Disease Progression in CNGA3 and CNGB3 Retinopathy; Characteristics of Slovenian Cohort and Proposed OCT Staging Based on Pooled Data from 126 Patients from 7 Studies

Achromatopsia has been proposed to be a morphologically predominately stable retinopathy with rare reports of progression of structural changes in the macula. A five-grade system of optical coherence tomography (OCT) features has been used for the classification of structural macular changes. However, their association with age remains questionable. We characterized the Slovenian cohort of 12 patients with pathogenic variants in CNGA3 or CNGB3 who had been followed up with OCT for up to 9 years. Based on observed structural changes in association with age, the following four-stage classification of retinal morphological changes was proposed: (I) preserved inner segment ellipsoid band (Ise), (II) disrupted ISe, (III) ISe loss and (IV) ISe and RPE loss. Data from six previously published studies reporting OCT morphology in CNGA3 and CNGB3 patients were additionally collected, forming the largest CNGA3/CNGB3 cohort to date, comprising 126 patients aged 1–71 years. Multiple regression analysis showed a significant correlation of OCT stage with age (p < 0.001) and no correlation with gene (p > 0.05). The median ages of patients with stages I–IV were 12 years, 23 years, 27 years and 48 years, respectively, and no patient older than 50 years had continuous ISe. Our findings suggest that achromatopsia presents with slowly but steadily progressive structural changes of the macular outer retinal layers. However, whether morphological changes in time follow the proposed four-stage linear pattern needs to be confirmed in a long-term study.     

A new carbohydrate-active oligosaccharide dehydratase is involved in the degradation of ulvan

Marine algae catalyze half of all global photosynthetic production of carbohydrates. Owing to their fast growth rates, Ulva spp. rapidly produce substantial amounts of carbohydrate-rich biomass and represent an emerging renewable energy and carbon resource. Their major cell wall polysaccharide is the anionic carbohydrate ulvan. Here, we describe a new enzymatic degradation pathway of the marine bacterium Formosa agariphila for ulvan oligosaccharides involving unsaturated uronic acid at the nonreducing end linked to rhamnose-3-sulfate and glucuronic or iduronic acid (Δ-Rha3S-GlcA/IdoA-Rha3S). Notably, we discovered a new dehydratase (P29_PDnc) acting on the nonreducing end of ulvan oligosaccharides, i.e., GlcA/IdoA-Rha3S, forming the aforementioned unsaturated uronic acid residue. This residue represents the substrate for GH105 glycoside hydrolases, which complements the enzymatic degradation pathway including one ulvan lyase, one multimodular sulfatase, three glycoside hydrolases, and the dehydratase P29_PDnc, the latter being described for the first time. Our research thus shows that the oligosaccharide dehydratase is involved in the degradation of carboxylated polysaccharides into monosaccharides.     

Continuous testing system for Baeyer-Villiger biooxidation using recombinant Escherichia coli expressing cyclohexanone monooxygenase encapsulated in polyelectrolyte complex capsules

An original strategy for universal laboratory testing of Baeyer-Villiger monooxygenases based on continuous packed-bed minireactor connected with flow calorimeter and integrated with bubble-free oxygenation is reported. Model enantioselective Baeyer-Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one as important chiral synthons for the synthesis of bioactive compounds were performed in the minireactor equipped with a column packed with encapsulated recombinant cells Escherichia coli overexpressing cyclohexanone monooxygenase. The cells were encapsulated in polyelectrolyte complex capsules formed by reaction of oppositely charged polymers utilizing highly reproducible and controlled encapsulation process. Encapsulated cells tested in minireactor exhibited high operational stability with 4 complete substrate conversions to products and 6 conversions above 80% within 14 repeated consecutive biooxidation tests. Moreover, encapsulated cells showed high enzyme stability during 91 days of storage with substrate conversions above 80% up to 60 days of storage. Furthermore, usable thermometric signal of Baeyer-Villiger biooxidation obtained by flow calorimetry using encapsulated cells was utilized for preparatory kinetic study in order to guarantee sub-inhibitory initial substrate concentration for biooxidation tests.     

Lateral drift correction in time-laps images by the particle-tracking algorithm

In long-term time-laps imaging of living cells, a significant lateral drift of the fluorescently labeled structures is often observed due to many reasons including superfusion of solution, temperature gradients, bolus addition of pharmacological agents and cell motility. We have detected lateral drift in long-term time-laps confocal imaging by tracking fluorescent puncta, which represent single exocytotic vesicles expressing synaptopHluorin (spH), a pH sensitive green fluorescence protein. Following the initial increase in fluorescence intensity due to alkalinization of vesicle lumen, the spH fluorescent puncta dimmed, which may be attributed to the resealing of the fusion pore and subsequent slow reacidification of the vesicle, or alternatively the dimming may be due to a significant lateral drift of the vesicle out of the region of interest (ROI). We identified and compensated the lateral drift by tracking particles present in the confocal images, without any additional mechanical and/or optical hardware components. The peak of the Gaussian two-dimensional (2D) curve fitted to the fluorescent particle intensity profile was recorded as the X and Y coordinates of the vesicle in each frame. The resulting coordinates of vesicle positions were averaged and rounded to the nearest pixel value, which was used to correct the drift in the time-laps images. In drift corrected time-laps images, the vesicle remained enclosed by the ROI, and the time dependent changes of spH fluorescence intensity averaged from the ROI remained at a constant level, revealing that endocytosis with subsequent slow reacidification of vesicles was an unlikely event.     

