Cryoprotection and banking of living cells in a 3D multiple emulsion‐based carrier

The ability to preserve stem cells/cells with minimal damage for short and long periods of time is essential for advancements in biomedical therapies and biotechnology. New methods of cell banking are continuously needed to provide effective damage prevention to cells. This paper puts forward a solution to the problem of the low viability of cells during cryopreservation in a traditional suspension and storage by developing innovative multiple emulsion‐based carriers for the encapsulation and cryopreservation of cells. During freezing‐thawing processes, irreversible damage to cells occurs as a result of the formation of ice crystals, cell dehydration, and the toxicity of cryoprotectant. The proposed method was effective due to the “flexible” protective structure of multiple emulsions, which was proven by a high cell survival rate, above 90%. Results make new contributions in the fields of cell engineering and biotechnology and contribute to the development of methods for banking biological material.     

The evolution of catalytic residues and enzyme mechanism within the bacterial nucleoside phosphorylase superfamily 1

Nucleoside phosphorylases are essential for the salvage and catabolism of nucleotides in bacteria and other organisms, and members of this enzyme superfamily have been of interest for the development of antimicrobial and cancer therapies. The nucleotide phosphorylase superfamily 1 encompasses a number of different enzymes which share a general superfold and catalytic mechanism, while they differ in the nature of the nucleophiles used and in the nature of characteristic active site residues. Recently, one subfamily, the uridine phosphorylases, has been subdivided into two types which differ with respect to the mechanism of transition state stabilization, as dictated by differences in critical amino acid residues. Little is known about the phylogenetic distribution and relationship of the two different types, as well as the relationship to other NP-1 superfamily members. Here comparative genomic analysis illustrates that UP-1s and UP-2s fall into monophyletic groups and are biased with respect to species representation. UP-1 evolved in Gram negative bacteria, while Gram positive species tend to predominantly contain UP-2. PNP (a sister clade to all UPs) contains both Gram positive and Gram negative species. The findings imply that the nucleoside phosphorylase superfamily 1 evolved through a series of three important duplications, leading to the separate, monophyletic enzyme families, coupled to individual lateral transfer events. Extensive horizontal transfer explains the occurrence of unexpected uridine phosphorylases in some genomes. This study provides a basis for understanding the evolution of uridine and purine nucleoside phosphorylases with respect to DNA/RNA metabolism and with potential utility in the design of antimicrobial and anti-tumor drugs.     

Beta diversity of geometrid moths (Lepidoptera: Geometridae) in an Andean montane rainforest

Abstract. Turnover in species composition of the extremely species‐rich family Geometridae (Lepidoptera) was investigated along an elevational gradient ranging from 1040 m to 2677 m above sea level. Moths were sampled using weak light traps (30 W) in three field periods in 1999 and 2000 in an Andean montane rainforest in the province of Zamora‐Chinchipe in southern Ecuador. A total of 13 938 specimens representing 1010 species were analysed. Similarities of ensembles of all geometrid moths and of the subfamilies Ennominae and Larentiinae were calculated using the NESS index (with m_max). Ordinations performed using nonmetric multidimensional scaling (NMDS) and correspondence analysis depicted a gradual change of the ensembles along the altitudinal gradient. Extracted ordination scores significantly correlate with altitude (?0.97 ≤ r ≤ ?0.95, P < 0.001) and with ambient air temperature (0.93 ≤ r ≤ 0.97, P < 0.001). Temperature is therefore assumed to be the most important abiotic determinant responsible for the species turnover among the moths. Matrix correlation tests were performed in order to compare faunal matrices with matrices derived from available environmental factors. Both tree diversity and vegetation structure significantly correlate with faunal data, but tree diversity explains considerably more of the data variability (range: Mantel r = 0.81–0.83, P < 0.001) than vegetation structure (range: Mantel r = 0.35, P < 0.005 to r = 0.43, P < 0.001). Tree diversity also changes gradually and scores of the first NMDS dimension are highly significantly correlated with altitude (r = 0.98, P < 0.001). A common underlying factor such as ambient temperature might also be responsible for such vegetation changes. Additionally, simulated model data was developed that assumed a constant turnover of moth species and equal elevational ranges of all species involved. Despite the simplicity of the models, they fit empirical data very well (Mantel r > 0.80 and P < 0.001 in all models).     

Biallelic CCM3 mutations cause a clonogenic survival advantage and endothelial cell stiffening

CCM3, originally described as PDCD10, regulates blood‐brain barrier integrity and vascular maturation in vivo. CCM3 loss‐of‐function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral‐ and plasmid‐free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss‐of‐function CCM3 variants into human ECs and studied the molecular and functional effects of long‐term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real‐time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3‐deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long‐term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3‐deficient dysfunctional ECs contributes to CCM formation.     

Production of chitinases by Aphanocladium album grown on crystalline and colloidal chitin

Abstract The deuteromycete Aphanocladium album produced two endochitinases (EC 3.2.1.14) with apparent isoelectric points of 7.1 and 7.6 and seven exochitinases (EC 3.2.1.30) with apparent isoelectric points ranging from 3.8 to 6.4 when grown on a colloidal chitin preparation. With crystalline chitin as carbon source, two endochitinases (p I 7.1 and 7.6) and only one exochitanase (p I 4.9) were detected. The exochitinase p I 4.9, which was produced with both substrates, has a relative molecular mass of 44 000. The different chitinases could be separated by chromatofocusing and their specific activities were determined.