Experience matters: prior exposure to plant toxins enhances diversity of gut microbes in herbivores

For decades, ecologists have hypothesised that exposure to plant secondary compounds (PSCs) modifies herbivore‐associated microbial community composition. This notion has not been critically evaluated in wild mammalian herbivores on evolutionary timescales. We investigated responses of the microbial communities of two woodrat species (Neotoma bryanti and N. lepida). For each species, we compared experienced populations that independently converged to feed on the same toxic plant (creosote bush, Larrea tridentata) to naïve populations with no exposure to creosote toxins. The addition of dietary PSCs significantly altered gut microbial community structure, and the response was dependent on previous experience. Microbial diversity and relative abundances of several dominant phyla increased in experienced woodrats in response to PSCs; however, opposite effects were observed in naïve woodrats. These differential responses were convergent in experienced populations of both species. We hypothesise that adaptation of the foregut microbiota to creosote PSCs in experienced woodrats drives this differential response.     

Axial stretch enhances sarcoplasmic reticulum Ca2+ leak and cellular Ca2+ reuptake in guinea pig ventricular myocytes: experiments and models

Cardiac cellular calcium (Ca2+) handling is the well-investigated mediator of excitation-contraction coupling, the process that translates cardiac electrical activation into mechanical events. The reverse--effects of mechanical stimulation on cardiomyocyte Ca2+ handling--are much less well understood, in particular during the inter-beat period, called 'diastole'. We have investigated the effects of diastolic length changes, applied axially using a pair of carbon fibres attached to opposite ends of Guinea pig isolated ventricular myocytes, on the availability of Ca2+ in the main cellular stores (the sarcoplasmic reticulum; SR), by studying the rest-decay of SR Ca2+ content [Ca2+]SR, and the reloading of the SR after prior depletion of Ca2+ from the cell. Cells were loaded with Fura-2 AM (an indicator of the cytosolic 'free' Ca2+ concentration, [Ca2+]i), and pre-conditioned by field-stimulation (2 Hz) at 37 degrees C, while [Ca2+]i transients and sarcomere length (SL) were recorded simultaneously. After reaching a steady state in the behaviour of observed parameters, stimulation was interrupted for between 5 and 60s, while cells were either held at resting length, or stretched (controlled to cause a 10% increase in SL, to aid inter-individual comparison). Thereafter, each cell was returned to its original resting length, followed by swift administration of 10mM of caffeine (in Na+/Ca2+-free solution), which causes the release of Ca2+ from the SR (caffeine), but largely prevents extrusion of Ca2+ from the cytosol to the cell exterior (Na+/Ca2+-free solution). By comparing the [Ca2+]i in cells exposed/not exposed to diastolic stretch of different duration, we assessed the rest-decay dynamics of [Ca2+]SR. To assess SR reloading after initial Ca2+ depletion, the same stretch protocol was implemented after prior emptying of the cell by application of 10mM of caffeine in normal Tyrode solution (which causes Ca2+ to be released from the SR and extruded from the cell via the Na+/Ca2+ exchanger; NCX). Axial stretch enhanced the rate of both rest-decay and reloading of [Ca2+]SR. Application of 40 microM streptomycin, a blocker of stretch-activated ion channels, did not affect the stretch-induced increase in SR reloading. This behaviour was reproduced in a computer simulation study, using a modified version of the 2006 Iribe-Kohl-Noble model of single cardiac myocyte Ca2+ handling, suggesting that stretch increases both Ca2+ leak from the SR and Ca2+ influx via the sarcolemma. This may have important implications for the mobilisation of Ca2+ in stretched cells, and could contribute to the regional 'matching' of individual cardiomyocyte contractility to dynamic, and regionally varying, changes in mechanical loads, such as diastolic pre-load, of cardiac tissue.     

