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The objective of this study was to investigate the effects of a milk byproduct–based starter pellet, high in lactose, on feed intakes, growth, and rumen development when calves were fed either a high or low level of milk. This was done by using a 2 × 2 factorial design, with 120 Holstein heifer calves randomly assigned to 1 of 2 milk treatments and 1 of 2 feed treatments upon enrollment at birth. Calves were either assigned to a low (LM) or high (HM) level of milk replacer by an automated milk-feeding system for individually-housed calves. The LM treatment calves were offered 6 L/d (150 g/L; 1.2 L, 5×/d), whereas HM treatment calves were offered 15 L/d (150 g/L; 3 L, 5×/d) of milk replacer. Calves were also assigned to a milk byproduct- (whey) based starter feed treatment (WF), or a grain-based starter ration (95% concentrate starter pellet, 5% chopped wheat straw) feed treatment (GF). All calves were offered their treatment feed starting on d 5, with water offered ad libitum from d 0. The WF treatment calves were offered 150 g/d of the milk byproduct–based pellet until they consumed the entirety for 2 out of 3 consecutive days. At this point, WF treatment calves received 150 g of the milk byproduct–based pellet top dressed onto the grain-based ration to allow for ad libitum feed consumption until weaned, when they only received the grain-based ration postweaning (d 64–77). Calves on the GF feed treatment received only the grain-based ration. All calves were gradually weaned from d 43 to 63. By design, calves on the HM treatment consumed greater levels of milk than LM treatment calves for the majority of the days in the preweaning phase (8.9 ± 0.11 vs. 5.5 ± 0.11 L/d; SE). Calves on the GF and WF treatments consumed similar amounts of milk in the preweaning phase (7.2 ± 0.11 vs. 7.2 ± 0.11 L/d); however, there was a milk and feed treatment interaction detected for milk consumption in the weaning phase, with HM-GF calves drinking more milk than the other treatments and HM-WF calves drinking more milk than both LM-GF and LM-WF calves. The WF treatment calves had greater solid feed intake during the preweaning phase than GF treatment calves, with no feed intake differences in the weaning or postweaning phases. The WF calves drank more water in the preweaning phase than GF calves. Average daily gain was similar throughout the study between the GF and WF treatment calves. Rumen physiological measures were all similar by the end of the trial (d 77) regardless of feed or milk treatment, suggesting similar rumen development postweaning. Overall, regardless of the level of milk fed, offering the milk byproduct–based starter pellet may increase preweaning feed intake, along with water consumption. However, both feed and milk treatments had similar performance and rumen development measures by the end of the trial.

Chronic myeloid leukemia (CML) is a hematological malignancy characterized by BCR-ABL1-derived permanent proliferation of myeloid progenitor cells. BCR-ABL1 tyrosine kinase inhibitors (TKI) are effective first-line therapeutic options to suppress tumor proliferation. However, TKI therapy is not always curative and drug-related side effects as well as drug resistance may evolve over time, necessitating salvage therapies. In this case report we present a 68-year-old woman who developed second- and third-generation TKI therapy resistance with BCR-ABL1T315I and BCR-ABL1E255V mutation. Considering contraindication for hematopoietic stem cell transplantation, we treated the patient in an individual treatment attempt with a third-generation TKI ponatinib in combination with palbociclib, a CDK4/CDK6 inhibitor, which has been shown to effectively inhibit proliferation of BCR-ABL1T315I-mutated cells in vitro. Our case study shows strong antineoplastic effects using this combination in an advanced CML patient resistant to ponatinib monotherapy as a fourth-line treatment. Combined administration of ponatinib/palbociclib at full dose showed almost a tenfold decrease (42.6 to 4.4 IS%) of BCR-ABL1-positive cells but with simultaneous hematopoietic toxicity, necessitating dose reduction. This combination treatment showed high clinical activity. However, biological activity needs to be further characterized in prospective clinical trials.

Summary Haemato‐oncological patients are at risk in case of severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection. Currently, vaccination is the best‐evaluated preventive strategy. In the present study, we aimed to assess serological response, predictive markers, and safety of BNT162b2 in haemato‐oncological patients. A total of 259 haemato‐oncological patients were vaccinated with two 30 µg doses of BNT162b2 administered 21 days apart. Serological response was assessed by ELECSYS® Anti‐SARS‐CoV‐2‐S immunoassay before vaccination, and at 3 and 7 weeks after the first dose (T1, T2). Safety assessment was performed. At T2 spike protein receptor binding domain (S/RBD) antibodies were detected in 71·4% of haematological and in 94·5% of oncological patients (P

Phytoplankton comprises a large fraction of the vertical carbon flux to deep water via the sinking of particulate organic matter (POM). However, despite the importance of phytoplankton in the coupling of benthic-pelagic productivity, the extent to which its deposition in the sediment affects bacterial dynamics at the water-sediment interface is poorly understood. Here, we conducted a microcosm experiment in which varying mixtures of diatom and cyanobacteria, representing phytoplankton-derived POM of differing quality, served as inputs to sediment cores. Characterization of 16S rRNA gene of the bacterial communities at the water-sediment interface showed that bacterial α-diversity was not affected by POM addition, while bacterial β-diversity changed significantly along the POM quality gradient, with the variation driven by changes in relative abundance rather than in taxon replacement. Analysing individual taxa abundances across the POM gradient revealed two distinct bacterial responses, in which taxa within either diatom- or cyanobacteria-favoured groups were more phylogenetically closely related to one another than other taxa found in the water. Moreover, there was little overlap in taxon identity between sediment and water communities, suggesting the minor role played by sediment bacteria in influencing the observed changes in bacterial communities in the overlying water. Together, these results showed that variability in phytoplankton-originated POM can impact bacterial dynamics at the water-sediment interface. Our findings highlight the importance of considering the potential interactions between phytoplankton and bacteria in benthic-pelagic coupling in efforts to understand the structure and function of bacterial communities under a changing climate.

Campylobacter cause gastroenteritis in humans and may be shed in the feces of livestock and poultry species, including cattle, chicken, turkey, and swine. However, a synthesis of the prevalence on farms in the United States and Canada is currently lacking. Thus, our objective was to conduct a systematic review and meta-analysis to estimate the prevalence of Campylobacter coli, Campylobacter jejuni, and Campylobacter spp. on livestock and poultry farms operated under commercial conditions in the United States and Canada. The relevant literature was identified and examined for eligibility based on a priori inclusion and exclusion criteria. Relevant data were extracted, and a meta-analysis was performed. The data were transformed using the Freeman-Tukey arcsine transformation to stabilize the variance. A separate meta-analysis was performed for each animal species, level of sampling (individual versus pooled), and species of Campylobacter, for a total of 29 meta-analyses. C. jejuni and Campylobacter spp. were present in all livestock and poultry species of interest, whereas C. coli was found in all species of interest with the exception of chickens. Furthermore, substantial heterogeneity was observed in most meta-analyses. In an attempt to account for this, subgroup analyses were performed on potential moderators. However, with the exception of beef cattle, where studies in feedlot cattle reported a consistently higher prevalence compared with adult cattle on pasture, significant heterogeneity remained in the majority of meta-analyses after accounting for potential moderators. The results of this review can be used to inform future risk assessment, consumer and producer awareness, and resource allocation, and identify gaps for future research.

Oncolytic viruses (OV) have been shown to activate the antitumor functions of specific immune cells like T cells. Here, we show OV can also reprogram tumor-associated macrophage (TAM) to a less immunosuppressive phenotype. Syngeneic, immunocompetent mouse models of primary breast cancer were established using PyMT-TS1, 4T1, and E0771 cell lines, and a metastatic model of breast cancer was established using the 4T1 cell line. Tumor growth and overall survival was assessed following intravenous administration of the OV, HSV1716 (a modified herpes simplex virus). Infiltration and function of various immune effector cells was assessed by NanoString, flow cytometry of dispersed tumors, and immunofluorescence analysis of tumor sections. HSV1716 administration led to marked tumor shrinkage in primary mammary tumors and a decrease in metastases. This was associated with a significant increase in the recruitment/activation of cytotoxic T cells, a reduction in the presence of regulatory T cells and the reprograming of TAMs towards a pro-inflammatory, less immunosuppressive phenotype. These findings were supported by in vitro data demonstrating that human monocyte-derived macrophages host HSV1716 replication, and that this led to immunogenic macrophage lysis. These events were dependent on macrophage expression of proliferating cell nuclear antigen (PCNA). Finally, the antitumor effect of OV was markedly diminished when TAMs were depleted using clodronate liposomes. Together, our results show that TAMs play an essential role in support of the tumoricidal effect of the OV, HSV1716—they both host viral replication via a novel, PCNA-dependent mechanism and are reprogramed to express a less immunosuppressive phenotype.

