Aldo-keto reductase inhibitors increase the anticancer effects of tyrosine kinase inhibitors in chronic myelogenous leukemia

Tyrosine kinase inhibitors (TKIs) are widely utilized in clinical practice to treat carcinomas, but secondary tumor resistance during chronic treatment can be problematic. AKR1B1 and AKR1B10 of the aldo-keto reductase (AKR) superfamily are highly expressed in cancer cells and are believed to be involved in drug resistance. The aim of this study was to understand how TKI treatment of chronic myelogenous leukemia (CML) cells changes their glucose metabolism and if inhibition of AKRs can sensitize CML cells to TKIs. K562 cells were treated with the TKIs imatinib, nilotinib, or bosutinib, and the effects on glucose metabolism, cell death, glutathione levels, and AKR levels were assessed. To assess glucose dependence, cells were cultured in normal and low-glucose media. Pretreatment with AKR inhibitors, including epalrestat, were used to determine AKR-dependence. Treatment with TKIs increased intracellular glucose, AKR1B1/10 levels, glutathione oxidation, and nuclear translocation of nuclear factor erythroid 2-related factor 2, but with minimal cell death. These effects were dependent on intracellular glucose accumulation. Pretreatment with epalrestat, or a selective inhibitor of AKR1B10, exacerbated TKI-induced cell death, suggesting that especially AKR1B10 was involved in protection against TKIs. Thus, by disrupting cell protective mechanisms, AKR inhibitors may render CML more susceptible to TKI treatments

Mice lacking nucleotide sugar transporter SLC35A3 exhibit lethal chondrodysplasia with vertebral anomalies and impaired glycosaminoglycan biosynthesis.

SLC35A3 is considered an uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter in mammals and regulates the branching of N-glycans. A missense mutation in SLC35A3 causes complex vertebral malformation (CVM) in cattle. However, the biological functions of SLC35A3 have not been fully clarified. To address these issues, we have established Slc35a3-/-mice using CRISPR/Cas9 genome editing system. The generated mutant mice were perinatal lethal and exhibited chondrodysplasia recapitulating CVM-like vertebral anomalies. During embryogenesis, Slc35a3 mRNA was expressed in the presomitic mesoderm of wild-type mice, suggesting that SLC35A3 transports UDP-GlcNAc used for the sugar modification that is essential for somite formation. In the growth plate cartilage of Slc35a3-/-embryos, extracellular space was drastically reduced, and many flat proliferative chondrocytes were reshaped. Proliferation, apoptosis and differentiation were not affected in the chondrocytes of Slc35a3-/-mice, suggesting that the chondrodysplasia phenotypes were mainly caused by the abnormal extracellular matrix quality. Because these histological abnormalities were similar to those observed in several mutant mice accompanying the impaired glycosaminoglycan (GAG) biosynthesis, GAG levels were measured in the spine and limbs of Slc35a3-/-mice using disaccharide composition analysis. Compared with control mice, the amounts of heparan sulfate, keratan sulfate, and chondroitin sulfate/dermatan sulfate, were significantly decreased in Slc35a3-/-mice. These findings suggest that SLC35A3 regulates GAG biosynthesis and the chondrodysplasia phenotypes were partially caused by the decreased GAG synthesis. Hence, Slc35a3-/- mice would be a useful model for investigating the in vivo roles of SLC35A3 and the pathological mechanisms of SLC35A3-associated diseases

Mice lacking nucleotide sugar transporter SLC35A3 exhibit lethal chondrodysplasia with vertebral anomalies and impaired glycosaminoglycan biosynthesis

SLC35A3 is considered an uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter in mammals and regulates the branching of N-glycans. A missense mutation in SLC35A3 causes complex vertebral malformation (CVM) in cattle. However, the biological functions of SLC35A3 have not been fully clarified. To address these issues, we have established Slc35a3–/–mice using CRISPR/Cas9 genome editing system. The generated mutant mice were perinatal lethal and exhibited chondrodysplasia recapitulating CVM-like vertebral anomalies. During embryogenesis, Slc35a3 mRNA was expressed in the presomitic mesoderm of wild-type mice, suggesting that SLC35A3 transports UDP-GlcNAc used for the sugar modification that is essential for somite formation. In the growth plate cartilage of Slc35a3–/–embryos, extracellular space was drastically reduced, and many flat proliferative chondrocytes were reshaped. Proliferation, apoptosis and differentiation were not affected in the chondrocytes of Slc35a3–/–mice, suggesting that the chondrodysplasia phenotypes were mainly caused by the abnormal extracellular matrix quality. Because these histological abnormalities were similar to those observed in several mutant mice accompanying the impaired glycosaminoglycan (GAG) biosynthesis, GAG levels were measured in the spine and limbs of Slc35a3–/–mice using disaccharide composition analysis. Compared with control mice, the amounts of heparan sulfate, keratan sulfate, and chondroitin sulfate/dermatan sulfate, were significantly decreased in Slc35a3–/–mice. These findings suggest that SLC35A3 regulates GAG biosynthesis and the chondrodysplasia phenotypes were partially caused by the decreased GAG synthesis. Hence, Slc35a3−/− mice would be a useful model for investigating the in vivo roles of SLC35A3 and the pathological mechanisms of SLC35A3-associated diseases

