Synthetic biology, genome engineering and directed evolution offer innumerable tools to expedite engineering of strains for optimising biosynthetic pathways. One of the most radical is SCRaMbLE, a system of inducible in vivo deletion and rearrangement of synthetic yeast chromosomes, diversifying the genotype of millions of Saccharomyces cerevisiae cells in hours. SCRaMbLE can yield strains with improved biosynthetic phenotypes but is limited by screening capabilities. To address this bottleneck, we combine automated sample preparation, an ultra-fast 84-second LC-MS method, and barcoded nanopore sequencing to rapidly isolate and characterise the best performing strains. Here, we use SCRaMbLE to optimise yeast strains engineered to produce the triterpenoid betulinic acid. Our semi-automated workflow screens 1,000 colonies, identifying and sequencing 12 strains with between 2- to 7-fold improvement in betulinic acid titre. The broad applicability of this workflow to rapidly isolate improved strains from a variant library makes this a valuable tool for biotechnology.
Christopher M. Jakobson, Richard She & Daniel F. Jarosz
Nature Communicationsvolume 10, Article number: 1222 (2019)
Quantitative genetics aims to map genotype to phenotype, often with the goal of understanding how organisms evolved. However, it remains unclear whether the genetic variants identified are exemplary of evolution. Here we analyzed progeny of two wild Saccharomyces cerevisiae isolates to identify 195 loci underlying complex metabolic traits, resolving 107 to single polymorphisms with diverse molecular mechanisms. More than 20% of causal variants exhibited patterns of emergence inconsistent with neutrality. Moreover, contrary to drift-centric expectation, variation in diverse wild yeast isolates broadly exhibited this property: over 30% of shared natural variants exhibited phylogenetic signatures suggesting that they are not neutral. This pattern is likely attributable to both homoplasy and balancing selection on ancestral polymorphism. Variants that emerged repeatedly were more likely to have done so in isolates from the same ecological niche. Our results underscore the power of super-resolution mapping of ecologically relevant traits in understanding adaptation and evolution.
R. Barer, A. E. Saunders-Singer
Factors which must be considered in the design of a low-power micromanipulator are discussed. A rough prototype model is described and it is shown how the final instrument was evolved from this. The instrument is completely three-dimensional, will follow a movement of the hand with
a reduction ratio of 4 to 1, and will “stay put” in any position. It is intended for use at magnifications up to 200 times, and the movement, as seen through a binocular dissection microscope, is not reversed.
Peter C Rickwood
American Mineralogist, Volume 62, pages 382-384, 1977
Micromanipulators used by life scientists have been evaluated for extracting minute grains (< 20 pm) from petrological thin sections. A pantograph design with a single control for all motion was found to be the most convenient, and sharpened hypodermic needles made ideal probes.
Vertebrate head is a complex structure that consists of various kinds of tissues and organs. Among them, cranial sensory organs and ganglions are derived from special cell groups, called
placodes. Based on the facts that they differentiated into multiple cell types and that are only found in vertebrate lineages, placodes are interesting subjects of studies from the perspectives of both ontogeny and phylogeny. Lines of studies have suggested that all placodes are derived from the pre-placodal ectoderm (PPE) that is boundary between future epidermis region and future neural region. A Xenopus gene Xhairy2 is, to our knowledge, the earliest gene that shows clear expression in PPE. The present study aimed at elucidating a function(s) of Xhairy2 in the formation of placodes by examining formation of cranial sensory organs. Morphological analyses of Xhairy2 morphants revealed that ocular lens was severely malformed though retinal structure looked normal. Vertebrate eyes are divided into retina and lens in terms of their origins: retina is formed from a part of diencephalons, while lens is developed from placode. Detailed marker gene analyses of eye in Xhairy2 morphants showed that the expression of all lens marker genes tested was reduced or abolished, while that of retinal marker genes was not affected. These results indicated that Xhairy2
was specifically required for lens development. Ectopic expression of p27xic1 by means of Xhairy2 knockdown was identified as a first trigger of lens malformation. First, apoptosis was induced in
Xhairy2 morphants, which is a known marker of ectopic expression of p27xic1. Second, overexpression of p27xic1 mimicked phenotypes of Xhairy2 morphants. Finally, the phenotypes of Xhairy2 knockdown were partially rescued by simultaneous knockdown of p27xic1. Since p27xic1 is a cell-cycle inhibitor, it was assumed that the number of lens precursor cells was drastically reduced. To test this assumption, cell proliferation within regions of lens induction was examined by means of BrdU incorporation analyses in Xhairy2 morphants. However, no significant changes were observed at the onset of lens induction. Furthermore, lens induction occurred normally in the embryos treated with drugs that directly inhibit cell cycle. Collectively, the results suggested that Xhairy2 repressed
the differentiation inducing activity, another activity of p27xic1. Placodes start differentiation long after the first fate decision. Therefore, it is quite important how they maintain stem-cell-like undifferentiated states. As Xhairy2 did not seem to be a master regulator of cascade of lens transcription factors and was shown to be involved in inhibition of differentiation via repression of p27xic1 expression, it was suggested that the undifferentiated states was actively maintained by early PPE expression of Xhairy2. The cascade of differentiation beginning with a signal input would normally work only when this kind of fundamental cell state, competence, exists.
Goodman MB, Lindsay TH, Lockery SR, Richmond JE.
Methods Cell Biol. 2012; 107: 409–436.
