An integrated microfluidics platform with high-throughput single-cell cloning array and concentration gradient generator for efficient cancer drug effect screening.

BACKGROUND: Tumor cell heterogeneity mediated drug resistance has been recognized as the stumbling block of cancer treatment. Elucidating the cytotoxicity of anticancer drugs at single-cell level in a high-throughput way is thus of great value for developing precision therapy. However, current techniques suffer from limitations in dynamically characterizing the responses of thousands of single cells or cell clones presented to multiple drug conditions. METHODS: We developed a new microfluidics-based "SMART" platform that is Simple to operate, able to generate a Massive single-cell array and Multiplex drug concentrations, capable of keeping cells Alive, Retainable and Trackable in the microchambers. These features are achieved by integrating a Microfluidic chamber Array (4320 units) and a six-Concentration gradient generator (MAC), which enables highly efficient analysis of leukemia drug effects on single cells and cell clones in a high-throughput way. RESULTS: A simple procedure produces 6 on-chip drug gradients to treat more than 3000 single cells or single-cell derived clones and thus allows an efficient and precise analysis of cell heterogeneity. The statistic results reveal that Imatinib (Ima) and Resveratrol (Res) combination treatment on single cells or clones is much more efficient than Ima or Res single drug treatment, indicated by the markedly reduced half maximal inhibitory concentration (IC50). Additionally, single-cell derived clones demonstrate a higher IC50 in each drug treatment compared to single cells. Moreover, primary cells isolated from two leukemia patients are also found with apparent heterogeneity upon drug treatment on MAC. CONCLUSION: This microfluidics-based "SMART" platform allows high-throughput single-cell capture and culture, dynamic drug-gradient treatment and cell response monitoring, which represents a new approach to efficiently investigate anticancer drug effects and should benefit drug discovery for leukemia and other cancers.

Lys63-linked ubiquitin chain adopts multiple conformational states for specific target recognition.

A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition.

Visualizing an ultra-weak protein-protein interaction in phosphorylation signaling.

Proteins interact with each other to fulfill their functions. The importance of weak protein-protein interactions has been increasingly recognized. However, owing to technical difficulties, ultra-weak interactions remain to be characterized. Phosphorylation can take place via a K(D)≈25 mM interaction between two bacterial enzymes. Using paramagnetic NMR spectroscopy and with the introduction of a novel Gd(III)-based probe, we determined the structure of the resulting complex to atomic resolution. The structure accounts for the mechanism of phosphoryl transfer between the two enzymes and demonstrates the physical basis for their ultra-weak interaction. Further, molecular dynamics (MD) simulations suggest that the complex has a lifetime in the micro- to millisecond regimen. Hence such interaction is termed a fleeting interaction. From mathematical modeling, we propose that an ultra-weak fleeting interaction enables rapid flux of phosphoryl signal, providing a high effective protein concentration.

Probing dynamics and mechanism of exchange process of quaternary ammonium dimeric surfactants, 14-s-14, in the presence of conventional surfactants.

