Understanding the molecular determinants of substrate and inhibitor specificities in the Carbapenemase KPC-2: exploring the roles of Arg220 and Glu276

β-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of β-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A β-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with β-lactams and β-lactamase inhibitors. To better understand the contribution of these residues to the catalytic process, we explored the roles of R220 and E276 in KPC-2, a class A β-lactamase that inactivates carbapenems and β-lactamase inhibitors. Our study reveals that substitutions at R220 of KPC-2 selectively impact catalytic activity toward substrates (50% or greater reduction in k_cat/K_m). In addition, we find that residue 220 is central to the mechanism of β-lactamase inhibition/inactivation. Among the variants tested at Ambler position 220, the R220K enzyme is relatively “inhibitor susceptible” (K_i of 14 ± 1 μM for clavulanic acid versus K_i of 25 ± 2 μM for KPC-2). Specifically, the R220K enzyme is impaired in its ability to hydrolyze clavulanic acid compared to KPC-2. In contrast, the R220M substitution enzyme demonstrates increased K_m values for β-lactamase inhibitors (>100 μM for clavulanic acid versus 25 ± 3 μM for the wild type [WT]), which results in inhibitor resistance. Unlike other class A β-lactamases (i.e., SHV-1 and TEM-1), the amino acid present at residue 276 plays a structural rather than kinetic role with substrates or inhibitors. To rationalize these findings, we constructed molecular models of clavulanic acid docked into the active sites of KPC-2 and the “relatively” clavulanic acid-susceptible R220K variant. These models suggest that a major 3.5-Å shift occurs of residue E276 in the R220K variant toward the active S70 site. We anticipate that this shift alters the shape of the active site and the positions of two key water molecules. Modeling also suggests that residue 276 may assist with the positioning of the substrate and inhibitor in the active site. These biochemical and molecular modeling insights bring us one step closer to understanding important structure-activity relationships that define the catalytic and inhibitor-resistant profile of KPC-2 and can assist the design of novel compounds.     

Enzymatic and Immunological Characterization of a New Cephalosporinase from Enterobacter aerogenes

A hospital strain of Enterobacter aerogenes (MULB 250) isolated from a urinary tract infection was found to be cephalosporin and ampicillin resistant and carbenicillin susceptible. The β-lactamase produced by this strain was extracted and purified by means of affinity chromatography, using a cephalosporin C-bound Sepharose 4B column. The purified enzyme was tested for hydrolysis of penicillin and various cephalosporins. The K_m value is 11.8 μM for benzyl penicillin and 130 μM for cephalosporin C. The isoelectric point of the enzyme is 9.3, and its molecular weight is 29,500 ± 1,000. Rabbit antiserum obtained against this MULB 250 β-lactamase showed no cross-reaction with other penicillinases or cephalosporinases in neutralization tests. Comparisons of results obtained with other β-lactamases, particularly from Enterobacter cloacae P99, indicate that the Enterobacter MULB 250 enzyme presents a typical cephalosporinase profile. As far as we know, this type of enzyme is relatively rare.     

Polished rice as natural sources of cancer-preventing geranylgeranoic acid

Geranylgeranoic acid, a 20-carbon polyprenoic acid (all-trans 3,7,11,15-tetramethyl-2,4,6,10,14-hexadecatetraenoic acid) and its derivatives were previously developed as synthetic “acyclic retinoids” for cancer chemoprevention. Recently, we demonstrated the natural occurrence of geranylgeranoic acid in various medicinal herbs (Shidoji and Ogawa, 2004). In this present study, we present several lines of evidence to demonstrate that geranylgeranyl diphosphate taken in foods could be metabolized to GGA through geranylgeraniol and geranylgeranyl aldehyde via the following steps: 1) The conversion from geranylgeranyl diphosphate to geranylgeraniol was demonstrated to occur by the action of bovine intestinal alkaline phosphatase, with a K_m of 46.1 µM. 2) Geranylgeraniol oxidase-mediated conversion of geranylgeraniol to geranylgeranyl aldehyde was revealed in rat liver homogenates, which activity was mainly localized in the mitochondrial fraction. The mitochondrial enzyme showed a K_m of 92.9 µM. 3) The conversion of geranylgeranyl aldehyde to geranylgeranoic acid by geranylgeranyl aldehyde dehydrogenase in rat liver homogenates was absolutely dependent on exogenously added NAD⁺ or NADP⁺. The K_m of the mitochondrial geranylgeranyl aldehyde dehydrogenase was 27.5 µM for geranylgeranyl aldehyde. Taken together, our data suggest that cancer preventive geranylgeranoic acid could be a physiological metabolite from commonly consumed foods.     

