A readily usable two‐photon fluorescence lifetime microendoscope

A two‐photon fluorescence lifetime (2P‐FLIM) microendoscope, capable of energetic metabolism imaging through the intracellular nicotinamide adenine dinucleotide (NADH) autofluorescence, at sub‐cellular resolution, is demonstrated. It exhibits readily usable characteristics such as convenient endoscope probe diameter (≈2 mm), fiber length (>5 m) and data accumulation rate (16 frames per second (fps)), leading to a FLIM refreshing rate of ≈0.1 to 1 fps depending on the sample. The spiral scanning image formation does not influence the instrument response function (IRF) characteristics of the system. Near table‐top microscope performances are achieved through a comprehensive system including a home‐designed spectro‐temporal pulse shaper and a custom air‐silica double‐clad photonic crystal fiber, which enables to reach up to 40 mW of ≈100 fs pulses @ 760 nm with a 80 MHz repetition rate. A GRadient INdex (GRIN) lens provides a lateral resolution of 0.67 μm at the focus of the fiber probe. Intracellular NADH fluorescence lifetime data are finally acquired on cultured cells at 16 fps.      

Phylogeny of spiny frogs Nanorana (Anura: Dicroglossidae) supports a Tibetan origin of a Himalayan species group

Recent advances in the understanding of the evolution of the Asian continent challenge the long‐held belief of a faunal immigration into the Himalaya. Spiny frogs of the genus Nanorana are a characteristic faunal group of the Himalaya–Tibet orogen (HTO). We examine the phylogeny of these frogs to explore alternative biogeographic scenarios for their origin in the Greater Himalaya, namely, immigration, South Tibetan origin, strict vicariance. We sequenced 150 Nanorana samples from 62 localities for three mitochondrial (1,524 bp) and three nuclear markers (2,043 bp) and complemented the data with sequence data available from GenBank. We reconstructed a gene tree, phylogenetic networks, and ancestral areas. Based on the nuDNA, we also generated a time‐calibrated species tree. The results revealed two major clades (Nanorana and Quasipaa), which originated in the Lower Miocene from eastern China and subsequently spread into the HTO (Nanorana). Five well‐supported subclades are found within Nanorana: from the East, Central, and Northwest Himalaya, the Tibetan Plateau, and the southeastern Plateau margin. The latter subclade represents the most basal group (subgenus Chaparana), the Plateau group (Nanorana) represents the sister clade to all species of the Greater Himalaya (Paa). We found no evidence for an east–west range expansion of Paa along the Himalaya, nor clear support for a strict vicariance model. Diversification in each of the three Himalayan subclades has probably occurred in distinct areas. Specimens from the NW Himalaya are placed basally relative to the highly diverse Central Himalayan group, while the lineage from the Tibetan Plateau is placed within a more terminal clade. Our data indicate a Tibetan origin of Himalayan Nanorana and support a previous hypothesis, which implies that a significant part of the Himalayan biodiversity results from primary diversification of the species groups in South Tibet before this part of the HTO was uplifted to its recent heights.     

Improvements in single-use bioreactor film material composition leads to robust and reliable Chinese hamster ovary cell performance

Single-use technologies, in particular disposable bioreactor bags, have become integral within the biopharmaceutical community. However, safety concerns arose upon the identification of toxic leachable compounds derived from the plastic materials. Although the leachable bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP) has been previously shown to inhibit CHO cell growth, it is critical to determine if other compounds like this are still present in subsequent generations of films for industrial application. This study compares the performance of CHO cells, CHO-K1, and CHO-DP12, cultured in media conditioned in an older single-use bioreactor (SUB) film (F-1) and a newer generation film (F-2) from the same vendor. CHO cells cultured in media conditioned for 7 days in the F-1 film demonstrated significantly reduced growth and antibody productivity profiles when compared to controls and media conditioned for the same time period in the newer F-2 film. Proteomic profiling of CHO cells cultured in the F-1 conditioned media identified differentially expressed proteins involved in oxidative stress response as well as compromised ATP synthesis. These potentially metabolically compromised cells exhibited reduced oxidative phosphorylation activity as well as lower glycolytic metabolism, characteristic of slower growing cells. Nonvolatile and metal leachables analysis of film extracts by LC–MS revealed a reduction in the abundance of the analyzed leachates from F-2 films when compared to F-1 films including bDtBPP, potentially explaining improved CHO cell growth in F-2 conditioned media. Furthermore, in vitro endocrine disruptor testing of the known leachable revealed this molecule to possess the potential to act as an androgen antagonist. This study demonstrates an improvement in the materials composition used in modern generations of SUBs for safe application in the bioprocess.     

