Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study.

BACKGROUND: Commercial anti-CD19 chimeric antigen receptor T-cell therapies (CART19) are efficacious against advanced B-cell non-Hodgkin lymphoma (NHL); however, most patients ultimately relapse. Several mechanisms contribute to this failure, including CD19-negative escape and CAR T dysfunction. All four commercial CART19 products utilize the FMC63 single-chain variable fragment (scFv) specific to a CD19 membrane-distal epitope and characterized by slow association (on) and dissociation (off) rates. We hypothesized that a novel anti-CD19 scFv that engages an alternative CD19 membrane-proximal epitope independent of FMC63 and that is characterized by faster on- and off-rates could mitigate CART19 failure and improve clinical efficacy. METHODS: We developed an autologous CART19 product with 4-1BB co-stimulation using a novel humanized chicken antibody (h1218). This antibody is specific to a membrane-proximal CD19 epitope and harbors faster on/off rates compared to FMC63. We tested h1218-CART19 in vitro and in vivo using FMC63-CART19-resistant models. We conducted a first-in-human multi-center phase I clinical trial to test AT101 (clinical-grade h1218-CART19) in patients with relapsed or refractory (r/r) NHL. RESULTS: Preclinically, h1218- but not FMC63-CART19 were able to effectively eradicate lymphomas expressing CD19 point mutations (L174V and R163L) or co-expressing FMC63-CAR19 as found in patients relapsing after FMC63-CART19. Furthermore, h1218-CART19 exhibited enhanced killing of B-cell malignancies in vitro and in vivo compared with FMC63-CART19. Mechanistically, we found that h1218-CART19 had reduced activation-induced cell death (AICD) and enhanced expansion compared to FMC63-CART19 owing to faster on- and off-rates. Based on these preclinical results, we performed a phase I dose-escalation trial, testing three dose levels (DL) of AT101 (the GMP version of h1218) using a 3 + 3 design. In 12 treated patients (7 DLBCL, 3 FL, 1 MCL, and 1 MZL), AT101 showed a promising safety profile with 8.3% grade 3 CRS (n = 1) and 8.3% grade 4 ICANS (n = 1). In the whole cohort, the overall response rate was 91.7%, with a complete response rate of 75.0%, which improved to 100% in DL-2 and -3. AT101 expansion correlates with CR and B-cell aplasia. CONCLUSIONS: We developed a novel, safe, and potent CART19 product that recognizes a membrane-proximal domain of CD19 with fast on- and off-rates and showed significant efficacy and promising safety in patients with relapsed B-cell NHL. TRIAL REGISTRATION: NCT05338931; Date: 2022-04-01.

Increasing use of therapeutic apheresis as a liver-saving modality.

INTRODUCTION: Therapeutic plasma exchange (TPE) is used for temporary support of liver function in patients presenting with early graft dysfunction after liver transplantation (LT) or liver surgery. We analyzed the effect of therapeutic apheresis on patients with liver disease. METHODS: Between January 2011 and August 2016, 93 apheresis procedures were performed for 26 patients at our institution. Anti-ABO isoagglutination immunoglobulin (Ig) M titer was checked using a type A and type B 3% red blood cell (RBC) suspension in saline with two-fold serial dilutions of patient serum. Anti-ABO isoagglutination IgG titer was checked by a type A and B 0.8% RBC suspension using a low-ionic strength/Coombs card. RESULTS: ABO-incompatible (ABOi) LT was the most common (n=10, 38.5%) indication for apheresis; early graft dysfunction after LT (n=8, 30.7%) was the second most common. Median initial IgM and IgG anti-ABO titers for ABOi LT recipients were 1:16 (range, 1:8-1:128) and 1:48 (range, 1:8-1:2048). We performed preoperative TPE in 10 recipients (median number of sessions, 1.5; range, 1-11). Among patients with early graft dysfunction, those who underwent living donor LT had better survival (4/4; 100%) than those who underwent nonliving donor LT (0/3; 0%). Patients who underwent living donor LT first and then additional LT also survived after three TPE sessions. CONCLUSION: Therapeutic apheresis is associated with a good survival rate and is essential for liver support in patients with early graft dysfunction after LT or posthepatectomy liver failure and during preparation for ABOi LT.

Effect of Rubus Occidentalis Extract on Metabolic Parameters in Subjects with Prediabetes: A Proof-of-concept, Randomized, Double-blind, Placebo-controlled Clinical Trial.