Thermally controlled biotransformation of glycyrrhizic acid via an asymmetric temperature-responsive polyurethane membrane

Separating a target product from a relatively complex bioreaction system is often difficult. In this work, a “smart” bioreaction system was developed by using the special characteristic of temperature-responsive polyurethane (TRPU). By combining solvent evaporation with a wet phase inversion technique, an asymmetric membrane consisting of an integral and dense skin layer supported by a porous sublayer was prepared from a thermally responsive polyurethane that experiences a sudden free volume increase upon heating through a phase transition temperature of 56 °C. Subsequently, the asymmetric TRPU membrane served as the carrier of an immobilized enzyme, wherein β-glucuronidase was multipoint-conjugated by using biotin and streptavidin on the porous sublayer. Then, the material-asymmetric TRPU membrane served jointly as the antennae as well as the actuator, which reversibly responds to temperature to switch (on–off) the access of the reactant glycyrrhizic acid (GL). Under the optimal temperature (40 °C) and pH (7.0) conditions, the immobilized β-glucuronidase contributed to almost 33% yield of glycyrrhetinic acid 3-O-mono-β-d-glucuronide (GAMG) of the isolated counterpart for the same concentration of substrate (250 mg L⁻¹) reaction for 24 h, while costing 1% of that of the isolated β-glucuronidase. Kinetic results showed that V_max and K_m values were 8.89 × 10³ mg L⁻¹ and 2.30 × 10³ mg L⁻¹ h⁻¹, respectively. The specific functional polymer-immobilized β-glucuronidase design serves as a bioreactor of GL into GAMG, as well as a separator deliberately irritated and controlled by temperature. This “smart” support material presents a potential facilitator for the separation of complex biotransformation reactions.

A “smart” bioreaction system was developed by using the special characteristic of a temperature-responsive polyurethane (TRPU). This “smart” support material presents a potential benefit of separation for complex biotransformation reactions.     

Changes in Prandial Glucagon Levels After a 2-Year Treatment With Vildagliptin or Glimepiride in Patients With Type 2 Diabetes Inadequately Controlled With Metformin Monotherapy

OBJECTIVE

To determine if the dipeptidyl peptidase-4 inhibitor vildagliptin more effectively inhibits glucagon levels than the sulfonylurea glimepiride during a meal.

RESEARCH DESIGN AND METHODS

Glucagon responses to a standard meal were measured at baseline and study end point (mean 1.8 years) in a trial evaluating add-on therapy to metformin with 50 mg vildagliptin b.i.d. compared with glimepiride up to 6 mg q.d. in type 2 diabetes (baseline A1C 7.3 ± 0.6%).

RESULTS

A1C and prandial glucose area under the curve (AUC)_{0–2 h} were reduced similarly in both groups, whereas prandial insulin AUC_{0–2 h} increased to a greater extent by glimepiride. Prandial glucagon AUC_{0–2 h} (baseline 66.6 ± 2.3 pmol · h⁻¹ · l⁻¹) decreased by 3.4 ± 1.6 pmol · h⁻¹ · l⁻¹ by vildagliptin (n = 137) and increased by 3.8 ± 1.7 pmol · h⁻¹ · l⁻¹ by glimepiride (n = 121). The between-group difference was 7.3 ± 2.1 pmol · h⁻¹ · l⁻¹ (P < 0.001).

CONCLUSIONS

Vildagliptin therapy but not glimepiride improves postprandial α-cell function, which persists for at least 2 years.Glucagon levels are increased in type 2 diabetes because of impaired glucose-mediated suppression of glucagon secretion resulting in increased hepatic glucose output with subsequent hyperglycemia (1). Improved glycemia by the dipeptidyl peptidase-4 inhibitor, vildagliptin (2), is mediated primarily by improved β- and α-cell sensitivity to glucose (3). As an add-on to metformin, vildagliptin displays equal efficacy as glimepiride, with the added benefits of a much lower risk of hypoglycemia and no weight gain (4). Here we report prandial assessments of glucagon levels and insulin secretion rates after up to ∼2 years of therapy with the two drugs.     

Synthesis of 2-ethoxycarbonylthieno[2,3-b]quinolines in biomass-derived solvent γ-valerolactone and their biological evaluation against protein tyrosine phosphatase 1B

A series of 2-ethoxycarbonylthieno[2,3-b]quinolines were synthesized in the bio-derived “green” solvent γ-valerolactone (GVL) and evaluated for their inhibitory activities against PTP1B, the representative compound 6a displayed an IC₅₀ value of 8.04 ± 0.71 μM with 4.34-fold preference over TCPTP. These results provided novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.

A series of 2-ethoxycarbonylthieno[2,3-b]quinolines were synthesized in the bio-derived “green” solvent γ-valerolactone and evaluated for their inhibitory activities against PTP1B, compound 6a displayed an IC₅₀ value of 8.04 ± 0.71 μM with 4.34-fold preference over TCPTP.     

