Further delineation of hydrophobic binding sites in dopamine D2/D3 receptors for N-4 substituents on the piperazine ring of the hybrid template 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol

Here we report a structure–activity relationship (SAR) study of analogues of 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol. Our SAR is focused on introduction of various substitutions in the piperazine ring of the hybrid template. The goal behind this study is to delineate the nature of the binding pocket for N-aryl substitution in the piperazine ring by observing the effect of various hydrophobic and other heteroaromatic substitutions on binding affinity (K_i), as measured with tritiated spiperone and HEK-293 cells expressing either D₂ or D₃ receptors. Functional activity of selected compounds was assessed with the GTPγS binding assay. Compound 8d was the most selective for the D₃ receptor in the spiperone binding assay. An interesting similarity in binding affinity was observed between isoquinoline derivative D-301 and the 2-substituted pyridine derivative 8d, suggesting the importance of relative spatial relationships between the N-atom of the ligand and the molecular determinants of the binding pocket in D₂/D₃ receptors. Functional activity assays demonstrated high potency and selectivity of (+)-8a and (?)-28b (D₂/D₃ (ratio of EC₅₀): 105 and 202, respectively) for the D₃ receptor and both compounds were more selective compared to the reference drug ropinirole (D₂/D₃ (ratio of EC₅₀): 29.5).     

Comparison of different DNA-extraction techniques to investigate the bacterial community of marine copepods

Marine zooplanktic organisms, such as copepods, are usually associated with large numbers of bacteria. Some of these bacteria live attached to copepods’ exoskeleton, while others prevail in their intestine and faecal pellets. Until now, general conclusions concerning the identity of these bacteria are problematic since the majority of previous studies focused on cultivable bacteria only. Hence, to date little is known on whether copepod genera or species harbour distinct bacterial populations and about the nature of this association. To shed more light on these copepod/bacteria consortia, the focus of this study was the development and evaluation of a suitable approach to extract bacterial DNA from different North Sea copepod genera. Furthermore, the bacterial DNA was analysed by PCR-DGGE and subsequent sequencing of excised bands. The result of this work was an appropriate extraction method for batches of ten to one copepod specimens and offered first insights as to which bacteria are attached to the copepods Acartia sp. and Temora sp. from Helgoland Roads (German Bight) and a laboratory-grown Acartia tonsa culture. It revealed the prevalence of Alphaproteobacteria.     

Rationale and design of EXPLORE: a randomized, prospective, multicenter trial investigating the impact of recanalization of a chronic total occlusion on left ventricular function in patients after primary percutaneous coronary intervention for acute ST-elevation myocardial infarction

Background

Hypothyroidism is a prevalent endocrine condition. Individuals with this disease are commonly managed through supplementation with synthetic thyroid hormone, with the aim of alleviating symptoms and restoring normal thyroid stimulating hormone levels. Generally this management strategy is effective and well tolerated. However, there is research to suggest that a significant proportion of hypothyroid sufferers are being inadequately managed. Furthermore, hypothyroid patients are more likely to have a decreased sense of well-being and more commonly experience constitutional and neuropsychiatric complaints, even with pharmacological intervention. The current management of hypothyroidism follows a biomedical model. Little consideration has been given to a biopsychosocial approach to this condition. Within the chiropractic profession there is growing support for the use of a biopsychosocial-based intervention called Neuro-Emotional Technique (NET) for this population.

Methods/Design

A placebo-controlled, single-blinded, randomised clinical pilot-trial has been designed to assess the influence of Neuro-Emotional Technique on a population with primary overt hypothyroidism. A sample of 102 adults (≥18 years) who meet the inclusion criteria will be randomised to either a treatment group or a placebo group. Each group will receive ten treatments (NET or placebo) over a six week period, and will be monitored for six months. The primary outcome will involve the measurement of depression using the Depression, Anxiety and Stress Scale (DASS). The secondary outcome measures to be used are; serum thyroid stimulating hormone, serum free-thyroxine, serum free-triiodothyronine, serum thyroid peroxidase auto-antibodies, serum thyroglobulin auto-antibodies as well as the measurement of functional health and well-being using the Short-Form-36 Version 2. The emotional states of anxiety and stress will be measured using the DASS. Self-measurement of basal heart rate and basal temperature will also be included among the secondary outcome measures. The primary and secondary measures will be obtained at commencement, six weeks and six months. Measures of basal heart rate and basal temperature will be obtained daily for the six month trial period, with recording to commence one week prior to the intervention.

Discussion

The study will provide information on the influence of NET when added to existing management regimens in individuals with primary overt hypothyroidism.

