Microbially induced precipitation of silica by anaerobic methane-oxidizing consortia and implications for microbial fossil preservation.

Authigenic carbonate minerals can preserve biosignatures of microbial anaerobic oxidation of methane (AOM) in the rock record. It is not currently known whether the microorganisms that mediate sulfate-coupled AOM-often occurring as multicelled consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB)-are preserved as microfossils. Electron microscopy of ANME-SRB consortia in methane seep sediments has shown that these microorganisms can be associated with silicate minerals such as clays [Chen et al., Sci. Rep. 4, 1-9 (2014)], but the biogenicity of these phases, their geochemical composition, and their potential preservation in the rock record is poorly constrained. Long-term laboratory AOM enrichment cultures in sediment-free artificial seawater [Yu et al., Appl. Environ. Microbiol. 88, e02109-21 (2022)] resulted in precipitation of amorphous silicate particles (~200 nm) within clusters of exopolymer-rich AOM consortia from media undersaturated with respect to silica, suggestive of a microbially mediated process. The use of techniques like correlative fluorescence in situ hybridization (FISH), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS), and nanoscale secondary ion mass spectrometry (nanoSIMS) on AOM consortia from methane seep authigenic carbonates and sediments further revealed that they are enveloped in a silica-rich phase similar to the mineral phase on ANME-SRB consortia in enrichment cultures. Like in cyanobacteria [Moore et al., Geology 48, 862-866 (2020)], the Si-rich phases on ANME-SRB consortia identified here may enhance their preservation as microfossils. The morphology of these silica-rich precipitates, consistent with amorphous-type clay-like spheroids formed within organic assemblages, provides an additional mineralogical signature that may assist in the search for structural remnants of microbial consortia in rocks which formed in methane-rich environments from Earth and other planetary bodies.

Abundant SAR11 viruses in the ocean.

Several reports proposed that the extraordinary dominance of the SAR11 bacterial clade in ocean ecosystems could be a consequence of unusual mechanisms of resistance to bacteriophage infection, including 'cryptic escape' through reduced cell size and/or K-strategist defence specialism. Alternatively, the evolution of high surface-to-volume ratios coupled with minimal genomes containing high-affinity transporters enables unusually efficient metabolism for oxidizing dissolved organic matter in the world's oceans that could support vast population sizes despite phage susceptibility. These ideas are important for understanding plankton ecology because they emphasize the potentially important role of top-down mechanisms in predation, thus determining the size of SAR11 populations and their concomitant role in biogeochemical cycling. Here we report the isolation of diverse SAR11 viruses belonging to two virus families in culture, for which we propose the name 'pelagiphage', after their host. Notably, the pelagiphage genomes were highly represented in marine viral metagenomes, demonstrating their importance in nature. One of the new phages, HTVC010P, represents a new podovirus subfamily more abundant than any seen previously, in all data sets tested, and may represent one of the most abundant virus subfamilies in the biosphere. This discovery disproves the theory that SAR11 cells are immune to viral predation and is consistent with the interpretation that the success of this highly abundant microbial clade is the result of successfully evolved adaptation to resource competition.

Roles of KChIP1 in the regulation of GABA-mediated transmission and behavioral anxiety.

K+ channel interacting protein 1 (KChIP1) is a neuronal calcium sensor (NCS) protein that interacts with multiple intracellular molecules. Its physiological function, however, remains largely unknown. We report that KChIP1 is predominantly expressed at GABAergic synapses of a subset of parvalbumin-positive neurons in the brain. Forced expression of KChIP1 in cultured hippocampal neurons increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs), reduced paired pulse facilitation of autaptic IPSCs, and decreases potassium current density. Furthermore, genetic ablation of KChIP1 potentiated potassium current density in neurons and caused a robust enhancement of anxiety-like behavior in mice. Our study suggests that KChIP1 is a synaptic protein that regulates behavioral anxiety by modulating inhibitory synaptic transmission, and drugs that act on KChIP1 may help to treat patients with mood disorders including anxiety.