Prevalence of Type 1 Diabetes Autoantibodies (GADA, IA2, and IAA) in Overweight and Obese Children

OBJECTIVE

Little is known about the prevalence of β-cell autoantibodies in children with excess body weight. The prevalence of type 1 diabetes autoantibodies and its relation with hyperglycemia was analyzed in 686 overweight/obese children and adolescents.

RESEARCH DESIGN AND METHODS

All children underwent an oral glucose tolerance test, and anti-GAD, anti-IA2, and anti-IAA autoantibodies were measured. Autoantibody prevalence was evaluated in 107 normal-weight children for comparison.

RESULTS

A single autoantibody was present in 2.18% of overweight/obese subjects and 1.86% normal-weight subjects (P = NS). Postload glycemia was significantly higher in antibody-positive children (133 ± 69.9 vs. 105.4 ± 17.7 mg/dl, P < 0.0001) compared with autoantibody-negative subjects. No difference in autoantibody distribution was seen when our cohort was stratified by age, sex, SDS-BMI, pubertal stage, and homeostasis model assessment–insulin resistance (HOMA-IR).

CONCLUSIONS

The 2.18% prevalence of type 1 diabetes autoantibodies is similar to that reported in nonobese children. This study provided evidence that excess body weight and insulin resistance do not influence autoantibody frequency.Over the last 60 years, a striking increase in the incidence of childhood type 1 diabetes has been observed consistently in almost all populations. EURODIAB (1) reported an overall increase of 3.2% per annum in Europe between 1989 and 1998. There have also been considerable changes in childhood nutrition, which have resulted in changes in growth. Increased weight, height, and BMI in children have all been associated with a higher risk of type 1 diabetes (2). The so-called “accelerator hypothesis” argues that obesity causing overworked β-cells underlies both type 1 and type 2 diabetes and that these “types” are only distinguished by how the body responds to this growth-induced β-cell stress. This hypothesis therefore attributes the rise in type 1 diabetes to an increase in child obesity (3). A variation of the hypothesis suggests that, once initiated, islet autoimmunity progresses more rapidly in the context of “overload” of the β-cells due to increased insulin resistance (4).Sardinia has one of the highest incidences of type 1 diabetes worldwide, second only to Finland (5). Moreover, Sardinian children and adolescents are experiencing the same increase in obesity as other European populations (6). To date, little is known on the prevalence of autoantibodies against β-cells in children with excess body weight.The aim of our study was to analyze the prevalence of type 1 diabetes autoantibodies in a cohort of Sardinian overweight/obese children and adolescents and to evaluate their distribution in relation to the presence of glucose abnormalities.     

Sustained weight loss following 12-month pramlintide treatment as an adjunct to lifestyle intervention in obesity