Background The medial prefrontal cortex (PFC) is crucial for regulating craving and alcohol seeking in alcohol use disorder (AUD) patients and alcohol seeking in animal models. Maladaptive changes in volitional ethanol (EtOH) intake have been associated with PFC function, yet synaptic adaptations within PFC have not been consistently detected in voluntary drinking rodent models. At least 80% of the neurons in PFC are glutamatergic pyramidal cells. Pyramidal cells provide the predominant cortical output to several brain regions relevant to AUD, including structures within the telencephalon (IT: e.g., basal ganglia, amygdala, other neocortical regions) and outside the telencephalon (ET: e.g., lateral hypothalamus, midbrain monoaminergic structures, thalamus). Methods In addition to their anatomical distinctions, studies from several laboratories have revealed that prefrontal cortical IT and ET pyramidal cells may be differentiated by specific electrophysiological parameters. These distinguishable parameters make it possible to readily classify pyramidal cells into separable subtypes. Here, we employed and validated the hyperpolarization sag ratio as a diagnostic proxy for separating ET (type A) and IT (type B) neurons. We recorded from deep-layer prelimbic PFC pyramidal cells of mice 1 day after 4 to 5 weeks of intermittent access (IA) EtOH exposure. Results Membrane properties were not altered by IA EtOH, but excitatory postsynaptic strength onto IT type B neurons was selectively enhanced in slices from IA EtOH mice. The increased excitatory drive was accompanied by enhanced mGlu2/3 receptor plasticity on IT type B neurons, providing a potential translational approach to mitigate cognitive and motivational changes to PFC function related to binge drinking. Conclusions Together, these studies provide insight into the specific PFC neurocircuits altered by voluntary drinking. In addition, the findings provide an additional rationale for developing compounds that potentiate mGlu2 and/or mGlu3 receptor function as potential treatments for AUD.

Although parasitism is one of the most prevalent interactions in nature, studies of aquatic food webs rarely include parasites. Syndiniales (Dinophyceae, Alveolata) is a diverse parasitic group of dinoflagellates, common in all marine environments, and are described as dominant components of pelagic ecosystems. However, their temporal dynamics, prevalence, and host-specificity are poorly known. Using DNA metabarcoding to explore trophic interactions of zooplankton, we found a high proportion of Syndiniales sequence reads associated with the targeted consumers. We observed the occurrence of Syndiniales in copepods, cladocerans, appendicularians, and polychaete larvae, ranging between 11 and 36% relative read abundance, encompassing 11 main putative clades. Zooplankton–Syndiniales interactions showed variability in occurrence across the taxa, but also certain host-specificity. The study suggests that the observed copepod–Syndiniales interactions can be both direct parasitic infections and the result of trophic transmission through potentially infected prey by Syndiniales. Given the quantitative importance of Syndiniales and zooplankton in marine environments, our findings emphasize that their interactions should be recognized as key players in the structure and connectivity of plankton food webs.

The risk that stoats can detect, and become averse to, human scent on traps, baits and tracking tunnels is an important but unsolved question. In this pilot study, the scent marking behaviour of th...

Stress is a major risk factor for the development and exacerbation of mood and anxiety disorders, and recent studies have suggested inflammatory contributions to the pathogenesis of depression. Interestingly, pharmacological inhibition of cyclooxygenase-2 (COX-2) has shown promise in the treatment of affective disorders in small scale clinical studies; however, the mechanisms by which COX-2 inhibition affects behavioral domains relevant to affective disorders are not well understood. Here, we examined the effects of pharmacological inhibition of COX-2 with the highly selective inhibitor Lumiracoxib (LMX) on anxiety-like behavior and in vivo basolateral amygdala (BLA) neural activity in response to acute restraint stress exposure. In male mice, pretreatment with LMX prevented the increase in BLA calcium transients induced by restraint stress and prevented anxiogenic behavior seen after restraint stress exposure. Specifically, acute injection of LMX 5mg kg(−1) reduced anxiety-like behavior in the light-dark box (LD) and elevated-zero maze (EZM). In addition, in vivo fiber photometry studies showed that acute stress increased calcium transients and the predicted action potential frequency of BLA neurons, which was also normalized by acute LMX pretreatment. These findings indicate pharmacological inhibition of COX-2 can prevent acute stress-induced increase in BLA cellular activity and anxiety-like behavior and provides insights into the neural mechanisms by which COX-2 inhibition could affect anxiety domain symptoms in patients with affective disorders.

Failed transfer of passive immunity (FTPI) in dairy calves has substantial health consequences, but there have been no recently published estimates of the prevalence of FTPI on Ontario dairy farms. The major objectives of this cross-sectional study were to determine the level of FTPI in dairy calves across the province of Ontario and to determine colostrum management practices used. A convenience sample of 109 dairy farms in Ontario were visited from June to August 2019, along with 4 auction facilities from January to February 2019. At the single visit to each dairy farm, an in-person questionnaire was administered, and all calves between 24 h and 9 d of age had a blood sample collected to determine the levels of serum total protein (STP). At the auction facility, a blood sample was collected on all calves that arrived on the day of the visit. The dairy farms were distributed throughout Ontario, and the farms milked on average 151 dairy cows. Of the 444 calves sampled on Ontario dairy farms, 46% were male, 54% were female, and the mean weight and age at sampling were 46.3 kg and 4.4 d, respectively. The mean STP of calves sampled on the dairy farm was 5.69 g/dL (standard deviation = 0.78), and 107 calves (24%) had FTPI. The likelihood of FTPI was not associated with sex (female vs. male, risk ratio = 0.89, 95% confidence interval = 0.64-1.24), age (per day, risk ratio = 1.02, 95% confidence interval = 0.94-1.11), or weight (per kg, risk ratio = 0.98, 95% confidence interval = 0.96-1.01). In contrast, at the auction facilities, 386 male dairy calves were sampled and had a mean STP of 5.79 g/dL (standard deviation = 0.83). Using a cut point of ≥5.2 g/dL, 91 calves (24%) had FTPI. From the in-person questionnaire administered on the dairy farms, it was reported that the median time to first feeding of colostrum was 2.5 h (range = 0-12 h). The farms reported providing a mean of 3.3, 2.9, and 3.4 L of colostrum from 0 to 6 h after birth, 6 to 12 h after birth, and 12 to 24 h after birth, respectively. Ninety-one farms (83%) fed colostrum from the dam of the calf as their predominant source of colostrum. The most common method used to deliver the first feeding of colostrum was a nipple bottle (89 farms, 82%). Twenty-seven farms (25%) reported managing colostrum differently for male calves, which included a different colostrum source, use of poorer quality colostrum, a smaller quantity of colostrum, a longer time from birth to feeding, and generally having a lower focus on colostrum management for male calves. The prevalence of FTPI on Ontario dairy farms appears to have decreased since previous estimates, and substantial room for improvement remains. Although no overall differences were found in FTPI between male and female calves, differential reported colostrum management by sex indicates this may be a risk for male calves on a proportion of Ontario dairy farms.

Knowledge of zooplankton in situ diet is critical for accurate assessment of marine ecosystem function and structure, but due to methodological constraints, there is still a limited understanding of ecological networks in marine ecosystems. Here, we used DNA-metabarcoding to study trophic interactions, with the aim to unveil the natural diet of zooplankton species under temporal variation of food resources. Several target consumers, including copepods and cladocerans, were investigated by sequencing 16S rRNA and 18S rRNA genes to identify prokaryote and eukaryote potential prey present in their guts. During the spring phytoplankton bloom, we found a dominance of diatom and dinoflagellate trophic links to copepods. During the summer period, zooplankton including cladocerans showed a more diverse diet dominated by cyanobacteria and heterotrophic prey. Our study suggests that copepods present trophic plasticity, changing their natural diet over seasons, and adapting their feeding strategies to the available prey spectrum, with some species being more selective. We did not find a large overlap of prey consumed by copepods and cladocerans, based on prey diversity found in their guts, suggesting that they occupy different roles in the trophic web. This study represents the first molecular approach to investigate several zooplankton-prey associations under seasonal variation, and highlights how, unlike other techniques, the diversity coverage is high when using DNA, allowing the possibility to detect a wide range of trophic interactions in plankton communities.

