect oligonucleotides

Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology and synthetic biology. Here, we show for the first time how recursion can be used to address this challenge and demonstrate a recursive procedure that constructs error‐free DNA molecules and their libraries from error‐prone oligonucleotides. Divide and Conquer (D&C), the quintessential recursive problem‐solving technique, is applied in silico to divide the target DNA sequence into overlapping oligonucleotides short enough to be synthesized directly, albeit with errors; error‐prone oligonucleotides are recursively combined in vitro, forming error‐prone DNA molecules; error‐free fragments of these molecules are then identified, extracted and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error‐free target molecule is formed. Our recursive construction procedure surpasses existing methods for de novo DNA synthesis in speed, precision, amenability to automation, ease of combining synthetic and natural DNA fragments, and ability to construct designer DNA libraries. It thus provides a novel and robust foundation for the design and construction of synthetic biological molecules and organisms.     

Detection and molecular characterization of 9,000-year-old Mycobacterium tuberculosis from a Neolithic settlement in the Eastern Mediterranean

Background

Mycobacterium tuberculosis is the principal etiologic agent of human tuberculosis. It has no environmental reservoir and is believed to have co-evolved with its host over millennia. This is supported by skeletal evidence of the disease in early humans, and inferred from M. tuberculosis genomic analysis. Direct examination of ancient human remains for M. tuberculosis biomarkers should aid our understanding of the nature of prehistoric tuberculosis and the host/pathogen relationship.

Methodology/Principal Findings

We used conventional PCR to examine bone samples with typical tuberculosis lesions from a woman and infant, who were buried together in the now submerged site of Atlit-Yam in the Eastern Mediterranean, dating from 9250-8160 years ago. Rigorous precautions were taken to prevent contamination, and independent centers were used to confirm authenticity of findings. DNA from five M tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex.

Conclusions/Significance

Human tuberculosis was confirmed by morphological and molecular methods in a population living in one of the first villages with evidence of agriculture and animal domestication. The widespread use of animals was not a source of infection but may have supported a denser human population that facilitated transmission of the tubercle bacillus. The similarity of the M. tuberculosis genetic signature with those of today gives support to the theory of a long-term co-existence of host and pathogen.     

Proline-Rich Peptide from the Coral Pathogen Vibrio shiloi That Inhibits Photosynthesis of Zooxanthellae

The coral-bleaching bacterium Vibrio shiloi biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29°C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH₄Cl, pure natural or synthetic toxin P (10 μM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH₄Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH₄Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH₃ into the cell. It is known that uptake of NH₃ into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by V. shiloi.     

Small-grained wild grasses as staple food at the 23 000-year-old site of Ohalo II,Israel

More than 16 000 grains of small-grained grasses were retrieved at Ohalo II, a submerged 23 000-year-old site on the shore of the Sea of Galilee, Israel. The grains were part of a very large archaeobotanical assemblage, unique for its period and region, as well as its exceptionally good preservation. This paper proposes that these grains were a staple food at Ohalo II, based on several lines of evidence: 1. the large number of grains found; 2. the fact that all grains were fully mature; and 3. ethnographic parallels for the use of small-grained grasses in hunter-gatherers’ societies as well as among present-day agriculturalists.     

Electron microscopical analysis of surface charge labelling density at various stages of the erythroid line

Summary With use of the positively charged, colloidal ferric oxide labelling technique for electron microscopy of sections of rabbit marrow, a reduction in labelling density on the surface of differentiating red cell precursors was demonstrated. The number of iron particles per unit length of membrane was counted. A progressive diminution in labelling density follows cell division, reaching a minimum in the orthochromatic erythroblast, from which the nucleus is expelled. A slight increase in charge density is noted in the reticulocyte, and further increase is observed with its maturation to the erythrocyte. The results indicate that biosynthesis of n-acetyl neuraminic acid stops at the earliest recognizable stage of erythroid differentiation.The Patrick E. Gorman Professor of Biological Ultrastructure.