Rubus occidentalis (RO) has beneficial effects on glucose and lipid profiles in vitro. The aim of the study was to investigate RO extract effect on metabolic parameters in prediabetic patients, adopting a 12-week, randomized, double-blind, placebo-controlled trial. Forty-four patients (age 59.0 ± 8.2 years, 70.5% females, HbA1c 5.8 ± 0.4%) were divided into placebo (n = 13), low-dose RO extract (LRE; n = 14), or high-dose RO extract (HRE; n = 17) groups. Either 900 or 1800 mg per day of RO extract was administered orally. Area under the curve for glucose obtained 2 h after a 75-g oral glucose tolerance test was significantly decreased in the HRE group, compared with the placebo group (-28.1 ± 42.4 vs. +13.4 ± 52.6 mg/dL, p < 0.05). Homoeostasis model assessment-B was increased (+17.11 ± 10.69, +5.24 ± 4.10, and +0.86 ± 6.01 in HRE, LRE, and placebo, respectively, p < 0.05). Serum levels of monocyte chemoattractant protein-1 and oxidized low-density lipoprotein were significantly decreased by treatment in a dose-dependent manner (monocyte chemoattractant protein-1: -35.0 ± 21.2, +8.4 ± 18.1, and +24.2 ± 14.5; oxidized low-density lipoprotein: -19.7 ± 8.5, -13.1 ± 7.2, and -2.2 ± 11.0 in the HRE, LRE, and placebo, respectively, p < 0.05). The results support the beneficial effects of RO extract on the control of glycemia and vascular inflammation in prediabetic patients. (ClinicalTrials.gov: NCT01964703). Copyright © 2016 John Wiley & Sons, Ltd.

Effects of black raspberry on lipid profiles and vascular endothelial function in patients with metabolic syndrome.

Black raspberry (Rubus occidentalis) has been known for its anti-inflammatory and anti-oxidant effects. However, short-term effects of black raspberry on lipid profiles and vascular endothelial function have not been investigated in patients with metabolic syndrome. Patients with metabolic syndrome (n = 77) were prospectively randomized into a group with black raspberry (n = 39, 750 mg/day) and a placebo group (n = 38) during a 12-week follow-up. Lipid profiles, brachial artery flow-mediated dilatation (baFMD), and inflammatory cytokines such as IL-6, TNF-α, C-reactive protein, adiponectin, sICAM-1, and sVCAM-1 were measured at the baseline and at the 12-week follow-up. Decreases from the baseline in the total cholesterol level (-22.8 ± 30.4 mg/dL vs. -1.9 ± 31.8 mg/dL, p < 0.05, respectively) and total cholesterol/HDL ratio (-0.31 ± 0.64 vs. 0.07 ± 0.58, p < 0.05, respectively) were significantly greater in the group with black raspberry than in the placebo group. Increases in baFMD at the 12-week follow-up were significantly greater in the group with black raspberry than in the placebo group (0.33 ± 0.44 mm vs. 0.10 ± 0.35 mm, p < 0.05, respectively). Decreases from the baseline in IL-6 (-0.4 ± 1.5 pg/mL vs. -0.1 ± 1.0 pg/mL, p < 0.05, respectively) and TNF-α (-2.9 ± 4.7 pg/mL vs. 0.1 ± 3.6 pg/mL, p < 0.05, respectively) were significantly greater in the group with black raspberry. The use of black raspberry significantly decreased serum total cholesterol level and inflammatory cytokines, thereby improving vascular endothelial function in patients with metabolic syndrome during the 12-week follow-up.

Caffeic Acid Enhances Anticancer Drug-induced Apoptosis in Acid-adapted HCT116 Colon Cancer Cells.

BACKGROUND/AIM: Apoptosis resistance in cancer cells adapted to acidic microenvironments poses a challenge for effective treatment. This study investigated the potential use of caffeic acid as an adjunct therapy to overcome drug resistance in colorectal cancer cells under acidic conditions. MATERIALS AND METHODS: Long-term exposure to low-pH conditions induced resistance in HCT116 colorectal cancer cells. The effects of caffeic acid on proliferation, clonogenicity, and apoptosis induction were assessed alone and in combination with oxaliplatin and 5-Fluorouracil. The signaling pathways involved in drug resistance were examined by assessing the activities of PI3K/Akt and ERK1/2. RESULTS: Caffeic acid inhibited the proliferation and clonogenicity of acid-adapted cancer cells, and enhanced apoptosis when combined with anticancer drugs. Mechanistically, caffeic acid attenuated the hyperactivation of the PI3K/Akt and ERK1/2 signaling pathways associated with drug resistance. CONCLUSION: Caffeic acid is a promising therapeutic agent for targeting resistant cancer cells in acidic microenvironments. Its ability to inhibit proliferation, sensitize cells to apoptosis, and modulate signaling pathways highlights its potential for overcoming drug resistance in cancer therapy.

