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The bulbourethral gland (or Cowper"s gland) plays an important role for fertility. It is, therefore, essential to have quantitative data about the morphological and histomorphological structure of this gland in nonpathological conditions. In the present study, Holstein bull"s bulbourethral glands were collected, and the volume of the glands, total epithelial cell number and epithelial cell"s nuclear volume was estimated for the first time by stereological methods. The smooth fractionator technique for epithelial cell counting was used. Epithelial cell"s nuclear volume was estimated on vertical sections. The mean number of the epithelial cells (coefficient of error; CE) and the mean epithelial cell"s nuclear volume were found 322x107 (0.1) and 59.1 µm3 (SD=3.7) respectively. Finally, the present studies stereological findings are in the acceptable range.

Presents abstracts for articles which appear in the April 1, 1998 issue of 'Cephalalgia.' Effective prophylactic therapy for cyclic vomiting in children using amitriptyline or cyproheptadine; Cerebral venous thrombosis and anticardioliptin antibodies; Giant cell (temporal) arteritis; Therapy of acute migraine attacks and migraine prophylaxis-guidelines of the German Migraine and Headache Society; Therapy of cluster headache guidelines of the German Migraine Headache-Society; Periodic syndrome and migraine in children and adolecents; Others.

Background: The use of medications for opioid use disorder such as methadone or buprenorphine is increasing among pregnant women. However, long-term effects of this treatment on the children's health are not well understood. A key challenge is distinguishing the effects of opioid exposure from other confounding factors associated with human opioid use, such as reduced maternal care. In this study, we therefore used a multi-risk factor design to examine anxiety-like behavior in rats prenatally exposed to methadone or buprenorphine, with or without maternal separation the first two weeks after birth., Methods: Female Sprague Dawley rats were exposed to methadone (10mg/kg/day), buprenorphine (1mg/kg/day) or sterile water throughout gestation. Half of the offspring in each litter experienced maternal separation for 3h per day from postnatal day 2 to 12. Male and female offspring (6-9 weeks) were tested in the open field, light-dark transition and elevated plus maze tests to assess anxiety-like behavior., Results: Offspring exposed to buprenorphine and not subjected to maternal separation displayed increased anxiety-like behavior in 3 out of 6 outcomes in the light-dark transition and elevated plus maze tests. Maternal separation did not exacerbate, but rather diminished this behavior. Males and females responded differently to methadone, with a trend towards reduced anxiety for males and increased anxiety for females., Conclusions: Prenatal exposure to methadone or buprenorphine may increase the risk of developing anxiety-like behavior later in life, but the effect depends on specific subgroup characteristics. Further research is required to draw definitive conclusions., Competing Interests: Declaration of Competing Interest No conflict declared., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The use and/or misuse of opioids by pregnant women would expose the fetuses to these drugs during critical stages of development with serious effects for the newborn, like the neonatal abstinence syndrome (NAS). We have revisited an established chicken model for NAS to describe the distribution of morphine and methadone to the brain and explore its validity as a valuable alternative to rodent models. For this purpose, chicken eggs were injected with a single dose of 10 mg/kg or 20 mg/kg morphine or 20 mg/kg methadone onto the chorioallantoic membrane (CAM) on embryonal day 13. Whole brains and lungs were harvested and the concentrations of morphine, methadone and their subsequent metabolites (morphine-3-glucuronide and EDDP, respectively) determined in the brain and lungs at different time points using LC-MS/MS. Morphine and methadone, as well as their metabolites, were detected both in the brain and lungs, with significantly higher concentrations in the lungs. Pharmacokinetic modelling showed that the distribution of morphine to the brain followed a first-order absorption with transit compartments and linear elimination, with concentrations linearly dependent on dose. Moreover, methadone, but not morphine, reduced μ receptor (the main morphine receptor) binding, which can be of relevance for opioid tolerance. The present study is the first to report the brain distribution of morphine, which can be described by standard pharmacokinetic processes, and methadone in the developing chicken embryo. The present findings supplement the already established model and support the use of this chicken model to study NAS., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ragnhild Elisabeth Paulsen reports financial support was provided by Anders Jahre's Foundation., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Immunotherapeutic interventions that block drug effects by binding drug molecules to specific antibodies in the bloodstream have shown promising effects in animal studies. For heroin, which effects are mainly mediated by the metabolites 6-acetylmorphine (6-AM; also known as 6-monoacetylmorphine or 6-MAM) and morphine, the optimal antibody specificity has been discussed. In rodents, 6-AM specific antibodies have been recommended based on the rapid metabolism of heroin to 6-AM in the bloodstream. Since the metabolic rate of heroin in blood is unsettled in humans, we examined heroin metabolism with state-of-the-art analytical methodology (UHPLC-MS/MS) in freshly drawn human whole blood incubated with a wide range of heroin concentrations (1-500 μM). The half-life of heroin was highly concentration dependent, ranging from 1.2-1.7 min for concentrations at or above 25 μM, and gradually increasing to approximately 20 min for 1 μM heroin. At concentrations that can be attained in the bloodstream shortly after an i.v. injection, approximately 70% was transformed into 6-AM within 3 min, similar to previous observations in vivo. Our results indicate that blood enzymes play a more important role for the rapid metabolism of heroin in humans than previously assumed. This points to 6-AM as an important target for an efficient immunotherapeutic approach to block heroin effects in humans., (© 2023 The Authors. Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Semen analysis is central in infertility investigation. Manual assessment of sperm motility according to the WHO recommendations is the golden standard, and extensive training is a requirement for accurate and reproducible results. Deep convolutional neural networks (DCNN) are especially suitable for image classification. In this study, we evaluated the performance of the DCNN ResNet-50 in predicting the proportion of sperm in the WHO motility categories. Two models were evaluated using tenfold cross-validation with 65 video recordings of wet semen preparations from an external quality assessment programme for semen analysis. The corresponding manually assessed data was obtained from several of the reference laboratories, and the mean values were used for training of the DCNN models. One model was trained to predict the three categories progressive motility, non-progressive motility, and immotile spermatozoa. Another model was used in predicting four categories, where progressive motility was differentiated into rapid and slow. The resulting average mean absolute error (MAE) was 0.05 and 0.07, and the average ZeroR baseline was 0.09 and 0.10 for the three-category and the four-category model, respectively. Manual and DCNN-predicted motility was compared by Pearson's correlation coefficient and by difference plots. The strongest correlation between the mean manually assessed values and DCNN-predicted motility was observed for % progressively motile spermatozoa (Pearson's r = 0.88, p < 0.001) and % immotile spermatozoa (r = 0.89, p < 0.001). For rapid progressive motility, the correlation was moderate (Pearson's r = 0.673, p < 0.001). The median difference between manual and predicted progressive motility was 0 and 2 for immotile spermatozoa. The largest bias was observed at high and low percentages of progressive and immotile spermatozoa. The DCNN-predicted value was within the range of the interlaboratory variation of the results for most of the samples. In conclusion, DCNN models were able to predict the proportion of spermatozoa into the WHO motility categories with significantly lower error than the baseline. The best correlation between the manual and the DCNN-predicted motility values was found for the categories progressive and immotile. Of note, there was considerable variation between the mean motility values obtained for each category by the reference laboratories, especially for rapid progressive motility, which impacts the training of the DCNN models., (© 2023. Springer Nature Limited.)