Sustained pediatric antimicrobial stewardship program with consultation to infectious diseases reduced carbapenem resistance and infection-related mortality

Objective: The impact of pediatric antimicrobial stewardship programs (ASP) on antimicrobial resistance (AMR) remains largely unknown. This study aimed to evaluate the AMR for carbapenem of Gram-negative bacilli (GNB) and carbapenem use with infectious diseases consultation after the implementation of an ASP. Methods: This quasi-experimental study was conducted at Tokyo Metropolitan Childrenâs Medical Center in Japan. The pre- and post-intervention periods were April 2010 to September 2011 and October 2011 to March 2017, respectively. The pre-intervention phase consisted of consultations with the infectious diseases service alone. The ASP was implemented during the post-intervention phase. The carbapenem resistance rates of GNB were calculated. The correlation between carbapenem resistance rates and carbapenem day of therapy (DOT) was examined. The outcome metrics were compared by average length of hospitalization, all-cause mortality, and infection-related mortality. Results: A positive correlation was observed between the carbapenem resistance rate in Pseudomonas aeruginosa and DOT (0.76, p = 0.04). The carbapenem resistance rate in P. aeruginosa (p < 0.01) and DOT (p < 0.01) decreased significantly in the post-intervention period. The length of hospitalization (p < 0.01) and infection-related mortality (p = 0.05) decreased in the post-intervention period. Conclusions: A sustained ASP with additional consultation with the infectious disease service reduced carbapenem use and resistance in P. aeruginosa, leading to favorable outcomes in terms of length of hospitalization and infection-related mortality. Keywords: Antimicrobial stewardship program, Antimicrobial resistance, Children, Pseudomonas aeruginosa, Carbapenem, Day of therap

Proliferation, apoptosis, and differentiation are not affected in <i>Slc35a3</i>^{<i>−/−</i>} chondrocytes.

(A) BrdU staining of the tibia sections from the BrdU-treated embryos at E18.5. RC, round chondrocyte zone; FC, flat chondrocyte zone. (B) Frequency of BrdU-positive cells in the RC and FC zones. (C) TUNEL staining of the hypertrophic chondrocyte zone in the tibia sections at E18.5. (D) Frequency of TUNEL-positive cells in the hypertrophic chondrocyte zone. (E) HE staining and in situ hybridization (ISH) images, produced using Col2a1 and Col10a1 probes of the tibia sections at E18.5. (F) Heights of Col2a1- and Col10a1-expressing zones (upper graphs), and the relative heights of Col2a1- and Col10a1-expressing zones normalized by the total heights of the growth plate (lower graphs) in the ISH images. Data are shown as the means ± standard deviation; n = 6 (B, D) and 4 (F). Combined values from three (B, D) and two (F) independent sections were compared. Two separate areas were counted in one section (B, D, F). P values were calculated using Student’s t-test. Ctrl, control (wild-type + Slc35a1+/−); N.S., not significant. Scale bars, 200 μm (A, E) and 100 μm (C).</p