Calreticulin affects fibronectin-based cell-substratum adhesion via the regulation of c-Src activity

Calreticulin is an endoplasmic reticulum Ca2+-storage protein, which influences gene expression and cell adhesion. In this study, we show that calreticulin induces fibronectin gene expression and matrix deposition, leading to differences in cell spreading and focal adhesion formation in cells differentially expressing calreticulin. We further show that these effects of calreticulin occur via a c-Src-regulated pathway and that c-Src activity is inversely related to calreticulin abundance. Since c-Src is an important regulator of focal contact turnover, we investigated the effect of c-Src inhibition on cells differentially expressing calreticulin. Inhibition of c-Src rescued the poorly adhesive phenotype of the calreticulin-underexpressing cells in that they became well spread, commenced formation of numerous focal contacts, and deposited a rich fibronectin matrix. Importantly, we show that c-Src activity is dependent on releasable Ca2+ from the endoplasmic reticulum, thus implicating Ca2+-sensitive pathways that are affected by calreticulin in cell-substratum adhesion. We propose that calreticulin affects fibronectin synthesis and matrix assembly via the regulation of fibronectin gene expression. In parallel, calcium-dependent effects of calreticulin on c-Src activity influence the formation and/or stability of focal contacts, which are instrumental in matrix assembly and remodeling.     

The renewal and differentiation of Isl1+ cardiovascular progenitors are controlled by a Wnt/beta-catenin pathway

Isl1(+) cardiovascular progenitors and their downstream progeny play a pivotal role in cardiogenesis and lineage diversification of the heart. The mechanisms that control their renewal and differentiation are largely unknown. Herein, we show that the Wnt/beta-catenin pathway is a major component by which cardiac mesenchymal cells modulate the prespecification, renewal, and differentiation of isl1(+) cardiovascular progenitors. This microenvironment can be reconstituted by a Wnt3a-secreting feeder layer with ES cell-derived, embryonic, and postnatal isl1(+) cardiovascular progenitors. In vivo activation of beta-catenin signaling in isl1(+) progenitors of the secondary heart field leads to their massive accumulation, inhibition of differentiation, and outflow tract (OFT) morphogenic defects. In addition, the mitosis rate in OFT myocytes is significantly reduced following beta-catenin deletion in isl1(+) precursors. Agents that manipulate Wnt signals can markedly expand isl1(+) progenitors from human neonatal hearts, a key advance toward the cloning of human isl1(+) heart progenitors.     

Using likelihood-free inference to compare evolutionary dynamics of the protein networks of H. pylori and P. falciparum

