Lats2/Kpm is required for embryonic development, proliferation control and genomic integrity

The Drosophila melanogaster warts/lats tumour suppressor has two mammalian counterparts LATS1/Warts-1 and LATS2/Kpm. Here, we show that mammalian Lats orthologues exhibit distinct expression profiles according to germ cell layer origin. Lats2(-/-) embryos show overgrowth in restricted tissues of mesodermal lineage; however, lethality ultimately ensues on or before embryonic day 12.5 preceded by defective proliferation. Lats2(-/-) mouse embryonic fibroblasts (MEFs) acquire growth advantages and display a profound defect in contact inhibition of growth, yet exhibit defective cytokinesis. Lats2(-/-) embryos and MEFs display centrosome amplification and genomic instability. Lats2 localizes to centrosomes and overexpression of Lats2 suppresses centrosome overduplication induced in wild-type MEFs and reverses centrosome amplification inherent in Lats2(-/-) MEFs. These findings indicate an essential role of Lats2 in the integrity of processes that govern centrosome duplication, maintenance of mitotic fidelity and genomic stability.     

Seeing 'where' through the ears: effects of learning-by-doing and long-term sensory deprivation on localization based on image-to-sound substitution

Background

Sensory substitution devices for the blind translate inaccessible visual information into a format that intact sensory pathways can process. We here tested image-to-sound conversion-based localization of visual stimuli (LEDs and objects) in 13 blindfolded participants.

Methods and Findings

Subjects were assigned to different roles as a function of two variables: visual deprivation (blindfolded continuously (Bc) for 24 hours per day for 21 days; blindfolded for the tests only (Bt)) and system use (system not used (Sn); system used for tests only (St); system used continuously for 21 days (Sc)). The effect of learning-by-doing was assessed by comparing the performance of eight subjects (BtSt) who only used the mobile substitution device for the tests, to that of three subjects who, in addition, practiced with it for four hours daily in their normal life (BtSc and BcSc); two subjects who did not use the device at all (BtSn and BcSn) allowed assessment of its use in the tasks we employed. The impact of long-term sensory deprivation was investigated by blindfolding three of those participants throughout the three week-long experiment (BcSn, BcSn/c, and BcSc); the other ten subjects were only blindfolded during the tests (BtSn, BtSc, and the eight BtSt subjects). Expectedly, the two subjects who never used the substitution device, while fast in finding the targets, had chance accuracy, whereas subjects who used the device were markedly slower, but showed much better accuracy which improved significantly across our four testing sessions. The three subjects who freely used the device daily as well as during tests were faster and more accurate than those who used it during tests only; however, long-term blindfolding did not notably influence performance.

Conclusions

Together, the results demonstrate that the device allowed blindfolded subjects to increasingly know where something was by listening, and indicate that practice in naturalistic conditions effectively improved “visual” localization performance.     

Effect of Nutrient Starvation on the Cellular Composition and Metabolic Capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.     

Thymic alterations in EphA4-deficient mice

In the present work, we have demonstrated in vivo an altered maturation of the thymic epithelium that results in defective T cell development which increases with age, in the thymus of Eph A4-deficient mice. The deficient thymi are hypocellular and show decreased proportions of double-positive (CD4+CD8+) cells which reach minimal numbers in 4-wk-old thymi. The EphA4 (-/-) phenotype correlates with an early block of T cell precursor differentiation that results in accumulation of CD44-CD25+ triple-negative cells and, sometimes, of CD44+CD25- triple-negative thymocytes as well as with increased numbers of apoptotic cells and an important reduction in the numbers of cycling thymocytes. Various approaches support a key role of the thymic epithelial cells in the observed phenotype. Thymic cytoarchitecture undergoes profound changes earlier than those found in the thymocyte maturation. Thymic cortex is extremely reduced and consists of densely packed thymic epithelial cells. Presumably the lack of forward Eph A4 signaling in the Eph A4 -/- epithelial cells affects their development and finally results in altered T cell development.     

The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues.     