Disbudding is a common procedure practiced in the dairy industry and is known to cause pain when performed without pain control. Dairy producers who disbud calves with caustic paste are less likely to provide pain control than those using cautery. Little research has been conducted on pain control for caustic paste disbudding and no studies have specifically examined calves under 9 d of age. The objective of this study was to evaluate the efficacy of local anesthesia and nonsteroidal anti-inflammatory drug analgesia on indicators of pain and inflammation in dairy calves disbudded using caustic paste. One hundred forty Holstein heifer calves 1 to 9 d of age were enrolled in 28 blocks and randomly allocated to 1 of 5 treatments: sham control (SH); positive control (POS); lidocaine cornual nerve block (LC); meloxicam (MEL); and lidocaine cornual nerve block plus meloxicam (LCM). We measured outcomes including serum cortisol and haptoglobin, pressure sensitivity, and lying behavior. Data were analyzed using mixed linear regression models with treatment as a fixed effect, baseline values as a covariate, and trial block as a random effect. Compared with the POS group, the LCM group had reduced serum cortisol at 15, 30, 45, and 60 min post-disbudding; cortisol values were not different between LC, LCM, and SH calves at these time points. At 60, 90, 120, and 180 min post-disbudding, LCM calves had reduced cortisol compared with LC calves, whereas, values did not differ between LCM and SH calves at these time points. At 3 to 4 d post-disbudding, the LCM group tended to have reduced haptoglobin, but no differences were found between groups at 180 min and 7 d post-disbudding. At 60, 90, and 120 min post-disbudding, LC and LCM treated calves had decreased pressure sensitivity compared with other groups. No differences were seen in pressure sensitivity between groups at 180 min, 3 to 4 or 7 d post-disbudding. No differences in lying behavior were found between treatment groups on any of the 7 d following disbudding. These findings demonstrate that the combination of a local anesthetic with a nonsteroidal anti-inflammatory drug is beneficial for reducing indicators of pain and inflammation in young calves disbudded with caustic paste.

Excitatory signaling mediated by NMDARs has been shown to regulate mood disorders. However, current treatments targeting NMDAR subtypes have shown limited success in treating patients, highlighting a need for alternative therapeutic targets. Here, we identify a role for GluN2D-containing NMDARs in modulating emotional behaviors and neural activity in the bed nucleus of the stria terminalis (BNST). Using a GluN2D KO mouse line (GluN2D−/−), we assessed behavioral phenotypes across tasks modeling emotional behavior. We then used a combination ofex vivoelectrophysiology andin vivofiber photometry to assess changes in BNST plasticity, cell-specific physiology, and cellular activity profiles. GluN2D−/−male mice exhibit evidence of exacerbated negative emotional behavior, and a deficit in BNST synaptic potentiation. We also found that GluN2D is functionally expressed on corticotropin-releasing factor (CRF)-positive BNST cells implicated in driving negative emotional states, and recordings in mice of both sexes revealed increased excitatory and reduced inhibitory drive onto GluN2D−/−BNST-CRF cellsex vivoand increased activityin vivo. Using a GluN2D conditional KO line (GluN2Dflx/flx) to selectively delete the subunit from the BNST, we find that BNST-GluN2Dflx/flxmale mice exhibit increased depressive-like behaviors, as well as altered NMDAR function and increased excitatory drive onto BNST-CRF neurons. Together, this study supports a role for GluN2D-NMDARs in regulating emotional behavior through their influence on excitatory signaling in a region-specific manner, and suggests that these NMDARs may serve as a novel target for selectively modulating glutamate signaling in stress-responsive structures and cell populations.SIGNIFICANCE STATEMENTExcitatory signaling mediated through NMDARs plays an important role in shaping emotional behavior; however, the receptor subtypes/brain regions through which this occurs are poorly understood. Here, we demonstrate that loss of GluN2D-containing NMDARs produces an increase in anxiety- and depressive-like behaviors in mice, deficits in BNST synaptic potentiation, and increased activity in BNST-CRF neurons known to drive negative emotional behavior. Further, we determine that deleting GluN2D in the BNST leads to increased depressive-like behaviors and increased excitatory drive onto BNST-CRF cells. Collectively, these results demonstrate a role for GluN2D-NMDARs in regulating the activity of stress-responsive structures and neuronal populations in the adult brain, suggesting them as a potential target for treating negative emotional states in mood-related disorders.

Objective: To review the metabolic and hormonal processes that regulate reproductive activity in dairy cattleduring the postpartum anestrus period.Design/methodology/approach: Analysis of scientific documents and systematization of information relatedto the reproductive physiology and management of dairy cattle in postpartum anestrus.Results: Postpartum anestrus is an event that occurs naturally and is necessary for restoration of the homeostasisof the cow’s body following calving. However, failure to reestablish this homeostasis actually increases itsduration and negatively affects the subsequent reproductive performance of the individual cow.Limitations on study/implications: Reproductive management decisions must be based on scientificknowledge.Findings/conclusions: Management of dairy cattle in the postpartum anestrus period should focus onreestablishment of the state of homeostasis and the subsequent reproductive activity, with nutritional andhormonal strategies implemented to induce cyclicity, ovulation and formation of the first corpus luteum in thatperiod, through application of exogenous hormones after day ten postpartum.

Mercury is a persistent, biomagnifying contaminant that can cause negative behavioral, immunological, and reproductive effects in wildlife and human populations. We examined the role of wetland water-management on mercury bioaccumulation in songbirds and ducks at Kellys Slough National Wildlife Refuge Complex, near Grand Forks, North Dakota USA. We assessed mercury concentrations in blood of wetland-foraging songbirds (80 common yellowthroats [Geothlypis trichas] and 14 Nelson's sparrows [Ammospiza nelsoni]) and eggs of upland-nesting ducks (28 gadwall [Mareca strepera], 19 blue-winged teal [Spatula discors], and 13 northern shoveler [S. clypeta]) across four wetland water-management classifications. Nelson's sparrow blood mercury concentrations were elevated (mean: 1.00 µg/g ww; 95% CL: 0.76-1.31) and similar to those reported 6 years previously. Mercury in songbird blood and duck eggs varied among wetland water-management classifications. Songbirds and ducks had 67% and 49% lower mercury concentrations, respectively, when occupying wetlands that were drawn down with water flow compared to individuals occupying isolated-depressional wetlands with no outflow. Additionally, songbirds within impounded and partially drawn-down wetland units with water flow had mercury concentrations that were 26-28% lower, respectively, than individuals within isolated-depressional wetlands with no outflow. Our results confirm that mercury concentrations in songbirds at Kellys Slough continue to be elevated and suggest that water-management could be an important tool for wetland managers to reduce bioaccumulation of mercury in birds.

Theory suggests that different taxa having colonized a similar, challenging environment will show parallel or lineage-specific adaptations to shared selection pressures, but empirical examples of parallel evolution in independent taxa are exceedingly rare. We employed comparative genomics to identify parallel and lineage-specific responses to selection within and among four species of North American sparrows that represent four independent, post-Pleistocene colonization events by an ancestral, upland subspecies and a derived salt marsh specialist. We identified multiple cases of parallel adaptation in these independent comparisons following salt marsh colonization, including selection of 12 candidate genes linked to osmoregulation. In addition to detecting shared genetic targets of selection across multiple comparisons, we found many novel, species-specific signatures of selection, including evidence of selection of loci associated with both physiological and behavioral mechanisms of osmoregulation. Demographic reconstructions of all four species highlighted their recent divergence and small effective population sizes, as expected given their rapid radiation into saline environments. Our results highlight the interplay of both shared and lineage-specific selection pressures in the colonization of a biotically and abiotically challenging habitat and confirm theoretical expectations that steep environmental clines can drive repeated and rapid evolutionary diversification in birds.

Background Relapse is a critical barrier to effective long-term treatment of alcoholism, and stress is often cited as a key trigger to relapse. Numerous studies suggest that stress-induced reinstatement to drug-seeking behaviors is mediated by norepinephrine (NE) and corticotropin-releasing factor (CRF) signaling interactions in the bed nucleus of the stria terminalis (BNST), a brain region critical to many behavioral and physiologic responses to stressors. Here, we sought to directly examine the effects of NE on BNST CRF neuron activity and determine whether these effects may be modulated by chronic intermittent EtOH (CIE) exposure or a single restraint stress. Methods Adult male CRF-tomato reporter mice were treatment-naive, or either exposed to CIE for 2 weeks or to a single 1-hour restraint stress. Effects of application of exogenous NE on BNST CRF neuron activity were assessed via whole-cell patch-clamp electrophysiological techniques. Results We found that NE depolarized BNST CRF neurons in naive mice in a β-adrenergic receptor (AR)-dependent mechanism. CRF neurons from CIE- or stress-exposed mice had significantly elevated basal resting membrane potential compared to naive mice. Furthermore, CIE and stress individually disrupted the ability of NE to depolarize CRF neurons, suggesting that both stress and CIE utilize β-AR signaling to modulate BNST CRF neurons. Neither stress nor CIE altered the ability of exogenous NE to inhibit evoked glutamatergic transmission onto BNST CRF neurons as shown in naive mice, a mechanism previously shown to be α-AR-dependent. Conclusions Altogether, these findings suggest that stress and CIE interact with β-AR signaling to modulate BNST CRF neuron activity, potentially disrupting the α/β-AR balance of BNST CRF neuronal excitability. Restoration of α/β-AR balance may lead to novel therapies for the alleviation of many stress-related disorders.