A manual assessment of sperm motility requires microscopy observation, which is challenging due to the fast-moving spermatozoa in the field of view. To obtain correct results, manual evaluation requires extensive training. Therefore, computer-aided sperm analysis (CASA) has become increasingly used in clinics. Despite this, more data is needed to train supervised machine learning approaches in order to improve accuracy and reliability in the assessment of sperm motility and kinematics. In this regard, we provide a dataset called VISEM-Tracking with 20 video recordings of 30 seconds (comprising 29,196 frames) of wet semen preparations with manually annotated bounding-box coordinates and a set of sperm characteristics analyzed by experts in the domain. In addition to the annotated data, we provide unlabeled video clips for easy-to-use access and analysis of the data via methods such as self- or unsupervised learning. As part of this paper, we present baseline sperm detection performances using the YOLOv5 deep learning (DL) model trained on the VISEM-Tracking dataset. As a result, we show that the dataset can be used to train complex DL models to analyze spermatozoa., (© 2023. The Author(s).)

We show that annihilation dynamics between oppositely charged optical vortex pairs can be manipulated by the initial size of the vortex cores, consistent with hydrodynamics. When sufficiently close together, vortices with strongly overlapped cores annihilate more quickly than vortices with smaller cores that must wait for diffraction to cause meaningful core overlap. Numerical simulations and experimental measurements for vortices with hyperbolic tangent cores of various initial sizes show that hydrodynamics governs their motion, and reveal distinct phases of vortex recombination; decreasing the core size of an annihilating pair can prevent the annihilation event.

