Patterned Stimulation of the Chrimson Opsin in Glutamatergic Motor Thalamus Neurons Improves Forelimb Akinesia in Parkinsonian Rats.

Parkinson's disease (PD) is a motor disorder charactertised by altered neural activity throughout the basal ganglia-thalamocortical circuit. Electrical deep brain stimulation (DBS) is efficacious in alleviating motor symptoms, but has several notable side-effects, most likely reflecting the non-specific nature of electrical stimulation and/or the brain regions targeted. We determined whether specific optogenetic activation of glutamatergic motor thalamus (Mthal) neurons alleviated forelimb akinesia in a chronic rat model of PD. Parkinsonian rats (unilateral 6-hydroxydopamine injection) were injected with an adeno-associated viral vector (AAV5-CaMKII-Chrimson-GFP) to transduce glutamatergic Mthal neurons with the red-shifted Chrimson opsin. Optogenetic stimulation with orange light at 15 Hz tonic and a physiological pattern, previously recorded from a Mthal neuron in a control rat, significantly increased forelimb use in the reaching test (p < 0.01). Orange light theta burst stimulation, 15 Hz and control reaching patterns significantly reduced akinesia (p < 0.0001) assessed by the step test. In contrast, forelimb use in the cylinder test was unaffected by orange light stimulation with any pattern. Blue light (control) stimulation failed to alter behaviours. Activation of Chrimson using complex patterns in the Mthal may be an alternative treatment to recover movement in PD. These vector and opsin changes are important steps towards translating optogenetic stimulation to humans.

Anaemia in cardiac surgery - a retrospective review of a centre's experience with a pre-operative intravenous iron clinic.

Pre-operative anaemia is associated with higher rates of transfusion and worse outcomes, including prolonged hospital stay, morbidity and mortality. Iron deficiency is associated with significantly lower haemoglobin levels throughout the peri-operative period and more frequent blood transfusion. Correction of iron stores before surgery forms part of the first pillar of patient blood management. We established a pre-operative anaemia clinic to aid identification and treatment of patients with iron deficiency anaemia scheduled for elective cardiac surgery. We present a retrospective observational review of our experience from January 2017 to December 2019. One-hundred and ninety patients received treatment with intravenous iron, a median of 21 days before cardiac surgery. Of these, 179 had a formal laboratory haemoglobin level measured before surgery, demonstrating a median rise in haemoglobin of 8.0 g.l-1 . Patients treated with i.v. iron demonstrated a significantly higher incidence of transfusion (60%) compared with the non-anaemic cohort (22%) during the same time period, p < 0.001. Significantly higher rates of new requirement for renal replacement therapy (6.7% vs. 0.6%, p < 0.001) and of stroke (3.7% vs. 1.2%, p = 0.010) were also seen in this group compared with those without anaemia, although there was no significant difference in in-hospital mortality (1.6% vs. 0.8%, p = 0.230). In patients where the presenting haemoglobin was less than 130 g.l-1 , but there was no intervention or treatment, there was no difference in rates of transfusion or of complications compared with the anaemic group treated with iron. In patients with proven iron deficiency anaemia, supplementation with intravenous iron showed only a modest effect on haemoglobin and this group still had a significantly higher transfusion requirement than the non-anaemic cohort. Supplementation with intravenous iron did not improve outcomes compared with patients with anaemia who did not receive intravenous iron and did not reduce peri-operative risk to non-anaemic levels. Questions remain regarding identification of patients who will receive most benefit, the use of concomitant treatment with other agents, and the optimum time frames for treatment in order to produce benefit in the real-world setting.

Experiences of breast cancer in Arab countries. A thematic synthesis.

BACKGROUND: Breast cancer is the most common cancer in women globally. Its negative effects on a woman's quality of life are related to the individual and socio-cultural factors. This review aimed to identify and synthesise the reported experiences and quality of life of women with breast cancer in Arab countries. METHODS: PubMed, Embase, Web of Science, SCOPUS, PsychInfo, CINAHL, Allied and Complementary Medicine Database, and Index Medicus for the Eastern Mediterranean Region were searched for articles published from start to March 2019 using PRISMA guidelines. These searches were complimented by citation tracking and  hand searching of relevant journals. A thematic synthesis was carried out on the 'findings/results' sections from the identified papers. RESULTS: Of 5228 records identified, 19 were included in the review which represented 401 women from 11 Arab countries. All used qualitative methods of data collection to produce rich descriptions of experiences. Thematic synthesis of the extracted data identified three major themes, Perceptions and reactions, Coping or enduring and Changing roles. CONCLUSIONS: This review provides a rich description of the reported quality of life and experiences of women with breast cancer in Arab countries. These are influenced by the women's and society's views of cancer, the women's role in society and family, religious faith and the healthcare context and access to treatment choices and information.

