A competing risk predictive model for kidney failure in patients with advanced chronic kidney disease.

BACKGROUND/PURPOSE: Predictive modeling aids in identifying patients at high risk of adverse events. Using routinely collected data, we report a competing risk prediction model for kidney failure. METHODS: A total of 5138 patients with CKD stages 3b-5 were included and randomized into the development and validation cohorts at a ratio of 7:3. The outcome was end-stage kidney disease, defined as the initiation of dialysis or kidney transplantation. All patients were followed-up until December 31, 2020. A Fine and Gray model was applied to estimate the sub-hazard ratio of kidney failure, with death as a competing event. RESULTS: In the development cohort, the mean age was 67.6 ± 13.9 years and 60 % were male. The mean index eGFR and median urinary protein-creatinine ratio (UPCR) were 26.5 ± 12.8 mL/min/1.73 m2 and 1051 mg/g, respectively. The median follow-up duration was 1051 days. The proportion of patients with kidney failure and death was 25.4 % and 14.1 %, respectively. Four models were applied, including eGFR, age, sex, UPCR, systolic and diastolic blood pressure, serum albumin, phosphate, uric acid, haemoglobin, and potassium levels had the best goodness of fit. All models had good discrimination with time-to-event c statistics of 0.89-0.95 in the development cohort and 0.86-0.95 in the validation cohort. The prediction models showed excellent and fairly good calibration at 2 and 5-year risk, respectively. CONCLUSION: Using real-world data, our competing risk model can accurately predict progression to kidney failure over 2 years in patients with advanced CKD.

CART Peptides Differently Regulate Firing Rates and GABAergic Synaptic Inputs of DMV Neurons Innervating the Stomach Antrum and Cecum of Adult Male Rats.

BACKGROUND/AIM: Central administration of cocaine- and amphetamine-regulated transcript peptides (CARTp) alters gastrointestinal motility and reduces food intake in rats. Since neurons in the dorsal motor nucleus of the vagus (DMV) receive GABAergic and glutamatergic inputs and innervate the smooth muscle of gastrointestinal organs, we hypothesized that CARTp acts on the DMV or presynaptic neurons. METHODS: We used 3,3'-dioctadecyloxa-carbocyanine perchlorate (DiO) retrograde tracing with electrophysiological methods to record DMV neurons innervating the stomach antrum or cecum in brainstem slices from adult rats. RESULTS: DiO application did not change the electrophysiological properties of DMV neurons. CART55-102 had no effect on the basal firing rates of neurons in either the stomach antrum-labeled group (SLG) or cecum-labeled group (CLG). When presynaptic inputs were blocked, CART55-102 further increased the firing rates of the SLG, suggesting a direct excitatory effect. Spontaneous inhibitory postsynaptic currents (sIPSCs) occurred at a higher frequency in SLG neurons than in CLG neurons. CART55-102 reduced the amplitude and the frequency of sIPSCs in SLG neurons dose-dependently, with higher doses also reducing spontaneous excitatory postsynaptic currents (sEPSCs). Higher doses of CART55-102 reduced sIPSC and sEPSC amplitudes in CLG neurons, suggesting a postsynaptic effect. In response to incremental current injections, the SLG neurons exhibited less increases in firing activity. Simultaneous applications of current injections and CART55-102 decreased the firing activity of the CLG. Therefore, stomach antrum-projecting DMV neurons possess a higher gating ability to stabilize firing activity. CONCLUSION: The mechanism by which CARTp mediates anorectic actions may be through a direct reduction in cecum-projecting DMV neuron excitability and, to a lesser extent, that of antrum-projecting DMV neurons, by acting on receptors of these neurons.

Developmental increase in hippocampal endocannabinoid mobilization: role of metabotropic glutamate receptor subtype 5 and phospholipase C.