Development of a radial pulse tonometric (RPT) sensor with a temperature compensation mechanism.

Several RPT sensors have been developed to acquire objective and quantitative pulse waves. These sensors offer improved performance with respect to pressure calibration, size and sensor deployment, but not temperature. Since most pressure sensors are sensitive to temperature, various temperature compensation techniques have been developed, but these techniques are largely inapplicable to RPT sensors due to the size restrictions of the sensor, and incompatibility between the compensation techniques and the RPT sensor. Consequently, in this paper a new RPT sensor comprising six piezoresistive pressure sensors and one thermistor has been developed through finite element analysis and then a suitable temperature compensation technique has been proposed. This technique compensates for temperature variations by using the thermistor and simple compensation equations. As verification of the proposed compensation technique, pulse waves of all types were successfully compensated for temperature changes.

Anticancer Effect of Gallic Acid on Acidity-Induced Invasion of MCF7 Breast Cancer Cells.

The acidic tumor environment has emerged as a crucial factor influencing the metastatic potential of cancer. We investigated the effect of an acidic environment on the acquisition of metastatic properties in MCF7 breast cancer cells and explored the inhibitory effects of gallic acid. Prolonged exposure to acidic culture conditions (over 12 weeks at pH 6.4) induced the acquisition of migratory and invasive properties in MCF7 cells, accompanied by increased expression of Matrix Metalloproteinase 2 and 9 (MMP2 and MMP9, respectively), together with alterations in E-cadherin, vimentin, and epithelial-to-mesenchymal transition markers. Gallic acid effectively inhibited the survival of acidity-adapted MCF7 (MCF7-6.4/12w) cells at high concentrations (>30 µM) and reduced metastatic characteristics induced by acidic conditions at low concentration ranges (5-20 µM). Moreover, gallic acid suppressed the PI3K/Akt pathway and the nuclear accumulation of ß-catenin, which were elevated in MCF7-6.4/12w cells. These findings highlight the potential of gallic acid as a promising therapeutic agent for metastatic traits in breast cancer cells under acidic conditions.

Gastrointestinal AA Amyloidosis following Recurrent SARS-CoV-2 Infection: A Case Report.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been associated with the overproduction of serum amyloid A protein, resulting in systemic AA amyloidosis. In this report, we describe a case of gastrointestinal (GI) AA amyloidosis following SARS-CoV-2 infection. A 75-year-old male presented to the emergency department with upper abdominal pain 6 weeks post kidney transplantation. He had a history of SARS-CoV-2 infection 4 weeks prior. On day 7 of hospitalization, while receiving conservative management, the patient developed symptoms of cough and fever, leading to a diagnosis of SARS-CoV-2 reinfection. The patient's abdominal pain persisted, and hematochezia developed on day 30 of hospitalization. Esophagogastroduodenoscopy and colonoscopy revealed multiple ulcers in the stomach and colon, with histologic findings revealing the presence of amyloid A. The patient was managed conservatively and was also given remdesivir for the SARS-CoV-2 infection. His clinical symptoms subsequently improved, and endoscopic findings demonstrated improvement in multiple gastric ulcers. GI amyloidosis may be a subacute complication following SARS-CoV-2 infection in immunocompromised patients.

Allylic C-H activations using Cu(II) 2-quinoxalinol salen and tert-butyl hydroperoxide.

Using a Cu(II) 2-quinoxalinol salen complex as the catalyst and tert-butyl hydroperoxide (TBHP) as the oxidant, allylic activations of olefin substrates can be converted to the corresponding enones or 1,4-enediones. Excellent yields can be achieved (up to 99%) within a very short reaction time and with great tolerance for additional functional groups. Possible mechanistic pathways have been characterized using Raman spectroscopy, cyclic voltammetry, and theoretical calculations.

Redox energetics of hypercloso boron hydrides B(n)H(n) (n = 6-13) and B12X12 (X = F, Cl, OH, and CH3).

