Reduced apoptosis in Chinese hamster ovary cells via optimized CRISPR interference

Chinese hamster ovary (CHO) cells are widely used for biopharmaceutical protein production. One challenge limiting CHO cell productivity is apoptosis stemming from cellular stress during protein production. Here we applied CRISPR interference (CRISPRi) to downregulate the endogenous expression of apoptotic genes Bak, Bax, and Casp3 in CHO cells. In addition to reduced apoptosis, mitochondrial membrane integrity was improved and the caspase activity was reduced. Moreover, we optimized the CRISPRi system to enhance the gene repression efficiency in CHO cells by testing different repressor fusion types. An improved Cas9 repressor has been identified by applying C-terminal fusion of a bipartite repressor domain, KRAB–MeCP2, to nuclease-deficient Cas9. These results collectively demonstrate that CHO cells can be rescued from cell apoptosis by targeted gene repression using the CRISPRi system.     

Understanding contact patterns of protein structures from protein contact map and investigation of unique patterns in the globin-like folded domains

Proteins are biochemical compounds made up of one or more polypeptides in a specific order, typically folded into a functionally active form. Proteins are categorized into four different structural classes according to the topology of α-helices and β-strands. In this study, we modeled these four structural classes as an undirected network depicting amino acids as nodes and interaction between them as edges. Results infer that basic protein classes can be easily recognized as well as distinguished by utilizing protein contact maps (PCM). Toward studying the globin-like fold, the helix-loop-helix region contacts were seen to be of a unique pattern, and these remained in all the folds. Further, the averaged diagonal contacts were analyzed and identified those contacts in α/β proteins were higher in comparison with the other class. Interesting, we noticed that anti-parallel beta sheets were dominant in all-β and α + β classes that lead to similar diagonal patterns. Network properties of all four basic classes were analyzed and found to possess small-world property. Findings infer that PCM may assist classify protein structure classes and it also helps in evaluating the predicted protein structures.     

Quorum sensing regulates ‘swim-or-stick’ lifestyle in the phycosphere

Interactions between phytoplankton and bacteria play major roles in global biogeochemical cycles and oceanic nutrient fluxes. These interactions occur in the microenvironment surrounding phytoplankton cells, known as the phycosphere. Bacteria in the phycosphere use either chemotaxis or attachment to benefit from algal excretions. Both processes are regulated by quorum sensing (QS), a cell–cell signalling mechanism that uses small infochemicals to coordinate bacterial gene expression. However, the role of QS in regulating bacterial attachment in the phycosphere is not clear. Here, we isolated a Sulfitobacter pseudonitzschiae F5 and a Phaeobacter sp. F10 belonging to the marine Roseobacter group and an Alteromonas macleodii F12 belonging to Alteromonadaceae, from the microbial community of the ubiquitous diatom Asterionellopsis glacialis. We show that only the Roseobacter group isolates (diatom symbionts) can attach to diatom transparent exopolymeric particles. Despite all three bacteria possessing genes involved in motility, chemotaxis, and attachment, only S. pseudonitzschiae F5 and Phaeobacter sp. F10 possessed complete QS systems and could synthesize QS signals. Using UHPLC–MS/MS, we identified three QS molecules produced by both bacteria of which only 3-oxo-C_16:1-HSL strongly inhibited bacterial motility and stimulated attachment in the phycosphere. These findings suggest that QS signals enable colonization of the phycosphere by algal symbionts.     

The leaf economics spectrum’s morning coffee: plant size-dependent changes in leaf traits and reproductive onset in a perennial tree crop

Background and AimsSize-dependent changes in plant traits are an important source of intraspecific trait variation. However, there are few studies that have tested if leaf trait co-variation and/or trade-offs follow a within-genotype leaf economics spectrum (LES) related to plant size and reproductive onset. To our knowledge, there are no studies on any plant species that have tested whether or not the shape of a within-genotype LES that describes how traits covary across whole plant sizes, is the same as the shape of a within-genotype LES that represents environmentally driven trait plasticity.MethodsWe quantified size-dependent variation in eight leaf traits in a single coffee genotype (Coffea arabica var. Caturra) in managed agroecosystems with different environmental conditions (light and fertilization treatments), and evaluated these patterns with respect to reproductive onset. We also evaluated if trait covariation along a within-genotype plant-size LES differed from a within-genotype environmental LES defined with trait data from coffee growing in different environmental conditions.Key ResultsLeaf economics traits related to resource acquisition – maximum photosynthetic rates (A) and mass-based leaf nitrogen (N) concentrations – declined linearly with plant size. Structural traits – leaf mass, leaf thickness, and leaf mass per unit area (LMA) – and leaf area increased with plant size beyond reproductive onset, then declined in larger plants. Three primary LES traits (mass-based A, leaf N and LMA) covaried across a within-genotype plant-size LES, with plants moving towards the ‘resource-conserving’ end of the LES as they grow larger; in coffee these patterns were nearly identical to a within-genotype environmental LES.ConclusionsOur results demonstrate that a plant-size LES exists within a single genotype. Our findings indicate that in managed agroecosystems where resource availability is high the role of reproductive onset in driving within-genotype trait variability, and the strength of covariation and trade-offs among LES traits, are less pronounced compared with plants in natural systems. The consistency in trait covariation in coffee along both plant-size and environmental LES axes indicates strong constraints on leaf form and function that exist within plant genotypes.     

No need for stepping stones: Direct,joint dispersal of the lichen‐forming fungus Mastodia tessellata (Ascomycota) and its photobiont explains their bipolar distribution

Aim

The hypotheses proposed to explain the high percentage of bipolar lichens in Antarctica have never been explicitly tested. We used the strictly bipolar, coastal lichenized fungus Mastodia tessellata (Verrucariaceae, Ascomycota) and its photobionts (Prasiola, Trebouxiophyceae, Chlorophyta) as model species to discern whether this extraordinary disjunction originated from vicariance or long‐distance dispersal.

Location

Coasts of Antarctica, Tierra del Fuego (Chile), Alaska (USA) and British Columbia (Canada).

Methods

Based on a comprehensive geographical (315 specimens and 16 populations from Antarctica, Tierra del Fuego and North America) and molecular sampling (three and four loci for the fungus and algae respectively), we implemented explicit Bayesian methods to compare alternative hypotheses of speciation and migration, and performed dating analyses for the fungal and algal partner, in order to infer the timing of the colonization events and the direction of gene flow among distant, disjunct areas.

Results

Mastodia tessellata comprises two fungal species which in turn associate with three photobiont lineages along the studied distribution range. Independent estimation of divergence ages for myco‐ and photobionts indicated a middle to latest Miocene species split in the Southern Hemisphere, and a late Miocene to Pleistocene acquisition of the bipolar distribution. Comparison of migration models and genetic diversity patterns suggested an austral origin for the bipolar species.

Main conclusions

The complex evolutionary history of Mastodia tessellata s.l. can be explained by a combination of vicariant and long‐distance dispersal mechanisms. We provide novel evidence of a pre‐Pleistocene long‐term evolution of lichens in Antarctica as well as for bipolar distributions shaped by Southern to Northern Hemisphere migratory routes without the need for stepping stones.