Endocannabinoids (eCBs) released from postsynaptic neurons mediate retrograde suppression of neurotransmitter release at central synapses. eCBs are crucial for establishing proper synaptic connectivity in the developing nervous system. Mobilization of eCBs is driven either by a rise in intracellular Ca(2+) (depolarization-induced suppression of inhibition, DSI) or postsynaptic G protein-coupled receptors (GPCRs) that activate phospholipase C beta (PLCß). To determine whether eCB mobilization changes between neonatal and juvenile ages, we used whole cell voltage-clamp recordings of CA1 neurons from rat hippocampal slices at postnatal days 1-18 (neonatal) and 19-43 (juvenile), because many neurophysiological parameters change dramatically between approximately postnatal days 18-20. We found that DSI was slightly greater in juveniles than in neonates, while eCB mobilization stimulated by GPCRs was unchanged. However, when DSI was elicited during GPCR activation, its increase was much greater in juveniles, suggesting that eCB mobilization caused by the synergy between the Ca(2+) and GPCR pathways is developmentally upregulated. Western blotting revealed significant increases in both metabotropic type glutamate receptor 5 (mGluR5) and PLCß1 proteins in juveniles compared with neonates. Responses to pharmacological activation or inhibition of PLC implied that eCB upregulation is associated with a functional increase in PLC activity. We conclude that synergistic eCB mobilization in hippocampal CA1 neurons is greater in juveniles than in neonates, and that this may result from increases in the mGluR5-PLCß1 eCB pathway. The data enhance our understanding of the developmental regulation of the eCB system and may provide insight into diseases caused by improper cortical wiring, or the impact of cannabis exposure during development.

Involvement of dopamine D2 receptor in the diurnal changes of tuberoinfundibular dopaminergic neuron activity and prolactin secretion in female rats.

BACKGROUND: An endogenous dopaminergic (DA) tone acting on D3 receptors has been shown to inhibit tuberoinfundibular (TI) DA neuron activity and stimulate prolactin (PRL) surge in the afternoon of estrogen-primed ovariectomized (OVX+E2) rats. Whether D2 receptor (D2R) is also involved in the regulation of TIDA and PRL rhythms was determined in this study. RESULTS: Intracerebroventricular (icv) injection of PHNO, a D2R agonist, in the morning inhibited TIDA and midbrain DA neurons' activities, and stimulated PRL secretion. The effects of PHNO were significantly reversed by co-administration of raclopride, a D2R antagonist. A single injection of raclopride at 1200 h significantly reversed the lowered TIDA neuron activity and the increased serum PRL level at 1500 h. Dopamine D2R mRNA expression in medial basal hypothalamus (MBH) exhibited a diurnal rhythm, i.e., low in the morning and high in the afternoon, which was opposite to that of TIDA neuron activity. The D2R rhythm was abolished in OVX+E2 rats kept under constant lighting but not in OVX rats with regular lighting exposures. Pretreatment with an antisense oligodeoxynucleotides (AODN, 10 µg/3 µl/day, icv) against D2R mRNA for 2 days significantly reduced D2R mRNAs in central DA neurons, and reversed both lowered TIDA neuron activity and increased serum PRL level in the afternoon on day 3. A diurnal rhythm of D2R mRNA expression was also observed in midbrain DA neurons and the rhythm was significantly knocked down by the AODN pretreatment. CONCLUSIONS: We conclude that a diurnal change of D2R mRNA expression in MBH may underlie the diurnal rhythms of TIDA neuron activity and PRL secretion in OVX+E2 rats.

Perinatal blockade of neuronal glutamine transport sex-differentially alters glutamatergic synaptic transmission and organization of neurons in the ventrolateral ventral media hypothalamus of adult rats.

Compared to male pups, perinatal female rats rely heavily on neuronal glutamine (Gln) transport for sustaining glutamatergic synaptic release in neurons of the ventrolateral ventral media nucleus of the hypothalamus (vlVMH). VMH mainly regulates female sexual behavior and increases glutamate release of perinatal hypothalamic neurons, permanently enhances dendrite spine numbers and is associated with brain and behavioral defeminization. We hypothesized that perinatal interruption of neuronal Gln transport may alter the glutamatergic synaptic transmission during adulthood. Perinatal rats of both sexes received an intracerebroventricular injection of a neuronal Gln uptake blocker, alpha-(methylamino) isobutyric acid (MeAIB, 5 mM), and were raised until adulthood. Whole-cell voltage-clamp recordings of miniature excitatory postsynaptic currents (mEPSCs) and evoked EPSCs (eEPSCs) of vlVMH neurons in adult rats with the perinatal pretreatment were conducted and neuron morphology was subjected to post hoc examination. Perinatal MeAIB treatment sex-differentially increased mEPSC frequency in males, but decreased mEPSC amplitude and synaptic Glu release in females. The pretreatment sex-differentially decreased eEPSC amplitude in males but increased AMPA/NMDA current ratio in females, and changed the morphology of vlVMH neurons of adult rats to that of the opposite sex. Most alterations in the glutamatergic synaptic transmission resembled the changes occurring during MeAIB acute exposure in perinatal rats of both sexes. We conclude that perinatal blockade of neuronal Gln transport mediates changes via different presynaptic and postsynaptic mechanisms to induce sex-differential alterations of the glutamatergic synaptic transmission and organization of vlVMH neurons in adult rats. These changes may be permanent and associated with brain and behavior feminization and/or defeminization in rats.

