Defective Mitochondrial Function In Vivo in Skeletal Muscle in Adults with Down’s Syndrome: A 31P-MRS Study

Down’s syndrome (DS) is a developmental disorder associated with intellectual disability (ID). We have previously shown that people with DS engage in very low levels of exercise compared to people with ID not due to DS. Many aspects of the DS phenotype, such as dementia, low activity levels and poor muscle tone, are shared with disorders of mitochondrial origin, and mitochondrial dysfunction has been demonstrated in cultured DS tissue. We undertook a phosphorus magnetic resonance spectroscopy (³¹P-MRS) study in the quadriceps muscle of 14 people with DS and 11 non-DS ID controls to investigate the post-exercise resynthesis kinetics of phosphocreatine (PCr), which relies on mitochondrial respiratory function and yields a measure of muscle mitochondrial function in vivo. We found that the PCr recovery rate constant was significantly decreased in adults with DS compared to non-DS ID controls (1.7±0.1 min⁻¹ vs 2.1±0.1 min⁻¹ respectively) who were matched for physical activity levels, indicating that muscle mitochondrial function in vivo is impaired in DS. This is the first study to investigate mitochondrial function in vivo in DS using ³¹P-MRS. Our study is consistent with previous in vitro studies, supporting a theory of a global mitochondrial defect in DS.     

Altered Antibody Profiles against Common Infectious Agents in Chronic Disease

Despite the important diagnostic value of evaluating antibody responses to individual human pathogens, antibody profiles against multiple infectious agents have not been used to explore health and disease mainly for technical reasons.  We hypothesized that the interplay between infection and chronic disease might be revealed by profiling antibodies against multiple agents. Here, the levels of antibodies against a panel of 13 common infectious agents were evaluated with the quantitative Luciferase Immunoprecipitation Systems (LIPS) in patients from three disease cohorts including those with pathogenic anti-interferon-γ autoantibodies (IFN-γ AAB), HIV and Sjögren’s syndrome (SjS) to determine if their antibody profiles differed from control subjects.  The IFN-γ AAB patients compared to controls demonstrated statistically higher levels of antibodies against VZV (p=0.0003), EBV (p=0.002), CMV (p=0.003), and C. albicans (p=0.03), but lower antibody levels against poliovirus (p=0.04). Comparison of HIV patients with blood donor controls revealed that the patients had higher levels of antibodies against CMV (p=0.0008), HSV-2 (p=0.0008), EBV (p=0.001), and C. albicans (p=0.01), but showed decreased levels of antibodies against coxsackievirus B4 (p=0.0008), poliovirus (p=0.0005),   and HHV-6B (p=0.002). Lastly, SjS patients had higher levels of anti-EBV antibodies (p=0.03), but lower antibody levels against several enteroviruses including a newly identified picornavirus, HCoSV-A (p=0.004), coxsackievirus B4 (p=0.04), and poliovirus (p=0.02). For the IFN-γ AAB and HIV cohorts, principal component analysis revealed unique antibody clusters that showed the potential to discriminate patients from controls.  The results suggest that antibody profiles against these and likely other common infectious agents may yield insight into the interplay between exposure to infectious agents, dysbiosis, adaptive immunity and disease activity.     

Integrins regulate centrosome integrity and astrocyte polarization following a wound

In response to a wound, astrocytes in culture extend microtubule‐rich processes and polarize, orienting their centrosomes and Golgi apparatus woundside. β1 Integrin null astrocytes fail to extend processes toward the wound, and are disoriented, and often migrate away orthogonal, to the wound. The centrosome is unusually fragmented in β1 integrin null astrocytes. Expression of a β1 integrin cDNA in the null background yields cells with intact centrosomes that polarize and extend processes normally. Fragmented centrosomes rapidly assemble following integrin ligation and cell attachment. However, several experiments indicated that cell adhesion is not necessary. For example, astrocytes in suspension expressing a chimeric β1 subunit that can be activated by an antibody assemble centrosomes suggesting that β1 activation is sufficient to cause centrosome assembly in the absence of cell adhesion. siRNA knockdown of PCM1, a major centrosomal protein, inhibits cell polarization, consistent with the notion that centrosomes are necessary for polarity and that integrins regulate polarity via centrosome integrity. Screening inhibitors of molecules downstream of integrins indicate that neither FAK nor ILK is involved in regulation of centrosome integrity. In contrast, blebbistatin, a specific inhibitor of non‐muscle myosin II (NMII), mimics the response of β1 integrin null astrocytes by disrupting centrosome integrity and cell polarization. Blebbistatin also inhibits integrin‐mediated centrosome assembly in astrocytes attaching to fibronectin, consistent with the hypothesis that NMII functions downstream of integrins in regulating centrosome integrity. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 73: 333–353, 2013.     

