Construction of stabilized proteins by combinatorial consensus mutagenesis

We constructed stabilized variants of beta-lactamase (BLA) from Enterobacter cloacae by combinatorial recruitment of consensus mutations. By aligning the sequences of 38 BLA homologs, we identified 29 positions where the E.cloacae gene differs from the consensus sequence of lactamases and constructed combinatorial libraries using mixtures of mutagenic oligonucleotides encompassing all 29 positions. Screening of 90 random isolates from these libraries identified 15 variants with significantly increased thermostability. The stability of these isolates suggest that all tested mutations make additive contributions to protein stability. A statistical analysis of sequence and stability data identified 11 mutations that made stabilizing contributions and eight mutations that destabilized the protein. A second-generation library recombining these 11 stabilizing mutations led to the identification of BLA variants that showed further stabilization. The most stable variant had a mid-point of thermal denaturation (Tm) that was 9.1 degrees C higher than the starting molecule and contained eight consensus mutations. Incubation of three stabilized BLA variants with several proteases showed that all tested isolates have significantly increased resistance to proteolysis. Our data demonstrate that combinatorial consensus mutagenesis (CCM) allows the rapid generation of protein variants with improved thermal and proteolytic stability.     

A de novo formulation of metformin using chitosan-based nanomicelles for potential diabetes therapy

A de novo formulation of metformin (MET) was developed through the physical loading of drug into a chitosan-grafted-[poly(acryl amide)-block-poly(acrylic acid)] [CS-g-(PAAm-b-PAA)] terpolymer. For this purpose, CS was functionazed with phthalic anhydride followed by 4-cyano, 4-[(phenylcarbothioyl)sulfanyl]pentanoic acid to produc a macro-RAFT agent (CS-CTA). Afterward, acryl amide and acrylic acid monomers were graft and block copolymerized onto the synthesized CS-CTA through a reversible addition–fragmentation chain transfer (RAFT) polymerization technique to afford CS-g-PAAm copolymer and CS-g-(PAAm-b-PAA) terpolymer, respectively. The fabricated CS-g-(PAAm-b-PAA) terpolymer was loaded with MET as an anti-diabetic drug, and its drug release behavior was evaluated in the body simulated environment. As results, it was concluded that the fabricated CS-g-(PAAm-b-PAA) nanosystem has high potential as de novo drug delivery system (DDS) for diabetes therapy, mainly due to controlled drug release profile in comparison with conventional formulations of MET. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 48037.     

The Effect of Ozone Gas on IL-1β and IL-10 Levels of Gingival Crevicular Fluid in Aggressive Periodontitis Patients

ABSTRACT

Although the use of ozone therapy in dentistry has become widespread, the number of controlled clinical trials evaluating its effectiveness in periodontal therapy is limited. The aim of this study was to evaluate the efficacy of ozone treatment, that is used in concert with scaling and root planning (SRP), on clinical periodontal parameters and to analyze its effect on cytokine levels of GCF in aggressive periodontitis patients. Totally, 27 patients with aggressive periodontitis were randomly selected into groups of treatment with either subgingival SRP followed by application of ozone with a periodontal probe (SRP+ozone) or subgingival SRP followed by irrigation with serum irrigation (SRP-control). The following parameters were evaluated at baseline (T0), and 6 weeks (T1): plaque index (PI); gingival index (GI); probing pocket depth (PPD), clinical attachment loss (CAL), GCF volume, GCF Interleukin-1β (IL-1β), and GCF Interleukin-10 (IL-10) cytokine levels. There was a significant difference in terms of clinical periodontal parameters before and after treatment in both groups. When comparing between groups, there was no significant difference between the treatment methods after 6 weeks with respect to the PI, PPD, CAL, and GCF IL-10 levels. In contrast, GI, GCF volume, and GCF IL-1β levels were statistically significantly different between the two groups at the 6th week after treatment. Application of ozone as an adjunctive therapy to SRP was shown to provide a statistically significant improvement in treatment results compared to SRP plus serum irrigation.     

Inhibition of Carbonic Anhydrase IX Promotes Apoptosis through Intracellular pH Level Alterations in Cervical Cancer Cells

Carbonic anhydrase IX (CAIX) is a hypoxia-related protein that plays a role in proliferation in solid tumours. However, how CAIX increases proliferation and metastasis in solid tumours is unclear. The objective of this study was to investigate how a synthetic CAIX inhibitor triggers apoptosis in the HeLa cell line. The intracellular effects of CAIX inhibition were determined with AO/EB, AnnexinV-PI, and γ-H2AX staining; measurements of intracellular pH (pHi), reactive oxygen species (ROS), and mitochondrial membrane potential (MMP); and analyses of cell cycle, apoptotic, and autophagic modulator gene expression (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin, and LC3), caspase protein level (pro-caspase 3 and cleaved caspase-3, -8, -9), cleaved PARP activation, and CAIX protein level. Sulphonamide CAIX inhibitor E showed the lowest IC₅₀ and the highest selectivity index in CAIX-positive HeLa cells. CAIX inhibition changed the morphology of HeLa cells and increased the ratio of apoptotic cells, dramatically disturbing the homeostasis of intracellular pHi, MMP and ROS levels. All these phenomena consequent to CA IX inhibition triggered apoptosis and autophagy in HeLa cells. Taken together, these results further endorse the previous findings that CAIX inhibitors represent an important therapeutic strategy, which is worth pursuing in different cancer types, considering that presently only one sulphonamide inhibitor, SLC-0111, has arrived in Phase Ib/II clinical trials as an antitumour/antimetastatic drug.     