Elimination of rat spinal substance P receptor bearing neurons dissociates cardiovascular and nocifensive responses to nicotinic agonists.

Intrathecal (IT) delivery of nicotinic agonists evokes dose dependent nocifensive behavior and cardiovascular responses. Previous studies suggested that these effects may be attenuated by the loss of substance P positive (sP(+)) primary afferents. To further characterize these cell systems, we examined the effect of selectively destroying neurokinin 1 receptor bearing (NK1-r(+)) dorsal horn neurons on IT nicotinic agonist evoked responses. In the dorsal spinal cord, confocal immunohistochemical microscopy revealed that nAChR subunits (alpha3, alpha4, alpha5, beta2 and beta4), NeuN B (neuronal marker) and NK1-r were all co-expressed in the superficial dorsal horn; however alpha3, alpha5, beta2 and beta4 exhibited the highest degree of colocalization with NK1-r expressing neurons. After intrathecal substance P-saporin (sP-SAP), NK1-r(+) cell bodies and dendrites in the superficial dorsal horn were largely abolished. The greatest loss in co-expression of nAChR subunits with NK1-r was observed with alpha3, alpha5, beta2 and beta4 subunits. Following intrathecal sP-SAP, the nocifensive responses to all nicotinic agonists were reduced; however, in contrast, while cardiovascular responses evoked by IT nicotine were unaltered, IT cytisine and epibatidine exhibited enhanced tachycardia and pressor responses. These results indicate subunit-specific relationships between the NK1-r and nicotinic receptor systems. The loss of nocifensive activity after destruction of the NK1-r bearing cells in spite of the persistence of nicotinic subunits on other cells, emphasizes the importance of the superficial marginal neuron in mediating these nicotinic effects. Further, the exaggerated cardiovascular responses to cytisine following loss of NK1-r bearing cells suggest the presence of a nicotinic receptor-mediated stimulation of inhibitory circuits at the spinal level.

Automated most-probable loss tomography of thick selectively stained biological specimens with quantitative measurement of resolution improvement.

We describe the technique and application of energy filtering, automated most-probable loss (MPL) tomography to intermediate voltage electron microscopy (IVEM). We show that for thick, selectively stained biological specimens, this method produces a dramatic increase in resolution of the projections and the computed volumes versus standard unfiltered transmission electron microscopy (TEM) methods. This improvement in resolution is attributed to the reduction of chromatic aberration, which results from the large percentage of inelastic electron-scattering events for thick specimens. These improvements are particularly evident at the large tilt angles required to improve tomographic resolution in the z-direction. This method effectively increases the usable thickness of selectively stained samples that can be imaged at a given accelerating voltage by dramatically improving resolution versus unfiltered TEM and increasing signal-to-noise versus zero-loss imaging, thereby expanding the utility of the IVEM to deliver information from within specimens up to 3 microm thick.

Ablation of primary afferent terminals reduces nicotinic receptor expression and the nociceptive responses to nicotinic agonists in the spinal cord.