Alcohol use disorder (AUD) is associated with substantial morbidity, mortality, and societal cost, and pharmacological treatment options are limited. The endogenous cannabinoid (eCB) signaling system is critically involved in reward processing, and alcohol intake is positively correlated with release of the eCB ligand 2-arachidonoylglycerol (2-AG) within the reward neurocircuitry. Here we show that genetic and pharmacological inhibition of diacylglycerol lipase (DAGL), the rate-limiting enzyme in the synthesis of 2-AG, reduces alcohol consumption in a variety of preclinical mouse models, ranging from a voluntary free-access model to aversion-resistant drinking and dependence-like drinking induced via chronic intermittent ethanol vapor exposure. DAGL inhibition during either chronic alcohol consumption or protracted withdrawal did not elicit anxiogenic and depression-like behavioral effects. Last, DAGL inhibition also prevented ethanol-induced suppression of GABAergic transmission onto midbrain dopamine neurons, providing mechanistic insight into how DAGL inhibition could affect alcohol reward. These data suggest that reducing 2-AG signaling via inhibition of DAGL could represent an effective approach to reducing alcohol consumption across the spectrum of AUD severity.

Campylobacter is the second leading cause of foodborne illness in the United States. Although many food production animals carry Campylobacter as commensal bacteria, consumption of poultry is the main source of human infection. Previous research suggests that the biology of Campylobacter results in complete flock colonization within days. However, a recent systematic review found that the on-farm prevalence of Campylobacter varies widely, with some flocks reporting low prevalence. We hypothesized that the low prevalence of Campylobacter in some flocks may be driven by a delayed introduction of the pathogen. The objectives of this study were to (a) develop a deterministic compartmental model that represents the biology of Campylobacter, (b) identify the parameter values that best represent the natural history of the pathogen in poultry flocks and (c) examine the possibility that a delayed introduction of the pathogen is sufficient to replicate the observed low prevalence examples documented in the literature. A deterministic compartmental model was developed to examine the dynamics of Campylobacter in chicken flocks over a 56-day time period prior to movement to the abattoir. The model outcome of interest was the final population prevalence of Campylobacter at day 56. The resulting model that incorporated a high transmission rate (β = 1.04) was able to reproduce the wide range of prevalence estimates observed in the literature when pathogen introduction time is varied. Overall, we established that the on-farm transmission rate of Campylobacter in chickens is likely high and can result in complete colonization of a flock when introduced early. However, delaying the time at which the pathogen enters the flock can reduce the prevalence observed at 56 days. These results highlight the importance of enforcing strict biosecurity measures to prevent or delay the introduction of the bacteria to a flock.

Alternative pathways of energy transfer guarantee the functionality and productivity in marine food webs that experience strong seasonality. Nevertheless, the complexity of zooplankton interactions is rarely considered in trophic studies because of the lack of detailed information about feeding interactions in nature. In this study, we used DNA metabarcoding to highlight the diversity of trophic niches in a wide range of micro- and mesozooplankton, including ciliates, rotifers, cladocerans, copepods and their prey, by sequencing 16- and 18S rRNA genes. Our study demonstrates that the zooplankton trophic niche partitioning goes beyond both phylogeny and size and reinforces the importance of diversity in resource use for stabilizing food web efficiency by allowing for several different pathways of energy transfer. We further highlight that small, rarely studied zooplankton (rotifers and ciliates) fill an important role in the Baltic Sea pelagic primary production pathways and the potential of ciliates, rotifers and crustaceans in the utilization of filamentous and picocyanobacteria within the pelagic food web. The approach used in this study is a suitable entry point to ecosystem-wide food web modelling considering species-specific resource use of key consumers.

Red blood cell transfusions (RBCT) remain the cornerstone of supportive care in lower-risk myelodysplastic syndrome (LRMDS) [1]. Transfusion dependency in LRMDS patients is associated with inferior outcomes, mainly attributed to severe bone marrow failure [2]. However, iron toxicity, due to frequent RBCT or ineffective erythropoiesis, may be an additional negative prognostic factor [3,4,5,6]. Recently, much progress has been made in unraveling the iron metabolism. The peptide hormone hepcidin is the key regulator by inhibiting iron uptake through degradation of ferroportin, a cellular iron exporter [7]. Erythroferrone and GDF15, produced by erythroblasts, inhibit hepcidin production, which leads to increased uptake and cellular release of iron for the purpose of erythropoiesis [8]. The pathophysiology of iron metabolism in MDS is still not completely understood. Exceedingly high reactive oxygen species (ROS) levels are associated with iron toxicity, disease development, and progression in MDS patients [9,10,11,12]. Malondialdehyde (MDA), resulting from lipid peroxidation of polyunsaturated fatty acids, is a biomarker of oxidative stress [10, 12]. Currently, little is known about the prognostic impact of ROS in MDS patients. The aim of this study is twofold: (1) describe iron and oxidative stress parameters over time in LRMDS patients and (2) to assess their effect on overall and progression-free survival. The EUMDS registry prospectively collects observational data on newly diagnosed LRMDS patients from 148 centers in 16 countries in Europe and Israel as of January 2008. All patients provided informed consent. Clinical data were collected at baseline and at each six-monthly follow-up visit. Serum samples were collected prospectively at each visit from 256 patients included in six participating countries. Conventional iron parameters were measured with routine assays. We additionally analyzed hepcidin, growth differentiation factor 15 (GDF15), soluble transferrin receptor (sTfR), non-transferrin bound iron (NTBI), labile plasma iron (LPI), and MDA. Subjects were prospectively followed until death, loss to follow-up, or withdrawal of consent. All iron parameters were measured centrally at the department of Laboratory Medicine of the Radboudumc, Nijmegen, The Netherlands. Serum samples were collected just prior to transfusion in transfusion-dependent patients and stored at −80 °C. Details on the assays and reference ranges of hepcidin, GDF15, sTfR, NTBI, LPI, and MDA are provided in the supplement. The Spearman rank test was used to evaluate correlations between iron parameters. We stratified the results by transfusion dependency per visit and the presence of ring sideroblasts. When evaluating temporal changes in iron parameters, with linear quantile mixed models, we excluded patients from the timepoint they received iron chelation therapy. Overall survival (OS) was defined as the time from MDS diagnosis to death or, in case of progression-free survival, to date of progression or death; patients still alive at the end of follow-up were censored. Time-dependent Kaplan–Meier curves and cox proportional hazards models were used. In total, 256 consecutive patients, were included in this study. Over five six-monthly visits, 1040 samples were collected. Table 1 describes the patient characteristics. Most patients without ring sideroblasts were transfusion-independent at diagnosis (nonRS-TI; 55.9%), 18.8% with ring sideroblasts were transfusion-independent (RS-TI), 18.4% without ring sideroblasts were transfusion-dependent (nonRS-TD), and 7% with ring sideroblasts were transfusion-dependent patients (RS-TD). The median follow-up time was 6.6 years (95% CI 5.9–7.0). LPI was positively correlated with transferrin saturation (TSAT) (r = 0.15, p < 0.001, Fig. S1). LPI values increased exponentially at TSAT values above 80%. This effect was most pronounced in the transfusion-dependent groups, but also observed in the RS-TI group. MDA was weakly correlated with NTBI (r = 0.09, p = 0.069) and negatively correlated with hemoglobin level (r = −0.1, p = 0.033). GDF15 and hepcidin were negatively correlated in the RS-TI and nonRS-TD group and significantly negatively correlated in the RS-TD group (r = −0.34, p = 0.007, Fig. S2). Serum ferritin levels were elevated in all subgroups with a mean value of 858 µg/L at visit 5. The highest serum ferritin levels were observed in the RS-TD group (mean value at visit 5: 2092 µg/L, Table S1). Serum ferritin increased significantly per visit in the RS-TD group (beta 454.46 µg/L; 95% CI 334.65–574.27), but not in the other groups (Table S2). All subgroups, except for the nonRS-TI, had elevated TSAT levels. TSAT levels were most markedly increased in the RS-TD group with a mean TSAT of 88% at visit 5 (Table S1). In both transfusion-dependent groups the median increase per visit was significant (Table S2). LPI was elevated in the RS-TD group exclusively with a mean value of 0.59 µmol/L at visit 5 (Table S1). NTBI was elevated in all subgroups, with the highest values in the RS-TD group (Table S1). The increase in median NTBI level was significant in both transfusion-dependent groups (Table S2). Hepcidin levels were markedly elevated in the nonRS-TD group. Interestingly, hepcidin levels were lower in the RS-TD group, probably reflecting ineffective erythropoiesis, likewise supported by lower hepcidin/ferritin ratios in RS groups (Table S1). Median hepcidin levels increased over time in the transfusion-dependent subgroups only (Table S2). GDF15 levels, analyzed in the light of its potential role in hepcidin suppression, were increased in all subgroups (Table S1). The RS subgroups had higher GDF15 levels compared to the nonRS groups, reflecting increased erythropoiesis. Mean sTfR levels were within the reference range in all subgroups except for the RS-TI group, which showed elevated levels, reflecting...

