Toxic but Drank: Gustatory Aversive Compounds Induce Post-ingestional Malaise in Harnessed Honeybees

BACKGROUND: Deterrent substances produced by plants are relevant due to their potential toxicity. The fact that most of these substances have an unpalatable taste for humans and other mammals contrasts with the fact that honeybees do not reject them in the range of concentrations in which these compounds are present in flower nectars. Here we asked whether honeybees detect and ingest deterrent substances and whether these substances are really toxic to them. RESULTS: We show that pairing aversive substances with an odor retards learning of this odor when it is subsequently paired with sucrose. Harnessed honeybees in the laboratory ingest without reluctance a considerable volume (20 µl) of various aversive substances, even if some of them induce significant post-ingestional mortality. These substances do not seem, therefore, to be unpalatable to harnessed bees but induce a malaise-like state that in some cases results in death. Consistently with this finding, bees learning that one odor is associated with sugar, and experiencing in a subsequent phase that the sugar was paired with 20 µl of an aversive substance (devaluation phase), respond less than control bees to the odor and the sugar. Such stimulus devaluation can be accounted for by the malaise-like state induced by the aversive substances. CONCLUSION: Our results indicate that substances that taste bitter to humans as well as concentrated saline solutions base their aversive effect on the physiological consequences that their ingestion generates in harnessed bees rather than on an unpalatable taste. This conclusion is only valid for harnessed bees in the laboratory as freely-moving bees might react differently to aversive compounds could actively reject aversive substances. Our results open a new possibility to study conditioned taste aversion based on post-ingestional malaise and thus broaden the spectrum of aversive learning protocols available in honeybees

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Electrophysiological studies of odor structure-activity relationships of the antennal benzoic-acid receptor cell of the female of the silk moth Bombyx mori L

El objetivo de este trabajo fue caracterizar las respuestas electrofisiológicas de la célula receptora olfativa al ácido benzoico, presentes en las hembras de la mariposa del gusano de la seda Bombyx mori. Nuestros estudios permiten concluir que la célula receptora al ácido benzoico es un receptor especialista ya que responde máximamente a su compuesto llave y tiene un espectro idéntico para compuestos menos efectivos. Mediante estudios de estructura-actividad con substituyentes halogenados del ácido benzoico, se determinó que un factor crítico para el reconocimiento del sitio receptor es la generación de un efecto inductivo por el halógeno substituyente. Este efecto es mayor para halógenos más electronegativos (flúor) y para la posición meta. Cuando fue estimulada con iodo benzeno, la célula receptora al ácido benzoico respondió con una inhibición durante el estímulo y con una excitación post-estímulo. En base a diversos experimentos propusimos un modelo para la acción de sustancias excitatorias e inhibitorias a nivel receptor olfativo. En dicho modelo, sustancias como el iodo benceno actuarían directamente sobre los canales iónicos (proceso inhibitorio rápido y transitorio, no dependiente de segundos mensajeros), o sobre el sitio receptor del ácido benzoico (proceso excitatorio posterior y dependiente de segundos mensajeros). La célula receptora al ácido benzoico mostró un aumento de su actividad basal en el aire del laboratorio sin filtrar. Por medio de experimentos combinados de cromatografía gaseosa- electroantenograma y registro de células únicas encontramos que dicho aire contenía ácido benzoico y benzaldehido (estimulantes de la célula receptora al ácido benzoico en la sensilia tricodea) y nonanal (estimulante de la sensilia coelocónica). El umbral de la célula receptora al ácido benzoico se halla por debajo de una carga de estímqu de 0.01 μg de ácido benzoico por papel de filtro. Se analizó la composición química del meconio, producto de excreción de la pupa y único estímulo natural capaz de activar a la célula receptora del ácido benzoico. El meconio está compuesto por una gran cantidad de ácido succínico y ácido benzoico y cantidades menores de otros ácidos. La cantidad estimada de ácido benzoico presente en el meconio de machos y hembras fue 8.4 μg y 4.2 μg por 100 μl de meconio, respectivamente. De los componentes hallados, el único componente activador de la célula receptora al ácido benzoico fue el ácido benzoico. Sin embargo cuando éste fue adicionado al meconio, la respuesta disminuyó significativamente. Concluimos que el meconio obstruye estructuralmente el papel activador del ácido benzoico. Así, el ácido benzoico relevante para la hembra de B. mori no es aquel que está presente en el meconio. Fuentes alternativas de ácido benzoico en la planta de mora serían los frutos mismos de la mora y/o la reacción de defensa de la hoja que resulta en la producción de ácido benzoico como fitolexina. En ambos casos el ácido benzoico sería señal de buena “calidad”de planta y por ende de sustrato adecuado para la oviposición. Estas respuestas pueden sin embargo haber sido modificadas por innumerables generaciones de selección y cultivo de B. mori en sericultura.The aim of this work was to characterize the electrophysiological responses of the olfactory benzoic-acid receptor cell, present in the females of the silk moth Bombyx mori. Our studies allow to characterize the benzoic-acid cell as a specialist as it responds maximally to its cue compound and has a similar response spectrum for less effective compounds. By means of studies of structure-activity relationships performed with halogenated substitutes of benzoic acid, we determined that the generation of an inductive effect by the halogen is a critical factor for the recognition of the odor molecule by the receptor site. Such an effect increased for the more electronegative halogens (fluoride) and for the meta position. When the benzoic-acid receptor cell was stimulated with iodine benzene, it was first showed inhibition during the stimulation and then an excitation post-stimulation. On the basis of various experiments we sμggested a model for the action of excitatory and inhibitory substances at the olfactory receptor level. In such a model, substances like the iodine benzene would act directly on the ion channels (inhibitory,fast, transient process that would not depend on second messengers), or on the benzoic-acid receptor site (excitatory, post-stimulation process that would depend on second messengers). The benzoic-acid receptor cell showed an increase of its basal activity when exposed to the unfiltered air of the laboratory. By means of experiments combining gas chromatography, electroantenograms and single-cell recordings we determined that the air of the laboratory contained benzoic acid and benzaldehyde (stimulants of the benzoic-acid cell in the sensilla trichoidea), and nonanal (stimulant of the sensilla coeloconica). The response threshold of the benzoic-acid receptor cell for was estimated to be below 0.01 μg of benzoic acid per filter paper. We analyzed the chemical composition of the meconium, an excretion product of the pupae and the only stimulus known capable of activating the benzoic-acid cell. It is constituted by large amounts of succinic and benzoic acid and by minor quantities of other acids. The amount of benzoic acid estimated per 100 μl meconium was 8.4 μg and 4.2 μg for the males and for the females, respectively. From the substances found, the only one capable of activating maximally the benzoic acid cell was benzoic acid. However, when it was added to or mixed with meconium, the response of the benzoic-acid cell significantly decreased. We concluded that the meconium structurally obstructs the excitatory role of benzoic acid. Thus, the benzoic acid that is relevant for the female of B. mori is not that naturally occurring in the meconium. Alternative sources of benzoic acid in the mulberry are the fruits themselves and/or the defense reaction to fungal infection of the leaves, which result in the release of benzoic acid as a phytolexin. In both cases the benzoic acid would signalize a plant of “good quality” and therefore an appropriated oviposition substrate. However, these responses may have been lost due to the innumerous generations of selection and domestication related to the exploitation of B. mori in sericulture.Fil:de Brito Sanchez, María Gabriela. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Devaluation of sucrose 1 M.