OBJECTIVE—To assess long-term weight loss efficacy and safety of pramlintide used at different dosing regimens and in conjunction with lifestyle intervention (LSI).RESEARCH DESIGN AND METHODS—In a 4-month, double-blind, placebo-controlled, dose-ranging study, 411 obese subjects were randomized to receive pramlintide (six arms: 120, 240, and 360 μg b.i.d. and t.i.d.) or placebo in conjunction with a structured LSI program geared toward weight loss. Of the 4-month evaluable subjects (n = 270), 77% opted to continue preexisting treatment during an 8-month single-blind extension (LSI geared toward weight maintenance).RESULTS—At month 4, mean weight loss from baseline in the pramlintide arms ranged from 3.8 ± 0.7 to 6.1 ± 0.8 kg (2.8 ± 0.8 kg with placebo). By month 12, initial 4-month weight loss was regained in the placebo group but was maintained in all but the 120-μg b.i.d. group. Placebo-corrected weight loss with 120 μg t.i.d. and 360 μg b.i.d. averaged 3.2 ± 1.2 kg (3.1 ± 1.1% body wt) and 3.3 ± 1.1 kg (3.1 ± 1.0% body wt), respectively, at month 4 (both P < 0.01; 4-month evaluable n = 270) and 6.1 ± 2.1 kg (5.6 ± 2.1% body wt) and 7.2 ± 2.3 kg (6.8 ± 2.3% body wt), respectively, at month 12 (both P < 0.01; 12-month evaluable n = 146). At month 12, 40 and 43% of subjects treated with 120 μg t.i.d. and 360 μg b.i.d., respectively, achieved ≥10% weight loss (vs. 12% for placebo). Nausea, the most common adverse event with pramlintide in the 4-month study (9–29% pramlintide vs. 2% placebo), was generally mild to moderate and occurred in <10% of subjects during the extension.CONCLUSIONS—When used over 12 months as an adjunct to LSI, pramlintide treatment, with low-dose three-times-daily or higher-dose two-times-daily regimens, helped obese subjects achieve greater initial weight loss and enhanced long-term maintenance of weight loss.To date, efforts to develop obesity pharmacotherapies aimed at reducing food intake and body weight have largely focused on small-molecule anorectics, an approach that has repeatedly been hampered by safety concerns (1). Peptide hormones originating from pancreas and gut regulate meal size and body weight by acting as short-term (episodic) signals (2). In contrast to small molecules, peptide hormones do not readily diffuse the blood-brain barrier to penetrate the entire central nervous system. Moreover, they act by enhancing signaling through specific, naturally occurring pathways that regulate food intake as opposed to acting more generally on multiple neuronal processes, for example, by altering synaptic concentrations of neurotransmitters. Based on these characteristics, peptide hormone therapeutics are potential alternatives to centrally-acting small-molecule anorectics.Amylin, a 37–amino acid β-cell hormone cosecreted with insulin in response to meals, reduces food intake and body weight in rodents and may fulfill the criteria for a peripheral satiation hormone (3–6). Pramlintide, a synthetic analog of human amylin, has been extensively studied as an antihyperglycemic treatment and is currently under investigation as a potential treatment for obesity.In two studies in obese subjects, pramlintide (120 μg single doses or 180 μg t.i.d. before meals for 6-weeks) reduced ad libitum food intake (7,8). Compared with placebo, pramlintide significantly reduced 24-h caloric intake (by ∼500–750 kcal) and caloric intake at a highly palatable fast-food challenge (by ∼20%) and improved control of eating, evidenced by a 45% reduction in binge-eating score (8).Pramlintide''s weight effects in obese subjects were initially assessed in a 16-week, randomized, double-blind, placebo-controlled, nonforced dose-escalation study. In this study, in which 88% of subjects escalated to the maximum dose (240 μg t.i.d.), pramlintide induced a placebo-corrected reduction in weight of 3.7% (P < 0.001), with 31% of pramlintide-treated subjects achieving ≥5% weight loss (versus 2% for placebo; P < 0.001) (9). Although these findings established a solid proof of concept for the antiobesity potential of pramlintide, the study was limited to 4 months and did not employ lifestyle intervention (LSI), and subjects were not randomly assigned to different pramlintide doses or dose frequencies.To evaluate the weight loss efficacy and safety of pramlintide across a range of doses, across different dose frequencies, in conjunction with LSI, and over 1 year, we conducted a 4-month dose-ranging study (main study) evaluating six pramlintide arms (120, 240, and 360 μg b.i.d. and t.i.d.) in conjunction with lifestyle intervention (LSI) and then implemented an 8-month single-blind extension protocol in which subjects continued their preassigned treatment.     

Self-radiolysis of tritiated water. 4. The scavenging effect of azide ions (N3−) on the molecular hydrogen yield in the radiolysis of water by 60Co γ-rays and tritium β-particles at room temperature

The effect of the azide ion N₃⁻ on the yield of molecular hydrogen in water irradiated with ⁶⁰Co γ-rays (∼1 MeV Compton electrons) and tritium β-electrons (mean electron energy of ∼7.8 keV) at 25 °C is investigated using Monte Carlo track chemistry simulations in conjunction with available experimental data. N₃⁻ is shown to interfere with the formation of H₂ through its high reactivity towards hydrogen atoms and, but to a lesser extent, hydrated electrons, the two major radiolytic precursors of the H₂ yield in the diffusing radiation tracks. Chemical changes are observed in the H₂ scavengeability depending on the particular type of radiation considered. These changes can readily be explained on the basis of differences in the initial spatial distribution of primary radiolytic species (i.e., the structure of the electron tracks). In the “short-track” geometry of the higher “linear energy transfer” (LET) tritium β-electrons (mean LET ∼5.9 eV nm⁻¹), radicals are formed locally in much higher initial concentration than in the isolated “spurs” of the energetic Compton electrons (LET ∼0.3 eV nm⁻¹) generated by the cobalt-60 γ-rays. As a result, the short-track geometry favors radical–radical reactions involving hydrated electrons and hydrogen atoms, leading to a clear increase in the yield of H₂ for tritium β-electrons compared to ⁶⁰Co γ-rays. These changes in the scavengeability of H₂ in passing from tritium β-radiolysis to γ-radiolysis are in good agreement with experimental data, lending strong support to the picture of tritium β-radiolysis mainly driven by the chemical action of short tracks of high local LET. At high N₃⁻ concentrations (>1 M), our H₂ yield results for ⁶⁰Co γ-radiolysis are also consistent with previous Monte Carlo simulations that suggested the necessity of including the capture of the precursors to the hydrated electrons (i.e., the short-lived “dry” electrons prior to hydration) by N₃⁻. These processes tend to reduce significantly the yields of H₂, as is observed experimentally. However, this dry electron scavenging at high azide concentrations is not seen in the higher-LET ³H β-radiolysis, leading us to conclude that the increased amount of intra-track chemistry intervening at early time under these conditions favors the recombination of these electrons with their parent water cations at the expense of their scavenging by N₃⁻.