Neural stem cells (NSCs) derived from human induced pluripotent stem cells were used to investigate effects of exposure to the food contaminant acrylamide (AA) and its main metabolite glycidamide (GA) on key neurodevelopmental processes. Diet is an important source of human AA exposure for pregnant women, and AA is known to pass the placenta and the newborn may also be exposed through breast feeding after birth. The NSCs were exposed to AA and GA (1 ×10 -8 - 3 ×10 -3 M) under 7 days of proliferation and up to 28 days of differentiation towards a mixed culture of neurons and astrocytes. Effects on cell viability was measured using Alamar Blue™ cell viability assay, alterations in gene expression were assessed using real time PCR and RNA sequencing, and protein levels were quantified using immunocytochemistry and high content imaging. Effects of AA and GA on neurodevelopmental processes were evaluated using endpoints linked to common key events identified in the existing developmental neurotoxicity adverse outcome pathways (AOPs). Our results suggest that AA and GA at low concentrations (1 ×10 -7 - 1 ×10 -8 M) increased cell viability and markers of proliferation both in proliferating NSCs (7 days) and in maturing neurons after 14-28 days of differentiation. IC 50 for cell death of AA and GA was 5.2 × 10 -3 M and 5.8 × 10 -4 M, respectively, showing about ten times higher potency for GA. Increased expression of brain derived neurotrophic factor (BDNF) concomitant with decreased synaptogenesis were observed for GA exposure (10 -7 M) only at later differentiation stages, and an increased number of astrocytes (up to 3-fold) at 14 and 21 days of differentiation. Also, AA exposure gave tendency towards decreased differentiation (increased percent Nestin positive cells). After 28 days, neurite branch points and number of neurites per neuron measured by microtubule-associated protein 2 (Map2) staining decreased, while the same neurite features measured by βIII-Tubulin increased, indicating perturbation of neuronal differentiation and maturation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The fatty acid composition of spermatozoa has been shown to be important for their function, and L-carnitine is crucial for fatty acid metabolism. Its levels in the seminal plasma positively correlate with semen quality, whereas high body mass index (BMI) is associated with both reduced semen quality and altered sperm fatty acid composition. Here, we examined the associations between free seminal L-carnitine levels and sperm fatty acid composition as well as BMI. Semen samples were collected and analyzed from 128 men with unknown fertility status and with BMI ranging from 19 kg m -2 to 63 kg m -2 . Sperm fatty acid composition was assessed by gas chromatography, while free seminal L-carnitine analysis was performed using high-performance liquid chromatography. Multiple linear regression analysis showed a positive correlation of free seminal L-carnitine levels with the amount of sperm palmitic acid (β = 0.21; P = 0.014), docosahexaenoic acid (DHA; β = 0.23; P = 0.007), and total n-3 polyunsaturated fatty acids (β = 0.23; P = 0.008) and a negative correlation of free seminal L-carnitine levels with lignoceric acid (β = -0.29; P = 0.001) and total n-6 polyunsaturated fatty acids (β = -0.24; P = 0.012) when adjusted for covariates. There was no relationship between free seminal L-carnitine levels and BMI. Since free seminal L-carnitine levels are associated with semen quality, the absence of a correlation with BMI suggests that reduced semen quality in obese men is independent of seminal L-carnitine., Competing Interests: None

We show that a two-dimensional hydrodynamics model provides a physical explanation for the splitting of higher-charge optical vortices under elliptical deformations. The model is applicable to laser light and quantum fluids alike. The study delineates vortex breakups from vortex unions under different forms of asymmetry in the beam, and it is also applied to explain the motion of intact higher-charge vortices.