In this Article, we investigated effects of different types of conventional surfactants on exchange dynamics of quaternary ammonium dimeric surfactants, with chemical formula C(14)H(29)N(+)(CH(3))(2)- (CH(2))(s)-N(+)(CH(3))(2)C(14)H(29)·2Br(-), or 14-s-14 for short. Two nonionic surfactants, TritonX-100 (TX-100) and polyethylene glycol (23) laurylether (Brij-35), and one cationic surfactant, n-tetradecyltrimethyl ammonium bromide (TTAB), and one ionic surfactant, sodium dodecyl sulfate (SDS) were chosen as typical conventional surfactants. Exchange rates of 14-s-14 (s = 2, 3, and 4) between the micelle form and monomer in solution were detected by two NMR methods: one-dimensional (1D) line shape analysis and two-dimensional (2D) exchange spectroscopy (EXSY). Results show that the nonionic surfactants (TX-100 and Brij-35), the cationic surfactant (TTAB), and the ionic surfactant (SDS) respectively accelerated, barely influenced, and slowed the exchange rate of 14-s-14. The effect mechanism was investigated by the self-diffusion experiment, relaxation time measurements (T(2)/T(1)), the fluorescence experiment (I(1)/I(3)) and observed chemical shift variations. Results reveal that, nonionic conventional surfactants (TX-100 and Brij-35) loosened the molecule arrangement and decreased hydrophobic interactions in the micelle, and thus accelerated the exchange rate of 14-s-14. The cationic conventional surfactant (TTAB) barely changed the molecule arrangement and thus barely influenced the exchange rate of 14-s-14. The ionic conventional surfactant (SDS) introduced the electrostatic attraction effect, tightened the molecule arrangement, and increased hydrophobic interactions in the micelle, and thus slowed down the exchange rate of 14-s-14. Additionally, the two-step exchange mechanism of 14-s-14 in the mixed solution was revealed through interesting variation tendencies of exchange rates of 14-s-14.

Structural and functional diversity of acidic scorpion potassium channel toxins.

BACKGROUND: Although the basic scorpion K(+) channel toxins (KTxs) are well-known pharmacological tools and potential drug candidates, characterization the acidic KTxs still has the great significance for their potential selectivity towards different K(+) channel subtypes. Unfortunately, research on the acidic KTxs has been ignored for several years and progressed slowly. PRINCIPAL FINDINGS: Here, we describe the identification of nine new acidic KTxs by cDNA cloning and bioinformatic analyses. Seven of these toxins belong to three new α-KTx subfamilies (α-KTx28, α-KTx29, and α-KTx30), and two are new members of the known κ-KTx2 subfamily. ImKTx104 containing three disulfide bridges, the first member of the α-KTx28 subfamily, has a low sequence homology with other known KTxs, and its NMR structure suggests ImKTx104 adopts a modified cystine-stabilized α-helix-loop-ß-sheet (CS-α/ß) fold motif that has no apparent α-helixs and ß-sheets, but still stabilized by three disulfide bridges. These newly described acidic KTxs exhibit differential pharmacological effects on potassium channels. Acidic scorpion toxin ImKTx104 was the first peptide inhibitor found to affect KCNQ1 channel, which is insensitive to the basic KTxs and is strongly associated with human cardiac abnormalities. ImKTx104 selectively inhibited KCNQ1 channel with a K(d) of 11.69 µM, but was less effective against the basic KTxs-sensitive potassium channels. In addition to the ImKTx104 toxin, HeTx204 peptide, containing a cystine-stabilized α-helix-loop-helix (CS-α/α) fold scaffold motif, blocked both Kv1.3 and KCNQ1 channels. StKTx23 toxin, with a cystine-stabilized α-helix-loop-ß-sheet (CS-α/ß) fold motif, could inhibit Kv1.3 channel, but not the KCNQ1 channel. CONCLUSIONS/SIGNIFICANCE: These findings characterize the structural and functional diversity of acidic KTxs, and could accelerate the development and clinical use of acidic KTxs as pharmacological tools and potential drugs.

NMR study of the dynamics of cationic gemini surfactant 14-2-14 in mixed solutions with conventional surfactants.

Three kinds of conventional surfactants, namely, two nonionic surfactants [polyethylene glycol (23) lauryl ether (Brij-35) and Triton X-100 (TX-100)], one cationic surfactant [n-tetradecyltrimethyl ammonium bromide (TTAB)], and an anionic surfactant [sodium n-dodecyl sulfate (SDS)}, were mixed into the quaternary ammonium gemini surfactant [C(14)H(29)N(+)(CH(3))(2)](2)(CH(2))(2).2Br(-) (14-2-14) in aqueous solution. The exchange rate constants between 14-2-14 molecules in the mixed micelles and those in the bulk solution were detected using two nuclear magnetic resonance (NMR) methods: one-dimensional (1D) line shape analysis and two-dimensional (2D) exchange spectroscopy (EXSY). The results obtained from these two methods were consistent. Both showed that mixing a nonionic conventional surfactant, either Brij-35 or TX-100, enhanced the exchange process between the 14-2-14 molecules in the mixed micelles and those in the bulk solution. In contrast, the anionic surfactant SDS and the cationic surfactant TTAB slowed the process slightly.