The effect of the azide ion on the yield of molecular hydrogen in water irradiated with ⁶⁰Co γ-rays and tritium β-electrons at 25 °C is investigated using Monte Carlo track chemistry simulations.     

RNA Aptamer Blockade of Osteopontin Inhibits Growth and Metastasis of MDA-MB231 Breast Cancer Cells

Osteopontin (OPN) is a secreted phosphoprotein which mediates tumorigenesis, local growth, and metastasis in a variety of cancers. It is a potential therapeutic target for the regulation of cancer metastasis. RNA aptamer technology targeting OPN may represent a clinically viable therapy. In this study, we characterize the critical sequence of an RNA aptamer, termed OPN-R3, directed against human OPN. It has a K_d of 18 nmol/l and binds specifically to human OPN as determined by RNA electrophoretic mobility assays. In MDA-MB231 human breast cancer cells examined under fluorescence microscopy, OPN-R3 ablates cell surface binding of OPN to its cell surface CD44 and α_vβ₃ integrin receptors. Critical enzymatic components of the OPN signal transduction pathways, PI3K, JNK1/2, Src and Akt, and mediators of extracellular matrix degradation, matrix metalloproteinase 2 (MMP2) and uroplasminogen activator (uPA), are significantly decreased following exposure to OPN-R3. OPN-R3 inhibits MDA-MB231 in vitro adhesion, migration, and invasion characteristics by 60, 50, and 65%, respectively. In an in vivo xenograft model of breast cancer, OPN-R3 significantly decreases local progression and distant metastases. On the basis of this “proof-of-concept” study, we conclude that RNA aptamer targeting of OPN has biologically relevance for modifying tumor growth and metastasis.     

Supramolecular interaction of sanguinarine dye with sulfobutylether-β-cyclodextrin: modulation of the photophysical properties and antibacterial activity

The noncovalent host–guest interaction of sanguinarine (SGR), a benzophenanthridine alkaloid, with a nontoxic, water soluble sulfobutylether-β-cyclodextrin (SBE₇βCD, commercially available as Captisol) macrocyclic host has been investigated using ground-state optical absorption, and steady-state and time-resolved fluorescence measurements. The pH-dependent changes in the absorbance of the dye at 327 nm showed a pK_a value of 7.5, which has been shifted to 8.1 in the presence of SBE₇βCD. The changes in the pK_a values, absorption and fluorescence spectra, and fluorescence lifetime values of these two forms of SG with SBE₇βCD indicate complex formation between them. The cationic form shows 3 times higher interaction towards SEB₇βCD (K = 1.2 × 10⁴ M⁻¹) as compared to the neutral form (K = 3.9 × 10³ M⁻¹) which leads to a moderate upward pK_a shift (pK_a values of SGR shifted by more than 0.6 units). The subsequent fluorescence “turn off” was demonstrated to be responsive to chemical stimuli, such as metal ions (Ca²⁺ ions). Upon addition of Ca²⁺ ions, nearly quantitative dissociation of the complex was established to regenerate the free dye and result in fluorescence “turn on”. Apart from improving the stability under ambient light conditions, the upward pK_a shift of SGR in the presence of SBE₇βCD results in increasing the antibacterial activity of the SBE₇βCD:SGR complex compared to that of the free dye towards four pathogenic micro-organisms at the physiological pH range. This work further compares SGR interaction with parent β-cyclodextrin.

The noncovalent host-guest interaction of sanguinarine (SGR) with a nontoxic, water soluble sulfobutylether-beta-cyclodextrin macrocyclic host modulates the photophysical properties, improves the photostability and antibacterial activity of SGR.     

Correction: The synthesis of calcium arsenate@iron arsenate coating materials and their application for arsenic-containing wastewater treatment

Correction for ‘The synthesis of calcium arsenate@iron arsenate coating materials and their application for arsenic-containing wastewater treatment’ by Yang Wang et al., RSC Adv., 2020, 10, 719–723.

The authors regret that there were errors in the Characterization of samples section of the original article, in Section 3.1 on page 720 of the original article. These errors are detailed below.The sentence beginning, “As is known, the main peaks of scorodite (FeAsO₄·2H₂O) are located at approximately 30.1°, 27.8°, 16.9° and 10.9°…” should read, “As is known, the main peaks of CaHAsO₄ are located at approximately 30.1°, 27.8°, 16.9° and 10.9°, which can be indexed as the (221), (220), (101) and (020) lattice planes of CaHAsO₄ (JCPDS no. 18-0288), respectively.”The sentence beginning, “As is known, the main peaks of scorodite located at approximately 30.8° and 31.7°…” should read, “As is known, the main peaks of Ca₃(AsO₄)₂ located at approximately 30.8° and 31.7°, can be indexed as the (020) and (021) lattice planes of Ca₃(AsO₄)₂ (JCPDS no. 01-0933).”The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.     