Methiopropamine is a novel psychoactive substance (NPS) that is associated with several cases of clinical toxicity, yet little information is available regarding its neuropharmacological properties. Here, we employed in vitro and in vivo methods to compare the pharmacokinetics and neurobiological effects of methiopropamine and its structural analog methamphetamine. Methiopropamine was rapidly distributed to the blood and brain after injection in C57BL/6 mice, with a pharmacokinetic profile similar to that of methamphetamine. Methiopropamine induced psychomotor activity, but higher doses were needed (E max 12.5 mg/kg; i.p.) compared to methamphetamine (E max 3.75 mg/kg; i.p.). A steep increase in locomotor activity was seen after a modest increase in the methiopropamine dose from 10 to 12.5 mg/kg, suggesting that a small increase in dosage may engender unexpectedly strong effects and heighten the risk of unintended overdose in NPS users. In vitro studies revealed that methiopropamine mediates its effects through inhibition of norepinephrine and dopamine uptake into presynaptic nerve terminals (IC 50 = 0.47 and 0.74 µM, respectively), while the plasmalemmal serotonin uptake and vesicular uptake are affected only at high concentrations (IC 50 > 25 µM). In summary, methiopropamine closely resembles methamphetamine with regard to its pharmacokinetics, pharmacodynamic effects and mechanism of action, with a potency that is approximately five times lower than that of methamphetamine.

Aims: To study whether the preparation procedure, and its acidic and heating conditions, used by heroin users to prepare heroin for intravenous administration affects the final composition of the fluid to be injected., Methods: Samples from different seizures of illegal heroin provided by the Norwegian police were prepared by adding water and ascorbic acid before heating under controlled conditions in the laboratory. Further, three seizures were prepared with different amounts of ascorbic or citric acid relative to their diacetylmorphine content. Pure diacetylmorphine base or salt was also submitted to the procedure applying two different heating intensities. The seizures and the final product after preparation were analysed for diacetylmorphine, 6-acetylmorphine and morphine using liquid chromatography with tandem mass spectrometry (LC-MS-MS)., Results: After preparation, a decrease of 19.8% (25th and 75th percentiles = -29.2 and -15.3) in the initial diacetylmorphine content was observed. Both the 6-acetylmorphine and morphine content increased but, due to their low content in the initial product, diacetylmorphine still represented 83.9% (25th and 75th percentiles = 77.3 and 88.0) of the sum of these three opioids in the final solution. The loss of water during preparation caused an increase in the concentration of diacetylmorphine, 6-acetylmorphine and morphine, depending on the heating intensity applied. The content of these opioids was affected by the quantity and type of acid added in relation to the heroin purity and the level of diacetylmorphine dissolved being proportional to the amount of ascorbic acid, but not citric acid, in the sample with high heroin purity., Conclusions: Preparation of heroin for intravenous injection appears to change the amount or concentration of diacetylmorphine and its active metabolites, 6-acetylmorphine and morphine in the final product, depending on heroin purity, amount and type of acid used or heating conditions. These circumstances can contribute to unintentional variations in the potency of the final injected solution, and therefore affect the outcome after injection., (© 2021 The Authors. Addiction published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

Introduction: Rodent models are routinely used to assess the safety and developmental toxicity of pharmaceuticals, along with analysis of their distribution. These models require sacrifice of parent females, have challenges in the estimation of the number of embryos and stage of development, and are expensive and time-consuming. In this study, we used fertilized chicken eggs as an alternative model to address drug distribution to the developing brain of two antiepileptic drugs, valproic acid (VPA) and lamotrigine (LTG) at two developmental stages., Methods: VPA or LTG was injected into the allantois of the egg on embryonic day 13 (E13) or E16. Whole chicken brains were harvested at time-points of 5 min to 24 h and the concentrations of the drugs determined using GC/MS and LC-MS/MS, for VPA and LTG, respectively., Results: VPA and LTG had distinct absorption and elimination phases and were found in the brain as early as 5-15 min after injection. Both drugs reached the brain in clinically relevant concentrations, with C max 10-30% of the calculated concentration assuming uniform distribution throughout the egg. LTG concentrations were higher when injected at E13 compared to E16., Conclusion: The chicken embryo model may be a suitable alternative animal model for preclinical drug distribution studies. It enables to easily approach antenatal development on an individual level, with a precise number of experimental animals, high reproducibility and low time and cost. Knowledge of the concentrations reaching the brain at different developmental stages with different drugs is important for the planning and interpretation of neurodevelopmental toxicity studies., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

A variety of nucleophilic aromatic substitution reactions were carried out mechanochemically to great advantage. On average, reactions rates were nine-times faster. The corresponding kinetic studies presented provide the clearest head-to-head kinetic comparisons between mechanochemical and conventional systems at identical temperatures. Attempts are provided at classifying the kinetics of one example. Removal of polar, protic solvents from these reactions presents environmental benefits to a reaction class whose kinetics are heavily dependent on such solvents.