Different residues in channel turret determining the selectivity of ADWX-1 inhibitor peptide between Kv1.1 and Kv1.3 channels.

The low selectivity of Kv1 peptide inhibitors for specific isoforms makes them poor candidates for the development of theraputics. Using combined approaches, we showed that the Kv1 turret is the critical determinant for ADWX-1 peptide inhibitor selectivity of Kv1.3 over Kv1.1. Mutation of Kv1.1 turret residues to match the sequence of Kv1.3 lead to increased inhibition of Kv1.1 activity. These studies may lead to improvements in peptide inhibitor drug development.

1H NMR study on pre-micellization of quaternary ammonium gemini surfactants.

Two quaternary ammonium Gemini surfactant series, 12-s-12, ([C(12)H(25)N+ (CH(3))(2)](2)(CH(2))(s).(2)Br(-)) and 14-s-14 ([C(14)H(29)N(+)(CH(3))(2)](2)(CH(2))(s).(2)Br(-)), where s = 2, 3, and 4, have been studied by the use of (1)H NMR in aqueous solution at concentrations below their critical micelle concentrations (CMC) at 25 degrees C. The appearance of a second set of peaks for the 14-s-14 series and the changes in chemical shifts, line widths, and line shapes of the 12-s-12 series with increasing concentration below the CMC are interpreted as evidence for the formation of premicelle aggregates (oligomers) that appear at approximately one-half their CMC values. Self-diffusion coefficients (D) and transverse relaxation times (T(2)) have also been detected and support the results obtained by (1)H NMR.

Polyaspartamide gadolinium complexes containing sulfadiazine groups as potential macromolecular MRI contrast agents.

Both diethylenetriaminepentaacetic acid (DTPA) and sulfadiazine (SD) were incorporated into polyaspartamides with different side chains, including poly-alpha,beta-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA), poly-alpha,beta-[N- (3-hydroxypropyl)-L-aspartamide] (PHPA), poly-alpha,beta-[N-(2-aminoethy1)-L-aspartamide] (PAEA), poly-alpha,beta-[N-(4-aminobuty1)-L-aspartamide] (PABA), and poly-alpha,beta-[N-(6-aminohexyl)-L-aspartamide] (PAHA). The polyaspartamide ligands containing DTPA and SD groups were further reacted with gadolinium chloride to give the corresponding macromolecular gadolinium complexes. Experimental data of 1H NMR, IR, UV, and elemental analysis exhibited the formation of the polyaspartamide ligands and gadolinium complexes. Relaxivity studies indicated that the macromolecular chelates possess higher relaxivities than that of the clinically used Gd-DTPA. MR imaging showed that the macromolecular chelate PAEA-Gd-DTPA-SD greatly enhanced the contrast of MR images of hepatoma in the lower limb of mice and provided prolonged intravascular duration. Thus the polyaspartamide gadolinium complex containing SD groups is expected to be used as the potential macromolecular MRI contrast agents for hepatoma in mice.

Synthesis and evaluation of gadolinium complexes based on PAMAM as MRI contrast agents.