The reduction potentials (E°(Red) versus SHE) of hypercloso boron hydrides B(n)H(n) (n = 6-13) and B(12)X(12) (X = F, Cl, OH, and CH(3)) in water have been computed using the Conductor-like Polarizable Continuum Model (CPCM) and the Solvation Model Density (SMD) method for solvation modeling. The B3LYP/aug-cc-pvtz and M06-2X/aug-cc-pvtz as well as G4 level of theory were applied to determine the free energies of the first and second electron attachment (ΔG(E.A.)) to boron clusters. The solvation free energies (ΔG(solv)) greatly depend on the choice of the cavity set (UAKS, Pauling, or SMD) while the dependence on the choice of exchange/correlation functional is modest. The SMD cavity set gives the largest ΔΔG(solv) for B(n)H(n)(0/-) and B(n)H(n)(-/2-) while the UAKS cavity set gives the smallest ΔΔG(solv) value. The E°(Red) of B(n)H(n)(-/2-) (n = 6-12) with the G4/M06-2X(Pauling) (energy/solvation(cavity)) combination agrees within 0.2 V of experimental values. The experimental oxidative stability (E(1/2)) of B(n)X(n)(2-) (X = F, Cl, OH, and CH(3)) is usually located between the values predicted using the B3LYP and M06-2X functionals. The disproportionation free energies (ΔG(dpro)) of 2B(n)H(n)(-) → B(n)H(n) + B(n)H(n)(2-) reveal that the stabilities of B(n)H(n)(-) (n = 6-13) to disproportionation decrease in the order B(8)H(8)(-) > B(9)H(9)(-) > B(11)H(11)(-) > B(10)H(10)(-). The spin densities in B(12)X(12)(-) (X = F, Cl, OH, and CH(3)) tend to delocalize on the boron atoms rather than on the exterior functional groups. The partitioning of ΔG(solv)(B(n)H(n)(2-)) over spheres allows a rationalization of the nonlinear correlation between ΔG(E.A.) and E°(Red) for B(6)H(6)(-/2-), B(11)H(11)(-/2-), and B(13)H(13)(-/2-).

Histone H4 deacetylation down-regulates catalase gene expression in doxorubicin-resistant AML subline.

We explored if epigenetic mechanisms could be involved in the down-regulated expression of catalase gene (CAT) in the doxorubicin-resistant acute myelogenous leukemia (AML)-2/DX100 cells. Down-regulated CAT expression in AML-2/DX100 cells was completely recovered after treatment of hydrogen peroxide (H(2)O(2)) and histone deacetylase inhibitor, trichostatin A (TSA) but was increased slightly by the treatment of DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-AdC). Bisulfite-sequencing PCR revealed that a CpG island of CAT was not methylated in AML-2/DX100 cells. Chromatin immunoprecipitation assay confirmed that acetylation of histone H4 in AML-2/DX100 cells significantly decreased as compared with that in AML-2/WT cells, which was significantly increased by TSA more than 5-AdC. Meanwhile, overexpression of other up-regulated peroxidase genes appears to make compensation for decreased H(2)O(2)-scavenging activity for the down-regulated CAT expression in AML-2/DX100 cells. These results suggest that histone H4 deacetylation is responsible for the down-regulated CAT expression in AML-2/DX100 cells, which are well adapted to oxidative stress.

Molecular Basis of Resveratrol-Induced Resensitization of Acquired Drug-Resistant Cancer Cells.

Multidrug resistance (MDR) to anticancer drugs remains a serious obstacle to the success of cancer chemotherapy. Resveratrol, a polyphenol, present in natural products exerts anticancer activity and acts as a potential MDR inhibitor in various drug-resistant cancer cells. In the process of resensitization of drug-resistant cancer cells, resveratrol has been shown to interfere with ABC transporters and drug-metabolizing enzymes, increase DNA damage, inhibit cell cycle progression, and induce apoptosis and autophagy, as well as prevent the induction of epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs). This review summarizes the mechanisms by which resveratrol counteracts MDR in acquired drug-resistant cancer cell lines and provides a critical basis for understanding the regulation of MDR as well as the development of MDR-inhibiting drugs.

Lipid raft-dependent death receptor 5 (DR5) expression and activation are critical for ursodeoxycholic acid-induced apoptosis in gastric cancer cells.