Sugar feeding protects against arboviral infection by enhancing gut immunity in the mosquito vector Aedes aegypti

As mosquito females require a blood meal to reproduce, they can act as vectors of numerous pathogens, such as arboviruses (e.g. Zika, dengue and chikungunya viruses), which constitute a substantial worldwide public health burden. In addition to blood meals, mosquito females can also take sugar meals to get carbohydrates for their energy reserves. It is now recognised that diet is a key regulator of health and disease outcome through interactions with the immune system. However, this has been mostly studied in humans and model organisms. So far, the impact of sugar feeding on mosquito immunity and in turn, how this could affect vector competence for arboviruses has not been explored. Here, we show that sugar feeding increases and maintains antiviral immunity in the digestive tract of the main arbovirus vector Aedes aegypti. Our data demonstrate that the gut microbiota does not mediate the sugar-induced immunity but partly inhibits it. Importantly, sugar intake prior to an arbovirus-infected blood meal further protects females against infection with arboviruses from different families. Sugar feeding blocks arbovirus initial infection and dissemination from the gut and lowers infection prevalence and intensity, thereby decreasing the transmission potential of female mosquitoes. Finally, we show that the antiviral role of sugar is mediated by sugar-induced immunity. Overall, our findings uncover a crucial role of sugar feeding in mosquito antiviral immunity which in turn decreases vector competence for arboviruses. Since Ae. aegypti almost exclusively feed on blood in some natural settings, our findings suggest that this lack of sugar intake could increase the spread of mosquito-borne arboviral diseases.     

Antibody-dependent cellular cytotoxicity and natural killer cytotoxicity of peritoneal cells from nude mice to herpes simplex virus-infected cells

Nude BALB/c mice (athymic) were more susceptible to fatal herpes simplex virus (HSV) than normal BALB/c mice (P = 0.002). The peritoneal cells of nude mice mediated levels of antibody-dependent cellular cytotoxicity (ADCC) of equal or greater magnitude than cells from normal BALB/c, heterozygote nu/+, or C57BL/6 mice. Unstimulated natural killer cytotoxicity of peritoneal cells from nude mice was higher (P less than 0.05) than that mediated by cells from C57BL/6 mice. Nude mice failed to make anti-HSV ADCC antibody 6 to 14 days post HSV inoculation, at times when nu/+, BALB/c, and C57BL/6 mice produced antibody. Passive reconstitution of nude mice with high titer intraperitoneal anti-HSV immune globulin provided circulating anti-HSV ADCC antibody and significant protection against lethal HSV infection.     

Nitrogen isotopic fractionation and 18O exchange in relation to the mechanism of denitrification of nitrite by Pseudomonas stutzeri

Two types of mechanisms for the enzymatic reduction of NO2- to N2O have been proposed. In one, two NO2- ions are reduced in parallel, with the nitrogen-nitrogen bond formed from reduced intermediates. In the second, the two NO2- ions enter the reaction sequentially, with the nitrogen of at least one of the two having a valence of 3+ when the nitrogen-nitrogen bond is formed. Our objective was to distinguish between these two types of mechanism. Toward that end, the exchange of 18O from H2O to NO2- and the overall nitrogen isotopic fractionation factor (beta obs) were measured. The rate of exchange of oxygen from H2O to NO2-, resulting from a protonation-dehydration step preceding reductive events in both mechanisms, was less than 10% of the rate of denitrification at both low and high [NO2-]. The value of beta obs was 1.010 +/- 0.001 and 1.020 +/- 0.001 at low and high [NO2-], respectively. Expressions for beta obs, as a function of the measured rate of entry of oxygen from H2O into NO2-, were derived for both types of mechanism. The measured dependence of beta obs on substrate concentration, as constrained by the 18O exchange data, is inconsistent with the first type of mechanism, but consistent with the second type. Thus, by combining nitrogen isotopic fractionation and 18O exchange data, we rule out any mechanism in Pseudomonas stutzeri in which NO2- ions are reduced in parallel, with the nitrogen-nitrogen bond being formed from reduced intermediates.     

Effect of exercise-induced hyperventilation on airway resistance and cycling endurance