Plastome variation and phylogeny of Taxillus (Loranthaceae).

Several molecular phylogenetic studies of the mistletoe family Loranthaceae have been published such that now the general pattern of relationships among the genera and their biogeographic histories are understood. Less is known about species relationships in the larger (> 10 species) genera. This study examines the taxonomically difficult genus Taxillus composed of 35-40 Asian species. The goal was to explore the genetic diversity present in Taxillus plastomes, locate genetically variable hotspots, and test these for their utility as potential DNA barcodes. Using genome skimming, complete plastomes, as well as nuclear and mitochondrial rDNA sequences, were newly generated for eight species. The plastome sequences were used in conjunction with seven publicly available Taxillus sequences and three sequences of Scurrula, a close generic relative. The Taxillus plastomes ranged from 121 to 123 kbp and encoded 90-93 plastid genes. In addition to all of the NADH dehydrogenase complex genes, four ribosomal genes, infA and four intron-containing tRNA genes were lost or pseudogenized in all of the Taxillus and Scurrula plastomes. The topologies of the plastome, mitochondrial rDNA and nuclear rDNA trees were generally congruent, though with discordance at the position of T. chinensis. Several variable regions in the plastomes were identified that have sufficient numbers of parsimony informative sites as to recover the major clades seen in the complete plastome tree. Instead of generating complete plastome sequences, our study showed that accD alone or the concatenation of accD and rbcL can be used in future studies to facilitate identification of Taxillus samples and to generate a molecular phylogeny with robust sampling within the genus.

Application of traditional clinical pathology quality control techniques to molecular pathology.

Many molecular diagnostic laboratories have evolved from research laboratories, initially performing low numbers of homebrew assays, but many laboratories now perform more kit-based assays, with ever increasing test volumes. One such assay is assessment of bone marrow transplantation engraftment. Allogeneic bone marrow transplantation is performed primarily in the treatment of hematological malignancies. Monitoring of engraftment was traditionally evaluated using minisatellites (variable number tandem repeats) and Southern blotting, but most laboratories now use Food and Drug Administration-cleared microsatellite (short tandem repeats) kits to assess the extent of engraftment. With the increase in equipment reliability, the use of kit-based assays, and the desire to provide the highest quality clinical data, we began applying traditional clinical pathology quality control tools to the molecular diagnostics laboratory. In this study, we demonstrate this approach using a microsatellite-based bone marrow engraftment assay. We analyzed control samples (pure and mixed) for two different microsatellites to establish quality control parameters and constructed Levey-Jennings charts to monitor both the precision and accuracy of this assay. By incorporating these tools into an overall quality assurance program, a laboratory can identify systematic errors and perform corrective actions before actual assay failure, thereby improving the quality of patient care.

Comparison of the CEDIA and MEIA assays for measurement of tacrolimus in organ transplant recipients.

