A new allele with abnormal cyclic-AMP phosphodiesterase activity in Phycomyces

Summary A mutant of the fungus Phycomyces blakesleeanus (Burgeff), C21 (madA7) that was isolated for its abnormal phototropism (Bergman et al. 1973) carries a secondary mutation pde-1 which is unlinked to the madA gene. The pde-1 allele causes the loss of about 80% of the cAMP phosphodiesterase activity. This allele is not essential for the photoreactions of the mycelium or the sporangiophore, and the bulk activity of the phosphodiesterase appears to play no role in the phototransduction pathway of Phycomyces.     

Determination of the membrane topology of PORCN,an O-acyl transferase that modifies Wnt signalling proteins

Wnt gradients elicit distinct cellular responses, such as proliferation, specification, differentiation and survival in a dose-dependent manner. Porcupine (PORCN), a membrane-bound O-acyl transferase (MBOAT) that resides in the endoplasmic reticulum, catalyses the addition of monounsaturated palmitate to Wnt proteins and is required for Wnt gradient formation and signalling. In humans, PORCN mutations are causal for focal dermal hypoplasia (FDH), an X-linked dominant syndrome characterized by defects in mesodermal and endodermal tissues. PORCN is also an emerging target for cancer therapeutics. Despite the importance of this enzyme, its structure remains poorly understood. Recently, the crystal structure of DltB, an MBOAT family member from bacteria, was solved. In this report, we use experimental data along with homology modelling to DltB to determine the membrane topology of PORCN. Our studies reveal that PORCN has 11 membrane domains, comprising nine transmembrane spanning domains and two reentrant domains. The N-terminus is oriented towards the lumen while the C-terminus is oriented towards the cytosol. Like DltB, PORCN has a funnel-like structure that is encapsulated by multiple membrane-spanning helices. This new model for PORCN topology allows us to map residues that are important for biological activity (and implicated in FDH) onto its three-dimensional structure.     

Plant DNases are potent therapeutic agents against Echis carinatus venom-induced tissue necrosis in mice

Echis carinatus envenomation leads to severe tissue necrosis at the bitten site by releasing DNA from immune cells that blocks the blood flow. An earlier report has shown that exogenous DNase 1 offers protection against such severe local tissue necrosis. Tricosanthus tricuspidata is a medicinal plant and the paste prepared from its leaves has been used extensively for the treatment of snakebite-induced tissue necrosis. Most studies including reports from our laboratory focused on plant secondary metabolite as therapeutic molecules against snakebite envenomation. However, the involvement of hydrolytic enzymes including DNase in treating snake venom-induced tissue necrosis has not been addressed. Several folk medicinal plants used against snakebite treatment showed the presence of DNase activity and found to be rich in T. tricuspidata. Further, purified T. tricuspidata DNase showed a single sharp peak in reversed-phase high-performance liquid chromatography (RP-HPLC) with an apparent molecular mass of 17 kDa. T. tricuspidata DNase exhibited potent DNA degrading activity performed using agarose gel electrophoresis, spectrophotometric assay, and DNA zymography. In addition, purified DNase from T. tricuspidata was able to neutralize E. carinatus venom-induced mouse tail tissue necrosis and normalized elevated serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels 30 minutes post venom injection. T. tricuspidata DNase was also able to reverse E. carinatus venom-induced histopathological changes and collagen depletion in mice tail tissue. All these observed pharmacological actions of T. tricuspidata DNase were inhibited by sodium fluoride (NaF). This study provides scientific validation of the traditional use of T. tricuspidata leaf paste in the healing of snakebite-induced tissue necrosis and might be exploited to treat snake venom-induced local toxicity.     