Association between Ocular Bacterial Carriage and Follicular Trachoma Following Mass Azithromycin Distribution in The Gambia

Background

Trachoma, caused by ocular Chlamydia trachomatis infection, is the leading infectious cause of blindess, but its prevalence is now falling in many countries. As the prevalence falls, an increasing proportion of individuals with clinical signs of follicular trachoma (TF) is not infected with C. trachomatis. A recent study in Tanzania suggested that other bacteria may play a role in the persistence of these clinical signs.

Methodology/Principal Findings

We examined associations between clinical signs of TF and ocular colonization with four pathogens commonly found in the nasopharnyx, three years after the initiation of mass azithromycin distribution. Children aged 0 to 5 years were randomly selected from 16 Gambian communitites. Both eyes of each child were examined and graded for trachoma according to the World Health Organization (WHO) simplified system. Two swabs were taken from the right eye: one swab was processed for polymerase chain reaction (PCR) using the Amplicor test for detection of C. trachomatis DNA and the second swab was processed by routine bacteriology to assay for the presence of viable Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus and Moraxella catarrhalis. Prevalence of TF was 6.2% (96/1538) while prevalence of ocular C. trachomatis infection was 1.0% (16/1538). After adjustment, increased odds of TF were observed in the presence of C. trachomatis (OR = 10.4, 95%CI 1.32–81.2, p = 0.03), S. pneumoniae (OR = 2.14, 95%CI 1.03–4.44, p = 0.04) and H. influenzae (OR = 4.72, 95% CI 1.53–14.5, p = 0.01).

Conclusions/Significance

Clinical signs of TF can persist in communities even when ocular C. trachomatis infection has been controlled through mass azithromycin distribution. In these settings, TF may be associated with ocular colonization with bacteria commonly carried in the nasopharnyx. This may affect the interpretation of impact surveys and the determinations of thresholds for discontinuing mass drug administration.     

Synthesis and Evaluation of Biphenyl Compounds as Kinesin Spindle Protein Inhibitors

Kinesin spindle protein (KSP), an ATP‐dependent motor protein, plays an essential role in bipolar spindle formation during the mitotic phase (M phase) of the normal cell cycle. KSP has emerged as a novel target for antimitotic anticancer drug development. In this work, we synthesized a range of new biphenyl compounds and investigated their properties in vitro as potential antimitotic agents targeting KSP expression. Antiproliferation (MTT (=3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide)) assays, combined with fluorescence‐assisted cell sorting (FACS) and Western blot studies analyzing cell‐cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Structural variants revealed that functionalization of biphenyl compounds with bulky aliphatic or aromatic groups led to a loss of activity. However, replacement of the urea group with a thiourea led to an increase in antiproliferative activity in selected cell lines. Further studies using confocal fluorescence microscopy confirmed that the most potent biphenyl derivative identified thus far, compound 7 , exerts its pharmacologic effect specifically in the M phase and induces monoaster formation. These studies confirm that chemical scope remains for improving the potency and treatment efficacy of antimitotic KSP inhibition in this class of biphenyl compounds.     

Adhesion and motility of fouling diatoms on a silicone elastomer

Recent demands for non-toxic antifouling technologies have led to increased interest in coatings based on silicone elastomers that ‘release’ macrofouling organisms when hydrodynamic conditions are sufficiently robust. However, these types of coatings accumulate diatom slimes, which are not released even from vessels operating at high speeds ( > 30 knots). In this study, adhesion strength and motility of three common fouling diatoms (Amphora coffeaeformis var. perpusilla (Grunow) Cleve, Craspedostauros australis Cox and Navicula perminuta Grunow) were measured on a polydimethylsiloxane elastomer (PDMSE) and acid-washed glass. Adhesion of the three species was stronger to PDMSE than to glass but the adhesion strengths varied. The wall shear stress required to remove 50% of cells from PDMSE was 17 Pa for Craspedostauros, 24 Pa for Amphora and >> 53 Pa for Navicula; the corresponding values for glass were 3, 10 and 25 Pa. In contrast, the motility of the three species showed little or no correlation between the two surfaces. Craspedostauros moved equally well on glass and PDMSE, Amphora moved more on glass initially before movement ceased and Navicula moved more on PDMSE before movement ceased. The results show that fouling diatoms adhere more strongly to a hydrophobic PDMSE surface, and this feature may contribute to their successful colonization of low surface energy, foul-release coatings. The results also indicate that diatom motility is not related to adhesion strength, and motility does not appear to be a useful indicator of surface preference by diatoms.     