Integrated palliative care in Europe: a qualitative systematic literature review of empirically-tested models in cancer and chronic disease.

BACKGROUND: Integrated Palliative Care (PC) strategies are often implemented following models, namely standardized designs that provide frameworks for the organization of care for people with a progressive life-threatening illness and/or for their (in)formal caregivers. The aim of this qualitative systematic review is to identify empirically-evaluated models of PC in cancer and chronic disease in Europe. Further, develop a generic framework that will consist of the basis for the design of future models for integrated PC in Europe. METHODS: Cochrane, PubMed, EMBASE, CINAHL, AMED, BNI, Web of Science, NHS Evidence. Five journals and references from included studies were hand-searched. Two reviewers screened the search results. Studies with adult patients with advanced cancer/chronic disease from 1995 to 2013 in Europe, in English, French, German, Dutch, Hungarian or Spanish were included. A narrative synthesis was used. RESULTS: 14 studies were included, 7 models for chronic disease, 4 for integrated care in oncology, 2 for both cancer and chronic disease and 2 for end-of-life pathways. The results show a strong agreement on the benefits of the involvement of a PC multidisciplinary team: better symptom control, less caregiver burden, improvement in continuity and coordination of care, fewer admissions, cost effectiveness and patients dying in their preferred place. CONCLUSION: Based on our findings, a generic framework for integrated PC in cancer and chronic disease is proposed. This framework fosters integration of PC in the disease trajectory concurrently with treatment and identifies the importance of employing a PC-trained multidisciplinary team with a threefold focus: treatment, consulting and training.

Effect of dietary chicory on boar taint.

Following preliminary screening and feeding trials on farms supplying a commercial abattoir, 360 entire male pigs were used to evaluate the effects of different percentages of chicory (Cichorium intybus L.) on levels of boar taint compounds and sensory aspects in backfat. Pigs were fed 0, 3, 6 or 9% chicory in the diet, 30 pigs being sampled at 3 different times: initially to measure basal levels of skatole and androstenone and after 1 and 2 weeks on the test diets. Cooked samples of backfat were presented to a trained sensory panel for "sniff" tests. Chicory fed at 9% for 2 weeks reduced skatole levels significantly (P<0.001), with 0.55 of pigs below 0.05 µg/g, typical of levels in castrated males. Abnormal odour scores were significantly lower for pigs in this group compared with 0% pigs (P<0.001), however, androstenone concentration was significantly higher in this group after the 2 week feeding period (P<0.005). Thus, feeding 9% chicory for 2 weeks was effective in reducing backfat skatole concentrations and abnormal odour scores of cooked fat but not androstenone concentration.

A national audit of pollen immunotherapy for children in the United Kingdom: patient selection and programme safety.

BACKGROUND: Specific immunotherapy (SIT) is an effective treatment for grass and/or tree pollen-induced severe allergic rhinoconjunctivitis. However, there are limited detailed data on the use of immunotherapy in children in the United Kingdom. OBJECTIVES: We audited NHS paediatric practice against current national guidelines to evaluate patient selection, SIT modalities and adverse events (AEs). METHODS: Paediatricians offering pollen SIT were identified through the British Society of Allergy and Clinical Immunology Paediatric Allergy Group (BSACI-PAG) and the database of SIT providers compiled for the Royal College of Physicians and Royal College of Pathologists 2010 joint working group. Standardized proformas were returned by 12 of 20 centres (60%), including 12 of 14 centres offering subcutaneous immunotherapy (SCIT) (85%). RESULTS: Three hundred and twenty-three children, with mean age 11 years at initiation (69% boys), had undergone 528 SIT cycles (SCIT 31%) over 10 years. Fifty-five percent of all patients had asthma. Among SCIT programmes 24.5% patients had perennial (± seasonal) asthma; 75.6% of asthmatics undertaking SCIT had treatments at BTS/SIGN step 2 or above. AEs occurred frequently (50.4% of all SIT cycles) but were mild. In sublingual immunotherapy (SLIT) treatment, local intraoral immediate reactions were most common (44.9% SLIT cycles), as compared with delayed reactions around the injection site in SCIT (28.3% SCIT cycles). An asthma diagnosis had no impact on the number of cycles with AEs, or the severity reported. Few cycles (2.9%) were discontinued as a result of AE(s). CONCLUSIONS AND CLINICAL RELEVANCE: Pollen SIT is available across England, though small numbers of children are being treated. Current national guidelines to exclude asthmatic children in SIT programmes are not being adhered to by most specialist paediatric allergy centres. SCIT and SLIT has been well tolerated. Review of patient selection criteria is needed and may allow greater use of this therapeutic option in appropriate clinical settings.