Diethylenetriaminepentaacetic acid (DTPA) and pyridoxamine (PM) were incorporated into the amine groups on the surface of ammonia-core poly(amidoamine) dendrimers (PAMAM, Generation 2.0-5.0) to obtain dendritic ligands. These dendritic ligands were reacted with gadolinium chloride to yield the corresponding dendritic gadolinium (Gd) complexes. The dendritic ligands and their gadolinium complexes were characterized by(1)HNMR, IR, UV and elemental analysis. Relaxivity studies showed that the dendritic gadolinium complexes possessed higher relaxation effectiveness compared with the clinically used Gd-DTPA. After administration of the dendritic gadolinium complexes (0.09 mmol kg(-1) ) to rats, magnetic resonance imaging of the liver indicated that the dendritic gadolinium complexes containing pyridoxamine groups enhanced the contrast of the MR images of the liver, provided prolonged intravascular duration and produced highly contrasted visualization of blood vessels.

Correlation of behavior changes and BOLD signal in Alzheimer-like rat model.

To explore a potential means for the early diagnosis of Alzheimer disease, we studied the relationship of resting T2* signal and tau hyperphosphorylation/spatial memory deficit. The rat model with tau hyperphosphorylation and spatial memory deficit was established by bilateral hippocampi injection of isoproterenol (IP). Then, the correlative alteration between resting T2* signal and spatial memory retention was assessed with blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) study and Morris Water Maze test, and Western blot was employed to confirm tau hyperphosphorylation. The analysis showed following results. (1) Tau phosphorylation at Ser396/Ser404 and Ser199/Ser202 was significantly increased in IP-injected rats as detected by PHF-1 and tau-1, respectively. (2) An AD-like spatial memory retention disturbance was induced at 24 h after isoproterenol injection. (3) A sensitivity threshold of resting T2* signal intensity, which separated the IP-treated rats from vehicle control, was obtained by applying linear regression analysis, and an estimated sensitivity statistical threshold was at 32.62. These results suggest that resting T2* signal may serve as a noninvasive quantitative marker in predicting AD-like spatial memory deficits and tau hyperphosphorylation.

[Propagation of brain injuries from artificial focus into the opposite hemisphere at the early stage of rat electrogenic epilepsy identified by histology and magnetic resonance image].

The purpose of this work was to study the characteristics of rat brain abnormalities at two hemispheres at the early stage of electrogenic epilepsy. Experiments were performed on 37 male Sprague-Dawley rats. Chronically repetitive tetanization (60 Hz, 2 s, 0.4 - 0.6 mA) was used to stimulate the right dorsal hippocampus (DHPC) of the rat brain once a day for 2, 4, 6, 8 or 10 d, respectively. The T(2) weighted magnetic resonance image (T(2)-WI) were obtained from each experimental rat at the end of the experiments. Histological sections were obtained after experimentation. The results showed that the main pathologic changes at the early stage of epilepsy included: (1) T(2)-WI hyperintensification, the histological enlargement of lateral ventricle (LV) and pathological hyperplasia of ventricular choroidea plexus occurred. The pathological hyperplasia was symmetric in two hemispheres, but the LV enlargement was not. (2) Histologically enlarged LV area showed a resemblance to T(2)-WI hyperintensive area. Compared with the control rats, large T(2)-WI hyperintensive area (P=0.0259; P=0.0184; P=0.0184; P=0.0404; P=0.0259) and histologically enlarged LV area (P=0.0210; P=0.01; P=0.0100; P=0.0152) were present in chronically tetanized rats. (3) Dynamic characteristics of histologically enlarged LV area resembled to those of T(2)-WI hyperintensity area in chronically tetanized rats at different stimulating day. Lateralization of T(2)-WI hyperintensity was in accordance with that of T(2)-WI abnormal area and of histologically enlarged LV. These abnormalities were severe on the contralateral side on the stimulating day 6, or on the ipsilateral side on the stimulating day 10. These results imply characteristic propagation of brain abnormalities crossing to the opposite hemisphere at the early stage of an electrogenic rat epilepsy.

[Effects of Angelica sinensis injection on the neuronal metabolites and blood flow speed within reperfusion following the ischemic cerebral injury in rats].