Language Metaphors of Life

We discuss the difference between formal and natural languages, and argue that should the language metaphor have any foundation, it’s analogy with natural languages that should be taken into account. We discuss how such operation like reading, writing, sign, interpretation, etc., can be applied in the realm of the living and what can be gained, by such an approach, in order to understand the phenomenon of life.     

High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae?

Evidence is presented that xylose metabolism in the anaerobic cellulolytic fungus Piromyces sp. E2 proceeds via a xylose isomerase rather than via the xylose reductase/xylitol-dehydrogenase pathway found in xylose-metabolising yeasts. The XylA gene encoding the Piromyces xylose isomerase was functionally expressed in Saccharomyces cerevisiae. Heterologous isomerase activities in cell extracts, assayed at 30 degrees C, were 0.3-1.1 micromol min(-1) (mg protein)(-1), with a Km for xylose of 20 mM. The engineered S. cerevisiae strain grew very slowly on xylose. It co-consumed xylose in aerobic and anaerobic glucose-limited chemostat cultures at rates of 0.33 and 0.73 mmol (g biomass)(-1) h(-1), respectively.     

Self-organization of domain structures by DNA-loop-extruding enzymes

The long chromosomal DNAs of cells are organized into loop domains much larger in size than individual DNA-binding enzymes, presenting the question of how formation of such structures is controlled. We present a model for generation of defined chromosomal loops, based on molecular machines consisting of two coupled and oppositely directed motile elements which extrude loops from the double helix along which they translocate, while excluding one another sterically. If these machines do not dissociate from DNA (infinite processivity), a disordered, exponential steady-state distribution of small loops is obtained. However, if dissociation and rebinding of the machines occurs at a finite rate (finite processivity), the steady state qualitatively changes to a highly ordered ‘stacked’ configuration with suppressed fluctuations, organizing a single large, stable loop domain anchored by several machines. The size of the resulting domain can be simply regulated by boundary elements, which halt the progress of the extrusion machines. Possible realizations of these types of molecular machines are discussed, with a major focus on structural maintenance of chromosome complexes and also with discussion of type I restriction enzymes. This mechanism could explain the geometrically uniform folding of eukaryote mitotic chromosomes, through extrusion of pre-programmed loops and concomitant chromosome compaction.     

Species–area relationships across four trophic levels – decreasing island size truncates food chains

That larger areas will typically host more diverse ecological assemblages than small ones has been regarded as one of the few fundamental ‘laws’ in ecology. Yet, area may affect not only species diversity, but also the trophic structure of the local ecological assemblage. In this context, recent theory on trophic island biogeography offers two clear‐cut predictions: that the slope of the species–area relationship should increase with trophic rank, and that food chain length (i.e. the number of trophic levels) should increase with area. These predictions have rarely been verified in terrestrial systems. To offer a stringent test of key theory, we focused on local food chains consisting of trophic specialists: plants, lepidopteran herbivores, and their primary and secondary parasitoids. For each of these four trophic levels, we surveyed species richness across a set of 20 off‐shore continental islands spanning a hundred‐fold range in size. We then tested three specific hypotheses: that species richness is affected by island size, that the slope of the species–area curve is related to trophic rank, and that such differences in slope translate into variation in food chain length with island size. Consistent with these predictions, estimates of the species–area slope steepened from plants through herbivores and primary parasitoids to secondary parasitoids. As a result of the elevated sensitivity of top consumers to island size, food chain length decreased from large to small islands. Since island size did not detectably affect the ratio between generalists and specialists among either herbivores (polyphages vs oligophages) or parasitoids (idiobionts vs koinobionts), the patterns observed seemed more reflective of changes in the overall number of nodes and levels in local food webs than of changes in their linking structure. Overall, our results support the trophic‐level hypothesis of island biogeography. Per extension, they suggest that landscape modification may imperil food web integrity and vital biotic interactions.     

The annexins of Dictyostelium

Annexins are a highly conserved ubiquitous family of Ca2+- and phospholipid-binding proteins present in nearly all eukaryotic cells. Analysis of the Dictyostelium genome revealed the presence of two annexin genes, the annexin C1 gene (nxnA) giving rise to two isoforms of 47 and 51 kDa (previously synexin), and the annexin C2 gene (nxnB) coding for a 56-kDa protein with 33% sequence identity to annexin C1. Annexin C2 is expressed at very low and constant levels throughout development. Quantification by real-time PCR indicated that it is present in about 35-fold lower amounts compared to annexin C1. We have used a GFP-tagged annexin C2 to study its cellular distribution and dynamics. In cell fractionation studies, annexin C2 cofractionates with annexin C1 and is enriched in the 100,000 g pellet. Like annexin C1, GFP-AnxC2 stains the plasma membrane. In addition it is present in the perinuclear region and overlaps to some degree with the Golgi apparatus, whereas annexin C1 is present on intracellular membranes resembling endosomal membranes and in the nucleus. Annexin C2 is not observed in the nucleus. An annexin C1 mutant (SYN-) which shows a defect during multicellular development can be rescued by full-length annexin C1, whereas overexpression of GFP-AnxC2 did not rescue the developmental defect The data support the concept that annexins, although having a highly conserved structure, participate in different functions in a cell.