A variety of studies indicate that spinal nicotinic acetylcholine receptors modulate the behavioral and autonomic responses elicited by afferent stimuli. To examine the location of and role played by particular subtypes of nicotinic receptors in mediating cardiovascular and nociceptive responses, we treated neonatal and adult rats with capsaicin to destroy C-fibers in primary afferent terminals. Reduction of C-fiber terminals was ascertained by the loss of isolectin B4, CGRP and vanilloid receptors as monitored by immunofluorescence. Receptor autoradiography shows a reduction in number of epibatidine binding sites following capsaicin treatment. The reduction is particularly marked in the dorsal horn and primarily affects the class of high affinity epibatidine binding sites thought to modulate nociceptive responses. Accompanying the loss of terminals and nicotinic binding sites were significant reductions in the expression of alpha 3, alpha 4, alpha 5, beta 2 and beta 4 nicotinic receptor subunits in the superficial layers of the spinal cord as determined by antibody staining and confocal microscopy. The loss of nicotinic receptors that follows capsaicin treatment results in attenuation of the nociceptive responses to both spinal cytisine and epibatidine. Capsaicin treatment also diminishes the capacity of cytisine to desensitize nicotinic receptors mediating nociception, but it shows little effect on intrathecal nicotinic agonist elicited pressor and heart rate responses. Hence, our data suggest that alpha 3, alpha 4, alpha 5, beta 2 and beta 4 subunits of nicotinic receptors are localized in the spinal cord on primary afferent terminals that mediate nociceptive input. A variety of convergent data based on functional studies and subunit expression suggest that alpha 3 and alpha 4, in combination with beta 2 and alpha 5 subunits, form the majority of functional nicotinic receptors on C-fiber primary afferent terminals. Conversely, spinal nicotinic receptors not located on C-fibers play a primary role in the spinal pathways evoking spinally coordinated autonomic cardiovascular responses.

NF-M is an essential target for the myelin-directed "outside-in" signaling cascade that mediates radial axonal growth.

Neurofilaments are essential for acquisition of normal axonal calibers. Several lines of evidence have suggested that neurofilament-dependent structuring of axoplasm arises through an "outside-in" signaling cascade originating from myelinating cells. Implicated as targets in this cascade are the highly phosphorylated KSP domains of neurofilament subunits NF-H and NF-M. These are nearly stoichiometrically phosphorylated in myelinated internodes where radial axonal growth takes place, but not in the smaller, unmyelinated nodes. Gene replacement has now been used to produce mice expressing normal levels of the three neurofilament subunits, but which are deleted in the known phosphorylation sites within either NF-M or within both NF-M and NF-H. This has revealed that the tail domain of NF-M, with seven KSP motifs, is an essential target for the myelination-dependent outside-in signaling cascade that determines axonal caliber and conduction velocity of motor axons.

Nicotinic acetylcholine receptor distribution in relation to spinal neurotransmission pathways.

Neuronal nicotinic receptors (nAChR) are pentameric assemblies of subunits of a gene family where specified combinations of alpha and beta subunits form functional receptors. To extend our understanding of the role of spinal nAChR in the processing of sensory stimuli and regulation of autonomic and motor responses, we initiated investigations to localize nAChR subunit expression within discrete spinal regions and cell types. High-affinity epibatidine binding was present in the superficial dorsal and ventral horns, the mediolateral and central canal regions. RT-PCR identified transcripts for alpha3, alpha4, alpha5, beta2, and beta4 in both spinal cord parenchyma and dorsal root ganglia (DRG). Our affinity-purified antibodies against alpha3, alpha4, alpha5, beta2, and beta4 subunits identified specific protein bands of appropriate molecular mass (preadsorbed with the respective antigens) in specific tissues and cells that express nicotinic receptors, including the spinal cord and DRG neurons. Having established the absence of crossreactivity with related subunits, specific fluorescence labeling of nerve terminals and cell bodies was achieved and correlated with the distribution of defined marker proteins and nicotinic receptor binding sites determined autoradiographically. Our findings indicate that alpha3, alpha4, alpha5, beta2, and beta4 subunits are all expressed on primary afferents (IB4-positive terminals) in the spinal cord. The predominant presynaptic (synaptophysin colocalization) labeling is in the superficial layer of the dorsal horn. These receptor subunits, except for beta4, are also present in postsynaptic autonomic (anti-bNOS-positive) and somatic motor neurons (anti-VAChT-positive). The alpha3, alpha5, and beta2 subunits showed additional staining in glial (anti-GFAP-positive) cells. These studies reveal a dense and distinguishable distribution of nAChR subunits in the spinal cord and point toward future therapeutic targeting for specific spinal actions.

5-HT2a receptors in rat sciatic nerves and Schwann cell cultures.