Arming Cytokine-induced Killer Cells With Chimeric Antigen Receptors: CD28 Outperforms Combined CD28–OX40 “Super-stimulation”

Cytokine-induced killer (CIK) cells raised interest for use in cellular antitumor therapy due to their capability to recognize and destroy autologous tumor cells in a HLA-independent fashion. The antitumor attack of CIK cells, predominantly consisting of terminally differentiated CD8⁺CD56⁺ cells, can be improved by redirecting by a chimeric antigen receptor (CAR) that recognizes the tumor cell and triggers CIK cell activation. The requirements for CIK cell activation were, however, so far less explored and are likely to be different from those of “younger” T cells. We revealed that CD28 and OX40 CARs produced higher interferon- secretion as compared with the first-generation ζ-CAR; CD28-ζ and the third-generation CD28-ζ–OX40 CAR, however, performed similar in modulating most CIK cell effector functions. Compared with the CD28-ζ CAR, however, the CD28-ζ–OX40 CAR accelerated terminal maturation of CD56⁺ CIK cells producing high frequencies in activation-induced cell death (AICD) and reduced antitumor efficiency in vivo. Consequently, CD28-ζ CAR CIK cells of CD56⁻ phenotype were superior in redirected tumor cell elimination. CAR-mediated CIK cell activation also increased antigen-independent target cell lysis; the CD28-ζ CAR was more efficient than the CD28-ζ–OX40 CAR. Translated into therapeutic strategies, CAR-redirected CIK cells benefit from CD28 costimulation; “super-costimulation” by the CD28-ζ–OX40 CAR, however, performed less in antitumor efficacy due to increased AICD.     

Nonstoichiometric oxygen in Mn–Ga–O spinels: reduction features of the oxides and their catalytic activity

The subject of this study was the content of oxygen in mixed oxides with the spinel structure Mn_1.7Ga_1.3O₄ that were synthesized by coprecipitation and thermal treatment in argon at 600–1200 °C. The study revealed the presence of excess oxygen in “low-temperature” oxides synthesized at 600–800 °C. The occurrence of superstoichiometric oxygen in the structure of Mn_1.7Ga_1.3O_4+δ oxide indicates the formation of cationic vacancies, which shows up as a decreased lattice parameter in comparison with “high-temperature” oxides synthesized at 1000–1200 °C; the additional negative charge is compensated by an increased content of Mn³⁺ cations according to XPS. The low-temperature oxides containing excess oxygen show a higher catalytic activity in CO oxidation as compared to the high-temperature oxides, the reaction temperature was 275 °C. For oxides prepared at 600 and 800 °C, catalytic activity was 0.0278 and 0.0048 cm³ (CO) per g per s, and further increase in synthesis temperature leads to a drop in activity to zero. The process of oxygen loss by Mn_1.7Ga_1.3O_4+δ was studied in detail by TPR, in situ XRD and XPS. It was found that the hydrogen reduction of Mn_1.7Ga_1.3O_4+δ proceeds in two steps. In the first step, excess oxygen is removed, Mn_1.7Ga_1.3O_4+δ → Mn_1.7Ga_1.3O₄. In the second step, Mn³⁺ cations are reduced to Mn²⁺ in the spinel structure with a release of manganese oxide as a single crystal phase, Mn_1.7Ga_1.3O₄ → Mn₂Ga₁O₄ + MnO.

The hydrogen reduction of Mn_1.7Ga_1.3O_4+δ proceeds in two steps. In the first step, excess oxygen is removed, Mn_1.7Ga_1.3O_4+δ → Mn_1.7Ga_1.3O₄. In the second step, Mn³⁺ cations are reduced to Mn²⁺ in the spinel structure and formation of MnO, Mn_1.7Ga_1.3O₄ → Mn₂Ga₁O₄ + MnO.     

