The archaeal highly thermostable GH35 family ß-galactosidase DaßGal has a unique seven domain protein fold.

The most extensively studied ß-d-galactosidases (EC3.2.1.23) belonging to four glycoside hydrolase (GH) families 1, 2, 35, and 42 are widely distributed among Bacteria, Archaea and Eukaryotes. Here, we report a novel GH35 family ß-galactosidase from the hyperthermophilic Thermoprotei archaeon Desulfurococcus amylolyticus (DaßGal). Unlike fungal monomeric six-domain ß-galactosidases, the DaßGal enzyme is a dimer; it has an extra jelly roll domain D7 and three composite domains (D4, D5, and D6) that are formed by the distantly located polypeptide chain regions. The enzyme possesses a high specificity for ß-d-galactopyranosides, and its distinguishing feature is the ability to cleave pNP-ß-d-fucopyranoside. DaßGal efficiently catalyzes the hydrolysis of lactose at high temperatures, remains stable and active at 65 °Ð¡, and retains activity at 95 °Ð¡ with a half-life time value equal to 73 min. These properties make archaeal DaßGal a more attractive candidate for biotechnology than the widely used fungal ß-galactosidases.

Direct Determination of the Structure of Single Biopolymer Molecules Using Nanopore Sequencing.

This review highlights operational principles, features, and modern aspects of the development of third-generation sequencing technology of biopolymers focusing on the nucleic acids analysis, namely the nanopore sequencing system. Basics of the method and technical solutions used for its realization are considered, from the first works showing the possibility of creation of these systems to the easy-to-handle procedure developed by Oxford Nanopore Technologies company. Moreover, this review focuses on applications, which were developed and realized using equipment developed by the Oxford Nanopore Technologies, including assembly of whole genomes, methagenomics, direct analysis of the presence of modified bases.

Laparoscopic intracorporeal ileal ureter replacement: multi-institutional data from 102 patients.

OBJECTIVE: To present long-term results of our laparoscopic intracorporeal ileal ureter replacement (LIUR) cohort, including more complex cases of laparoscopic ileocalycostomy. MATERIAL AND METHODS: We collected records of patients undergoing LIUR. Follow-up included a chemical profile and urine cultures. Imaging consisted of renal ultrasonography, excretory urography, cystography, and computer tomographic or magnetic resonance urography. RESULTS: One hundred and two patients were included. Stricture location was left (46.1%), right (39.2%), or bilateral (14.7%). No open conversion was performed. Seventy-four patients (72.5%) underwent a total ureteral unit removal. The mean operative time was 289.4 (120 - 680) minutes. The estimated blood loss was 185.2 (10-400) mL. Three patients had intraoperative complications, and fifteen had early postoperative complications. The mean postoperative hospital stay was 12.2 (7-35) days. The mean follow-up duration period was 37.7 (12-162) months. Most patients' follow-up was uneventful (88%), and seven patients presented with Grade 2 late complications. CONCLUSIONS: Intracorporeal laparoscopic ileal ureteral replacement in cases of extensive ureteral lesions offers optimal long-term outcomes and a low complication rate. Ileocalycostomy constitutes a viable option in the small group of patients with long proximal ureteral strictures and intrarenal pelvis.

Laparoscopic totally intracorporeal ileal ureter replacement: a multi-institutional study.

OBJECTIVE: To prove the feasibility, as well as the reproducibility of laparoscopic totally intracorporeal ileal ureter replacement (LIUR), by presenting a multicenter patient cohort with a long follow-up. MATERIAL AND METHODS: Records of patients undergoing different types of ureteral replacements have been collected. Follow-up included a chemical profile and urine cultures. Imaging consisted of renal ultrasonography and excretory urography, as well as a cystography or an isotopic renography when indicated. RESULTS: Forty patients were included in the study. Twelve underwent a right, 20 a left, and eight a bilateral laparoscopic ureteral replacement. The mean procedure time was 335 (150-680) minutes and the mean estimated blood loss was 221 (50-400) mL. Only three patients presented intraoperative complications, which were managed immediately, and three patients presented a Clavien III postoperative complication. Abdominal drains and nephrostomy tubes were removed after 24-36 h and 7-10 days, respectively. The mean hospital stay was 13.5 (10-35) days. Follow-up was at least six months. CONCLUSIONS: LIUR constitutes a feasible and reproducible method for the restoration of long ureteral defects.

Functionalization of polyacrylamide for nanotrapping positively charged biomolecules.

Engineering new materials which are capable of trapping biomolecules in nanoscale quantities, is crucial in order to achieve earlier diagnostics in different diseases. This article demonstrates that using free radical copolymerization, polyacrylamide can be successfully functionalized with specific synthons for nanotrapping positively charged molecules, such as numerous proteins, through electrostatic interactions due to their negative charge. Specifically, two functional random copolymers, acrylamide/acrylic acid (1) and acrylamide/acrylic acid/N-(pyridin-4-yl-methyl)acrylamide (2), whose negative net charges differ in their water solutions, were synthetized and their ability to trap positively charged proteins was studied using myoglobin as a proof-of-concept example. In aqueous solutions, copolymer 1, whose net charge for a 100 chain fragment (Q pH 6/M) is -1.323 × 10-3, interacted with myoglobin forming a stable monodisperse nanosuspension. In contrast, copolymer 2, whose value of Q pH 6/M equals -0.361 × 10-3, was not able to form stable particles with myoglobin. Nevertheless, thin films of both copolymers were grown using a dewetting process, which exhibited nanoscale cavities capable of trapping different amounts of myoglobin, as demonstrated by bimodal AFM imaging. The simple procedures used to build protein traps make this engineering approach promising for the development of new materials for biomedical applications where trapping biomolecules is required.

Thermostability improvement of Aspergillus awamori glucoamylase via directed evolution of its gene located on episomal expression vector in Pichia pastoris cells.

Novel thermostable variants of glucoamylase (GA) from filamentous fungus Aspergillus awamori X100 were constructed using the directed evolution approach based on random mutagenesis by error-prone PCR of the catalytic domain region of glucoamylase gene located on a new episomal expression vector pPEHα in Pichia pastoris cells. Out of 3000 yeast transformants screened, six new thermostable GA variants with amino acid substitutions Val301Asp, Thr390Ala, Thr390Ala/Ser436Pro, Leu7Met/His391Tyr, Asn9His/Ile82Phe and Ser8Arg/Gln338Leu were identified and studied. To estimate the effect of each substitution in the double mutants, we have constructed the relevant single mutants of GA by site-directed mutagenesis and analyzed their thermal properties. Results of the analysis showed that only Ile82Phe and Ser8Arg substitutions by themselves increased enzyme thermostability. While the substitutions Leu7Met, Asn9His and Gln338Leu decreased the thermal stability of GA, the synergistic effect of double mutant variants Leu7Met/His391Tyr, Asn9His/Ile82Phe and Ser8Arg/Gln338Leu resulted in significant thermostability improvement as compared to the wild type GA. Thr390Ala and Thr390Ala/Ser436Pro mutant variants revealed the highest thermostability with free activation energy changes ΔΔG of 2.99 and 3.1 kJ/mol at 80°C, respectively.