OBJECTIVES: Tacrolimus is widely used in organ transplantation. We evaluated 2 immunoassays, CEDIA and MEIA, for the measurement of whole blood tacrolimus concentrations. METHODS: For each assay the following were evaluated: total precision, limit of detection (analytical sensitivity), limit of quantitation (functional sensitivity), linearity, and accuracy. Patient correlation studies were performed, comparing each assay with liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Total precision for MEIA, corresponding to mean concentrations of 6.8 and 22.5 ng/ml, was 17.4 and 11.9%, respectively. The limit of detection was 0.9 ng/ml, and the limit of quantitation was 4.7 ng/ml. Total precision for CEDIA, corresponding to mean concentrations of 5.3 and 19.9 ng/ml, was 20.6 and 6.3%, respectively. The limit of detection was 0.8 ng/ml, with a limit of quantitation of 4.9 ng/ml. Analysis of proficiency material demonstrated acceptable performance for both assays. In addition, both assays were acceptably linear over their respective reportable ranges. Analysis of patient correlation data using Passing-Bablok analysis and Bland-Altman plots demonstrated a positive average bias for both assays versus LC-MS/MS results. CONCLUSION: Based on our evaluation, both assays demonstrated acceptable performance for use in clinical monitoring of tacrolimus.

The glutamine-glutamate cycle regulates synaptic glutamate release in the ventrolateral ventromedial nucleus of the hypothalamus of perinatal female rats.

The astrocytic glutamine (Gln)-glutamate (Glu) cycle (GGC) supplies Gln for the regulation of glutamatergic synaptic transmission (GST) in the adult hippocampus. Increased synaptic Glu release in the perinatal ventrolateral ventromedial nucleus of the hypothalamus (vlVMH) modulates sexual differentiation, however, whether GGC regulates GST in the perinatal vlVMH has not been determined. Sex differences in oestradiol (E2 ) levels exist in the neonatal hypothalamus, and E2 increases levels of glutamine synthetase and glutaminase, two key enzymes involved in the GGC. Thus, it is hypothesised that sexually dimorphic phenotypes may exist in glutamatergic synapses associated with the GGC in the vlVMH in perinatal rats. Whole-cell voltage-clamp recordings in vlVMH neurones in brain slices from male and female pups revealed that pharmacological disruption of the GGC by α-(methylamino) isobutyric acid (5 mmol L-1 ), which blocks neuronal Gln uptake; or by l-methionine sulphoximine (1.5 mmol L-1 ), which inhibits astrocytic Gln synthesis, decreased miniature excitatory postsynaptic current (mEPSC) amplitudes in female but not male pups. By contrast, GGC interruptions decreased evoked (e)EPSC amplitudes in both sexes following increased synaptic activity produced by a period of stimulation. In male pups, the decreased eEPSCs were attributable to reduced Glu release, as assessed by paired-pulse stimulations, whereas, in female pups, they were attributable to decreased Glu content in the synaptic vesicles, as measured by strontium-evoked mEPSCs. The l-methionine sulphoximine-mediated decrease in eEPSCs was rapidly rescued by exogenous Gln in female but not male pups. The reductions in mEPSCs and eEPSCs in female pups were accompanied by enhanced blocking effects of the low-affinity Glu AMPA receptor antagonist, γ-d-glutamylglycine, consistent with diminished Glu release. In conclusion, female, but not male pups, rely on constitutive astrocytic Gln for sustained synaptic Glu release in the vlVMH. This glutamatergic synaptic phenotype may be associated with brain and behaviour feminisation and/or defeminisation in rats.

Dynamic regulation of synaptic GABA release by the glutamate-glutamine cycle in hippocampal area CA1.

Vesicular GABA and intraterminal glutamate concentrations are in equilibrium, suggesting inhibitory efficacy may depend on glutamate availability. Two main intraterminal glutamate sources are uptake by neuronal glutamate transporters and glutamine synthesized through the astrocytic glutamate-glutamine cycle. We examined the involvement of the glutamate-glutamine cycle in modulating GABAergic synaptic efficacy. In the absence of neuronal activity, disruption of the glutamate-glutamine cycle by blockade of neuronal glutamine transport with alpha-(methylamino) isobutyric acid (MeAIB; 5 mM) or inhibition of glutamine synthesis in astrocytes with methionine sulfoximine (MSO; 1.5 mM) had no effect on miniature IPSCs recorded in hippocampal area CA1 pyramidal neurons. However, after a period of moderate synaptic activity, application of MeAIB, MSO, or dihydrokainate (250 microM; an astrocytic glutamate transporter inhibitor) significantly reduced evoked IPSC (eIPSC) amplitudes. The MSO effect could be reversed by exogenous application of glutamine (5 mM), whereas glutamine could not rescue the eIPSC decreases induced by the neuronal glutamine transporter inhibitor MeAIB. The activity-dependent reduction in eIPSCs by glutamate-glutamine cycle blockers was accompanied by an enhanced blocking effect of the low-affinity GABA(A) receptor antagonist, TPMPA [1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid], consistent with diminished GABA release. We further corroborated this hypothesis by examining MeAIB effects on minimal stimulation-evoked quantal IPSCs (meIPSCs). We found that, in MeAIB-containing medium, moderate stimulation induced depression in potency of meIPSCs but no change in release probability, consistent with reduced vesicular GABA content. We conclude that the glutamate-glutamine cycle is a major contributor to synaptic GABA release under physiological conditions, which dynamically regulates inhibitory synaptic strength.