Limb length discrepancy after total knee arthroplasty: A systematic review and meta-analysis

BACKGROUNDLimb length discrepancy (LLD) after total knee arthroplasty (TKA) has been considered as one of the reasons for the unsatisfactory outcome. However, there is no consensus about the extent of LLD that can be considered as clinically relevant.AIMTo evaluate the incidence of radiographic LLD and its impact on functional outcome following TKA.METHODSAll randomized-controlled trial and observational studies on LLD in TKA, published till 22^nd June 2020, were systematically searched and reviewed. The primary outcome was “limb lengthening or LLD after TKA”. The secondary outcomes included “assessment of LLD in varus/valgus deformity” and “impact of LLD on the functional outcome”. RESULTSOf 45 retrieved studies, qualitative and quantitative assessment of data was performed from eight studies and six studies, respectively. Five studies (n = 1551) reported the average limb lengthening of 5.98 mm. The LLD after TKA was ranging from 0.4 ± 10 mm to 15.3 ± 2.88 mm. The incidence of postoperative radiographic LLD was reported in 44% to 83.3% of patients. There was no difference in the preoperative and postoperative LLD (MD -1.23; 95%CI: -3.72, 1.27; P = 0.34). Pooled data of two studies (n = 219) revealed significant limb lengthening in valgus deformity than varus (MD -2.69; 95%CI: -5.11, 0.27; P = 0.03). The pooled data of three studies (n = 611) showed significantly worse functional outcome in patients with LLD of ≥ 10 mm compared to < 10 mm (standard MD 0.58; 95%CI: 0.06, 1.10; P = 0.03). CONCLUSIONLimb lengthening after TKA is common, and it is significantly more in valgus than varus deformity. Significant LLD (≥ 10 mm) is associated with suboptimal functional outcome.     

Structure of CARB-4 and AER-1 CarbenicillinHydrolyzing β-Lactamases

We determined the nucleotide sequences of bla_CARB-4 encoding CARB-4 and deduced a polypeptide of 288 amino acids. The gene was characterized as a variant of group 2c carbenicillin-hydrolyzing β-lactamases such as PSE-4, PSE-1, and CARB-3. The level of DNA homology between the bla genes for these β-lactamases varied from 98.7 to 99.9%, while that between these genes and bla_CARB-4 encoding CARB-4 was 86.3%. The bla_CARB-4 gene was acquired from some other source because it has a G+C content of 39.1%, compared to a G+C content of 67% for typical Pseudomonas aeruginosa genes. DNA sequencing revealed that bla_AER-1 shared 60.8% DNA identity with bla_PSE-3 encoding PSE-3. The deduced AER-1 β-lactamase peptide was compared to class A, B, C, and D enzymes and had 57.6% identity with PSE-3, including an STHK tetrad at the active site. For CARB-4 and AER-1, conserved canonical amino acid boxes typical of class A β-lactamases were identified in a multiple alignment. Analysis of the DNA sequences flanking bla_CARB-4 and bla_AER-1 confirmed the importance of gene cassettes acquired via integrons in bla gene distribution.Penicilloyl serine transferases, routinely called β-lactamases, cleave the cyclic amide bond of β-lactam antibiotics via the formation of a serine ester-linked penicilloyl enzyme giving a product devoid of antibacterial activity (46). A close inspection of databases indicated that in the last 3 years, a collection of at least 150 DNA sequences from plasmid-mediated and chromosomal bla genes has been acquired. Analysis of deduced peptides confirmed that most have conserved motifs typical of serine active-site enzymes that are divided into three major classes (classes A, C, and D) on the basis of a level of amino acid sequence identity of more than 20% between members in each class (10).In 1969, a β-lactamase was found in Pseudomonas aeruginosa Dalgleish, it was noticed to be “markedly active against carbenicillin,” and the enzyme was named PSE-4 (13, 32). As other β-lactamase enzymes were found, it was noticed that some β-lactamases have better activities than others against carbenicillin. All β-lactamases except class C enzymes hydrolyze carbenicillin at very different levels; class C enzymes hydrolyse it poorly. Genes encoding enzymes similar to PSE-4 were subsequently discovered in other bacterial species and are now known to be part of multidrug resistance transposons (24). In addition to the four original β-lactamases called PSE-1, PSE-2, PSE-3, and PSE-4, a plethora of plasmid-mediated enzymes capable of hydrolyzing carbenicillin at a high rate, such as LCR-1 (10), AER-1 (16), CARB-3 (22), NPS-1 (26), CARB-5 (35), and CARB-4 (36), were identified; but these enzymes have subtle differences in their biochemical properties and in their substrate profiles (7). The amino acid sequences of PSE-1 (17), PSE-2 (18), PSE-3 (8), PSE-4 (4), and CARB-3 (23) have been compared to those of other class A and class D enzymes, and it has been confirmed that PSE-2 (OXA-10) is a class D enzyme (10).The relationship of CARB-4 to other plasmid-mediated β-lactamases has been tested by determining the neutralization of benzylpenicillin-hydrolyzing activity with antisera prepared against purified the TEM-1, OXA-4, and CARB-3 β-lactamases (36). Antisera prepared with CARB-3 antigen inactivated the CARB-4 β-lactamase as well as the PSE-1, PSE-4, and CARB-3 enzymes (22, 36). The bla_CARB-3 and bla_CARB-4 genes are localized within transposons Tn1413 (7 kb) and Tn1408 (25 kb), respectively; these mobile elements were from plasmids isolated from bacterial strains of distinct origins (24, 27, 47).Unusual β-lactamases such as a metalloenzyme have been reported in Aeromonas hydrophila, a water-borne, gram-negative rod known to be highly resistant to β-lactam antibiotics, including carbenicillin (42). A carbenicillin-hydrolyzing β-lactamase has been discovered in an isolate of A. hydrophila from blood (16). The substrate profile of the AER-1 enzyme resembled those of plasmid-mediated carbenicillin-hydrolyzing enzymes, but it had a different isoelectric point (pI 5.9) and molecular mass (29 kDa); these values are reminiscent of those for BRO-1 (pI 5.45), PSE-1 (pI 5.7), CARB-3 (pI 5.75), and CARB-5 (pI 5.35). The gene coding for AER-1 is part of the Ω7711 unit which is IncP mobilizable but RecA dependent and which inserts only between purC and guaB at a specific site in the Escherichia coli chromosome (16). The Ω7711 unit cotransfers resistance to the antibiotics chloramphenicol, streptomycin, and sulfonamide; the transfer of multidrug resistance and insertion at a unique site are properties analogous to those of Tn7.In the study described in this report, we have focused on a carbenicillin-hydrolyzing enzyme identified from a clinical isolate, P. aeruginosa p83372, containing the pUD12 plasmid and producing CARB-4. This enzyme has an acidic isoelectric point (pI 4.3) and hydrolyzes carbenicillin very efficiently (36). We also present the nucleotide sequences of bla_AER-1 and bla_CARB-4, including flanking sequences containing integrons that explain their distribution and presence in different mobile genetic elements (15). We compared the deduced AER-1 and CARB-4 polypeptides with those of other group 2c enzymes (7) via a multiple alignment.     