<p>The graph shows the performance (percentage of proboscis extension responses or PER) during (<b>a</b>) an odor-sucrose association in which the response to the odor (conditioned stimulus or CS) was quantified, and during (<b>b,c</b>) a test phase following a devaluation phase in which responses to the sugar (US; see <b>b</b>) and to the odor (CS see <b>c</b>) were quantified in paired and unpaired groups of bees experiencing or not an association between sugar and either distilled water, quinine 10 mM, LiCl 140 mM or amygdaline 1 mM (319 bees in total). (<b>a</b>) All bees learned the odor-fructose association. The graph shows the pooled acquisition performance of all eight groups of bees. (<b>b</b>) Ingestion of quinine, LiCl and amygdaline did not diminish US responsiveness with respect to a water control. Bees responded maximally to sucrose (100% PER). Responses of paired and unpaired groups were similar. (<b>c</b>) Ingestion of quinine, LiCl and amygdaline decreased CS responsiveness with respect of a water control. Responses to a novel odor remained low and equivalent in all groups. Different letters indicate significant between-group differences.</p

Insulin effects on honeybee appetitive behaviour

Worker honeybees (Apis mellifera) carry out multiple tasks throughout their adult lifespan. It has been suggested that the insulin/insulin-like signalling pathway participates in regulating behavioural maturation in eusocial insects. Insulin signalling increases as the honeybee worker transitions from nurse to food processor to forager. As behavioural shifts require differential usage of sensory modalities, our aim was to assess insulin effects on olfactory and gustatory responsiveness as well as on olfactory learning in preforaging honeybee workers of different ages. Adults were reared in the laboratory or in the hive. Immediately after being injected with insulin or vehicle (control), and focusing on the proboscis extension response, bees were tested for their spontaneous response to odours, sucrose responsiveness and ability to discriminate odours through olfactory conditioning. Bees injected with insulin have higher spontaneous odour responses. Sucrose responsiveness and odour discrimination are differentially affected by treatment according to age: whereas insulin increases gustatory responsiveness and diminishes learning abilities of younger workers, it has the opposite effect on older bees. In summary, insulin can improve chemosensory responsiveness in young workers, but also worsens their learning abilities to discriminate odours. The insulin signalling pathway is responsive in young workers, although they are not yet initiating outdoor activities. Our results show strong age-dependent effects of insulin on appetitive behaviour, which uncover differences in insulin signalling regulation throughout the honeybee workeŕs adulthood.Fil: Mengoni Goñalons, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Guiraud, Marie. Universitéde Toulouse; FranciaFil: De Brito Sanchez, María Gabriela. Universitéde Toulouse; FranciaFil: Farina, Walter Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Ranking of sugar solutions by harnessed bees.