The purpose of the present study was to investigate the effect of exercise induced hyperventilation and hypocapnia on airway resistance (R _aw), and to try to answer the question whether a reduction of R _aw is a mechanism contributing to the increase of endurance time associated with a reduction of exercise induced hyperventilation as for example has been observed after respiratory training. Eight healthy volunteers of both sexes participated in the study. Cycling endurance tests (CET) at 223 (SD 47) W, i.e. at 74 (SD 5)% of the subject's peak exercise intensity, breathing endurance tests and body plethysmograph measurements of pre- and postexercise R _aw were carried out before and after a 4-week period of respiratory training. In one of the two CET before the respiratory training CO₂ was added to the inspired air to keep its end-tidal concentration at 5.4% to avoid hyperventilatory hypocapnia (CO₂-test); the other test was the control. The pre-exercise values of specific expiratory R _aw were 8.1 (SD 2.8), 6.8 (SD 2.6) and 8.0 (SD 2.1) cm H₂O · s and the postexercise values were 8.5 (SD 2.6), 7.4 (SD 1.9) and 8.0 (SD 2.7) cm H₂O · s for control CET, CO₂-CET and CET after respiratory training, respectively, all differences between these tests being nonsignificant. The respiratory training significantly increased the respiratory endurance time during breathing of 70% of maximal voluntary ventilation from 5.8 (SD 2.9) min to 26.7 (SD 12.5) min. Mean values of the cycling endurance time (t _cend) were 22.7 (SD 6.5) min in the control, 19.4 (SD 5.4) min in the CO₂-test and 18.4 (SD 6.0) min after respiratory training. Mean values of ventilation ( _E) during the last 3␣min of CET were 123 (SD 35.8) l · min⁻¹ in the control, 133.5 (SD 35.1) l · min⁻¹ in the CO₂-test and 130.9 (SD 29.1) l · min⁻¹ after respiratory training. In fact, six subjects ventilated more and cycled for a shorter time, whereas two subjects ventilated less and cycled for a longer time after the respiratory training than in the control CET. In general, the subjects cycled longer the lower the _E, if all three CET are compared. It is concluded that R _aw measured immediately after exercise is independent of exercise-induced hyperventilation and hypocapnia and is probably not involved in limiting t _cend, and that t _cend at a given exercise intensity is shorter when _E is higher, no matter whether the higher _E occurs before or after respiratory training or after CO₂ inhalation. Accepted: 11 September 1996     

Alteration in liver pyruvate kinase protein and catalytic activity upon starvation and refeeding

Liver pyruvate kinase (L-type isozyme) was purified from the livers of rats fed a high carbohydrate, low protein diet for 4 days. The protein was homogeneous as judged by polyacrylamide-gel electrophoresis with and without added sodium dodecyl sulfate and as judged by high speed sedimentation and low speed equilibrium centrifugation. The specific activity of the purified protein was 190–220 international units (IU)/mg. A precipitating antiserum directed specifically against liver pyruvate kinase was obtained from rabbits and was used to determine the amount of liver pyruvate kinase protein present in the 80,000g supernatant fraction of rat liver homogenates in response to the dietary status of the animal. Rats maintained on a high carbohydrate, low protein diet for 4 days prior to sacrifice have at least 20 mg of precipitable liver pyruvate kinase protein per liver. Starvation of the animal results in a marked reduction in liver pyruvate kinase so that by 3 days of starvation less than 7 mg of liver pyruvate kinase protein per liver remains. Refeeding the animal a high carbohydrate, low protein diet results in a return of the liver pyruvate kinase protein to the prestarvation level of 20 mg per liver. The liver pyruvate kinase activity per liver varies in the same direction as does the liver pyruvate kinase protein but does not parallel the change in protein. Animals fed a high carbohydrate, low protein diet for 4 days have 60–70 IU/mg of liver pyruvate kinase protein whereas animals starved for periods exceeding 30 h have greater than 100 IU/mg of liver pyruvate kinase protein. Refeeding starved animals with a high carbohydrate, low protein diet initially causes a large increase in activity per milligram of liver pyruvate kinase protein followed by a return of this value to the prestarvation level. The observed rise in the ratio of activity per milligram of liver pyruvate kinase protein during starvation suggests a modification in the enzyme protein resulting either in an increase in the specific activity of the enzyme or in a decrease in the affinity of the enzyme for the antibody.     

Zebrin II / Aldolase C Expression in the Cerebellum of the Western Diamondback Rattlesnake (Crotalus atrox)

Aldolase C, also known as Zebrin II (ZII), is a glycolytic enzyme that is expressed in cerebellar Purkinje cells of the vertebrate cerebellum. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with high ZII expression (ZII+), alternating with stripes of Purkinje cells with little or no expression (ZII-). The patterns of ZII+ and ZII- stripes in the cerebellum of birds and mammals are strikingly similar, suggesting that it may have first evolved in the stem reptiles. In this study, we examined the expression of ZII in the cerebellum of the western diamondback rattlesnake (Crotalus atrox). In contrast to birds and mammals, the cerebellum of the rattlesnake is much smaller and simpler, consisting of a small, unfoliated dome of cells. A pattern of alternating ZII+ and ZII- sagittal stripes cells was not observed: rather all Purkinje cells were ZII+. This suggests that ZII stripes have either been lost in snakes or that they evolved convergently in birds and mammals.