Mutational analysis of circulating tumour (ct) DNA holds promise as an effective tool to predict the course of metastatic breast cancer (MBC). In the present study we used targeted next generation sequencing of ctDNA to evaluate the impact of cancer driven mutations on the prognosis of MBC. The study included 59 oestrogen receptor-positive (ER+), HER2-negative MBC patients. Sequencing analysis was performed in ESR1, PIK3CA, ERBB2, PTEN, TP53, KRAS, HRAS, NRAS, and AR. At baseline, patients started receiving either chemotherapy (34%; n = 20) or cyclin-dependent kinase 4/6 inhibitor therapy in combination with endocrine therapy (CDK4/6i+ET; 66%; n = 39). Overall, 64.4% (n = 38) of the patients carried at least one pathogenic or likely-pathogenic mutation. Number of ctDNA mutations was significantly linked with worse progression free survival (PFS; p = 0.003) and overall survival (OS; p = 0.007). Furthermore, ctDNA load, defined by the number of mutant ctDNA molecules per mL plasma, significantly correlated with PFS (p p = 0.001). Furthermore, mutational status of ESR1 and TP53 significantly predicted PFS (p = 0.024 and p = 0.035, respectively) and OS (p p = 0.035, respectively). These results emphasizes the clinical value of ctDNA mutational analysis in the management of advanced breast cancer.

Novel drug leads for malaria therapy are urgently needed because of the widespread emergence of resistance to all available drugs. Screening of the Harbor Branch enriched fraction library against the Plasmodium falciparum chloroquine-resistant strain (Dd2) followed by bioassay-guided fractionation led to the identification of two potent antiplasmodials, a novel diterpene designated as bebrycin A (1) and the known C21 degraded terpene nitenin (2). A SYBR Green I assay was used to establish a Dd2 EC50 of 1.08 ± 0.21 and 0.29 ± 0.02 µM for bebrycin A and nitenin, respectively. Further analysis was then performed to assess the stage specificity of the inhibitors antiplasmodial effects on the Dd2 intraerythrocytic life cycle. Exposure to bebrycin A was found to block parasite maturation at the schizont stage if added any time prior to late schizogony at 42 hours post invasion, (HPI). In contrast, early life cycle exposure to nitenin (prior to 18 HPI) was identified as crucial to parasite inhibition, suggesting nitenin may target the maturation of the parasite during the transition from ring to early trophozoite (6–18 HPI), a novel property among known antimalarials.

Multidrug-resistant Gram-negative bacteria pose a global threat to human health. The AcrB efflux pump confers inherent and evolved drug resistance to Enterobacterales, including Escherichia coli and Salmonella enterica serovar Typhimurium., In the fight against antibiotic resistance, drugs that target resistance mechanisms in bacteria can be used to restore the therapeutic effectiveness of antibiotics. The multidrug resistance efflux complex AcrAB-TolC is the most clinically relevant efflux pump in Enterobacterales and is a target for drug discovery. Inhibition of the pump protein AcrB allows the intracellular accumulation of a wide variety of antibiotics, effectively restoring their therapeutic potency. To facilitate the development of AcrB efflux inhibitors, it is desirable to discover the native substrates of the pump, as these could be chemically modified to become inhibitors. We analyzed the native substrate profile of AcrB in Escherichia coli MG1655 and Salmonella enterica serovar Typhimurium SL1344 using an untargeted metabolomics approach. We analyzed the endo- and exometabolome of the wild-type strain and their respective AcrB loss-of-function mutants (AcrB D408A) to determine the metabolites that are native substrates of AcrB. Although there is 95% homology between the AcrB proteins of S. Typhimurium and E. coli, we observed mostly different metabolic responses in the exometabolomes of the S. Typhimurium and E. coli AcrB D408A mutants relative to those in the wild type, potentially indicating a differential metabolic adaptation to the same mutation in these two species. Additionally, we uncovered metabolite classes that could be involved in virulence of S. Typhimurium and a potential natural substrate of AcrB common to both species.

The dorsal region of the bed nucleus of the stria terminalis (dBNST) receives substantial dopaminergic input which overlaps with norepinephrine input implicated in stress responses. Using ex vivo fast scan cyclic voltammetry in male C57BL6 mouse brain slices, we demonstrate that electrically stimulated dBNST catecholamine signals are of substantially lower magnitude and have slower uptake rates compared with caudate signals. Dopamine terminal autoreceptor activation inhibited roughly half of the catecholamine transient, and noradrenergic autoreceptor activation produced an ∼30% inhibition. Dopamine transporter blockade with either cocaine or GBR12909 significantly augmented catecholamine signal duration. We optogenetically targeted dopamine terminals in the dBNST of transgenic (TH:Cre) mice of either sex and, using ex vivo whole-cell electrophysiology, we demonstrate that optically stimulated dopamine release induces slow outward membrane currents and an associated hyperpolarization response in a subset of dBNST neurons. These cellular responses had a similar temporal profile to dopamine release, were significantly reduced by the D2/D3 receptor antagonist raclopride, and were potentiated by cocaine. Using in vivo fiber photometry in male C57BL/6 mice during training sessions for cocaine conditioned place preference, we show that acute cocaine administration results in a significant inhibition of calcium transient activity in dBNST neurons compared with saline administration. These data provide evidence for a mechanism of dopamine-mediated cellular inhibition in the dBNST and demonstrate that cocaine augments this inhibition while also decreasing net activity in the dBNST in a drug reinforcement paradigm. SIGNIFICANCE STATEMENT The dorsal bed nucleus of the stria terminalis (dBNST) is a region highly implicated in mediating stress responses; however, the dBNST also receives dopaminergic inputs from classically defined drug reward pathways. Here we used various techniques to demonstrate that dopamine signaling within the dBNST region has inhibitory effects on population activity. We show that cocaine, an abused psychostimulant, augments both catecholamine release and dopamine-mediated cellular inhibition in this region. We also demonstrate that cocaine administration reduces population activity in the dBNST, in vivo. Together, these data support a mechanism of dopamine-mediated inhibition within the dBNST, providing a means by which drug-induced elevations in dopamine signaling may inhibit dBNST activity to promote drug reward.

Human primary skeletal myoblasts can be isolated from diagnostic muscle biopsy specimens and are routinely stored in biobanks. These myoblasts often represent the only available cell type from a specific patient, thereby serving as a potentially valuable cell source for induced pluripotent stem cell (iPSC) generation and disease modeling. Myoblasts can be reprogrammed into iPSCs through ectopic overexpression of critical transcription factors using genome-integration and integration-free methodologies. Furthermore, reprogramming efficiency can be enhanced by small molecule inhibitors. Combined with recent advances in genome editing, patient-specific iPSC lines serve as an invaluable resource for disease modeling of muscular dystrophies, and offer substantial promise in regenerative and personalized medicine. In this chapter, we discuss myoblast reprogramming into iPSCs; the use of genome editing tools for studying the pathogenesis of muscular dystrophy in patient-derived iPSCs; and potential future applications of genome editing in iPSCs for the treatment of Duchenne muscular dystrophy.