AIM: To investigate the effects of Angelica sinensis injection on the neuronal metabolites and blood flow speed within reperfusion in the ischemic cerebral injury of rats. METHODS: Sixty-nine male Sprague Dawley rats with an average body weight of 150 to 170 g were used, and were randomly divided into three groups: sham operation group (n = 4), ischemia injury group (n = 30) underwent an operation of ischemic brain injury, Angelica-treated group (n = 35) underwent the same operation and received the treatment of Angelica sinensis injection (5 g/kg bw, i. p). The right middle cerebral artery occlusion (MCAO) of both ischemia injury group and Angelica-treated group rats was induced by 5/0 nylon suture for 2 hours. The reperfusion was conducted for three to four hours and five to six hours respectively following MCAO. T2 weighted-imaging (T2WI) and 1H magnetic resonance spectroscopy (1H MRS) were performed, to study the changes in imaging and neuronal metabolites N-acetyl aspartate (NAA), creatine/phosphocreatine (Cr/ PCr) and choline (Cho) following cerebral ischemia. The changes in blood flow speed were measured by laser Doppler flowmetry. The surface vascular density in right hemisphere were calculated. RESULTS: The hyperintense signals and volume in the right cerebrum in Angelica-treated group decreased compared with those of the ischemia injury group, the T2 values were decreased, and the level of NAA increased, the ratio of Cr/NAA and Cho/NAA decreased. The blood flow speed in Angelica-treated group was improved. The length of brain surface vessels in group C increased. CONCLUSION: The Angelica sinensis injection enhanced the blood circulation in the ischemic brain, improved the neuronal metabolisms.

[The effects of Erigeron breviscapus preparation on the imaging and neuronal metabolites at the early time points after reperfusion following the ischemic cerebral injury in rats].

OBJECTIVE: To investigate the effects of Erigeron breviscapus preparation on the imaging and neuronal metabolites after reperfusion in the ischemic cerebral injury in rats. METHOD: Twenty-three male Sprague Dawley rats with an average body weight of (165 +/- 15) g (mean +/- S) were used, and were randomly divided into two groups: group A rats (n = 11) underwent an operation of ischemic brain injury, group B rats (n = 12) underwent the same operation and received the treatment of Erigeron breviscapus preparation (1.5 mg.kg-1 weight, i.p.). The right middle cerebral artery occlusion (MCAO) of rats in both groups was induced by 5/0 nylon suture for 2 hours. The reperfusion was conducted for four hours and six hours respectively following MCAO. T2 weighted-imaging (T2WI) and 1H-magnetic resonance spectroscopy (1H-MRS) were performed, to study the changes of the imaging and the neuronal metabolites N-acetyl aspartate (NAA), creatine/phosphocreatine (Cr/PCr), choline (Cho) and lactose (Lac) in cerebrum following cerebral ischemia. RESULT: The hyperintense signals in the right cerebrum in group B decreased compared with those in group A, the T2 values decreased, the level of NAA increased, the ratio of Cr/NAA and Cho/NAA decreased, and no lactose was observed. The brain surface vessels of rats in group B were in the state of dilation. CONCLUSION: Erigeron breviscapus preparation is beneficial to the reestablishment of the blood circulation in the ischemic brain, and to the improvement of the neuronal metabolism and survival.

A study on human urine in a high-selenium area of China by 1H-NMR spectroscopy.

In this study, high-resolution 600-MHz 1H-NMR (nuclear magnetic resonance) spectroscopies were used to compare the urinary metabolic profiles of healthy humans and humans in a high-selenium area of China. NMR biomarkers for renal and liver lesions were observed by comparing the urine 1H-NMR spectra. In urinary excretion, the concentrations in human urine samples of formate, lactate, acetate, hippurate, and alanine in overexposure to selenium were increased, whereas citrate, creatine, and TMAO excretion were decreased compared with that of the healthy human--some of them even disappeared. An interesting result was the appearance of formate in urine, which has previously been shown to lead to acidosis and chronic renal failure and interfere with the lumen and proximal tubular cells. The level of creatine was associated with the seminal activity. The changes of acetate and citrate may explain the disorder of the cellular energy metabolism caused by selenium, and the changes of other amino acids were a result of the reuptake of these compounds that had been blocked in the glomerulus and proximal tubule. The results elucidate the renal/liver lesion in humans in high-selenium area by 1H-NMR spectroscopy and offer the molecular basic of selenium toxicity.

