The Ancient Chemistry of Avoiding Risks of Predation and Disease

Illness, death, and costs of immunity and injury strongly select for avoidance of predators or contagion. Ants, cockroaches, and collembola recognize their dead using unsaturated fatty acids (e.g., oleic or linoleic acid) as “necromone” cues. Ants, bees, and termites remove dead from their nests (necrophoric behavior) whereas semi-social species seal off corpses or simply avoid their dead or injured (necrophobic behavior). Alarm and avoidance responses to exudates from injured conspecifics are widespread. This involves diverse pheromones, complex chemistry and learning. We hypothesized that necromones are a phylogenetically ancient class of related signals and predicted that terrestrial Isopoda (that strongly aggregate and lack known dispersants) would avoid body fluids and corpses using fatty acid “necromones.” Isopods were repelled by crushed conspecifics (blood), intact corpses, and alcohol extracts of bodies. As predicted, the repellent fraction contained oleic and linoleic acids and authentic standards repelled several isopod species. We further predicted a priori that social caterpillars (lacking known dispersants) would be repelled by their own body fluids and unsaturated fatty acids. Both tent caterpillars and fall webworms avoided branches treated with conspecific body fluid. Oleic and linoleic acids were also strongly avoided by both species. Necromone signaling appears widespread and likely traces to aquatic ancestors pre-dating the divergence of the Crustacea and Hexapoda at least 420 million years ago.     

Long-Term Survival of Salmonella enterica Serovar Typhimurium Reveals an Infectious State That Is Underrepresented on Laboratory Media Containing Bile Salts

Cells in desiccated Salmonella enterica serovar Typhimurium rdar (red, dry, and rough) morphotype colonies were examined for culturability and infectivity after 30 months. Culturability decreased only 10-fold; however, cells were underrepresented on Salmonella selective media containing bile salts. These cells were mildly attenuated compared to the infectivity of freshly grown cells but still able to cause systemic infections in mice.Salmonella enterica serovar Typhimurium (hereafter referred to as S. Typhimurium) is a gram-negative enteric pathogen of humans and other mammalian species. S. Typhimurium causes self-limiting gastroenteritis in humans and typhoid-like fever in mice (4). When propagated on the surface of nutrient-limited laboratory media, Salmonella cells form patterned colonies (1) involving the production of an extracellular matrix comprised of thin aggregative fimbriae (Tafi or curli) and cellulose (16, 21) and other polysaccharides. This rdar morphotype is characterized by the formation of red, dry, and rough colonies on solid agar medium containing Congo red as an indicator dye (8). Cells within rdar colonies are resistant to desiccation and commonly used disinfectants (14, 18), and the propensity to form the rdar morphotype is conserved throughout the salmonellae (7, 19). These findings, coupled with the observations that neither Tafi nor cellulose is required for virulence (13, 17), suggest that the rdar phenotype may contribute to the environmental survival of Salmonella (17).Previous experiments by our group established that approximately 10% (∼10⁸) of bacteria within a rdar morphotype colony were able to survive a 9-month period of starvation and desiccation (18). In this study, we examined survival after a longer time period of 30 months. S. Typhimurium ATCC 14028 cells were grown at 28°C for 4 days on tryptone agar, at which time colonies were detached from the agar surface and stored at room temperature in sterile, 24-well plates, as previously described (18). The results were compared to those for planktonic S. Typhimurium cells grown overnight in 1% tryptone and washed with distilled water prior to storage on plastic. Surprisingly, the number of viable cells recovered from rdar colonies after 30 months was similar to that obtained at the 9-month time point (Fig. ​(Fig.1).1). In contrast, planktonic S. Typhimurium cells exhibited a decrease in viability of 4 orders of magnitude within 14 days. However, if planktonic cells were stored in water or isotonic saline, the viability decreased by only 2 orders of magnitude after 84 days (Fig. ​(Fig.1).1). This finding indicated that desiccation rather than starvation was the primary cause of bacterial death under these conditions. The survival after 30 months of isogenic ΔcsgD mutant cells, which are deficient in extracellular matrix production (8, 18), was reduced 25-fold compared to that of wild-type cells (data not shown), confirming that cellulose and Tafi polymers have an important role in long-term survival. Together, these results indicated that extreme longevity of cells within rdar colonies is a morphotype-specific phenomenon rather than a general property of S. Typhimurium ATCC 14028.Open in a separate windowFIG. 1.Long-term survival of S. Typhimurium cells in rdar morphotype colonies or from planktonic culture under conditions of desiccation and nutrient absence. Approximately 10⁹ CFU of planktonic cells (▴) or cells within rdar colonies (•) were placed on sterile plastic, or planktonic cells were stored in sterile water (▪). At time points shown, cells were rehydrated, homogenized, and serially diluted before being grown on LB medium to enumerate the CFU. Data points represent averages and error bars show the standard deviations of the results from at least three biological samples.Under most laboratory conditions, S. Typhimurium is resistant to bile concentrations exceeding 60% (wt/vol) (3, 5, 6, 15); therefore, many selective media used in microbiological laboratories for culturing Salmonella contain bile salts. During storage on plastic with no nutrients or water, cells within rdar colonies became sensitive to sodium cholate and sodium deoxycholate (bile salts) over time (Table ​(Table1).1). As expected, freshly grown cells (exponential or stationary phase) did not display bile sensitivity (data not shown). The culturability of cells in 30-month-old colonies on media containing bile salts was significantly reduced compared to that of cells from 2-day-old or 2-week-old rdar colonies or colonies that were lyophilized for 1 week (Table ​(Table1).1). In contrast, culturability was not reduced to the same extent when aged cells were cultured on media that did not contain bile salts (brilliant green agar or XLD and LSA without bile salts [see Table ​Table11 for media]). Within diagnostic laboratories, bacterial isolates in environmental samples are commonly incubated in recovery broth prior to enumeration on selective medium (2). When cells derived from 30-month-old rdar colonies were incubated overnight in buffered peptone water, they recovered their resistance to bile salts (data not shown).