Attenuated PLD1 association and signalling at the H452Y polymorphic form of the 5-HT2A receptor

The 5-HT_2A receptor (5-HT_2AR) is implicated in psychotropic changes within the central nervous system (CNS). A number of polymorphisms have been reported in the 5-HT_2AR gene; one of these results in a non-synonymous change, H452Y, in the carboxy-terminal tail of the receptor protein. The minor allele (9% occurrence) has been statistically associated with CNS dysfunction such as impaired memory processing and resistance to neuroleptic treatment in schizophrenic patients. We investigated the impact of H452Y mutation of the 5-HT_2AR expressed in COS7 cells on distinctly coupled intracellular signalling pathways from the receptor, focusing on the heterotrimeric G protein-independent phospholipase D (PLD) pathway, compared to the conventional Gq/11-linked phospholipase C (PLC) pathway. The H452Y mutation selectively attenuated PLD signalling, which as in the wild-type receptor, was mediated by a molecular complex involving PLD1 docked to the receptor's carboxy-terminal tail domain. Co-immunoprecipitation and GST-fusion protein experiments revealed that the H452Y mutation selectively reduced PLD1 binding to the receptor. Experiments with blocking peptides to mimic short sections of the 5-HT_2AR tail sequence revealed that the peptide spanning residue 452 strongly reduced PLD but not PLC responses of the receptor. Similar observations were made when assessing both PLD responses and PLD-dependent cellular proliferation elicited by activation of 5-HT_2ARs natively expressed in MCF-7 cells. Overall these findings indicate that the H452Y polymorphic variant of the 5-HT_2AR displays selective disruption of its PLD signalling pathway. This may potentially play a role in the CNS dysfunction associated with the H452Y allele of the 5-HT_2AR.     

Amino acid analysis of spider dragline silk using H NMR

The amino acid composition of Nephila clavipes dragline silk fiber was determined by conducting ¹H nuclear magnetic resonance (NMR) spectroscopy experiments on acid-hydrolyzed material. N. clavipes dragline silk was found to consist of 43.0 ± 0.6% Gly, 29.3 ± 0.2% Ala, 9.1 ± 0.1% Glx, 4.0 ± 0.1% Leu, 3.3 ± 0.1% Tyr, 3.4 ± 0.2% Ser, 2.7 ± 0.1% Pro, 2.1 ± 0.1% Arg, 1.07 ± 0.05% Asx, 0.96 ± 0.05% Val, 0.48 ± 0.03% Thr, 0.35 ± 0.03% Phe, and 0.28 ± 0.03% Ile. Compared with standard chromatography-based amino acid analysis (AAA), the chemical resolution of NMR allows for an amino acid solution to be characterized without separation and is shown to provide considerably higher precision. This allows for more accurate statistics on the variability of amino acids in spider dragline silk. In general, this ¹H NMR AAA technique is applicable to a large range of proteins and peptides for precise composition characterization, especially when the precise content of a minor component is critical and relatively large amounts of sample are available (microgram to milligram quantities).     

Metapopulation Dynamics Enable Persistence of Influenza A,Including A/H5N1, in Poultry

Highly pathogenic influenza A/H5N1 has persistently but sporadically caused human illness and death since 1997. Yet it is still unclear how this pathogen is able to persist globally. While wild birds seem to be a genetic reservoir for influenza A, they do not seem to be the main source of human illness. Here, we highlight the role that domestic poultry may play in maintaining A/H5N1 globally, using theoretical models of spatial population structure in poultry populations. We find that a metapopulation of moderately sized poultry flocks can sustain the pathogen in a finite poultry population for over two years. Our results suggest that it is possible that moderately intensive backyard farms could sustain the pathogen indefinitely in real systems. This fits a pattern that has been observed from many empirical systems. Rather than just employing standard culling procedures to control the disease, our model suggests ways that poultry production systems may be modified.     

Assimilation of Diazotrophic Nitrogen into Pelagic Food Webs

The fate of diazotrophic nitrogen (N_D) fixed by planktonic cyanobacteria in pelagic food webs remains unresolved, particularly for toxic cyanophytes that are selectively avoided by most herbivorous zooplankton. Current theory suggests that N_D fixed during cyanobacterial blooms can enter planktonic food webs contemporaneously with peak bloom biomass via direct grazing of zooplankton on cyanobacteria or via the uptake of bioavailable N_D (exuded from viable cyanobacterial cells) by palatable phytoplankton or microbial consortia. Alternatively, N_D can enter planktonic food webs post-bloom following the remineralization of bloom detritus. Although the relative contribution of these processes to planktonic nutrient cycles is unknown, we hypothesized that assimilation of bioavailable N_D (e.g., nitrate, ammonium) by palatable phytoplankton and subsequent grazing by zooplankton (either during or after the cyanobacterial bloom) would be the primary pathway by which N_D was incorporated into the planktonic food web. Instead, in situ stable isotope measurements and grazing experiments clearly documented that the assimilation of N_D by zooplankton outpaced assimilation by palatable phytoplankton during a bloom of toxic Nodularia spumigena Mertens. We identified two distinct temporal phases in the trophic transfer of N_D from N. spumigena to the plankton community. The first phase was a highly dynamic transfer of N_D to zooplankton with rates that covaried with bloom biomass while bypassing other phytoplankton taxa; a trophic transfer that we infer was routed through bloom-associated bacteria. The second phase was a slowly accelerating assimilation of the dissolved-N_D pool by phytoplankton that was decoupled from contemporaneous variability in N. spumigena concentrations. These findings provide empirical evidence that N_D can be assimilated and transferred rapidly throughout natural plankton communities and yield insights into the specific processes underlying the propagation of N_D through pelagic food webs.