<p>The graph shows the percentage of proboscis extension responses (PER) upon antennal stimulation with fructose 1,66 M, glucose 1,66 M and sucrose 1 M. Each sugar was assayed with a different group of bees experiencing also a control stimulation with distilled water control (n = 30 each). Bees responded significantly more to the sugar than to the water. The preference ranking was fructose < glucose < sucrose. Different letters indicate significant between-group differences.</p

Devaluation of fructose 1.66 M.

<p>The graph shows the performance (percentage of proboscis extension responses or PER) during (<b>a</b>) an odor-fructose association in which the response to the odor (conditioned stimulus or CS) was quantified, and during (<b>b,c</b>) a test phase following a devaluation phase in which responses to the sugar (US; see <b>b</b>) and to the odor (CS see <b>c</b>) were quantified in paired and unpaired groups of bees experiencing or not an association between sugar and either distilled water, quinine 10 mM, LiCl 140 mM or amygdaline 1 mM (319 bees in total). (<b>a</b>) All bees learned the odor-fructose association. The graph shows the pooled acquisition performance of all eight groups of bees. (<b>b</b>) Ingestion of quinine, LiCl and amygdaline decreased US responsiveness with respect to a water control. Responses of paired and unpaired groups were similar. (<b>c</b>) Ingestion of quinine, LiCl and amygdaline decreased CS responsiveness with respect of a water control. Responses to a novel odor remained low and equivalent in all groups. Different letters indicate significant between-group differences.</p

Kaplan–Meier curves of survival for harnessed honeybees following feeding of aversive compounds.

<p>(<b>a</b>) <b><i>First series</i></b>. The probability of survival differed significantly between groups (long-rank test: <i>χ²</i> = 64.07, df:3, <i>p</i><0.0001). The group of honeybees having ingested NaCl 3 M (n = 30) and quinine 100 mM (n = 30) exhibited a significant decrease of their survival probability compared to the distilled water group (n = 30). The group having ingested salicine 100 mM (n = 30) had intermediate mortality levels and comparison to the distilled water group, which exhibited a low decrease of the probability of survival, was marginally non-significant (<i>Z</i> = 1.78, <i>p</i> = 0.07). (<b>b</b>) <b><i>Second series</i></b>. The probability of survival differed significantly between groups (long-rank test: <i>χ²</i> = 108.93, df:8, <i>p</i><0.0001). The group of bees having ingested sucrose 1 M group (n = 30) did not exhibit any variation of their probability of survival over time. The quinine 100 mM group (n = 30) experienced higher mortality than the distilled water group (n = 60). The quinine 10 mM (n = 60) and LiCl 140 mM (n = 60) groups experienced also induced higher mortality than the distilled water group. The amygdaline 1 mM group (n = 30) exhibited inetrmediate mortality compared to the the distilled water group. Mortality in the L-canavanine 40 mM (n = 30) and 100 mM (n = 30) groups was not significantly different from that of the distilled water group. The probability of survival from the groups having ingested mixtures of quinine 10 mM and sucrose 1 M (n = 30) and LiCL 140 mM and sucrose 1 M (n = 30) did not differ from that of the distilled water group.</p

International Nosocomial Infection Control Consortiu (INICC) report, data summary of 43 countries for 2007-2012. Device-associated module

We report the results of an International Nosocomial Infection Control Consortium (INICC) surveillance study from January 2007-December 2012 in 503 intensive care units (ICUs) in Latin America, Asia, Africa, and Europe. During the 6-year study using the Centers for Disease Control and Prevention's (CDC) U.S. National Healthcare Safety Network (NHSN) definitions for device-associated health care–associated infection (DA-HAI), we collected prospective data from 605,310 patients hospitalized in the INICC's ICUs for an aggregate of 3,338,396 days. Although device utilization in the INICC's ICUs was similar to that reported from ICUs in the U.S. in the CDC's NHSN, rates of device-associated nosocomial infection were higher in the ICUs of the INICC hospitals: the pooled rate of central line–associated bloodstream infection in the INICC's ICUs, 4.9 per 1,000 central line days, is nearly 5-fold higher than the 0.9 per 1,000 central line days reported from comparable U.S. ICUs. The overall rate of ventilator-associated pneumonia was also higher (16.8 vs 1.1 per 1,000 ventilator days) as was the rate of catheter-associated urinary tract infection (5.5 vs 1.3 per 1,000 catheter days). Frequencies of resistance of Pseudomonas isolates to amikacin (42.8% vs 10%) and imipenem (42.4% vs 26.1%) and Klebsiella pneumoniae isolates to ceftazidime (71.2% vs 28.8%) and imipenem (19.6% vs 12.8%) were also higher in the INICC's ICUs compared with the ICUs of the CDC's NHSN