Exploring the anthelmintic properties of Australian native shrubs with respect to their potential role in livestock grazing systems.

We measured in vitro anthelmintic activity in extracts from 85 species of Australian native shrub, with a view to identifying species able to provide a degree of worm control in grazing systems. Approximately 40% of the species showed significant activity in inhibiting development of Haemonchus contortus larvae. The most active extracts showed IC50 values of 60-300 microg/ml. Pre-incubation with polyvinylpolypyrrolidine removed the activity from some extracts, implicating tannins as the bioactive agent, while in other cases the pre-incubation had no effect, indicating the presence of other anthelmintic compounds. Plant reproductive maturity (onset of flowering or fruiting) was associated with increasing anthelmintic activity in some species. Variability was observed between plants of the same species growing in different environments, while variation between individual plants of the same species within a single field suggests the existence of distinct chemotypes. Significant activity against adult H. contortus worms in vitro was also demonstrated in a limited number of extracts tested against this life stage. Our study indicates that there is potential for Australian native shrubs to play an anthelmintic role in grazing systems, and highlights some plant biology factors which will need to be considered in order to maximize any anthelmintic effects.

Effect of dietary oil source on the flavour and the colour and lipid stability of lamb meat.

This study investigated the influence of five sources of dietary oil (linseed oil (LO), fish oil (FO), a protected lipid supplement (PLS, 18:2 to 18:3 ratio 3:1), fish oil/marine algae (FOMA) and PLSMA) on the colour and lipid stability of lamb muscle and the flavour of grilled loin chops. LO produced the highest proportion of 18:3n-3 in muscle phospholipid, the highest ratings for lamb flavour intensity and overall liking and the lowest ratings for abnormal flavour intensity. PLS increased the proportion of 18:2n-6 which reduced lamb flavour intensity and increased abnormal lamb flavour intensity. Diets containing FO or MA increased proportions of the longer chain n-3 fatty acids and similar reduced ratings for lamb flavour as the PLS diet. FO-containing diets increased fishy flavour notes, especially when in combination with MA. 'Putty' and 'fish oil' odours were recognised as being present more frequently in cooked subcutaneous lamb fat from lambs fed FO and FOMA than other diets. Lambs fed MA, FO and the combination of the two produced meat that was oxidatively less stable and had a reduced colour and lipid oxidative shelf-life, which was at least partially due to the lower vitamin E content of the muscle. These results have significant implications for the formulation of diets that may improve nutritional ratios in lamb meat but which adversely affect flavour and meat stability.

Flavour perception of oxidation in beef.

Lipid oxidation is a major factor in meat quality. In order to relate human perceptions of lipid oxidation, as determined by a trained taste panel, to a chemical measurement of oxidation, we studied meat from animals with a wide range of potential oxidation through differences in their PUFA composition and by displaying the meat in high oxygen modified atmosphere packs for varying lengths of time. Meat was obtained from 73 Angus- and Charolais-cross steers from different trials that had been raised on 10 different diets: grass silage (high in C18:3, n-3), cereal concentrate (high in C18:2, n-6), three diets with 3% added fat consisting of three levels of protected lipid supplement (high in C18:2, n-6 and C18:3, n-3, ratio 1:1), a control with Megalac(®) (relatively saturated), three diets with three levels of inclusion of protected fish oil (high in C20:5 n-3 and C22:6 n-3) plus a constant amount of unprotected fish oil and a final diet with an unprotected fish oil control. The longissimus dorsi muscle was excised from the left carcass side, aged vacuum packaged for 10-13 days depending on the projects and frozen for less than eight months. TBARS and sensory analyses were performed on steaks displayed for 0, 4 or 9 days under simulated retail conditions, exposed to light in modified atmosphere packaging (CO(2):O(2); 25:75). Meat oxidation increased throughout display for each of the diets, as shown by a rise in TBARS values. This increase was not linear, differences between 0 and 4 days of display were smaller than between 4 and 9 days of display. The lowest TBARS and lowest increment occurred in the two control diets and the grass-fed animals, probably due to the more saturated fat of meat from animals fed the control diets and the higher content of vitamin E. Sensory attributes were also influenced by time of display. Positive attributes, such as beef flavour or overall liking, decreased throughout display, whereas negative attributes, such as abnormal and rancid flavours, increased. The correlations between sensory and analytical attributes were high. TBARS were a good predictor of the perception of rancidity (Spearman's rho=0.84). Panellist preferences were related to the presence of beef flavour (rho=0.93) and to the absence of abnormal (rho=-0.88) and rancid flavours (rho=-0.83). Under the experimental conditions used, a TBARS value of around 2 could be considered the limiting threshold for the acceptability of oxidised beef.