Ursodeoxycholic acid (UDCA) is known as a suppressor of cholestatic liver diseases and colorectal cancer development. Here, we demonstrate that UDCA induces apoptosis without necrotic features in SNU601, SNU638, SNU1 and SNU216 human gastric cancer cells, implying its possible use as an effective chemotherapeutic agent in treatment of gastric cancer. UDCA-induced apoptosis was dominantly mediated by an extrinsic pathway dependent on caspase-8, -6 and -3. UDCA increased expression of death receptor 5 [(DR5), also known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2], and this DR appeared to be responsible for UDCA-induced apoptosis, as evidenced by DR5 knockdown. UDCA triggered formation of lipid rafts that played crucial roles in UDCA-induced apoptotic actions. Lipid rafts were required not only for provision of a proper site for DR5 action but also for mediation of DR5 expression. In addition, reactive oxygen species (ROS) and protein kinase C (PKC) δ appeared to be implicated in UDCA-induced raft-dependent DR5 expression. Our results indicate that UDCA-induced apoptosis is mediated by DR5 expression, which is regulated by the raft formation/ROS production/PKCδ activation pathway and DR5 localization into lipid rafts in gastric cancer cells. Tumor-suppressive activity of UDCA was confirmed in an in vivo system: UDCA (120 mg/kg/day) significantly decreased tumor growth in gastric cancer xenograft mice. Taken together, our results demonstrate that UDCA can be used as a potent chemotherapeutic agent for treatment of gastric cancer.

Dissolution thermochemistry of alkali metal dianion salts (M2X1, M = Li+, Na+, and K+ with X = CO3(2-), SO4(2-), C8H8(2-), and B12H12(2-)).

The dissolution Gibbs free energies (ΔG°(diss)) of salts (M(2)X(1)) have been calculated by density functional theory (DFT) with Conductor-like Polarizable Continuum Model (CPCM) solvation modeling. The absolute solvation free energies of the alkali metal cations (ΔG(solv)(M(+))) come from the literature, which coincide well with half reduction potential versus SHE data. For solvation free energies of dianions (ΔG(solv)(X(2-))), four different DFT functionals (B3LYP, PBE, BVP86, and M05-2X) were applied with three different sets of atomic radii (UFF, UAKS, and Pauling). Lattice free energies (ΔG(latt)) of salts were determined by three different approaches: (1) volumetric, (2) a cohesive Gibbs free energy (ΔG(coh)) plus gaseous dissociation free energy (ΔG(gas)), and (3) the Born-Haber cycle. The G4 level of theory, electron propagator theory, and stabilization by dielectric medium were used to calculate the second electron affinity to form the dianions CO(3)(2-) and SO(4)(2-). Only the M05-2X/Pauling combination with the three different methods for estimating ΔG(latt) yields the expected negative dissolution free energies (ΔG°(diss)) of M(2)SO(4). Salts with large dianions like M(2)C(8)H(8) and M(2)B(12)H(12) reveal the limitation of using static radii in the volumetric estimation of lattice energies. The value of ΔE(coh) was very dependent on the DFT functional used.

Dependence of pKa on solute cavity for diprotic and triprotic acids.

A systematic study of ΔG(aq)/pK(a) for monoprotic, diprotic, and triprotic acids has been carried out based on DFT/aug-cc-pVTZ combined with CPCM and SMD solvation modeling. All DFT/cavity set combinations considered showed similar accuracy for ΔG(aq)(1)/pK(a1) (70% within ±2.5 kcal mol(-1) of experiment) while only the M05-2X/Pauling cavity combination gave reasonable results for ΔG(aq)(2)/pK(a2) when both pK(a) values are separated by more than three units (70% within ±5.0 kcal mol(-1) of experiment). The choice of experimental data is critical to the interpretation of the calculated accuracy especially for several inorganic acids. For the calculation of ΔG(aq)(3)/pK(a3), the larger experimental uncertainty and an unrealistic orbital population of diffuse function for trianions in the gas phase hinders an evaluation of the predictive performance. We find the M05-2X functional with the Pauling cavity set is the best choice for ΔG(aq)(2)/pK(a2) prediction in aqueous media while all DFT/cavity sets considered were competitive for ΔG(aq)(1)/pK(a1).

Extracellular Acidity-mediated Expression of cPLA2γ Confers Resistance in Gastric Cancer Cells.