Gene duplication with subsequent interaction divergence is one of the primary driving forces in the evolution of genetic systems. Yet little is known about the precise mechanisms and the role of duplication divergence in the evolution of protein networks from the prokaryote and eukaryote domains. We developed a novel, model-based approach for Bayesian inference on biological network data that centres on approximate Bayesian computation, or likelihood-free inference. Instead of computing the intractable likelihood of the protein network topology, our method summarizes key features of the network and, based on these, uses a MCMC algorithm to approximate the posterior distribution of the model parameters. This allowed us to reliably fit a flexible mixture model that captures hallmarks of evolution by gene duplication and subfunctionalization to protein interaction network data of Helicobacter pylori and Plasmodium falciparum. The 80% credible intervals for the duplication–divergence component are [0.64, 0.98] for H. pylori and [0.87, 0.99] for P. falciparum. The remaining parameter estimates are not inconsistent with sequence data. An extensive sensitivity analysis showed that incompleteness of PIN data does not largely affect the analysis of models of protein network evolution, and that the degree sequence alone barely captures the evolutionary footprints of protein networks relative to other statistics. Our likelihood-free inference approach enables a fully Bayesian analysis of a complex and highly stochastic system that is otherwise intractable at present. Modelling the evolutionary history of PIN data, it transpires that only the simultaneous analysis of several global aspects of protein networks enables credible and consistent inference to be made from available datasets. Our results indicate that gene duplication has played a larger part in the network evolution of the eukaryote than in the prokaryote, and suggests that single gene duplications with immediate divergence alone may explain more than 60% of biological network data in both domains.     

A cis-element in the 5' untranslated region of the preproinsulin mRNA (ppIGE) is required for glucose regulation of proinsulin translation

Insulin production in pancreatic beta cells is predominantly regulated through glucose control of proinsulin translation. Previously, this was shown to require sequences within the untranslated regions (UTRs) of the preproinsulin (ppI) mRNA. Here, those sequences were found to be sufficient for specific glucose-regulated proinsulin translation. Furthermore, an element 40-48 bp from the 5' end of the ppI mRNA specifically bound a factor present in islets of Langerhans. Glucose-responsive factor binding to this cis-element exhibited temporal and glucose-concentration-dependent patterns that paralleled proinsulin biosynthesis. Mutating this cis-element abolished the ability of ppI mRNA UTRs to confer glucose regulation upon translation. Like the rat 5'UTR, the human ppI 5'UTR conferred glucose regulation of translation. However alternative splicing of the human 5'UTR that disrupts the cis-element abolished glucose-regulated translation. These data indicate that glucose regulation of cis-element/trans-acting factor interaction is a key component of the mechanism by which glucose regulates insulin production.     

Early queen infection shapes developmental dynamics and induces long-term disease protection in incipient ant colonies

Infections early in life can have enduring effects on an organism's development and immunity. In this study, we show that this equally applies to developing ‘superorganisms’––incipient social insect colonies. When we exposed newly mated Lasius niger ant queens to a low pathogen dose, their colonies grew more slowly than controls before winter, but reached similar sizes afterwards. Independent of exposure, queen hibernation survival improved when the ratio of pupae to workers was small. Queens that reared fewer pupae before worker emergence exhibited lower pathogen levels, indicating that high brood rearing efforts interfere with the ability of the queen's immune system to suppress pathogen proliferation. Early-life queen pathogen exposure also improved the immunocompetence of her worker offspring, as demonstrated by challenging the workers to the same pathogen a year later. Transgenerational transfer of the queen's pathogen experience to her workforce can hence durably reduce the disease susceptibility of the whole superorganism.     

Live-imaging provides an atlas of cellular growth dynamics in the stamen

Development of multicellular organisms is a complex process involving precise coordination of growth among individual cells. Understanding organogenesis requires measurements of cellular behaviors over space and time. In plants, such a quantitative approach has been successfully used to dissect organ development in both leaves and external floral organs, such as sepals. However, the observation of floral reproductive organs is hampered as they develop inside tightly closed floral buds, and are therefore difficult to access for imaging. We developed a confocal time-lapse imaging method, applied here to Arabidopsis (Arabidopsis thaliana), which allows full quantitative characterization of the development of stamens, the male reproductive organs. Our lineage tracing reveals the early specification of the filament and the anther. Formation of the anther lobes is associated with a temporal increase of growth at the lobe surface that correlates with intensive growth of the developing locule. Filament development is very dynamic and passes through three distinct phases: (1) initial intense, anisotropic growth, and high cell proliferation; (2) restriction of growth and proliferation to the filament proximal region; and (3) resumption of intense and anisotropic growth, displaced to the distal portion of the filament, without cell proliferation. This quantitative atlas of cellular growth dynamics provides a solid framework for future studies into stamen development.