Molecular and morphological diversity of Trebouxia microalgae in sphaerothallioid Circinaria spp. lichens1

Three vagrant (Circinaria hispida, Circinaria gyrosa, and Circinaria sp. ‘paramerae’) and one crustose (semi‐vagrant, Circinaria sp. ‘oromediterranea’) lichens growing in very continental areas in the Iberian Peninsula were selected to study the phycobiont diversity. Mycobiont identification was checked using nrITS DNA barcoding: Circinaria sp. ‘oromediterranea’ and Circinaria sp. ‘paramerae’ formed a new clade. Phycobiont diversity was analyzed in 50 thalli of Circinaria spp. using nrITS DNA and LSU rDNA, with microalgae coexistence being found in all the species analyzed by Sanger sequencing. The survey of phycobiont diversity showed up to four different Trebouxia spp. as the primary phycobiont in 20 thalli of C. hispida, in comparison with the remaining Circinaria spp., where only one Trebouxia was the primary microalga. In lichen species showing coexistence, some complementary approaches are needed (454 pyrosequencing and/or ultrastructural analyses). Five specimens were selected for high‐throughput screening (HTS) analyses: 22 Trebouxia OTUs were detected, 10 of them not previously known. TEM analyses showed three different cell morphotypes (Trebouxia sp. OTU A12, OTU S51, and T. cretacea) whose ultrastructure is described here in detail for the first time. HTS revealed a different microalgae pool in each species studied, and we cannot assume a specific pattern between these pools and the ecological and/or morphological characteristics. The mechanisms involved in the selection of the primary phycobiont and the other microalgae by the mycobiont are unknown, and require complex experimental designs. The systematics of the genus Circinaria is not yet well resolved, and more analyses are needed to establish a precise delimitation of the species.     

Systemic and mucosal antibody response in experimental Chlamydia pneumoniae infection of mice

Chlamydia pneumoniae is a common human respiratory pathogen, and sera from infected individuals recognize several proteins of C. pneumoniae. We produced C. pneumoniae-specific proteins in a Bacillus subtilis expression system. We then used these recombinant C. pneumoniae proteins and purified C. pneumoniae elementary bodies as antigens in enzyme immunoassays to assess the kinetics and protein specificity of the systemic and mucosal antibody responses induced by C. pneumoniae intranasal infection in BALB/c mice. The systemic antibodies in mice recognized strong 'key' immunogens of Chlamydia, Omp2 and Hsp60, but weakly targeted the MOMP protein, the major immunogen in chlamydial species other than C. pneumoniae. The IgA antibodies in bronchial secretions specifically recognized the putative surface protein of C. pneumoniae, Omp4. Our preliminary observations point to the necessity of further characterization of the mucosal antibody response during C. pneumoniae infection.     

Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.     

Plant transformation by particle bombardment of embryogenic pollen

Summary Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 10⁴ pollen grains were GUS⁺. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS⁺ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.Abbreviations GUS ß-glucuronidase - CaMV Cauliflower Mosaic Virus - MCS multicellular structure - NPTII neomycin phosphotransferase - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide - DAPI 4,6-diamidino-2-phenylindole - Tris Tris(hydroxymethyl)aminomethane hydrochloride - EDTA ethylenedinitrilo tetraacetic acid, disodium salt dihydrate     

Enhanced evapotranspiration was observed during extreme drought from Miscanthus,opposite of other crops

The impact of extreme drought and heat stress that occurred in the Midwestern U.S. in 2012 on evapotranspiration (ET), net ecosystem productivity (NEP), and water‐use efficiency (WUE) of three perennial ecosystems (switchgrass, miscanthus, prairie) and a maize/soybean agroecosystem was studied as part of a long‐term experiment. Miscanthus had a slower initial response but an eventually drastic ET as drought intensified, which resulted in the largest water deficit among the crops. The substantially higher ET at peak drought was likely supplied by access to deep soil water, but suggests that stomatal conductance of miscanthus during the drought may respond differently than the other ecosystems, consistent with an anisohydric strategy. While there was a discrepancy in the water consumption of maize and switchgrass/prairie in the early time of drought, all these ecosystems followed a water‐saving strategy when drought intensified. The gross primary production (GPP) of miscanthus dropped, but was reversible, when temperature reached 40 °C and still provided the largest total GPP among the ecosystems. Increased ET for miscanthus during 2012 resulted a large decline in ecosystem WUE compared to what was observed in other years. The biophysical responses of miscanthus measured during an extreme, historic drought suggest that this species can maintain high productivity longer than other ecosystems during a drought at the expense of water use. While miscanthus maintained productivity during drought, recovery lagged associated with depleted soil moisture. The enhanced ET of miscanthus may intensify droughts through increase supply of deep soil moisture to the atmosphere.