Double contrast barium enema sensitivity: a comparison of studies by radiographers and radiologists.

PURPOSE: A retrospective study of histologically proven cases of colorectal cancer (CRC) was performed to assess whether the sensitivity of the radiographer-performed double contrast barium enema (DCBE) differed from that of the radiologist-performed study. MATERIALS AND METHODS: Histologically proven cases of CRC were reviewed over a 3-year period to ascertain whether: the diagnosis had been made by DCBE in the 3 years before histological diagnosis; the lesion had been correctly diagnosed; the examination had been performed by a radiologist or radiographer. RESULTS: In the 3-year period there were 478 cases with histologically proven CRC. Of these, 239 (50%) had undergone DCBE as the initial radiological investigation of the colon. Sixty-four examinations had been performed by radiographers. A correct diagnosis was made in 58 cases (90.6%), the report was equivocal in one case (1.6%), there were four false-negatives (6.25%), and one case was abandoned (1.6%). One hundred and seventy-five examinations were performed by radiologists. A correct diagnosis was made in 157 cases (89.7%), the report was equivocal in one case (0.6%), there were 16 false-negatives (9.1%), and one case was abandoned (0.6%). CONCLUSION: A sensitivity of 90.6% for radiographer-performed studies compared favourably with 89.7% for radiologist-performed studies and supports the practice of radiographers undertaking barium enemas.

The phosphorus budget of the Thame catchment, Oxfordshire: 2. Modelling.

The phosphorus budget of the River Thame was modelled at a daily time scale, using estimates of diffuse and point source contributions of discharge. The model simulated suspended sediment (SS), soluble unreactive phosphorus (SUP), soluble reactive phosphorus (SRP) and particulate phosphorus (PP) concentrations within the main river and major tributaries. Diffuse source estimates of phosphorus loads were based on characteristic losses from identified main landscape classes, with hydrology described by a simple conceptual storage model. In-stream flow was modelled using a kinematic wave equation. Transfer of suspended sediment and phosphorus components was approximated by advection. In-stream sources and sinks included uptake and release of soluble reactive phosphorus by bed sediment, instant equilibration between SRP and the PP concentration on suspended sediment, and flow-related entrainment and deposition of suspended sediment. Simulations at sites within the catchment were compared with measurements made in 1998-1999. Results showed the P budget is dominated by mixing of diffuse and point source water, but some within-river processes have been shown to be capable of significantly influencing SRP concentrations. The development of a sediment entrainment and deposition component of the model has proved particularly valuable in emulating the hysteretic relationship between discharge and suspended sediment concentration in the river. It also provides a measure of available bed sediment.

Self-priming of retroviral minus-strand strong-stop DNAs.

After minus-strand strong-stop DNA (-sssDNA) is synthesized, the RNA template is degraded by the RNase H activity of the reverse transcriptase (RT), generating a single-stranded DNA. The 3' end of -sssDNA from HIV-1 can form a hairpin; this hairpin will self-prime in vitro. We previously used a model substrate, -R ssDNA, which corresponds to the 3' end of the -sssDNA of HIV-1, to show that the self-priming of this model substrate could be prevented by annealing a 17-nt-long DNA oligonucleotide to the 3' end of -R ssDNA in the presence of HIV-1 nucleocapsid (NC) protein. Similar model substrates were prepared for HIV-2 and HTLV-1; the R regions of these two viruses are longer and form more complex structures than the R region of the HIV-1 genome. However, the size of the R region and the complexity of the secondary structures they can form do not affect self-priming or its prevention. The efficiency of the self-priming is related to the relative stabilities of the conformations of -R ssDNA that can and cannot induce self-priming. For the three viruses (HIV-1, HIV-2, and HTLV-1), the size of the DNA oligonucleotide needed to block self-priming in the presence of NC is similar to the expected size of the piece of RNA left after degradation of the RNA template during reverse transcription. We also found that when the 3' end of -R ssDNA is annealed to a complementary DNA oligonucleotide, it is a good substrate for efficient nonspecific strand transfer to other single-stranded DNA molecules.

Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells.

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after, but not before, 14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair.

All thalamocortical neurones possess a T-type Ca2+ 'window' current that enables the expression of bistability-mediated activities.

1. The existence of a non-negligible steady-state ('window') component of the low threshold, T-type Ca2+current (IT) and an appropriately large ratio of IT to ILeak conductance (i.e. gT/gLeak) have been shown to underlie a novel form of intrinsic bistability that is present in about 15 % of thalamocortical (TC) neurones. 2. In the present experiments, the dynamic clamp technique was used to introduce into mammalian TC neurones in vitro either an artificial, i.e. computer-generated, IT in order to enhance endogenous IT, or an artificial inward ILeak to decrease endogenous ILeak. Using this method, we were able to investigate directly whether the majority of TC neurones appear non-bistable because their intrinsic ionic membrane properties are essentially different (i.e. presence of a negligible IT 'window' component), or simply because they possess a gT or gLeak conductance that is insufficiently large or small, respectively. 3. The validity of the dynamic clamp arrangement and the accuracy of artificial IT were confirmed by (i) recreating the low threshold calcium potential (LTCP) with artificial IT following its block by Ni2+ (0.5-1 mM), and (ii) blocking endogenous LTCPs with an artificial outward IT. 4. Augmentation of endogenous IT by an artificial analog or introduction of an artificial inward ILeak transformed all non-bistable TC neurones to bistable cells that expressed the full array of bistability-mediated behaviours, i.e. input signal amplification, slow oscillatory activity and membrane potential bistability. 5. These results demonstrate the existence of a non-negligible IT 'window' component in all TC neurones and suggest that rather than being a novel group of neurones, bistable cells are merely representative of an interesting region of dynamical modes in the (gT, gLeak) parameter space that may be expressed under certain physiological or pathological conditions by all TC neurones and other types of excitable cells that possess an IT 'window' component with similar biophysical properties.

Differential RNA display identifies novel genes associated with decreased vitamin D receptor expression.

To characterize further the function of the intracellular vitamin D receptor (VDR), we have developed stable transfectant variants of a vitamin D-responsive cell line (U937) which express either decreased or increased numbers of VDR. In this study we have analyzed changes in gene expression associated with this variable VDR expression. Initial experiments indicated that a 50% decrease in VDR levels was associated with a 2-fold increase in cell proliferation and a similar rise in c-myc mRNA expression. Further studies were carried out using differential RNA display (DD). Sequence analysis of DD products revealed two cDNAs with identity to known gene products: the catalytic sub-unit of DNA-protein kinase (DNA-PK(CS)), and the peroxisomal enzyme 17beta-hydroxysteroid dehydrogenase type IV (17beta-HSD IV). Northern analysis confirmed that expression of both mRNAs was reduced in cells with decreased numbers of VDR. Down-regulation of 17beta-HSD IV mRNA expression was associated with enhanced estradiol inactivation by U937 cells, suggesting a link between estrogenic pathways and cell proliferation. Further Northern analyses indicated that there was no significant change in 17beta-HSD IV or DNA-PK(CS) mRNA levels following treatment with 1,25(OH)2D3, although expression of both genes varied with changes in cell proliferation. These data suggest that, in addition to its established role as a hormone-dependent trans-activator, VDR may influence gene expression by ligand-independent mechanisms.

Dynamic clamp study of Ih modulation of burst firing and delta oscillations in thalamocortical neurons in vitro.