The study of depriving cells from background ionizing radiation for the past decades has provided valuable insights into its role in cellular homeostasis control. To explore the existence of such response in eukaryotic cells, we grew Chinese hamster (Cricetulus griseus) V79 cells for 23 days using three different dose rates: 0.91 (below background), 35 (surface control) and 72 nGy h−1 (underground KCl-amended control). We did not observe a significant difference in cell number during the course of the experiment. However, cells grown at below background showed significantly lower viability compared to those grown at both control levels after 5 days of incubation and lasted, intermittently, for up to 21 days. We also observed a clear differentiation between the underground and the surface controls that could be explained by the variety of radiation sources present during cell growth under unshielded conditions. To explore the molecular mechanisms for these responses we performed transcriptome analysis on samples collected on days 2 and 5, but only samples from day 5 resulted in significant regulation. Gene enrichment analysis revealed two control-dependent general transcriptional responses. When compared the underground-KCl control, below-background cells showed the upregulation of processes intended for the response to drugs, metals and mechanical stimuli. In comparison, the response relative to the surface control was characterized by the upregulation of responses to organic substances and abiotic stimuli involved in the regulation of signaling, as well as to cell proliferation and homeostatic control of the number of cell processes.

IntroductionThe ability for ecosystems to maintain productivity and functionality under seasonal and long term changes in resource availability relies on the diversity of functional groups (Cadotte et al. 2011). In marine food webs, functionally diverse assemblages of heterotrophic bacteria, heterotrophic protists and zooplankton transfer the organic matter from primary producers to higher trophic levels (Sommer 1989). Zooplankton regulate the flow of energy and matter in the food web through several mechanisms including grazing, respiration, excretion, and as food to support higher trophic levels (Calbet & Landry 2004; Mitra & Davis 2010; Steinberg & Landry 2017). Variation in temporal abundance, feeding traits, size, phenotypic plasticity, growth rate and predation resistance all contribute to the total diversity of zooplankton functional groups in marine food webs (Petchey & Gaston 2006). A high diversity of functional groups contributes to a large variety of resource use that is crucial for the maintenance of ecosystem services under changing conditions (Cadotte et al. 2011). To generate accurate predictions of vulnerability and estimate the resilience of marine ecosystems, a mechanistic understanding of resource use by zooplankton is needed (Bindoff et al. 2019). However, most trophic studies are based on size or phylogeny, and the complexity of zooplankton interactions is rarely considered in trophic studies because of the lack of detailed information about feeding interactions in nature. Consequently, the functional diversity of the zooplankton community and their ability to exploit similar resources is typically not accurately considered (Mitra et al. 2014).The diversity of zooplankton allows for maintaining the biomass of fish stocks over the seasons by a shift from a phytoplankton to detritus-based food webs at times when the biomass of phytoplankton is low or inedible (D’Alelio et al. 2016). While crustacean zooplankton (e.g. copepods and cladocerans) constitute the primary link between phytoplankton and planktivorous fish (Cushing 1990), microzooplankton (i.e. heterotrophic flagellates, ciliates and rotifers) can at times dominate ocean’s carbon respiration in productive coastal ecosystems(Sherr & Sherr 2002; Calbet & Landry 2004). By utilizing matter recycled by heterotrophic bacteria in the microbial loop (Azamet al. 1983), the microzooplankton serve as an additional link between primary producers and crustacean zooplankton (Gifford 1991). The possibility to switch between alternative food web states may be particularly critical in coastal ecosystems that experience an increase in filamentous cyanobacteria due to climate warming (Paerl & Huisman 2008; Cloern et al. 2016).While most trophic studies have clustered zooplankton into broad phylogenetic groups (Mitra et al. 2014), recent studies suggest that models based on traits, particularly size, reflect the true ecosystem structure more effectively (Sommer & Stibor 2002; Boyceet al. 2015). However, none of these approaches consider the entire functional group diversity of zooplankton. As an example, the rotifer phylum contains members of different size classes (belonging to both the micro- and mesozooplankton) (Arndt 1993), as well as organisms with different feeding behaviors such as micro-filtering feeders (Pourriot 1977), selective feeders (Bogdan et al. 1980; Bogdan & Gilbert 1982; Gilbert & Jack 1993), and in some cases even carnivores (Gilbert 1980). Similarly, copepods and cladocerans can perform different feeding strategies ranging from feeding-current feeding to passive/active ambush feeding (Kiorboe 2011), utilizing a wide spectrum of resources.The difficulty in resolving plankton food webs lies within method limitations. Traditional methods to study plankton food webs, such as grazing dilution techniques (Landry & Hassett 1982), biogeochemical tracers or microscopic observations (Post 2002), may not display its full complexity with enough resolution, and has created a biased knowledge towards larger organisms in the food web that are more frequently studied (Gutierrez-Rodriguez et al. 2014). Molecular techniques, including DNA sequencing targeting plankton communities, has highlighted the complexity and diversity of plankton interactions on a global scale (Lima-Mendez et al. 2015). Further, DNA metabarcoding of gut content or selected organisms has proven to be a useful tool for resolving trophic interactions (Pompanon et al.2012) and for zooplankton, barcoding of whole organisms can resolve both trophic, parasitic and mutualistic interactions among crustacean zooplankton (De Corte et al. 2017; Zamora-Terol et al.2020). To our knowledge no study has so far aimed to estimate the diversity of functional groups of zooplankton spanning both phylum and size, using targeted DNA metabarcoding.In this study, we aimed to investigate the functional group diversity of the most abundant zooplankton genera in the Baltic Sea, a temperate coastal sea with strong seasonal variability, and where both micro- and mesozooplankton are at times dominating with well-defined abundance peaks (Fig. 1). We hypothesize that diet composition between zooplankton consumers constitutes a more realistic proxy for functional diversity compared to size and phylogeny. By sequencing 18S rRNA and16S rRNA barcoding genes, we analyzed zooplankton-associated prey of selected individuals of different size classes including a ciliate and rotifers, and compared them with the most abundant crustacean zooplankton (copepods and cladocerans). We demonstrate a larger functional group diversity in resource use within zooplankton in the Baltic Sea than previously acknowledged. The functional group diversity goes beyond both phylogenetic diversity and size and is crucial for the understanding of key ecological processes and maintenance of ecosystem functions.

Compound-specific isotope analyses (CSIA) of fatty acids (FA) constitute a promising tool for tracing energy flows in food-webs. However, past applications of FA-specific carbon isotope analyses have been restricted to a relatively coarse food-source separation and mainly quantified dietary contributions from different habitats. Our aim was to evaluate the potential of FA-CSIA to provide high-resolution data on within-system energy flows using algae and zooplankton as model organisms. First, we investigated the power of FA-CSIA to distinguish among four different algae groups, namely cyanobacteria, chlorophytes, haptophytes and diatoms. We found substantial within-group variation but also demonstrated that δ 13 C of several FA (e.g. 18:3 ω 3 or 18:4 ω 3) differed among taxa, resulting in group-specific isotopic fingerprints. Second, we assessed changes in FA isotope ratios with trophic transfer. Isotope fractionation was highly variable in daphnids and rotifers exposed to different food sources. Only δ 13 C of nutritionally valuable poly-unsaturated FA remained relatively constant, highlighting their potential as dietary tracers. The variability in fractionation was partly driven by the identity of food sources. Such systematic effects likely reflect the impact of dietary quality on consumers' metabolism and suggest that FA isotopes could be useful nutritional indicators in the field. Overall, our results reveal that the variability of FA isotope ratios provides a substantial challenge, but that FA-CSIA nevertheless have several promising applications in food-web ecology. This article is part of the theme issue ‘The next horizons for lipids as ‘trophic biomarkers’: evidence and significance of consumer modification of dietary fatty acids’.

Background Dysregulation of arousal is symptomatic of numerous psychiatric disorders. Previous research has shown that the activity of dopamine (DA) neurons in the ventral periaqueductal gray (vPAG) tracks with arousal state, and lesions of vPAGDA cells increase sleep. However, the circuitry controlling these wake-promoting DA neurons is unknown. Methods This study combined designer receptors exclusively activated by designer drugs (DREADDs), behavioral pharmacology, electrophysiology, and immunoelectron microscopy in male and female mice to elucidate mechanisms in the vPAG that promote arousal. Results Activation of locus coeruleus projections to the vPAG or vPAGDA neurons induced by DREADDs promoted arousal. Similarly, agonist stimulation of vPAG alpha1-adrenergic receptors (α1ARs) increased latency to fall asleep, whereas α1AR blockade had the opposite effect. α1AR stimulation drove vPAGDA activity in a glutamate-dependent, action potential–independent manner. Compared with other dopaminergic brain regions, α1ARs were enriched on astrocytes in the vPAG, and mimicking α1AR transmission specifically in vPAG astrocytes via Gq-DREADDS was sufficient to increase arousal. In general, the wake-promoting effects observed were not accompanied by hyperactivity. Conclusions These experiments revealed that vPAG α1ARs increase arousal, promote glutamatergic input onto vPAGDA neurons, and are abundantly expressed on astrocytes. Activation of locus coeruleus inputs, vPAG astrocytes, or vPAGDA neurons increase sleep latency but do not produce hyperactivity. Together, these results support an arousal circuit whereby noradrenergic transmission at astrocytic α1ARs activates wake-promoting vPAGDA neurons via glutamate transmission.