TABLE 1.

Relative recovery of S. Typhimurium ATCC 14028 on common Salmonella selective media

Acidosis-Induced Zinc-Dependent Death of Cultured Cerebellar Granule Neurons

Severe acidosis caused death of cultured cerebellar granule neurons (CGNs). Acidosis was accompanied by a progressive increase of the intracellular zinc ions ([Zn²⁺]_i) and decrease of [Ca²⁺]_i. Zn²⁺ chelator, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), prevented the increase of [Zn²⁺]_i and acidosis-induced neuronal death. However, neuronal death was insensitive to blockade of ASIC1 channels with amiloride, as CGNs display considerably lower expression of ASIC1a than other neurons. The antioxidant trolox and menadione significantly protected neurons from acidotic death. Earlier, we demonstrated that menadione rescues neurons from the deleterious effect of inhibition of mitochondrial complex I (Isaev et al. Neuroreport 15:2227–2231, 2004). We speculate that excessive Zn²⁺-dependent production of reactive oxygen species by mitochondrial complex I may be a general motive for the induction of cell death in CGNs under acidotic conditions.     

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.Download video file.^{(59M, mov)}     

New or little-known species of the genus Amphimenes Bates, 1873 (Coleoptera, Carabidae, Lebiinae) from Vietnam

Twelve new species of the genus Amphimenes Bates, 1873 are described from Vietnam: Amphimenes maculatussp. n., Amphimenes bidoupensissp. n., Amphimenes gracilissp. n., Amphimenes montanussp. n., Amphimenes giganteussp. n., Amphimenes mediussp. n., Amphimenes minutussp. n., Amphimenes rufipessp. n., Amphimenes reflexicollissp. n., Amphimenes planicollissp. n., Amphimenes nitidussp. n., and Amphimenes kabakovisp. n.. Amphimenes rugulipennis Bates, 1892, comb. n., is transferred from the genus Brachichila Chaudoir, 1869 and is redescribed from fresh material. A key to all congeners is provided, these being arranged into five new species-groups. Adults of the rugulipennis- and piceolus-groups show subcortical habits, while those of the medius-, rufipes- and planicollis- groupsare herpetobiotic, resulting in such morphological adaptations as partly reduced eyes, missing wings and adnate elytra.     

Generation of dopamine neurons from human embryonic stem cells in vitro

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth.     