An NIR-based PAT approach for real-time control of loading in Protein A chromatography in continuous manufacturing of monoclonal antibodies

Control of column loading in Protein A chromatography is a crucial part of development of robust and flexible process platforms for continuous production of monoclonal antibody (mAb) products. In this paper, we propose a control system that uses near infrared spectroscopy (NIRS) flow cells to accomplish the above. Two applications have been demonstrated using a periodic counter-current continuous chromatography setup. The first application involves use of single NIR flow cell before the inlet of the loading column to measure the concentration of mAb in the harvested broth. Measurement was in real-time (every 3 s) and within ±0.05 mg/ml, significantly better than making UV-based concentration estimations. The second application involved use of an additional NIR flow cell at the outlet of the loading column to measure column breakthrough in real time. The concentration data was transferred to a Python-based monitoring and control algorithm layered over a Cadence BioSMB system. The program could successfully run a three-column periodic counter current method on the BioSMB whereas controlling loading to ensure optimal resin utilization in each loading cycle phase based on precharacterized dynamic binding capacity models, whereas maintaining periodic elutions. The system was tested with multiple perturbations in harvest concentration, modeled after deviations that could arise downstream of a perfusion cell culture system. The results show that the proposed control is a spectroscopy-based process analytical technology tool that facilitates real time monitoring and control of loading in process chromatography. It is adaptable to any continuous chromatography equipment and is very well suited for implementation in a continuous mAb production train.     

Environmental cycles regulate development time via circadian clock mediated gating of adult emergence

Background

Previous studies have implicated a role for circadian clocks in regulating pre-adult development of organisms. Among them two approaches are most notable: 1) use of insects whose clocks have different free-running periods and 2) imposition of artificial selection on either rate of development, timing of emergence or circadian period in laboratory populations. Using these two approaches, influence of clock on rate of development has been elucidated. However, the contribution of circadian clocks in determining time taken for pre-adult development has remained unclear. Here we present results of our studies aimed to understand this influence by examining populations of fruit flies carrying three different alleles of the period gene and hence having different free-running periods. We tried to achieve similarity of genetic background among the three strains while also ensuring that they harbored sufficient variation on loci other than period gene.

Results

We find that under constant conditions, flies with long period have slower development whereas in presence of light-dark cycles (LD) of various lengths, the speed of development for each genotype is influenced by whether their eclosion rhythms can entrain to them. Under LD 12:12 (T24), where all three strains entrain, they do not show any difference in time taken for emergence, whereas under LD 10:10 (T20) where long period flies do not entrain and LD 14:14 (T28) where short period flies do not entrain, they have slower and faster pre-adult development, respectively, compared to the controls. We also show that a prior stage in development namely pupation is not rhythmic though time taken for pupation is determined by both the environmental cycle and period allele.

Conclusion

We discuss how in presence of daily time cues, interaction of the cyclic environmental factors with the clock determines the position and width of the gate available for a fly to emerge (duration of time within a cycle when adult emergence can occur) resulting in an altered developmental duration from that observed under constant conditions. We also discuss the relevance of genetic background influencing this regulation.

     

Genome evolution of a tertiary dinoflagellate plastid

The dinoflagellates have repeatedly replaced their ancestral peridinin-plastid by plastids derived from a variety of algal lineages ranging from green algae to diatoms. Here, we have characterized the genome of a dinoflagellate plastid of tertiary origin in order to understand the evolutionary processes that have shaped the organelle since it was acquired as a symbiont cell. To address this, the genome of the haptophyte-derived plastid in Karlodinium veneficum was analyzed by Sanger sequencing of library clones and 454 pyrosequencing of plastid enriched DNA fractions. The sequences were assembled into a single contig of 143 kb, encoding 70 proteins, 3 rRNAs and a nearly full set of tRNAs. Comparative genomics revealed massive rearrangements and gene losses compared to the haptophyte plastid; only a small fraction of the gene clusters usually found in haptophytes as well as other types of plastids are present in K. veneficum. Despite the reduced number of genes, the K. veneficum plastid genome has retained a large size due to expanded intergenic regions. Some of the plastid genes are highly diverged and may be pseudogenes or subject to RNA editing. Gene losses and rearrangements are also features of the genomes of the peridinin-containing plastids, apicomplexa and Chromera, suggesting that the evolutionary processes that once shaped these plastids have occurred at multiple independent occasions over the history of the Alveolata.