Fish larvae abundances, diversity and trophic position across shallow seagrass, coral reef and open water habitats were examined to characterize their distribution in coastal East Africa. Larvae were identified to family and analysed for abundance differences between sites and habitats, trophic level using stable-isotope analysis and parental spawning mode. Abundances differed greatly between sites with the highest numbers of larvae occurring in the open-water and seagrass habitats. Larval fish diversity was high across habitats with 51 families identified with small differences between sites and among habitats. Notably, larvae of abundant large herbivorous fishes present in reef and seagrass habitats were almost completely absent at all sampling locations. In the seagrass, demersal spawned larvae were more abundant compared with the reef and open-water habitats. Stable-isotope analysis revealed that fish larvae have a varied diet, occupying trophic level two to three and utilizing planktonic prey. This study offers new insights into distributional aspects of fish larvae along the East African coast where such information is sparse.

There is a strong need for management strategies that help reduce the incidence of disease and subsequent antimicrobial use in the veal industry. Biomarkers could serve as useful tools that allow producers to take preventative measures before clinical problems occur. The objective of this prospective cohort study was to identify biomarkers measured upon arrival associated with morbidity, mortality, and average daily gain (ADG) in grain-fed veal calves. Upon arrival at a grain-fed veal facility in Ontario, Canada, calves were weighed and health-scored using a standardized health-scoring system. Several metabolites were measured including creatine kinase (CK), cholesterol, haptoglobin, manganese, serum total protein, iron, cobalt, zinc, selenium, molybdenum, and IgG. Farm personnel treated calves according to their farm protocol and weighed them at 78 d after arrival. Performance, treatment, and mortality records from the facility were used for analysis. Multivariable Cox proportional hazard models were created to evaluate metabolic biomarkers associated with morbidity and mortality. A mixed linear regression model was created to determine biomarkers associated with ADG. A total of 992 male dairy calves were evaluated at arrival from January to December 2017. Of the calves evaluated, 74 calves (7.5%) died and 877 (88.4%) were treated for illness over the 11-wk period under observation. Higher levels of haptoglobin and molybdenum were associated with a greater hazard of morbidity, whereas higher weight upon arrival and higher levels of both CK and IgG were associated with a reduced hazard of morbidity. For mortality, higher weight upon arrival and higher levels of cholesterol and IgG were associated with a lower hazard of mortality occurring over the 78-d period of observation. Higher weight, cholesterol, copper, CK, iron, and IgG were associated with increased ADG, whereas increased zinc and haptoglobin were negatively associated with ADG. These results demonstrate that certain biomarkers could be used to identify high-risk calves when measured upon arrival at a veal facility. Future research should focus on determining the utility of these biomarkers to selectively target intervention strategies to improve ADG and reduce morbidity and mortality.

Ant mimicry, i.e., the mimicking of ant workers by another organism, is a widespread phenomenon among different groups of Euarthropoda, including spiders and different insect species. One example of ant mimicry occurs among praying mantises (Mantodea); here the first stage nymphs have been recorded to perform ant mimicry. In this study, we investigated different nymphal instars of Miomantis binotata for possible morphological similarities to ants. The different instars were compared as stages supposed to perform ant mimicry would differ morphologically from those stages not supposed to resemble ants. The specimens were investigated under different microscopic settings and measurements were performed. Our results do not show significant differences concerning morphological measurements or shape of structures between the different nymphal instars of M. binotata. One prominent difference between stage one nymphs and later stages occurs in the colouration of the body, which is very dark in the earliest nymph. This difference might explain why young nymphs of Miomantis binotata are interpreted as ant-mimicking, despite the apparent lack of other morphological resemblances., Fragmenta Entomologica, Vol. 52 No. 1 (2020)

The bed nucleus of the stria terminalis (BNST) is emerging as a critical region in multiple psychiatric disorders including anxiety, PTSD, and alcohol and substance use disorders. In conjunction with growing knowledge of the BNST, an increasing number of studies examine connections of the BNST and how those connections impact BNST function. The importance of this BNST network is highlighted by rodent studies demonstrating that projections from other brain regions regulate BNST activity and influence BNST-related behavior. While many animal and human studies replicate the components of the BNST network, to date, structural connections between the BNST and insula have only been described in rodents and have yet to be shown in humans. In this study, we used probabilistic tractography to examine BNST-insula structural connectivity in humans. We used two methods of dividing the insula: 1) anterior and posterior insula, to be consistent with much of the existing insula literature; and 2) eight subregions that represent informative cytoarchitectural divisions. We found evidence of a BNST-insula structural connection in humans, with the strongest BNST connectivity localized to the anteroventral insula, a region of agranular cortex. BNST-insula connectivity differed by hemisphere and was moderated by sex. These results translate rodent findings to humans and lay an important foundation for future studies examining the role of BNST-insula pathways in psychiatric disorders.

Food quantity–quality interactions determine growth rates and reproductive success of consumers and thereby regulate community dynamics and food web structure. Predator–prey models that shape our conceptual understanding of foraging ecology typically rely on the parametrization of fixed consumer responses to either food quantity or food quality. In nature, however, consumers optimize their fitness by responding simultaneously to changes in food quantity and quality. Therefore, we assessed consumer responses to changing food environments using a new fitness optimization model that accounted for food quality–quantity interactions to better capture the regulatory flexibility of consumers. Our simulations demonstrated that the impact of food quality on important consumer traits can be altered or even reversed by changes in food quality. Low food quality, for example, affected feeding rates negatively at low food concentrations but triggered surplus feeding at high food concentrations. The scope of surplus feeding was thereby mainly dependent on dynamics of nutrient digestion and in contrast to previous assumptions, energy costs of feeding played a minor role. Further, the regulation of digestive enzyme production, a crucial factor determining assimilation efficiencies, was strongly dependent on whether nonessential or essential nutrients were limiting growth. Consequently, not only the degree but also the type of nutrient limitation mediated the impact of the food environment on consumers’ fitness. At the community level, food quality was key in shaping predator–prey biomass ratios. High food qualities resulted in top‐heavy systems with larger consumer than prey biomass. Decreases of prey digestibility or the availability of essential nutrients, however, triggered a switch from inverted to classical pyramid shapes of bi‐trophic systems. The impact of food quantity on trophic transfer and emerging structural ecosystem properties thus critically hinges on behavioral and physiological responses of consumers. The inclusion of the regulatory flexibility of consumers is therefore an essential next step to improve predator–prey models and our conceptual understanding of trophic interactions.

Background: The pretreatment De Ritis ratio [aspartate transaminase (AST)/alanine transaminase (ALT)] has been shown to be an adverse prognostic marker in various cancer entities. However, its relevance to advanced pancreatic ductal adenocarcinoma (PDAC) has not yet been studied. In the present study we investigated the AST/ALT ratio as a possible predictor of treatment response and disease outcome in patients with advanced PDAC treated with first-line gemcitabine/nab-paclitaxel. Methods: A post hoc analysis of a prospective, multicenter, noninterventional study was performed. A total of 202 patients with advanced PDAC treated with first-line gemcitabine/nab-paclitaxel for whom the AST/ALT ratio was measured were included in this analysis. Results: Median and 1-year progression-free survival estimates were 4.8 months and 5.1%, respectively in patients with an AST/ALT ratio above the 75th percentile of its distribution, and 6.0 months and 18.7%, respectively in patients with an AST/ALT ratio less than or equal to this cutoff, respectively (log-rank p = 0.004). In univariable Cox regression, a doubling of the AST/ALT ratio was associated with a 1.4-fold higher relative risk of progression or death [hazard ratio = 1.38, 95% confidence interval (CI): 1.06–1.80, p = 0.017]. The prognostic association was also found in multivariable analysis adjusting for Eastern Cooperative Oncology Group performance status and lung metastases (hazard ratio per AST/ALT ratio doubling = 1.32, 95% CI: 1.00–1.75, p = 0.047). In treatment response analysis, a doubling of the AST/ALT ratio was associated with a 0.5-fold lower odds of objective response (odds ratio = 0.54, 95% CI: 0.31–0.94, p = 0.020). Conclusions: The pretreatment serum AST/ALT ratio predicts poor disease outcome and response rate in patients with advanced PDAC treated with gemcitabine/nab-paclitaxel and might represent a novel and inexpensive marker for individual risk assessment in the treatment of pancreatic cancer.