Hydration and packing along the folding pathway of SH3 domains by pressure-dependent NMR

The volumetric properties associated with protein folding transitions reflect changes in protein packing and hydration of the states that participate in the folding reaction. Here, NMR spin relaxation techniques are employed to probe the folding-unfolding kinetics of two SH3 domains as a function of pressure so that the changes in partial molar volumes along the folding pathway can be measured. The two domains fold with rates that differ by approximately 3 orders of magnitude, so their folding dynamics must be probed using different NMR relaxation experiments. In the case of the drkN SH3 domain that folds via a two-state mechanism on a time scale of seconds, nitrogen magnetization exchange spectroscopy is employed, while for the G48M mutant of the Fyn SH3 domain where the folding occurs on the millisecond time scale (three-step reaction), relaxation dispersion experiments are utilized. The NMR methodology is extremely sensitive to even small changes in equilibrium and rate constants, so reliable estimates of partial molar volumes can be obtained using low pressures (1-120 bar), thus minimizing perturbations to any of the states along the folding reaction coordinate. The volumetric data that were obtained are consistent with a similar folding mechanism for both SH3 domains, involving early chain compaction to states that are at least partially hydrated. This work emphasizes the role of NMR spin relaxation in studying dynamic processes over a wide range of time scales.     

A new group B adenovirus receptor is expressed at high levels on human stem and tumor cells

CD46 is used by human group B adenoviruses (Ads) as a high-affinity attachment receptor. Here we show evidence that several group B Ads utilize an additional receptor for infection of human cells, which is different from CD46. We tentatively named this receptor receptor X. Competition studies with unlabeled and labeled Ads, recombinant Ad fiber knobs, and soluble CD46 and CD46 antibodies revealed three different subgroups of group B Ads, in terms of their receptor usage. Group I (Ad16, -21, -35, and -50) nearly exclusively uses CD46. Group II (Ad3, -7p, and -14) utilizes receptor X and not CD46. Group III (Ad11p) uses both CD46 and the alternative receptor X. Interaction of group II and III Ads with receptor X occurs via the fiber knob. Receptor X is an abundantly expressed glycoprotein that interacts with group II and III Ads at relatively low affinity in a Ca(2+)-dependent manner. This receptor is expressed at high levels on human mesenchymal and undifferentiated embryonic stem cells, as well as on human cancer cell lines. These findings have practical implications for stem cell and gene therapy.     

Dimeric calcium complexes of arabinan-rich pectic polysaccharides from Olea europaea L. cell walls

The study carried out in this work concerns the pectic polysaccharides of olive cell walls as present in olive pulp and that remained entrapped in the cellulosic residue after sequential extraction of the cell wall material (CWM) with imidazole, carbonate and KOH aqueous solutions. These polymers, obtained after neutralisation and dialysis of an aqueous suspension of the residue (sn-CR fraction), extracted with 4 M KOH, were arabinan-rich pectic polysaccharides. They accounted for 11–19% of the total pectic polysaccharides found in the olive pulp cell walls of fruits collected in two years and in three stages of ripening (green, cherry and black). The analysis by powder X-ray diffraction highlighted the existence, in all sn-CR fractions, of crystalline phases related with the presence of calcium-pectic polysaccharide complexes (CPPC) occurring in an amorphous carbohydrate network. The relative crystallinity of the CPPC varied linearly with the Ca²⁺/GalA molar ratio until a maximum of 0.57. Size-exclusion chromatography showed that sn-CR fractions possessed a bimodal molecular weight distribution. The lower molecular weight fraction of sn-CR (M_w = 70–135 kDa) was independent on the ripening stage of olive fruit, whereas the higher molecular weight fraction showed values of 1.1, 0.6–0.9 and 0.5–0.7 MDa, respectively, for green, cherry and black olives. Treatment of the sn-CR pectic polysaccharides with a 2 M imidazole solution disrupted the CPPC crystalline network showing the loss of low molecular weight galacturonan-rich material during dialysis (12–14 kDa cut off) and the decrease of molecular weight of the polymers to roughly half. These results allowed to infer the presence of oligogalacturonides held within cell walls by calcium ions and that the pectic polysaccharides of sn-CR fraction occurred in olive pulp cell walls as calcium bridged macrodimers.