Pharmacological approaches and optical recordings have shown that Schwann cells of a myelinating phenotype are activated by 5-HT upon its interaction with the 5-HT(2A) receptor (5-HT(2A)R). In order to further characterize the expression and distribution of this receptor in Schwann cells, we examined rat sciatic nerve and cultured rat Schwann cells using probes specific to 5-HT(2A)R protein mRNA. We also examined the endogenous sources of 5-HT in rat sciatic nerve by employing both histochemical stains and an antibody that specifically recognizes 5-HT. Rat Schwann cells of a myelinating phenotype contained both 5-HT(2A)R protein and mRNA. In the healthy adult rat sciatic nerve, 5-HT(2A)Rs were evenly distributed along the outermost portion of the Schwann cell plasma membrane and within the cytoplasm. The most prominent source of 5-HT was within granules of the endoneurial mast cells, closely juxtaposed to Schwann cells within myelinating sciatic nerves. These results support the hypothesis that the 5-HT receptors expressed by rat Schwann cells in vivo are activated by the release of 5-HT from neighboring mast cells.

Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein.

The recent characterization of an acetylcholine binding protein (AChBP) from the fresh water snail, Lymnaea stagnalis, shows it to be a structural homolog of the extracellular domain of the nicotinic acetylcholine receptor (nAChR). To ascertain whether the AChBP exhibits the recognition properties and functional states of the nAChR, we have expressed the protein in milligram quantities from a synthetic cDNA transfected into human embryonic kidney (HEK) cells. The protein secreted into the medium shows a pentameric rosette structure with ligand stoichiometry approximating five sites per pentamer. Surprisingly, binding of acetylcholine, selective agonists, and antagonists ranging from small alkaloids to larger peptides results in substantial quenching of the intrinsic tryptophan fluorescence. Using stopped-flow techniques, we demonstrate rapid rates of association and dissociation of agonists and slow rates for the alpha-neurotoxins. Since agonist binding occurs in millisecond time frames, and the alpha-neurotoxins may induce a distinct conformational state for the AChBP-toxin complex, the snail protein shows many of the properties expected for receptor recognition of interacting ligands. Thus, the marked tryptophan quenching not only documents the importance of aromatic residues in ligand recognition, but establishes that the AChBP will be a useful functional as well as structural surrogate of the nicotinic receptor.

Overexpression of the CT GalNAc transferase in skeletal muscle alters myofiber growth, neuromuscular structure, and laminin expression.

Carbohydrates have been shown to mediate or modulate a number of important events in the development of the nervous system; however, there is little evidence that they participate directly in the development of synapses. One carbohydrate structure that is likely to be important in synaptic development of the neuromuscular junction is the CT carbohydrate antigen [GalNAcbeta1,4[NeuAcalpha2,3]Galbeta1(-3GalNAc or -4GlcNAc)]. The synaptic localization of the CT antigen is due to the presence of the terminal beta1,4 GalNAc linkage, and such linkages are localized to the neuromuscular junction in many species. Here we show that an enzyme that can create the synaptic CT structure, the CT GalNAc transferase, is also confined to the neuromuscular junction in mice. Using transgenic mice, we show that overexpression of the CT GalNAc transferase in extrasynaptic regions in skeletal myofibers caused as much as a 60% reduction in the diameter of adult myofibers and an order of magnitude increase in satellite cells. Neuromuscular junctions of transgenic mice had severely reduced numbers of secondary folds, Schwann cell processes were present in the synaptic cleft, and secondary folds were often misaligned with active zones. In addition, multiple presynaptic specializations occurred on individual myofibers. In addition, some normally synaptic proteins, including laminin alpha4, laminin alpha5, utrophin, and NCAM, were expressed along extrasynaptic regions of myofibers. One of the muscle proteins that displayed increased glycosylation with the CT antigen in the transgenic mice was alpha-dystroglycan. These experiments provide the first in vivo evidence that a synaptic carbohydrate antigen has important roles in the development of the neuromuscular synapse and suggest that the CT antigen is involved in controlling the expression of synaptic molecules.