The dynamic clamp technique was used in thalamocortical neurons of the rat and cat dorsal lateral geniculate nucleus in vitro to investigate the effects of the hyperpolarization-activated cation current, Ih, and of its neuromodulation on burst firing and delta oscillations. Specific block of endogenous Ih using 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyridinium chloride (ZD7288) (300 microM) abolished the depolarizing "sag" response to negative current steps, markedly increased the latency and shortened the duration of the low-threshold Ca2+ potentials, and decreased the number of action potentials in the burst evoked by the low-threshold Ca2+ potential. Subsequent introduction of artificial Ih using the dynamic clamp re-instated the "sag" and all the original properties of the low-threshold Ca2+ potential. In the absence of ZD7288, introduction of artificial outward Ih with the intention of abolishing endogenous Ih removed the depolarizing "sag" and produced similar effects on the low-threshold Ca2+ potentials as those observed during the pharmacological block of Ih. Application of ZD7288 to thalamocortical neurons displaying delta oscillations led to a reduction in the voltage range of their existence or to a complete cessation of this behaviour. A subsequent introduction of artificial Ih re-enabled the generation of delta oscillations. In the presence of ZD7288, physiologically relevant positive shifts in the voltage-dependence of artificial Ih increased the amplitude and duration of the low-threshold Ca2+ potential and increased the likelihood of delta oscillations while negative shifts had opposite effects. These results highlight the important difference between the dependence of burst firing and oscillations on membrane potential and their dependence on the properties of Ih, and demonstrate that the modulation by Ih of low-threshold Ca2+ potentials and burst firing in thalamocortical neurons, as well as the ability of these neurons to generate delta oscillations, is more elaborate than previously described.

Analysis and biophysical interpretation of bistable behaviour in thalamocortical neurons.

Thalamocortical neurons display a wide spectrum of activity patterns that are the expressions of the non-linear interactions between the various voltage-gated ion channels. Here, we show how bistable behaviour can emerge in these neurons, and how it is brought about by the steady-state residual ("window") component of IT, the low-threshold Ca2+ current. In particular, we present results that describe the dependence of bistability on two system parameters: the injected current and the leakage conductance. In addition, we provide a biophysical interpretation of these results by means of the properties of the electrical circuit representing the neuron membrane.

In vitro anti-HIV activity of biflavonoids isolated from Rhus succedanea and Garcinia multiflora.

Eleven biflavonoids, including amentoflavone (1), agathisflavone (2), robustaflavone (3), hinokiflavone (4), volkensiflavone (5), morelloflavone (7), rhusflavanone (9), succedaneaflavanone (10), GB-1a (11), GB-1a 7"-O-beta-glucoside (13), and GB-2a (14) isolated from Rhus succedanea and Garcinia multiflora, as well as their methyl ethers, volkensiflavone hexamethyl ether (6), morelloflavone heptamethyl ether (8), and GB-1a hexamethyl ether (12), were evaluated for their anti-HIV-1 RT activity. The results indicated that compounds 3 and 4 demonstrated similar activity against HIV-1 reverse transcriptase (RT), with IC50 values of 65 microM. Compounds 1, 2, 7, 11, and 14 were moderately active against HIV-1 RT, with IC50 values of 119 microM, 100 microM, 116 microM, 236 microM, and 170 microM, respectively. Morelloflavone (7) also demonstrated significant antiviral activity against HIV-1 (strain LAV-1) in phytohemagglutinin-stimulated primary human peripheral blood mononuclear cells at an EC50 value of 6.9 microM and a selectivity index value of approximately 10. The other biflavonoids were either weakly active, inactive, or not selective against HIV-1 in human lymphocytes.

The 'window' component of the low threshold Ca2+ current produces input signal amplification and bistability in cat and rat thalamocortical neurones.