Trial Registration

ANZCTR Number: 12607000040460     

Quantitative cortical synapse proteomics of a transgenic migraine mouse model with mutated CaV2.1 calcium channels

Familial hemiplegic migraine type 1 (FHM1) is caused by missense mutations in the CACNA1A gene that encodes the α1A pore‐forming subunit of Ca_V2.1 Ca²⁺ channels. Knock‐in (KI) transgenic mice expressing Ca_V2.1 Ca²⁺ channels with a human pathogenic FHM1 mutation reveal enhanced glutamatergic neurotransmission in the cortex. In this study, we employed an iTRAQ‐based LC‐LC MS/MS approach to identify differentially expressed proteins in cortical synapse proteomes of Cacna1a R192Q KI and wild‐type mice. All expression differences determined were subtle and in the range of 10–30%. Observed upregulated proteins in the mutant mice are involved in processes, such as neurite outgrowth and actin dynamics, vesicle turnover, and glutamate transporters. Our data support the view that in Cacna1a R192Q KI mice, several compensatory mechanisms counterbalancing a dysregulated glutamatergic signaling have come into effect. We propose that such adaptation mechanisms at the synapse level may play a role in the pathophysiology of FHM and possibly in the common forms of migraine.     

Mitochondrial dysfunction: A potential link between neuroinflammation and neurodegeneration?

Dysfunctional mitochondria are thought to play a cardinal role in the pathogenesis of various neurological disorders, such as multiple sclerosis, Alzheimer’s disease, Parkinson’s disease and stroke. In addition, neuroinflammation is a common denominator of these diseases. Both mitochondrial dysfunction and neuroinflammatory processes lead to increased production of reactive oxygen species (ROS) which are detrimental to neurons. Therefore, neuroinflammation is increasingly recognized to contribute to processes underlying neurodegeneration. Here we describe the involvement of mitochondrial (dys)function in various neurological disorders and discuss the putative link between mitochondrial function and neuroinflammation.     

Dynamics of light-induced activation in the PAS domain proteins LOV2 and PYP probed by time-resolved tryptophan fluorescence

Light-induced activation of the LOV2-Jα domain of the photoreceptor phototropin from oat is believed to involve the detachment of the Jα helix from the central β-sheet and its subsequent unfolding. The dynamics of these conformational changes were monitored by time-resolved emission spectroscopy with 100 ns time resolution. Three transitions were detected during the LOV2-Jα photocycle with time constants of 3.4 μs, 500 μs, and 4.3 ms. The fastest transition is due to the decay of the flavin phosphorescence in the transition of the triplet LOV(L)(660) state to the singlet LOV(S)(390) signaling state. The 500 μs and 4.3 ms transitions are due to changes in tryptophan fluorescence and may be associated with the dissociation and unfolding of the Jα helix, respectively. They are absent in the transient absorption signal of the flavin chromophore. The tryptophan fluorescence signal monitors structural changes outside the chromophore binding pocket and indicates that there are at least three LOV(S)(390) intermediates. Since the 500 μs and 4.3 ms components are absent in a construct without the Jα helix and in the mutant W557S, the fluorescence signal is mainly due to tryptophan 557. The kinetics of the main 500 μs component is strongly temperature dependent with activation energy of 18.2 kcal/mol suggesting its association with a major structural change. In the structurally related PAS domain protein PYP the N-terminal cap dissociates from the central β-sheet and unfolds upon signaling state formation with a similar time constant of ～1 ms. Using transient fluorescence we obtained a nearly identical activation energy of 18.5 kcal/mol for this transition.     

Pennisetum setaceum or Pennisetum advena cultivars,what ornamental do we have in our garden

Pennisetum Rich. or following recent taxonomic insights Cenchrus L. is a genus with some 120 species worldwide, especially in warm areas. The genus includes some crops, some ornamentals but mostly species that are considered weedy. The name of one of the weedy species Pennisetum setaceum (Forssk.) Chiov. is also found on labels of ornamental grasses as P. setaceum “Rubrum.” It has been debated to belong to a species on its own Pennisetum advena Wipff & Veldkamp or Cenchrus advena (Wipff & Veldkamp) Morrone, only known from cultivation, whereas others still adhere to a broader species concept of P. setaceum. The recent inclusion of P. setaceum on the EU List of Union concern has revitalized the discussion on this issue for commercial reasons. Based on a morphological and molecular comparison (ITS, rbcL, and the trnh‐psbA intergenic spacer sequences) of the type specimen of P. advena, five of its “cultivars” in trade and collections of P. setaceum from different regions of the world we conclude that plants currently in trade in Western Europe belong to a separate species P. advena. A drooping inflorescence is consistent as is the difference in width of the leaf blade, the leaf blade being flat or involute, the central vein being swollen or not, and the length of the stipe being 0.3–1.1 mm in P. advena and 1.1–3.1 mm in P. setaceum. On the chloroplast markers rbcL and trnH‐psbA, the species consistently differ in 2 and 4 base pairs, respectively. On the nuclear ITS sequence, there is only 90% overlap between the two species. This justifies these ornamentals to be excluded from the List of Union concern of EU regulation 1143/2014.     