S1 Raw images -

SLC35A3 is considered an uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter in mammals and regulates the branching of N-glycans. A missense mutation in SLC35A3 causes complex vertebral malformation (CVM) in cattle. However, the biological functions of SLC35A3 have not been fully clarified. To address these issues, we have established Slc35a3–/–mice using CRISPR/Cas9 genome editing system. The generated mutant mice were perinatal lethal and exhibited chondrodysplasia recapitulating CVM-like vertebral anomalies. During embryogenesis, Slc35a3 mRNA was expressed in the presomitic mesoderm of wild-type mice, suggesting that SLC35A3 transports UDP-GlcNAc used for the sugar modification that is essential for somite formation. In the growth plate cartilage of Slc35a3–/–embryos, extracellular space was drastically reduced, and many flat proliferative chondrocytes were reshaped. Proliferation, apoptosis and differentiation were not affected in the chondrocytes of Slc35a3–/–mice, suggesting that the chondrodysplasia phenotypes were mainly caused by the abnormal extracellular matrix quality. Because these histological abnormalities were similar to those observed in several mutant mice accompanying the impaired glycosaminoglycan (GAG) biosynthesis, GAG levels were measured in the spine and limbs of Slc35a3–/–mice using disaccharide composition analysis. Compared with control mice, the amounts of heparan sulfate, keratan sulfate, and chondroitin sulfate/dermatan sulfate, were significantly decreased in Slc35a3–/–mice. These findings suggest that SLC35A3 regulates GAG biosynthesis and the chondrodysplasia phenotypes were partially caused by the decreased GAG synthesis. Hence, Slc35a3−/− mice would be a useful model for investigating the in vivo roles of SLC35A3 and the pathological mechanisms of SLC35A3-associated diseases.</div

Disaccharide composition of keratan sulfate (KS) disaccharides in the spine and limb.

Disaccharide composition of keratan sulfate (KS) disaccharides in the spine and limb.</p

HPLC profiles of the chondroitinase digests of GAG-peptide preparations from the spine and limbs.

The 2-aminobenzamide (2AB)-derivatives of the yielded CS/DS and HA disaccharides after digestion with a mixture of chondroitinases ABC and AC-II, were separated using anion-exchange HPLC on an amine-bound silica PA-G column with a linear gradient of NaH2PO4 as indicated by the dashed line for the analysis of CS/DS and HA. HPLC profiles of the spine (A, B) and limb (C, D) samples of control (A, C) and Slc35a3−/− mice (B and D). The elution positions of 2AB-labeled CS/DS disaccharide standards are indicated by numbered arrows: 1, ΔHexUA-GalNAc; 2, ΔHexUA-GalNAc(6S); 3, ΔHexUA-GalNAc(4S); 4, ΔHexUA(2S)-GalNAc(6S); 5, ΔHexUA(2S)-GalNAc(4S); 6, ΔHexUA-GalNAc(4S,6S); 7, ΔHexUA(2S)-GalNAc(4S,6S). Asterisk indicates the ΔHexUA-GlcNAc derived from HA. Abbreviations: CS, chondroitin sulfate; DS, dermatan sulfate; HA, hyaluronan, ΔHexUA, 4,5-unsaturated hexuronic acid; GalNAc, N-acetyl-D-galactosamine; GlcNAc, N-acetyl-D-glucosamine; 2S, 2-O-sulfate; 4S, 4-O-sulfate; 6S, 6-O-sulfate. (TIF)</p

<i>Slc35a3</i> mRNA was expressed in the presomitic mesoderm during somite formation.

Whole-mount in situ hybridization images of wild-type mouse embryos at E9.25 (somite 20–22) produced using the antisense (A, B) and the sense (C) probes against Slc35a3 mRNA. Lateral side view (A), and the magnified images of the tail region (B, C). NE: neuroepithelium, PSM: presomatic mesoderm. Scale bars: 500 μm. The same results were obtained from two independent experiments.</p

HPLC profiles of the heparinase digests of GAG-peptide preparations from the spine and limbs.

The 2-aminobenzamide (2AB)-derivatives of the yielded HS disaccharides after digestion with a mixture of heparinase-I, heparitinase-II, and heparinase-III, were separated using anion-exchange HPLC on an amine-bound silica PA-G column with a linear gradient of NaH2PO4 as indicated by the dashed line for the analysis of HS. HPLC profiles of the spine (A, B) and limb (C, D) samples of control (A, C) and Slc35a3−/− mice (B, D). The elution positions of 2AB-labeled HS disaccharide standards are indicated by numbered arrows: 1, ΔHexUA-GlcNAc; 2, ΔHexUA-GlcNAc(6S); 3, ΔHexUA-GlcN(NS); 4, ΔHexUA-GlcN(NS,6S); 5, ΔHexUA(2S)-GlcN(NS); 6, ΔHexUA(2S)-GlcN(NS,6S). Abbreviations: HS, heparan sulfate; ΔHexUA, 4,5-unsaturated hexuronic acid; GlcNAc, N-acetyl-D-glucosamine; GlcN, D-glucosamine; 2S, 2-O-sulfate; 4S, 4-O-sulfate; 6S, 6-O-sulfate; NS, 2-N-sulfate. (TIF)</p