Tumour suppressor function of RNase L in a mouse model.

RNase L is one of the key enzymes involved in the molecular mechanisms of interferon (IFN) actions. Upon binding with its activator, 5'-phosphorylated, 2'-5' oligoadenylates (2-5A), RNase L plays an important role in the antiviral and anti-proliferative functions of IFN, and exerts proapoptotic activity independent of IFN. In this study, we have found that RNase L retards proliferation in an IFN-dependent and independent fashion. To directly measure the effect of RNase L on tumour growth in the absence of other IFN-induced proteins, human RNase L cDNA was stably expressed in P-57 cells, an aggressive mouse fibrosarcoma cell line. Three clonal cell lines were isolated in which the overexpression of RNase L was 15-20-fold of the endogenous level. Groups of five nude mice were injected subcutaneously with either the human RNase L overexpressing clones (P-RL) or control cells transfected with an empty vector (P-Vec). Tumour growth by the two cell lines was monitored by measuring tumour volumes. In the P-RL group, tumour formation was significantly delayed and the tumours grew much slower compared to the control group. Morphologically, the P-RL tumour appeared to have more polygonal cells and increased single cell tumour necrosis. Interestingly, P-RL tumours eventually started to grow. Further analysis revealed, however, that these tumours no longer expressed ectopic RNase L. Our findings suggest that RNase L plays a critical role in the inhibition of fibrosarcoma growth in nude mice.

Enzymes and related proteins as cancer biomarkers: a proteomic approach.

BACKGROUND: The discovery of cancer biomarkers has become a major focus of cancer research, which holds promising future for early detection, diagnosis, monitoring disease recurrence and therapeutic treatment efficacy to improve long-term survival of cancer patients. Most of the functional information of the cancer-associated genes resides in the proteome. Since cancer is a complex disease, it might require a panel of multiple biomarkers in order to achieve sufficient clinical efficacy. METHODS: Serum/plasma is the most accessible biological specimen collected from patients. Therefore, serum proteomic diagnostics would be the most promising new test for cancer. With the advent of new and improved proteomic technologies, such as protein chips and mass spectrometry coupled with advanced bioinformatic tools, it is possible to develop potential cancer biomarkers. However, specimen collection, handling, study design and data analysis are essential components for successful biomarker discovery and validation. Multi-center case control study should be conducted with extensive clinical validation to minimize the impact of possible confounding variables (non-biological). CONCLUSIONS: Enzymes and related proteins, such as inhibitors, are promising candidates for cancer diagnostics.

Clioquinol effects on tissue chelatable zinc in mice.

Recent evidence for the involvement of zinc in the formation of beta-amyloid plaques in the brain in Alzheimer's disease has led to the establishment of new therapeutic strategies for the degenerative disorder based on metal chelation. The present experiment was conducted on a membrane-permeable zinc chelator, clioquinol (CQ), that has shown potential in initial studies on a mouse model of Alzheimer's disease [1]. The degree of chelatable zinc in mice treated with CQ, delivered by two different routes, was measured using complementary protocols for identifying chelatable zinc: 6-methoxy-8-quinolyl- p-toluenesulfonamide (TSQ) histofluorescence, and selenite autometalography. Mice injected intraperitoneally with CQ showed a dramatic reduction in chelatable zinc in brain, testis, and pancreas. In contrast, mice given CQ orally showed no significant change in levels of chelatable zinc in these tissues. This suggests that CQ administered orally to patients with Alzheimer's disease should not significantly perturb chelatable zinc levels in key organs and may be used over long periods without adverse endocrinological and reproductive effects related to zinc deficiency. In contrast, CQ injected intraperitoneally may be used not only as a tool for investigating chelatable zinc pools but also in a clinical context. For example, injected CQ could be employed in situations requiring the rapid buffering of excessive chelatable zinc following ischemic episodes or brain trauma. Thus, our findings indicate that CQ has considerable potential as a versatile scientific and clinical tool used for selective modulation of zinc pools.