Influence of HLA Class I and HLA-KIR Compound Genotypes on HIV-2 Infection and Markers of Disease Progression in a Manjako Community in West Africa

Overall, the time to AIDS after HIV-2 infection is longer than with HIV-1, and many individuals infected with HIV-2 virus remain healthy throughout their lives. Multiple HLA and KIR gene products have been implicated in the control of HIV-1, but the effect of variation at these loci on HIV-2 disease is unknown. We show here for the first time that HLA-B*1503 is associated significantly with poor prognosis after HIV-2 infection and that HLA-B*0801 is associated with susceptibility to infection. Interestingly, previous data indicate that HLA-B*1503 is associated with low viral loads in HIV-1 clade B infection but has no significant effect on viral load in clade C infection. In general, alleles strongly associated with HIV-1 disease showed no effect in HIV-2 disease. These data emphasize the unique nature of the effects of HLA and HLA/KIR combinations on HIV-2 immune responses relative to HIV-1, which could be related to their distinct clinical course.Since the first report of this virus in 1986, HIV-2 remains largely confined to West Africa (11). It shares between 30 and 60% nucleotide and amino acid homology with HIV-1 but differs greatly in pathogenicity and transmissibility (20). Studies on HIV-2 patients across West Africa have shown that some people remain uninfected despite repeated exposure (36), and a substantial proportion of infected people remain relatively healthy for a very long time with low plasma viral load and normal CD4⁺ T-cell counts, a characteristic of long-term nonprogressors (LTNPs) infected with HIV-1 (37). This is perhaps a reflection of an effective immune response mounted against the virus, including a vigorous CD8⁺ T-cell response (28), maintenance of HIV-specific CD4⁺ T-cell function (15), and the presence of a strong neutralizing antibody response in many subjects (4), features that are highly desirable for a successful HIV-1 vaccine. Thus, HIV-2 disease course provides a natural model for investigating mechanisms that control HIV infection, and a better understanding of these mechanisms might inform new strategies for HIV prevention and treatment.HLA class I molecules present antigenic epitopes to cytotoxic T cells and are central to the acquired immune response. A number of associations between HLA class I alleles and HIV disease outcomes have been reported (10), the most consistent being B*57 and B*27, which show strong protection across studies, and certain subtypes of B*35, which associate with more rapid progression (19). While several mother-infant studies have implicated sharing of certain HLA alleles in transmission of the virus from mother to infant (29, 30), there is no convincing data that particular HLA class I alleles protect against HIV infection in general.HLA class I allotypes also serve as ligands for killer cell immunoglobulin-like receptors (KIRs), which modulate natural killer (NK) cell function. KIRs are structurally similar to one another and can be divided into activating and inhibitory receptors. NK cells are key components of the innate immune system and constantly survey host cell surfaces for appropriate levels of HLA class I molecules through a network of NK cell receptors, including KIRs (26). Upon engagement with their ligand, inhibitory KIR suppress NK cell activity, but if the ligand is missing or has been downregulated on target cells, the threshold for NK cell activation is lowered, thus allowing for activation signals to dominate (23).HLA and KIR genes are found on chromosomes 6 and 19, respectively, so they segregate independently. As such, the genes/alleles for the corresponding receptor-ligand pair must be present to confer functionality, whereas presence of one without the other results in a null phenotype. A number of HLA and KIR gene products either individually or collectively has been implicated in the control of HIV-1 (9), but nothing is known of their role in HIV-2.Epidemiological data from Caio and other cohorts in West Africa (3, 39) indicate that HIV-2 infection in a substantial proportion of infected individuals is compatible with normal survival and without signs of immunodeficiency, suggesting distinct viral pathogenic mechanisms and protective host factors against HIV-2 relative to HIV-1. Here, we determined the HLA class I and KIR gene profiles of the Caio population (>95% Manjako) from Guinea-Bissau and investigated their effects on susceptibility to HIV-2 infection and disease progression.     

Quantitative understanding of the energy transfer between fluorescent proteins connected via flexible peptide linkers

The fusion of different protein domains via peptide linkers is a powerful, modular approach to obtain proteins with new functions. A detailed understanding of the conformational behavior of peptide linkers is important for applications such as fluorescence resonance energy transfer (FRET)-based sensor proteins and multidomain proteins involved in multivalent interactions. To investigate the conformational behavior of flexible glycine- and serine-containing peptide linkers, we constructed a series of fusion proteins of enhanced cyan and yellow fluorescent proteins (ECFP-linker-EYFP) in which the linker length was systematically varied by incorporating between 1 and 9 GGSGGS repeats. As expected, both steady-state and time-resolved fluorescence measurements showed a decrease in energy transfer with increasing linker length. The amount of energy transfer observed in these fusion proteins can be quantitatively understood by simple models that describe the flexible linker as a worm-like chain with a persistence length of 4.5 A or a Gaussian chain with a characteristic ratio of 2.3. The implications of our results for understanding the properties of FRET-based sensors and other fusion proteins with Gly/Ser linkers are discussed.