In recent years, the amount of data produced in the field of ART has increased exponentially. The diversity of data is large, ranging from videos to tabular data. At the same time, artificial intelligence (AI) is progressively used in medical practice and may become a promising tool to improve success rates with ART. AI models may compensate for the lack of objectivity in several critical procedures in fertility clinics, especially embryo and sperm assessments. Various models have been developed, and even though several of them show promising performance, there are still many challenges to overcome. In this review, we present recent research on AI in the context of ART. We discuss the strengths and weaknesses of the presented methods, especially regarding clinical relevance. We also address the pitfalls hampering successful use of AI in the clinic and discuss future possibilities and important aspects to make AI truly useful for ART., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Anthropogenic disturbances associated with urban ecosystems can create favorable conditions for populations of some invasive plant species. Light pollution is one of these disturbances, but how it affects the growth and establishment of invasive plant populations is unknown. Cheatgrass ( Bromus tectorum ) is a problematic invasive species where it has displaced native grassland communities in the United States, but to our knowledge, there have been no studies of the ecological factors that affect cheatgrass presence in urban ecosystems. We conducted field surveys in urban alleys in Denver, Colorado, to compare the presence of cheatgrass at sites with and without artificial light at night (hereafter artificial light) from streetlights. These streetlights are mounted on utility poles, which cause ground disturbance when installed in alleys; we were able to test the independent effect of poles on cheatgrass establishment because not all poles have streetlights on them. We found that cheatgrass was positively associated with the presence of streetlights and to a lesser extent poles. In addition to cheatgrass, we also found that other plants were positively associated with the presence of both poles and streetlights. Our results suggest that artificial light may benefit the occurrence of cheatgrass and other plant species in urban settings. While invasive populations of cheatgrass in wild habitats attract the most attention from managers, we suggest more consideration for this grass in urban environments where its growth and establishment benefit from anthropogenic changes., Competing Interests: None declared., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

There is a growing concern related to the use of opioid maintenance treatment during pregnancy. Studies in both humans and animals have reported reduced cognitive functioning in offspring prenatally exposed to methadone or buprenorphine; however, little is known about the neurobiological mechanisms underlying these impairments. To reveal possible neurobiological effects of such in utero exposure, we examined brain tissue from methadone- and buprenorphine-exposed rat offspring previously shown to display impaired learning and memory. We studied µ-opioid receptor (MOR) and N-methyl-D-aspartate receptor (NMDAR) binding in the rat offspring cerebrum during development and in the hippocampus at young adulthood. Moreover, we examined activation of the Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and the extracellular signal-regulated kinase (ERK), which are central in the downstream signaling of these receptors. The methadone- and buprenorphine-exposed rat pups displayed reduced MOR binding up to two weeks after birth, whereas the NMDAR binding was unaffected. Prenatal exposure to methadone or buprenorphine also resulted in decreased activation of CaMKII and/or ERK during development, while young adult offspring displayed increased hippocampal ERK activation. In conclusion, our findings suggest that prenatal exposure to exogenous opioids, such as methadone or buprenorphine, may disturb the endogenous opioid system during development, with long-term effects on proteins important for cognitive functioning., (© 2020 The Authors. International Journal of Developmental Neuroscience published by John Wiley & Sons Ltd on behalf of International Society for Developmental Neuroscience.)