Detection of an unkown peak at 1.2 ppm in proton magnetic resonance spectra of cats with cerebral ischemic necrosis.

OBJECTIVE: To confirm an unknown peak (Pu) in proton magnetic resonance spectra ((1)H-MRS) in cats with permanent focal cerebral ischemia, and surmise its potential value of application. METHODS: After focal cerebral ischemia, diffusion-weighted imaging (DWI) was used to identify regions of ischemia for (1)H-MRS voxel localization with the assistance of stereotaxic atlas of cat brain. (1)H-MRS was used to monitor the progression of focal cerebral ischemia in 6 cats over a period of 7 d following middle cerebral artery occlusion (MCAO). The changes in lactate, N-acetyl-aspartate (NAA), choline, creatine, and Pu were observed. RESULTS: In the involved regions, lactate was elevated almost immediately after the onset of cerebral ischemia, and NAA declined within several hours of acute infarction. The Pu at 1.2 ppm was persistently detected in the affected cerebral areas 2 to 7 d after MCAO. CONCLUSION: Pu has a close relationship with cerebral ischemia necrosis and may be a intrinsic production of the necrotic tissue, which can be utilized as a specific marker and therefore has important clinical diagnostic value.

NMR study on the low-affinity interaction of human serum albumin with diclofenac sodium.

The low-affinity interaction between human serum albumin (HSA) and Diclofenac sodium (DCF) was studied using NMR techniques. Both 13C-NMR chemical shift and linewidth show that the dichlorophenyl ring in DCF molecule plays a primary role in its interaction with HSA. Langmuir adsorption isotherm was applied to evaluate the association constant K and the number of binding sites n of the drug/HSA complex through (1)H-NMR spin-lattice relaxation measurement. The results indicate that Langmuir isotherm can perfectly explain the capacity of low-affinity binding of proteins for the ligands.

[Development and progression in rat brain abnormalities related to early stage of epilepsy measured by magnetic resonance image].

The purpose of the present study was to investigate the features of pathophysiological neural networks in rat temporal lobe epileptogenesis. To establish electrogenic epilepsy model, repetitive tetanization (60 Hz, 2 s, 0.4-0.6 mA) was delivered into the right dorsal hippocampus (HPC) of rat brain. Rats were divided into different groups. Experimental animals received tetanic stimulation once a day for 2, 4, 6, 8 or 10 days, respectively. Primary wet dog shakes (WEDS) of the animals were recorded daily during the stimulation to understand the development of behavioral seizures at early stage of epilepsy. The T(2)-weighted (T(2)-WI) spin-echo images were obtained from each experimental rat. The results demonstrated that T(2)-WI hyperintensity of experimental rats was observed in bilateral symmetric dorsal lateral ventricle (LV) areas at stimulating day 2 (n=4), in contralateral medial and ventral LV areas to the side of the electrode at stimulating day 6 (n=5), in contralateral ventral LV areas at stimulating day 8 (n=3), and in ipsilateral ventral LV areas at stimulating day 10 (n=4). Therefore the peak rate of primary WEDS appeared on stimulating day 4 in the experimental rats. Morphological identification demonstrated that the T(2)-WI signal abnormalities were related to the enlarged LV and pathological ventricular choroidea plexus hyperplasia. The results suggest that the development of rat brain abnormalities from dorsal LV to ventral LV at early stage of epilepsy can be measured by magnetic resonance image, which implies reorganization of pathophysiologically functional networks before kindling effect appear.