Pretreatment with antisense oligodeoxynucleotide against D(2) or D(3) receptor mRNA diminished dopamine's inhibitory effect on dorsomedial arcuate neurons in brain slices of estrogen-treated ovariectomized rats.

A high percentage of dopamine (DA)-responsive neurons has been repeatedly shown in hypothalamic dorsomedial arcuate nucleus (dmARN) using single-unit recording in brain slices. Both D(2) and D(3) receptors may be involved in the inhibitory action of DA as indicated by results obtained from using specific DA agonists and antagonists. To further delineate the DA receptor types involved, ovariectomized, estrogen-primed Sprague-Dawley rats pretreated with antisense oligodeoxynucleotide (ODN, 10 microg/3 microl, i.c.v.) against either D(2) or D(3) receptor mRNA for 2 days were used in this study for brain slice preparation. Rats pretreated with aCSF, random antisense or sense ODNs were used as controls. DA (5-50 nmol) inhibited a majority of dmARN neurons in brain slices prepared from rats pretreated with aCSF (71.4% of 35 U), random antisense/sense ODN for D(2) (67.6%, n=34), D(3) (59.5%, n=42), or D(2) plus D(3) (60.5%, n=38) mRNAs. In contrast, DA inhibited 43.6% (n=39) of dmARN neurons in slices prepared from D(2), and 38.5% (n=39) from D(3) antisense ODN-pretreated rats. Furthermore, in brain slices prepared from rats pretreated with combined D(2) and D(3) antisense ODNs, DA only inhibited 18.4% (n=38) of dmARN neurons. We conclude that both D(2) and D(3) receptors are involved in the action of DA on dmARN neurons in ovariectomized, estrogen-treated rats.

An endogenous dopaminergic tone acting on dopamine D3 receptors may be involved in diurnal changes of tuberoinfundibular dopaminergic neuron activity and prolactin secretion in estrogen-primed ovariectomized rats.

The diurnal rhythm of tuberoinfundibular dopaminergic (TIDA) neuron activity, i.e., high in the morning and low in the afternoon, is prerequisite for the afternoon prolactin (PRL) surge in proestrous and estrogen-primed ovariectomized (OVX) female rats. Whether dopamine acts via D(3) receptors in regulating the rhythmic TIDA neuron activity and PRL secretion in estrogen-primed OVX (OVX+E(2)) rats is the focus of this study. Intracerebroventricular (icv) injection of a D(3) receptor agonist, PD128907 (0.1-10 µg/3 µl), in the morning significantly reduced the basal activity of TIDA neurons and increased plasma PRL level. The effects of PD128907 were reversed by co-administration of U99194A, a D(3) receptor antagonist, but not by raclopride, a D(2) receptor antagonist. To determine whether endogenous dopamine acts on D(3) receptors involved in the diurnal changes of the activities, we used both U99194A, a D(3) receptor antagonist, and an antisense oligodeoxynucleotide (ODN) against D(3) receptor mRNA in the study. U99194A (0.1 µg/3 µl, icv) given at 1200 h significantly reversed the lowered TIDA neuron activity and the afternoon PRL surge at 1500 h. Moreover, OVX+E(2) rats pretreated with the antisense ODN (10 µg/3 µl, icv) for 2 days had the same effects as the D(3) receptor antagonist on TIDA neuron activity and the PRL surge. The same treatment with sense ODN had no effect. In conclusion, an endogenous DA tone may act on D(3) receptors to inhibit TIDA neuron activity and in turn stimulate the PRL surge in the afternoon of OVX+E(2) rats.

The spontaneous firing rates of dopamine-inhibited dorsomedial arcuate neurons exhibit a diurnal rhythm in brain slices obtained from ovariectomized plus estrogen-treated rats.