Activation of calcium/calmodulin-dependent protein kinase II (CaMKII), particularly its α isoform, is known to be important for neuronal processes central for learning and memory and has also been implicated in the maladaptive learning involved in drug addiction.Thr286 autophosphorylation of αCaMKII has been shown to be indispensable for establishment of cocaine-induced CPP (Easton et al., 2014). To study the contribution of CaMKII in opioid induced conditioned learning, we examined how establishment of conditioned place preference (CPP) induced by 10 or 30 μmol/kg morphine or its active metabolite morphine-6-glucuronide (M6G) affects the levels and Thr286 autophosphorylation of the α- and β-isoforms of CaMKII, as well as β-actin levels, in dorsal and ventral striatum and in hippocampus of mice. An acute and a sub-chronic treatment were used as controls. Whereas an acute single administration of morphine or M6G caused increases in CaMKII levels and phosphorylation at Thr286 and β-actin in striatal areas, CPP induced by these opioids was accompanied primarily by an increase in the protein levels of both CaMKII isoforms and β-actin in dorsal striatum and hippocampus. Decreases in CaMKII Thr286 phosphorylation were observed in dorsal striatum after the sub-chronic pharmacological treatment. Despite the changes observed in αCaMKII activity in wild type mice, morphine-induced CPP was not affected in αCaMKII T286A autophosphorylation-deficient mice. These results indicate that opioid-induced CPP is accompanied by activation of α- and βCaMKII in striatum and hippocampus, but, in opposition to what has been observed with cocaine, αCaMKII autophosphorylation is not essential for establishment of opioid-induced CPP., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Concerns have been raised about the use of opioid maintenance treatment (OMT) during pregnancy and negative effects for the offspring. While neonatal outcomes and short-term effects are relatively well described, studies examining long-term effects in adolescents and adults are absent. The aim of the present study was to examine effects on learning and memory in young adult rats prenatally exposed to methadone or buprenorphine., Methods: Female rats were implanted with a 28-day osmotic minipump delivering methadone (10 mg/kg/day), buprenorphine (1 mg/kg/day) or vehicle 5 days prior to mating. To examine possible effects on cognitive functioning, young adult offspring were included in three different behavioral tests that examine recognition memory, nonspatial, and spatial learning and memory. In addition, offspring growth and maternal behavior after birh were investigated., Results: Prenatal exposure to methadone or buprenorphine caused impaired recognition memory and nonspatial reference learning and memory in young adult rats compared with the vehicle-treated group. Methadone-exposed offspring, but not the buprenorphine-exposed, also showed reduced long-term spatial memory. We did not observe any changes in maternal behavior or offspring growth after prenatal exposure to methadone or buprenorphine, suggesting that the impaired cognitive functioning is due to the opioid exposure rather than reduced maternal caregiving., Conclusion: The present findings of long-term cognitive impairments in methadone- and buprenorphine-exposed offspring points to a negative impact of OMT on neurobiological development., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aim: To combine meta-analyses of multiple long-term outcomes in children prenatally exposed to methadone or buprenorphine through their mothers' Opioid maintenance therapy (OMT) with a systematic review of similar outcomes in experimental animals., Method: The Medline, Embase, Web of Science, CINAHL, Cochrane and Epistemonikos databases were searched through August 30, 2018. Clinical studies measuring effects on cognitive, behavioral or visual outcomes in 3 months or older children prenatally exposed to OMT and control group(s) were included for meta-analyses. Experimental animal studies with similar exposures and outcomes were included in a systematic review. The three authors independently performed abstract screenings and full-text reviews, and extracted the data. One author performed the meta-analyses., Results: The pooled results of the meta-analyses showed worse cognitive, psychomotor, behavioral, attentional and executive functioning, and affected vision in children born to mothers who were in OMT during pregnancy compared to children without prenatal drug exposure (overall effect size = 0.49, 95% confidence interval = 0.38, 0.59, p < 0.00001). Many of the experimental animal studies showed impaired outcomes after prenatal exposure to methadone or buprenorphine. The clinical results may be biased, e.g., with the OMT group having more concurrent risk factors than the unexposed comparison group. There are few studies of older children., Conclusion: Children born to mothers in OMT show worse outcomes for a number of different behaviors and impaired vision compared to children born to nonusers. Experimental animal studies indicate that there might be a causal relationship between prenatal methadone or buprenorphine exposure and subsequent negative outcomes., Competing Interests: Financial disclosure The authors have no financial relationships relevant to this article to disclose., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Question: Does adding mobilisation with movement (MWM) to usual care (ie, exercises plus advice) improve outcomes after immobilisation for a distal radius fracture?, Design: A prospective, multicentre, randomised, clinical trial with concealed allocation, blinding and intention-to-treat analysis., Participants: Sixty-seven adults (76% female, mean age 60 years) treated with casting after distal radius fracture., Intervention: The control group received exercises and advice. The experimental group received the same exercises and advice, plus supination and wrist extension MWM., Outcome Measures: The primary outcome was forearm supination at 4 weeks (immediately post-intervention). Secondary outcomes included wrist extension, flexion, pronation, grip strength, QuickDASH (Disabilities of Arm, Shoulder and Hand), Patient-Rated Wrist Evaluation (PRWE) and global rating of change. Follow-up time points were 4 and 12 weeks, with patient-rated measures at 26 and 52 weeks., Results: Compared with the control group, supination was greater in the experimental group by 12 deg (95% CI 5 to 20) at 4 weeks and 8 deg (95% CI 1 to 15) at 12 weeks. Various secondary outcomes were better in the experimental group at 4 weeks: extension (14 deg, 95% CI 7 to 20), flexion (9 deg, 95% CI 4 to 15), QuickDASH (-11, 95% CI -18 to -3) and PRWE (-13, 95% CI -23 to -4). Benefits were still evident at 12 weeks for supination, extension, flexion and QuickDASH. The experimental group were more likely to rate their global change as 'improved' (risk difference 22%, 95% CI 5 to 39). There were no clear benefits in any of the participant-rated measures at 26 and 52 weeks, and no adverse effects., Conclusion: Adding MWM to exercise and advice gives a faster and greater improvement in motion impairments for non-operative management of distal radius fracture., Registration: ACTRN12615001330538., (Copyright © 2020. Published by Elsevier B.V.)