BACKGROUND/AIM: Extracellular acidity, a characteristic of solid tumors, has been proposed to be a critical factor for aggravating tumor malignancy and conferring resistance to therapeutics. Recently, acidity has been implicated in inflammatory responses, which are mediated through active lipid metabolites in various human tissues. In the present study, we investigated whether acidity can affect lipid-mediated signaling, and found that phospholipase A2 (PLA2) activity increased at acidic pH in SNU601 and AGS gastric carcinoma cell lines. MATERIALS AND METHODS: To identify the PLA2 isoform that is responsible for the acidity-induced activity, we assessed mRNA levels of cPLA2 isotypes through real-time qPCR, and protein levels through immunoblot assay in cells cultured in acidic medium. RESULTS: It was found that acidic pH conditions markedly elevated the PLA2γ expression. A gene interference study using specific siRNA of cPLA2γ suggested that expression of cPLA2γ in acidic culture conditions may be associated with protection of cancer cells in acidic environment, as shown by cell viability and clonogenic assays. In addition, expression of cPLA2γ appeared to confer cell resistance to anticancer drugs under acidic pH conditions. CONCLUSION: Acidity-induced cPLA2γ expression may exert protective effects by imparting resistance to the gastric cancer cells under acidic environment.

An effective method for allylic oxidation of Delta5-steroids using tert-butyl hydroperoxide.

An allylic oxidation method for Delta(5)-steroids using TBHP as oxidant with a 2-quinoxalinol salen Cu(II) complex as catalyst is reported. A variety of Delta(5)-steroidal substrates are selectively oxidized to the corresponding enones. Excellent yields are achieved (up to 99% under optimized conditions) while significantly reducing reaction times required as compared to other current methods.

Mechanistic study of LiNH(2)BH(3) formation from (LiH)(4) + NH(3)BH(3) and subsequent dehydrogenation.

The formation of LiNH(2)BH(3) from (LiH)(4) and NH(3)BH(3) and the subsequent dehydrogenation have been studied computationally at the CCSD(T)/6-311++G(3d,2p)//MP2/6-311++G(2d,p) level. A cubic unit of (LiH)(4) is predicted to react readily with NH(3)BH(3) to form LiNH(2)BH(3) plus H(2). The (LiH)(4) tetramer enables dehydrogenation through the exchange of a hydride vertex of (LiH)(4) and NH(2)BH(3)(-) where NH(2)BH(3)(-) is formed when the hydride vertex of (LiH)(4) abstracts a proton from NH(3). The free energy of activation for loss of H(2) is reduced from 37.2 kcal/mol in NH(3)BH(3) to 11.0 kcal/mol in (LiH)(4) + NH(3)BH(3). Further, H(2) elimination from the (LiNH(2)BH(3))(2) dimer is predicted to be much easier than from the monomer which may suggest a cooperative H(2)-loss mechanism is possible in solid LiNH(2)BH(3). While two molecules of H(2) can be lost reversibly from (LiNH(2)BH(3))(2), loss of further H(2) molecules is more difficult but could occur if the lattice energy stabilization accompanying H(2) loss is sufficiently large.

Detection of drug transporter expression using a 25-multiplex RT-PCR assay.

Membrane transporters play important roles in mediating chemoresistance and chemosensitivity of tumor cells. Five sets of 5 genes were designed to simultaneously detect drug transporter expression in a single reaction tube using multiplex RT-PCR for 25 genes, including 17 ABC transporters, one non-ABC transporter, 4 SLC transporters, 2 copper transporters and one housekeeping gene. We optimized the multiplex RT-PCR conditions using chemosensitive cancer cells and then validated the system using chemoresistant cancer cells. This reliable multiplex RT-PCR assay can be utilized not only to investigate anticancer drug-resistance mechanisms but also to estimate the efficacy of anticancer chemotherapy in the clinic.

Endohedral hydrogen exchange reactions in C60 (nH2@C60, n = 1-5): comparison of recent methods in a high-pressure cooker.

The interactions of several H(2) molecules [(H(2))(n), n = 1-5] within C(60), C(70), and C(82) have been studied with several DFT methods as well as with MP2 and SCS-MP2. As expected, B3LYP significantly underestimates dispersion interactions, while the M05-2X and M06-2X methods are in much better agreement with MP2 and SCS-MP2 results. Degenerate hydrogen exchange reactions were calculated for 3H(2) --> 3H(2) inside C(60), C(70), and C(82). The free-energy barrier at 298 K for the hydrogen exchange reaction 3H(2) --> 3H(2) is reduced from 88.8 kcal/mol for the free reaction to 36.2 kcal/mol for the reaction within C(60), corresponding to a k(cat)/k(uncat) ratio of 10(36). Steric compression, dispersion, and a favorable entropy contribute similar increments to the reduction in the free-energy barrier.