The activity of tuberoinfundibular dopaminergic (TIDA) neurons exhibits a diurnal rhythm in female rats, as determined by neurochemical investigation. Whether the spontaneous firing rates of presumed TIDA neurons in the dorsomedial arcuate nucleus (dmARN) also exhibit a diurnal pattern has yet to be ascertained. Single-unit activities of 131 dmARN neurons were recorded in brain slices prepared from 83 ovariectomized plus estrogen-primed rats, and grouped according to their responses to dopamine and the time at which they were observed. In dopamine-inhibited dmARN neurons, significantly lower firing rates were observed in the afternoon compared to those recorded in the morning (2.51 ± 0.27 Hz, n=15, from 1130 to 1330 h vs. 1.08 ± 0.07 Hz, n=47, from 1430 to 1630 h). No such change was observed in dopamine-excited or nonresponsive dmARN neurons (1.83 ± 0.32 Hz, n=9 vs. 1.46 ± 0.17 Hz, n=21). Four dmARN neurons were continuously recorded from 1130 to 1600 h or even longer until 2000 h. The averaged firing rates decreased significantly between 1300 and 1600 h, two neurons were also inhibited by dopamine and a selective D(2) receptor agonist, PHNO, in both normal and low Ca(2+), high Mg(2+) perfusion mediums. This study revealed the existence of diurnal changes in the firing rates of dopamine-inhibited dmARN neurons. These results are strongly correlated with the rhythmic changes observed in TIDA neuronal activity determined through neurochemical methods.

Manipulation of mechanical compliance of elastomeric PGS by incorporation of halloysite nanotubes for soft tissue engineering applications.

Poly (glycerol sebacate) (PGS) is a promising elastomer for use in soft tissue engineering. However, it is difficult to achieve with PGS a satisfactory balance of mechanical compliance and degradation rate that meet the requirements of soft tissue engineering. In this work, we have synthesised a new PGS nanocomposite system filled with halloysite nanotubes, and mechanical properties, as well as related chemical characters, of the nanocomposites were investigated. It was found that the addition of nanotubular halloysite did not compromise the extensibility of material, compared with the pure PGS counterpart; instead the elongation at rupture was increased from 110 (in the pure PGS) to 225% (in the 20 wt% composite). Second, Young's modulus and resilience of 3-5 wt% composites were ∼0.8 MPa and >94% respectively, remaining close to the level of pure PGS which is desired for applications in soft tissue engineering. Third, an important feature of the 1-5 wt% composites was their stable mechanical properties over an extended period, which could allow the provision of reliable mechanical support to damaged tissues during the lag phase of the healing process. Finally, the in vitro study indicated that the addition of halloysite slowed down the degradation rate of the composites. In conclusion, the good compliance, enhanced stretchability, stable mechanical behavior over an extended period, and reduced degradation rates make the 3-5 wt% composites promising candidates for application in soft tissue engineering.

In vitro enzymatic degradation of poly (glycerol sebacate)-based materials.

Enzymatic degradation is a major feature of polyester implants in vivo. An in vitro experimental protocol that can simulate and predict the in vivo enzymatic degradation kinetics of implants is of importance not only to our understanding of the scientific issue, but also to the well-being of animals. In this study, we explored the enzymatic degradation of PGS-based materials in vitro, in tissue culture medium or a buffer solution at the pH optima and under static or cyclic mechanical-loading conditions, in the presence of defined concentrations of an esterase. Surprisingly, it was found that the in vitro enzymatic degradation rates of the PGS-based materials were higher in the tissue culture medium than in the buffered solution at the optimum pH 8. The in vitro enzymatic degradation rate of PGS-based biomaterials crosslinked at 125°C for 2 days was approximately 0.6-0.9 mm/month in tissue culture medium, which falls within the range of in vivo degradation rates (0.2-1.5mm/month) of PGS crosslinked at similar conditions. Enzymatic degradation was also further enhanced in relation to mechanical deformation. Hence, in vitro enzymatic degradation of PGS materials conducted in tissue culture medium under appropriate enzymatic conditions can quantitatively capture the features of in vivo degradation of PGS-based materials and can be used to indicate effective strategies for tuning the degradation rates of this material system prior to animal model testing.