Saccharopolyspora erythraea is used for industrial erythromycin production. To explore the physiological role of intracellular energy state in metabolic regulation by S. erythraea , we initially overexpressed the F 1 part of the endogenous F 1 F 0 -ATPase in the high yielding erythromycin producing strain E3. The F 1 -ATPase expression resulted in lower [ATP]/[ADP] ratios, which was accompanied by a strong increase in the production of a reddish pigment and a decreased erythromycin production. Subsequent transcriptional analysis revealed that the lower intracellular [ATP]/[ADP] ratios exerted a pleotropic regulation on the metabolism of S. erythraea . The lower [ATP]/[ADP] ratios induced physiological changes to restore the energy balance, mainly via pathways that tend to produce ATP or regenerate NADH. The F 1 -ATPase overexpression strain exhibited a state of redox stress, which was correlated to an alteration of electron transport at the branch of the terminal oxidases, and S. erythraea channeled the enhanced glycolytic flux toward a reddish pigment in order to reduce NADH formation. The production of erythromycin was decreased, which is in accordance with the net ATP requirement and the excess NADH formed through this pathway. Partial growth inhibition by apramycin increased the intracellular [ATP]/[ADP] ratios and demonstrated a positive correlation between [ATP]/[ADP] ratios and erythromycin synthesis. Finally, overexpression of the entire F 1 F 0 -ATPase complex resulted in 28% enhanced erythromycin production and markedly reduced pigment synthesis in E3. The work illustrates a feasible strategy to optimize the distribution of fluxes in secondary metabolism.

We propose the braiding of optical vortices in a laser beam with more than $ 2\pi $2π rotation by superposing Bessel modes with a plane wave. We experimentally demonstrate this by using a Bessel-Gaussian beam and a coaxial Gaussian, and we present measurements of three complete braids. The amount of braiding is fundamentally limited only by the numerical aperture of the system, and we discuss how braiding can be controlled experimentally for any number of vortices.

Background: The secondary alcohol 2-butanol has many important applications, e.g., as a solvent. Industrially, it is usually made by sulfuric acid-catalyzed hydration of butenes. Microbial production of 2-butanol has also been attempted, however, with little success as witnessed by the low titers and yields reported. Two important reasons for this, are the growth-hampering effect of 2-butanol on microorganisms, and challenges associated with one of the key enzymes involved in its production, namely diol dehydratase., Results: We attempt to link the metabolism of an engineered Lactococcus lactis strain, which possesses all enzyme activities required for fermentative production of 2-butanol from glucose, except for diol dehydratase, which acts on meso -2,3-butanediol (mBDO), with that of a Lactobacillus brevis strain which expresses a functional dehydratase natively. We demonstrate growth-coupled production of 2-butanol by the engineered L. lactis strain, when co-cultured with L. brevis . After fine-tuning the co-culture setup, a titer of 80 mM (5.9 g/L) 2-butanol, with a high yield of 0.58 mol/mol is achieved., Conclusions: Here, we demonstrate that it is possible to link the metabolism of two bacteria to achieve redox-balanced production of 2-butanol. Using a simple co-cultivation setup, we achieved the highest titer and yield from glucose in a single fermentation step ever reported. The data highlight the potential that lies in harnessing microbial synergies for producing valuable compounds., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