During winter at temperate and high latitudes, the low ambient temperatures, limited food supplies and short foraging periods mean small passerines show behavioural, morphological and physiological adaptations to reduce the risk of facing energy shortages. Peripheral tissues vasoconstrict in low ambient temperatures to reduce heat loss and cold injury. Peripheral vasoconstriction has been observed with food restriction in captivity but has yet to be explored in free-ranging animals. We experimentally food restricted both wild and captive great tits (Parus major) during winter months and measured surface temperatures of the bill and eye region using thermal imaging, to investigate whether birds show rapid local heterothermic responses, which may reduce their thermoregulatory costs when facing a perceived imminent food shortage. Our results of a continuously filmed wild population showed that bill temperature was immediately reduced in response to food restriction compared with when food was available ad libitum, an apparent autonomic response. Such immediacy implies a ‘pre-emptive’ response before the bird experiences any shortfalls in energy reserves. We also demonstrate temporal variation in vasoconstriction of the bill, with bill temperature gradually rising throughout the food restriction after the initial drop. Eye-region temperature in the wild birds remained at similar levels throughout food restriction compared with unrestricted birds, possibly reflecting the need to maintain steady circulation to the central nervous and visual systems. Our findings provide evidence that birds selectively allow the bill to cool when a predictable food supply is suddenly disrupted, probably as a means of minimising depletion of body reserves for a perceived future shortage in energy.

Purpose This paper aims to provide instructions on how to implement an adapted version of the standard repertory grid technique (VARGT). The purpose of which is to provide practitioners with a tool, which enables active engagement by participants in research and clinical practice. This tool has been used effectively with people convicted of stalking offences. Design/methodology/approach Repertory grids, developed from Kelly’s Personal Construct Theory (1955), had never been used with those who stalk, either clinically or in a research context. Visual and kinaesthetic adaptations were made to standard RGT procedures (Grice, 2002; Tan and Hunter, 2002), for use in a mixed methods research study (Wheatley, 2019, p. 77) due to expected challenges in engaging with this group. This manuscript presents theoretical underpinnings and step-by-step instructions for practical application. Findings The VARGT is easy to administer and produces rich data, in both qualitative and quantitative formats. This adapted approach encourages active participation and an interpreted therapeutic collaboration (Wheatley et al., 2020). Practical implications This novel technique has engaged men convicted of stalking offences collaboratively in research activities and showed potential for its use as a clinical tool. This instructional technical paper allows the technique to be replicated. Originality/value This novel technique has engaged men convicted of stalking offences collaboratively in research activities and showed potential for its use as a clinical tool. This instructional technical paper allows the technique to be replicated.

Substances available in nature with potential therapeutic effects are the subject of research that raises tremendous hopes for new challenges in medicine. Fungi are the most common organisms in the ecosystem and the most interesting in this respect. This review discusses two species of edible fungi, used for centuries in Eastern natural medicine, with the best-documented effect – Hericium erinaceus (He) and Trametes versicolor (Tv). The results of in vivo and in vitro studies conducted on mice and human cell lines demonstrate immunomodulatory, potentially, anticancer, anti-inflammatory and neuroregenerative effects of substances isolated from these fungi. The substances contained in the extracts of He and Tv seem to have immunomodulatory effects that may support chemotherapy. The use of these extracts is justified stronger than the other supportive treat ments based on supplements.

The aim of this study was to investigate the prognostic relevance of plasma amylase and lipase concerning survival of patients suffering from metastatic pancreatic cancer (PC). This retrospective study included 351 patients with metastatic PC, who were treated in a single academic institution. Cancer-specific survival (CSS) was analyzed using the Kaplan-Meier method. To further evaluate the prognostic significance of lipase and amylase, univariate and multivariate values were calculated using Cox proportional models. In univariate analysis, an increased amylase level was associated with shorter CSS in PC patients (hazard ratio HR = 1.258; 95% confidence interval CI = 1.011–1.566; p = 0.039). In multivariate analysis, including gender, age, CA19-9 and administration of chemotherapy, increased amylase levels prevailed as an independent prognostic factor for CSS (HR = 1.373; 95%CI = 1.004–1.878; p = 0.047). Plasma amylase was found to be an independent prognostic factor in patients with metastatic PC. The results indicate that amylase might represent a novel and useful marker for better patient stratification in PC management.

Background Endocannabinoid signaling plays an important role in regulating synaptic transmission in the striatum, a brain region implicated as a central node of dysfunction in autism spectrum disorder. Deficits in signaling mediated by the endocannabinoid 2-arachidonoylglycerol (2-AG) have been reported in mouse models of autism spectrum disorder, but a causal role for striatal 2-AG deficiency in phenotypes relevant to autism spectrum disorder has not been explored. Methods Using conditional knockout mice, we examined the electrophysiological, biochemical, and behavioral effects of 2-AG deficiency by deleting its primary synthetic enzyme, diacylglycerol lipase α (DGLα), from dopamine D1 receptor–expressing or adenosine A2a receptor–expressing medium spiny neurons (MSNs) to determine the role of 2-AG signaling in striatal direct or indirect pathways, respectively. We then used viral-mediated deletion of DGLα to study the effects of 2-AG deficiency in the ventral and dorsal striatum. Results Targeted deletion of DGLα from direct-pathway MSNs caused deficits in social interaction, excessive grooming, and decreased exploration of a novel environment. In contrast, deletion from indirect-pathway MSNs had no effect on any measure of behavior examined. Loss of 2-AG in direct-pathway MSNs also led to increased glutamatergic drive, which is consistent with a loss of retrograde feedback inhibition. Subregional DGLα deletion from the dorsal striatum produced deficits in social interaction, whereas deletion from the ventral striatum resulted in repetitive grooming. Conclusions These data suggest a role for 2-AG deficiency in social deficits and repetitive behavior, and they demonstrate a key role for 2-AG in regulating striatal direct-pathway MSNs.

Item does not contain fulltext Mutations in the glucocerebrosidase gene (GBA), which cause Gaucher disease, are also potent risk factors for Parkinson's disease. We examined whether a genetic burden of variants in other lysosomal storage disorder genes is more broadly associated with Parkinson's disease susceptibility. The sequence kernel association test was used to interrogate variant burden among 54 lysosomal storage disorder genes, leveraging whole exome sequencing data from 1156 Parkinson's disease cases and 1679 control subjects. We discovered a significant burden of rare, likely damaging lysosomal storage disorder gene variants in association with Parkinson's disease risk. The association signal was robust to the exclusion of GBA, and consistent results were obtained in two independent replication cohorts, including 436 cases and 169 controls with whole exome sequencing and an additional 6713 cases and 5964 controls with exome-wide genotyping. In secondary analyses designed to highlight the specific genes driving the aggregate signal, we confirmed associations at the GBA and SMPD1 loci and newly implicate CTSD, SLC17A5, and ASAH1 as candidate Parkinson's disease susceptibility genes. In our discovery cohort, the majority of Parkinson's disease cases (56%) have at least one putative damaging variant in a lysosomal storage disorder gene, and 21% carry multiple alleles. Our results highlight several promising new susceptibility loci and reinforce the importance of lysosomal mechanisms in Parkinson's disease pathogenesis. We suggest that multiple genetic hits may act in combination to degrade lysosomal function, enhancing Parkinson's disease susceptibility.

Background Nemaline myopathy (NEM) is one of the three major forms of congenital myopathy and is characterized by diffuse muscle weakness, hypotonia, respiratory insufficiency, and the presence of nemaline rod structures on muscle biopsy. Mutations in troponin T1 (TNNT1) is 1 of 10 genes known to cause NEM. To date, only homozygous nonsense mutations or compound heterozygous truncating or internal deletion mutations in TNNT1 gene have been identified in NEM. This extended family is of historical importance as some members were reported in the 1960s as initial evidence that NEM is a hereditary disorder. Methods Proband and extended family underwent Sanger sequencing for TNNT1. We performed RT‐PCR and immunoblot on muscle to assess TNNT1 RNA expression and protein levels in proband and father. Results We report a novel heterozygous missense mutation of TNNT1 c.311A>T (p.E104V) that segregated in an autosomal dominant fashion in a large family residing in the United States. Extensive sequencing of the other known genes for NEM failed to identify any other mutant alleles. Muscle biopsies revealed a characteristic pattern of nemaline rods and severe myofiber hypotrophy that was almost entirely restricted to the type 1 fiber population. Conclusion This novel mutation alters a residue that is highly conserved among vertebrates. This report highlights not only a family with autosomal dominant inheritance of NEM, but that this novel mutation likely acts via a dominant negative mechanism.