Genetically Engineered Human Islets Protected From CD8-mediated Autoimmune Destruction In Vivo

Islet transplantation is a promising therapy for type 1 diabetes, but graft function and survival are compromised by recurrent islet autoimmunity. Immunoprotection of islets will be required to improve clinical outcome. We engineered human β cells to express herpesvirus-encoded immune-evasion proteins, “immunevasins.” The capacity of immunevasins to protect β cells from autoreactive T-cell killing was evaluated in vitro and in vivo in humanized mice. Lentiviral vectors were used for efficient genetic modification of primary human β cells without impairing their function. Using a novel β-cell–specific reporter gene assay, we show that autoreactive cytotoxic CD8⁺ T-cell clones isolated from patients with recent onset diabetes selectively destroyed human β cells, and that coexpression of the human cytomegalovirus-encoded US2 protein and serine proteinase inhibitor 9 offers highly efficient protection in vitro. Moreover, coimplantation of these genetically modified pseudoislets with β-cell–specific cytotoxic T cells into immunodeficient mice achieves preserved human insulin production and C-peptide secretion. Collectively, our data provide proof of concept that human β cells can be efficiently genetically modified to provide protection from killing mediated by autoreactive T cells and retain their function in vitro and in vivo.     

The daily response for proton pump inhibitor treatment in Japanese reflux esophagitis and non-erosive reflux disease

We investigated comparison according to reflux esophagitis and non-erosive reflux disease about “daily” symptom improvement for proton pump inhibitor treatment. We enrolled 57 reflux esophagitis and 90 non-erosive reflux disease patients. They took rabeprazole 10 mg/day for 28 days and completed “daily” in the Frequency Scale for the Symptoms of GERD from baseline until day 14, and after 28 days of treatment. The efficacy endpoint was the improvement rates in Frequency Scale for the Symptoms of GERD, based on baseline. Frequency Scale for the Symptoms of GERD was decreased in reflux esophagitis and non-erosive reflux disease (p<0.001) and was significantly lower in reflux esophagitis than in non-erosive reflux disease from the first day of treatment (p<0.05). Symptomatic improvement rates were also significantly higher in reflux esophagitis (50.3 ± 44.9%) than in non-erosive reflux disease (31.7 ± 43.2%) from the first day of treatment (p<0.0001). The symptomatic improvement rates in reflux esophagitis were significant increased from the second day of treatment until after 28 days of treatment (p = 0.0006), however, these in non-erosive reflux disease were significant increased from third days until after 28 days of treatment (p = 0.0002). In non-erosive reflux disease, the improvement of dysmotility symptom was particularly gradual as well as of reflux symptom, too. As for results of prediction of proton pump inhibitor response (completed symptom resolution) form early symptom improvement within 1 week, it was able to predict proton pump inhibitor response from the symptom improvement rate on 3 days in reflux esophagitis and on day 7 in non-erosive reflux disease. In conclusion, the prediction of the proton pump inhibitor response in non-erosive reflux disease was slow in comparison with reflux esophagitis. The cause was gradual improvement of dysmotility symptom.