Water soluble nickel(0) and cobalt(0) nanoclusters stabilized by poly(4-styrenesulfonic acid-co-maleic acid): Highly active, durable and cost effective catalysts in hydrogen generation from the hydrolysis of ammonia borane

This paper reports the in-situ generation and catalytic activity of nickel(0) and cobalt(0) nanoclusters stabilized by poly(4-styrene sulfonic acid-co-maleic acid), PSSA-co-MA, in the hydrolysis of ammonia borane (AB). PSSA-co-MA stabilized nickel(0) (PSMA-Ni) and cobalt(0) nanoclusters (PSMA-Co) having average particle size of 2.1 ± 0.6 and 5.3 ± 1.6 nm, respectively, were generated by in-situ reduction of nickel(II) chloride or cobalt(II) chloride in an aquoues solution of NaBH₄/H₃NBH₃ in the presence of PSSA-co-MA. The in-situ generated nanoclusters were isolated from the reaction solution and characterized by UV-Vis, TEM, XRD and FT-IR techniques. Compared with the previous catalyst systems, PSMA-Ni and PSMA-Co are found to be highly active catalysts for hydrogen generation from the hydrolysis of AB with the turnover frequency values of 10.1 min⁻¹ for Ni and 25.7 min⁻¹ for Co. They are also very stable during the hydrolysis of AB providing 22450 and 17650 turnovers, respectively. The results of mercury poisoning experiments reveal that PSMA-Ni and PSMA-Co are heterogeneous catalysts in the hydrolysis of AB. Herein, we also report the results of a detailed kinetic study on the hydrogen generation from the hydrolysis of AB catalyzed by PSMA-Ni and PSMA-Co depending on catalyst concentration, substrate concentration, and temperature along with the activation parameters of catalytic hydrolysis of AB calculated from the kinetic data.     

Silica embedded cobalt(0) nanoclusters: Efficient, stable and cost effective catalyst for hydrogen generation from the hydrolysis of ammonia borane

Cobalt(0) nanoclusters embedded in silica (Co@SiO₂) were prepared by a facile two-step procedure. In the first step, the hydrogenphosphate anion (HPO₄²⁻) stabilized cobalt(0) nanoclusters were in situ generated from the reduction of cobalt(II) chloride during the hydrolysis of sodium borohydride (NaBH₄) in the presence of stabilizer. Next, HPO₄²⁻ anion-stabilized cobalt(0) nanoclusters were embedded in silica formed by in situ hydrolysis and condensation of tetraethylorthosilicate added as ethanol solution. Co@SiO₂ can be separated from the solution by vacuum filtration and characterized by UV-Vis electronic absorption spectroscopy, TEM, SEM-EDX, ATR-IR and ICP-OES techniques. Co@SiO₂ are found to be highly active and stable catalysts in the hydrolysis of ammonia borane (AB) even at low cobalt concentration and room temperature. They provide an initial turnover frequency of 13.3 min⁻¹ and 24,400 total turnovers over 52 h in the hydrolysis of AB at 25.0 ± 0.5 °C. Moreover, Co@SiO₂ retain 72% and 74% of the initial activity after ten runs recyclability and five cycles reusability test in the hydrolysis of AB, respectively. The kinetics of hydrogen generation from the hydrolysis of AB catalyzed by Co@SiO₂ was studied depending on the catalyst concentration, substrate concentration, and temperature. The activation parameters of this catalytic reaction were also determined from the evaluation of the kinetic data.     

Canonical inductor simulators with grounded capacitors using DCCII

Differential current conveyor (DCCII) can be defined as a second-generation current conveyor with current differencing capability. In this article, a BJT implementation of the DCCII is introduced. Also, to emphasise the advantage of this active element, two new inductance simulator circuits are proposed as application examples. Furthermore, the presented inductor simulator circuits employ a minimum number of elements and they enjoy having grounded capacitors. Experimental and simulation results are given to verify theoretical analyses.     

Enhanced dynamic range analog filter topologies with a notch/all-pass circuit example

The signal handling capability of the filters is called dynamic range. In this paper, a topological form for the synthesis of filters with high dynamic range is proposed. A biquad notch/all-pass filter is shown in conformity with the given topological form. It is shown that there is a trade-off between dynamic range and high input impedance property. The presented circuit is compared with other notch filters in the literature. It has less number of components, better high-frequency response and dynamic range compared to others. Since the circuit includes a minimum number of resistors, it can easily provide electronically tunable circuits through resistor/controlled current conveyor replacement. Simulations are performed to verify the theoretical results. Routh-Hurwitz stability analyses are also given.     

Broadband impedance matching via lossless unsymmetrical lattice networks

In this paper, a broadband impedance matching network (equalizer) design algorithm has been proposed. In the equalizer, a lossless unsymmetrical lattice network has been utilized. The branch impedances of the lattice network are considered as singly terminated lossless LC networks, since it is not desired to dissipate power in the equalizer. After giving the algorithm, its usage has been illustrated via an example.     

Comment on "Electronically tunable current-mode second-order universal filter using minimum elements"

This comment, relates to a recently published paper by M. Sagbas and K. Fidanboylu (see ibid., vol.40, p.2-4, 2004) which presents a current-mode second-order universal filter for realising lowpass, bandpass and highpass responses. This filter employs two second-generation current controlled conveyors (CCCIIs) and two capacitors. It is shown here that this circuit is not new, simply derived from another filter proposed by S. Ozoguz and C. Acar (see ibid., vol.33, p.948-9, 1997), employing CCCIIs instead of second-generation current conveyors (CCIIs).