Reversion mutation of cDNA CA8-204 minigene construct produces a truncated functional peptide that regulates calcium release in vitro and produces profound analgesia in vivo

Mammalian Genome - Intracellular calcium is critical in orchestrating neuronal excitability and analgesia. Carbonic anhydrase-8 (CA8) regulates intracellular calcium signaling through allosteric...     

Sympatric diversification vs. immigration: deciphering host‐plant specialization in a polyphagous insect,the stolbur phytoplasma vector Hyalesthes obsoletus (Cixiidae)

The epidemiology of vector transmitted plant diseases is highly influenced by dispersal and the host‐plant range of the vector. Widening the vector's host range may increase transmission potential, whereas specialization may induce specific disease cycles. The process leading to a vector's host shift and its epidemiological outcome is therefore embedded in the frameworks of sympatric evolution vs. immigration of preadapted populations. In this study, we analyse whether a host shift of the stolbur phytoplasma vector, Hyalesthes obsoletus from field bindweed to stinging nettle in its northern distribution range evolved sympatrically or by immigration. The exploitation of stinging nettle has led to outbreaks of the grapevine disease bois noir caused by a stinging nettle‐specific phytoplasma strain. Microsatellite data from populations from northern and ancestral ranges provide strong evidence for sympatric host‐race evolution in the northern range: Host‐plant associated populations were significantly differentiated among syntopic sites (0.054 < F_HT < 0.098) and constant over 5 years. While gene flow was asymmetric from the old into the predicted new host race, which had significantly reduced genetic diversity, the genetic identity between syntopic host‐race populations in the northern range was higher than between these populations and syntopic populations in ancestral ranges, where there was no evidence for genetic host races. Although immigration was detected in the northern field bindweed population, it cannot explain host‐race diversification but suggests the introduction of a stinging nettle‐specific phytoplasma strain by plant‐unspecific vectors. The evolution of host races in the northern range has led to specific vector‐based bois noir disease cycles.     

Looking inside the box: using Raman microspectroscopy to deconstruct microbial biomass stoichiometry one cell at a time

Stoichiometry of microbial biomass is a key determinant of nutrient recycling in a wide variety of ecosystems. However, little is known about the underlying causes of variance in microbial biomass stoichiometry. This is primarily because of technological constraints limiting the analysis of macromolecular composition to large quantities of microbial biomass. Here, we use Raman microspectroscopy (MS), to analyze the macromolecular composition of single cells of two species of bacteria grown on minimal media over a wide range of resource stoichiometry. We show that macromolecular composition, determined from a subset of identified peaks within the Raman spectra, was consistent with macromolecular composition determined using traditional analytical methods. In addition, macromolecular composition determined by Raman MS correlated with total biomass stoichiometry, indicating that analysis with Raman MS included a large proportion of a cell''s total macromolecular composition. Growth phase (logarithmic or stationary), resource stoichiometry and species identity each influenced each organism''s macromolecular composition and thus biomass stoichiometry. Interestingly, the least variable peaks in the Raman spectra were those responsible for differentiation between species, suggesting a phylogenetically specific cellular architecture. As Raman MS has been previously shown to be applicable to cells sampled directly from complex environments, our results suggest Raman MS is an extremely useful application for evaluating the biomass stoichiometry of environmental microorganisms. This includes the ability to partition microbial biomass into its constituent macromolecules and increase our understanding of how microorganisms in the environment respond to resource heterogeneity.     

Selective enrichment and molecular characterization of a previously uncultured Nitrospira-like bacterium from activated sludge

Previously uncultured nitrite-oxidizing bacteria affiliated to the genus Nitrospira have for the first time been successfully enriched from activated sludge from a municipal wastewater treatment plant. During the enrichment procedure, the abundance of the Nitrospira-like bacteria increased to approximately 86% of the total bacterial population. This high degree of purification was achieved by a novel enrichment protocol, which exploits physiological features of Nitrospira-like bacteria and includes the selective repression of coexisting Nitrobacter cells and heterotrophic contaminants by application of ampicillin in a final concentration of 50 microg ml(-1). The enrichment process was monitored by electron microscopy, fluorescence in situ hybridization (FISH) with rRNA-targeted probes and fatty acid profiling. Phylogenetic analysis of 16S rRNA gene sequences revealed that the enriched bacteria represent a novel Nitrospira species closely related to uncultured Nitrospira-like bacteria previously found in wastewater treatment plants and nitrifying bioreactors. The enriched strain is provisionally classified as 'Candidatus Nitrospira defluvii'.     

Rib lesions in skeletons from early neolithic sites in Central Germany: On the trail of tuberculosis at the onset of agriculture

As an infectious disease, tuberculosis (TB) is one of the major causes of death worldwide. Paleopathological and paleomicrobiological studies indicate a long standing association of the causative agent Mycobacterium tuberculosis and its human host. Since the occurrence and the epidemic spread of this pathogen seem to be closely linked to social and biological factors, it is of particular interest to understand better the role of TB during periods of social and nutritional change such as the Neolithic. In this study, 118 individuals from three sites in Saxony‐Anhalt (Germany) dating to the Linear Pottery Culture (5400–4800 BC) were examined macroscopically to identify TB related bone lesions. In two individuals, Pott's disease was detected. In addition, periosteal reactions of varying degrees and frequency were observed mainly along the neck of the ribs in 6.5% (2/31) of subadults and 35.1% (20/57) of adults, with one site standing out markedly. Rib lesions, however, are not specific indicators of TB as they can also be caused by other diseases; so additional investigations were undertaken using histology and micro‐CT scans to say more about the disease process. Supplementary molecular analyses indicate the presence of pathogens belonging to the Mycobacterium tuberculosis complex in individuals of all sites. Furthermore, we discuss the occurrence and spread of TB during the Neolithic with regard to nutritional aspects and possible risks of infection. The data presented provide important insights into the health status of Early Neolithic populations in Central Germany. Am J Phys Anthropol, 2012. © 2012 Wiley Periodicals, Inc.     

Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity

Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary. Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da. Using genome-wide linkage analyses, we discovered an association between nerve-injury-induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7. Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain.     

Unexpected diversity of chlorite dismutases: a catalytically efficient dimeric enzyme from Nitrobacter winogradskyi

Chlorite dismutase (Cld) is a unique heme enzyme catalyzing the conversion of ClO(2)(-) to Cl(-) and O(2). Cld is usually found in perchlorate- or chlorate-reducing bacteria but was also recently identified in a nitrite-oxidizing bacterium of the genus Nitrospira. Here we characterized a novel Cld-like protein from the chemolithoautotrophic nitrite oxidizer Nitrobacter winogradskyi which is significantly smaller than all previously known chlorite dismutases. Its three-dimensional (3D) crystal structure revealed a dimer of two identical subunits, which sharply contrasts with the penta- or hexameric structures of other chlorite dismutases. Despite a truncated N-terminal domain in each subunit, this novel enzyme turned out to be a highly efficient chlorite dismutase (K(m) = 90 μM; k(cat) = 190 s(-1); k(cat)/K(m) = 2.1 × 10(6) M(-1) s(-1)), demonstrating a greater structural and phylogenetic diversity of these enzymes than was previously known. Based on comparative analyses of Cld sequences and 3D structures, signature amino acid residues that can be employed to assess whether uncharacterized Cld-like proteins may have a high chlorite-dismutating activity were identified. Interestingly, proteins that contain all these signatures and are phylogenetically closely related to the novel-type Cld of N. winogradskyi exist in a large number of other microbes, including other nitrite oxidizers.     

Multiphasic Morphine Modulation of Substance P Release from Capsaicin-Sensitive Primary Afferent Fibers

Morphine produces a multiphasic modulation of K⁺-evoked substance P release from trigeminal slices and dorsal root ganglion neurons in culture. We now found that the C-fiber stimulant, capsaicin (1 M), evoked release of substance P that was inhibited, enhanced and inhibited by 0.1 nM, 1 M, and 10 M morphine, respectively. This morphine's multiphasic effect was blocked by naloxone (100 nM). Neonatal treatment with capsaicin produced thermal hypoalgesia and abolished the multiphasic effect of morphine on substance P release evoked by 50 mM K⁺. These findings suggest that the multiphasic modulation of substance P release by morphine is dependent on C-type afferents and may be of relevance to nociception.     

Carbonic Anhydrase-8 Regulates Inflammatory Pain by Inhibiting the ITPR1-Cytosolic Free Calcium Pathway

Calcium dysregulation is causally linked with various forms of neuropathology including seizure disorders, multiple sclerosis, Huntington’s disease, Alzheimer’s, spinal cerebellar ataxia (SCA) and chronic pain. Carbonic anhydrase-8 (Car8) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates intracellular calcium release fundamental to critical cellular functions including neuronal excitability, neurite outgrowth, neurotransmitter release, mitochondrial energy production and cell fate. In this report we test the hypothesis that Car8 regulation of ITPR1 and cytoplasmic free calcium release is critical to nociception and pain behaviors. We show Car8 null mutant mice (MT) exhibit mechanical allodynia and thermal hyperalgesia. Dorsal root ganglia (DRG) from MT also demonstrate increased steady-state ITPR1 phosphorylation (pITPR1) and cytoplasmic free calcium release. Overexpression of Car8 wildtype protein in MT nociceptors complements Car8 deficiency, down regulates pITPR1 and abolishes thermal and mechanical hypersensitivity. We also show that Car8 nociceptor overexpression alleviates chronic inflammatory pain. Finally, inflammation results in downregulation of DRG Car8 that is associated with increased pITPR1 expression relative to ITPR1, suggesting a possible mechanism of acute hypersensitivity. Our findings indicate Car8 regulates the ITPR1-cytosolic free calcium pathway that is critical to nociception, inflammatory pain and possibly other neuropathological states. Car8 and ITPR1 represent new therapeutic targets for chronic pain.     

Population Genomic Analysis of Ancient and Modern Genomes Yields New Insights into the Genetic Ancestry of the Tyrolean Iceman and the Genetic Structure of Europe

Genome sequencing of the 5,300-year-old mummy of the Tyrolean Iceman, found in 1991 on a glacier near the border of Italy and Austria, has yielded new insights into his origin and relationship to modern European populations. A key finding of that study was an apparent recent common ancestry with individuals from Sardinia, based largely on the Y chromosome haplogroup and common autosomal SNP variation. Here, we compiled and analyzed genomic datasets from both modern and ancient Europeans, including genome sequence data from over 400 Sardinians and two ancient Thracians from Bulgaria, to investigate this result in greater detail and determine its implications for the genetic structure of Neolithic Europe. Using whole-genome sequencing data, we confirm that the Iceman is, indeed, most closely related to Sardinians. Furthermore, we show that this relationship extends to other individuals from cultural contexts associated with the spread of agriculture during the Neolithic transition, in contrast to individuals from a hunter-gatherer context. We hypothesize that this genetic affinity of ancient samples from different parts of Europe with Sardinians represents a common genetic component that was geographically widespread across Europe during the Neolithic, likely related to migrations and population expansions associated with the spread of agriculture.