Patch-clamp electrophysiology is a technique of choice for the biophysical analysis of the function of nerve, muscle, and synapse in Caenorhabditis elegans nematodes. Considerable technical progress has been made in C. elegans electrophysiology in the decade since the initial publication of this technique. Today, most, if not all, electrophysiological studies that can be done in larger animal preparations can also be done in C. elegans. This chapter has two main goals. The first is to present to a broad audience the many techniques available for patch-clamp analysis of neurons, muscles, and synapses in C. elegans. The second is to provide a methodological introduction to the techniques for patch clamping C. elegans neurons and body-wall muscles in vivo, including emerging methods for optogenetic stimulation coupled with postsynaptic recording. We also present samples of the cell-intrinsic and postsynaptic ionic currents that can be measured in C. elegans nerves and muscles.
Nakayama K, Takebayashi Y, Hata K, Fujiwaki R, Iida K, Fukumoto M, Miyazaki K.
Br J Cancer. 2004 Mar 22; 90(6): 1204–1210.
Ovarian tumours of low malignant potential (LMP) are intermediate between adenomas and ovarian carcinomas. These tumours are often associated with a significantly better prognosis than ovarian carcinomas. However, a subset of these tumours can progress and become lethal. In order to seek sensitive diagnostic tools for monitoring patients after surgical operation, we performed a genome-wide scan for loss of heterozygosity (LOH) in 41 mucinous LMPs using 91 polymorphic microsatellite markers at an average interval of 50 cM across all of the human chromosomes and 25 LOH markers reportedly associated with ovarian carcinoma. In addition, we assessed whether clinicopathological parameters, microvessel density, Ki-67 labeling index, apoptotic index or p53 overexpression would be useful for predicting the postoperative outcome of LMP patients. Of the 116 markers examined, 19q12 and Xq11-12 showed significant correlation between postoperative progression-free survival time and LOH status (P<0.05). Patients with a high Ki-67 labeling index had a significantly poorer progression-free survival time than those with lower levels (P=0.042). Other clinicopathological factors and immunohistochemical analysis had no correlation with progression-free survival time in this series of patients. When the combination of LOH at 19q12 and/or Xq11-12 was assessed using Cox's regression analysis, patients with tumours that showed LOH at these positions were at greatest risk of progression (P=0.0073). These findings suggest that the identification of LOH at 19q12 and/or Xq11-12 in former mucinous LMP sites should alert the clinician to the presence of a potentially aggressive lesion in the coelomic epithelium, even if a distinction between second primary tumours or recurrence could not be determined.
Ioanna Antoniades, Panayiota Stylianou, and Paris A. Skourides
Developmental Cell 28, 70–80, January 13, 2014
Cilia have been associated with diverse developmental and physiological processes, and defects in cilia underlie a number of genetic conditions. Several lines of evidence support a critical role of the actin cytoskeleton in ciliogenesis and ciliary function. Here, we show that well-characterized focal adhesion (FA) proteins, including FAK, Paxillin, and Vinculin, associate with the basal bodies of multiciliated cells and form complexes (CAs) that interact with the actin cytoskeleton. FAK downregulation leads to ciliogenesis defects similar to those observed when the actin cytoskeleton is disrupted, including defects in basal body migration, docking, and spacing, suggesting that CAs link basal bodies to the actin cytoskeleton. The important role of FA proteins in
ciliogenesis leads us to propose that evolutionarily FA proteins, many of which are found in primitive flagellated unicellular eukaryotes, may have originally evolved to perform functions at flagella and were later co-opted for use in cell adhesion.
Patricia Stahn, Hubert H. Lim, Marius P. Hinsberger, Katharina Sorg, Lukas Pillong, Marc Kannengießer, Cathleen Schreiter, Hans-Jochen Foth, Achim Langenbucher, Bernhard Schick & Gentiana I. Wenzel
Scientific Reportsvolume 9, Article number: 4171 (2019)
Hearing impairment is one of the most common sensory deficits in humans. Hearing aids are helpful to patients but can have poor sound quality or transmission due to insufficient output or acoustic feedback, such as for high frequencies. Implantable devices partially overcome these issues but require surgery with limited locations for device attachment. Here, we investigate a new optoacoustic approach to vibrate the hearing organ with laser stimulation to improve frequency bandwidth, not requiring attachment to specific vibratory structures, and potentially reduce acoustic feedback. We developed a laser pulse modulation strategy and simulated its response at the umbo (1–10 kHz) based on a convolution-based model. We achieved frequency-specific activation in which non-contact laser stimulation of the umbo, as well as within the middle ear at the round window and otic capsule, induced precise shifts in the maximal vibratory response of the umbo and neural activation within the inferior colliculus of guinea pigs, corresponding to the targeted, modelled and then stimulated frequency. There was also no acoustic feedback detected from laser stimulation with our experimental setup. These findings open up the potential for using a convolution-based optoacoustic approach as a new type of laser hearing aid or middle ear implant.
This report covers the measurement and evaluation of the pinning pressure achieved by the PIXL Precision Colony Picker, produced by Singer Instruments.
The pinning pressure was measured using a bespoke test procedure, which replicated the standard pinning functionality of the PIXL. Over the span of 80 separate pinning events the PIXL achieved an average pressure of 6.97 ± 0.74 g/mm2, with a maximum recorded pressure of 8.531 g/mm2.