1. The mechanism underlying a novel form of input signal amplification and bistability was investigated by intracellular recording in rat and cat thalamocortical (TC) neurones maintained in slices and by computer simulation with a biophysical model of these neurones. 2. In a narrow membrane potential range centred around -60 mV, TC neurones challenged with small (10-50 pA), short (50-200 ms) current steps produced a stereotyped, large amplitude hyperpolarization (> 20 mV) terminated by the burst firing of action potentials, leading to amplification of the duration and amplitude of the input signal, that is hereafter referred to as input signal amplification. 3. In the same voltage range centred around -60 mV, single evoked EPSPs and IPSPs also produced input signal amplification, indicating that this behaviour can be triggered by physiologically relevant stimuli. In addition, a novel, intrinsic, low frequency oscillation, characterized by a peculiar voltage dependence of its frequency and by the presence of plateau potentials on the falling phase of low threshold Ca2+ potentials, was recorded. 4. Blockade of pure Na+ and K+ currents by tetrodotoxin (1 microM) and Ba2+ (0.1-2.0 mM), respectively, did not affect input signal amplification, neither did the presence of excitatory or inhibitory amino acid receptor antagonists in the perfusion medium. 5. A decrease in [Ca2+]o (from 2 to 1 mM) and an increase in [Mg2+]o (from 2 to 10 mM), or the addition of Ni2+ (2-3 mM), abolished input signal amplification, while an increase in [Ca2+]o (from 2 to 8 mM) generated this behaviour in neurones where it was absent in control conditions. These results indicate the involvement of the low threshold Ca2+ current (IT) in input signal amplification, since the other Ca2+ currents of TC neurones are activated at potentials more positive than -40 mV. 6. Blockade of the slow inward mixed cationic current (Ih) by 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium++ + chloride (ZD 7288)(100-300 microM) did not affect the expression of the large amplitude hyperpolarization, but abolished the subsequent repolarization to the original membrane potential. In this condition, therefore, input signal amplification was replaced by bistable membrane behaviour, where two stable membrane potentials separated by 15-30 mV could be switched between by small current steps. 7. Computer simulation with a model of a TC neurone, which contained only IT, Ih, K+ leak current (ILeak) and those currents responsible for action potentials, accurately reproduced the qualitative and quantitative properties of input signal amplification, bistability and low frequency oscillation, and indicated that these phenomena will occur at some value of the injected DC if, and only if, the 'window' component of IT (IT,Window) and the leak conductance (gLeak) satisfy the relation (dIT,Window/dV)max > gLeak. 8. The physiological implications of these findings for the electroresponsiveness of TC neurones are discussed, and, as IT is widely expressed in the central nervous system, we suggest that 'window' IT will markedly affect the integrative properties of many neurones.

On the nature of anomalous rectification in thalamocortical neurones of the cat ventrobasal thalamus in vitro.

1. Intracellular sharp electrode current clamp and discontinuous single electrode voltage clamp recordings were made from thalamocortical neurones (n = 57) of the cat ventrobasal thalamus in order to investigate the mechanism underlying anomalous rectification. 2. Under current clamp conditions, voltage-current (V-I) relationships in a potential range of -55 to -110 mV demonstrated anomalous rectification with two components: fast rectification, which controlled the peak of negative voltage deviations, and time-dependent rectification. Time-dependent rectification was apparent as a depolarizing sag generated during the course of negative voltage deviations, was first formed at potentials in the range -60 to -70 mV, and was sensitive to 3 mM Cs+ (n = 6). Similarly, under voltage clamp conditions, instantaneous and steady-state I-V relationships demonstrated anomalous rectification. A slowly activating inward current with an activation threshold in the range of -65 to -70 mV formed time-dependent rectification. This current was sensitive to Cs+ (3 mM) (n = 3) and had properties similar to the slow inward mixed cationic current (Ih). 3. 4-(N-Ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium++ + chloride (ZD 7288) (100-300 microM) irreversibly blocked time-dependent rectification mediated by Ih (n = 23 of 25 neurones), and led to a hyperpolarization of the resting membrane potential (6.8 +/- 0.5 mV). In the presence of ZD 7288, V-I and I-V relationships, exhibited fast anomalous rectification, first activated from potential more negative than -80 mV. 4. Ba2+ (100 microM) (n = 8), in the continuous presence of ZD 7288, reversibly linearized peak V-I and instantaneous I-V relationships over a potential range of -70 to -120 mV, and led to a membrane depolarization (13.3 +/- 4.2 mV) or tonic inward current (192 +/- 36 pA). 5. The co-application of ZD 7288 and Ba2+ revealed a depolarizing sag in negative voltage deviations under current clamp conditions, or a large inward current with kinetics two to three times slower than those of Ih under voltage clamp conditions. This novel form of time-dependent rectification was first apparent at potentials more negative than about -85 mV, was sensitive to 5 mM Cs+ (n = 4), and is termed Ih,slow. Ih,slow tail currents reversed between -65.3 and -56.6 mV (with potassium acetate electrodes, n = 3) or -57.6 and -50.3 mV (with KCl electrodes, n = 3). 6. Computer simulations confirmed that the pattern of anomalous rectification in thalamocortical neurones of the cat ventrobasal thalamus is mediated by the concerted action of Ih and a Ba(2+)-sensitive current with properties similar to an inwardly rectifying K+ current (IKIR).