We have previously demonstrated that heroin's first metabolite, 6-acetylmorphine (6-AM), is an important mediator of heroin's acute effects. However, the significance of 6-AM to the rewarding properties of heroin still remains unknown. The present study therefore aimed to examine the contribution of 6-AM to heroin-induced reward and locomotor sensitization. Mice were tested for conditioned place preference (CPP) induced by equimolar doses of heroin or 6-AM (1.25-5 μmol/kg). Psychomotor activity was recorded during the CPP conditioning sessions for assessment of drug-induced locomotor sensitization. The contribution of 6-AM to heroin reward and locomotor sensitization was further examined by pretreating mice with a 6-AM specific antibody (anti-6-AM mAb) 24 hours prior to the CPP procedure. Both heroin and 6-AM induced CPP in mice, but heroin generated twice as high CPP scores compared with 6-AM. Locomotor sensitization was expressed after repeated exposure to 2.5 and 5 μmol/kg heroin or 6-AM, but not after 1.25 μmol/kg, and we found no correlation between the expression of CPP and the magnitude of locomotor sensitization for either opioid. Pretreatment with anti-6-AM mAb suppressed both heroin-induced and 6-AM-induced CPP and locomotor sensitization. These findings provide evidence that 6-AM is essential for the rewarding and sensitizing properties of heroin; however, heroin caused stronger reward compared with 6-AM. This may be explained by the higher lipophilicity of heroin, providing more efficient drug transfer to the brain, ensuring rapid increase in the brain 6-AM concentration., (© 2019 Society for the Study of Addiction.)

Methods for automatic analysis of clinical data are usually targeted towards a specific modality and do not make use of all relevant data available. In the field of male human reproduction, clinical and biological data are not used to its fullest potential. Manual evaluation of a semen sample using a microscope is time-consuming and requires extensive training. Furthermore, the validity of manual semen analysis has been questioned due to limited reproducibility, and often high inter-personnel variation. The existing computer-aided sperm analyzer systems are not recommended for routine clinical use due to methodological challenges caused by the consistency of the semen sample. Thus, there is a need for an improved methodology. We use modern and classical machine learning techniques together with a dataset consisting of 85 videos of human semen samples and related participant data to automatically predict sperm motility. Used techniques include simple linear regression and more sophisticated methods using convolutional neural networks. Our results indicate that sperm motility prediction based on deep learning using sperm motility videos is rapid to perform and consistent. Adding participant data did not improve the algorithms performance. In conclusion, machine learning-based automatic analysis may become a valuable tool in male infertility investigation and research.

The use of opioids during pregnancy has been associated with neurodevelopmental toxicity in exposed children, leading to cognitive and behavioural deficits later in life. The N-methyl-D-aspartate receptor (NMDAR) subunit GluN2B plays critical roles in cerebellar development, and methadone has been shown to possess NMDAR antagonist effect. Consequently, we wanted to explore if prenatal opioid exposure affected GluN2B subunit expression and NMDAR function in rat and chicken cerebellum. Pregnant rats were exposed to methadone (10 mg/kg/day) or buprenorphine (1 mg/kg/day) for the whole period of gestation, using an osmotic minipump. To further examine potential effects of prenatal opioid exposure in a limited time window, chicken embryos were exposed to a 20 mg/kg dose of methadone or morphine on embryonic days 13 and 14. Western blot analysis of cerebella isolated from 14 days old rat pups exposed to buprenorphine showed significantly lower level of the GluN2B subunit, while the opioid exposed chicken embryo cerebellar GluN2B expression remained unaffected at embryonic day 17. However, we observed increased NMDA/glycine-induced calcium influx in cerebellar granule neurone cultures from opioid exposed chicken embryos. We conclude that prenatal opioid exposure leads to opioid receptor-dependent reduction in the postnatal expression of GluN2B in rat cerebella, and increase in NMDA-induced calcium influx in chicken embryo cerebella., (Copyright © 2019 ISDN. Published by Elsevier Ltd. All rights reserved.)

Experimental animal studies are valuable in revealing a causal relationship between prenatal exposure to opioid maintenance treatment (OMT) and subsequent effects; however, previous animal studies of OMT during pregnancy have been criticized for their lack of clinical relevance because of their use of high drug doses and the absence of pharmacokinetic data. Hence, the aim of this study was to determine blood and brain concentrations in rat dams, fetuses, and offspring after continuous maternal exposure to methadone or buprenorphine during gestation and to examine the offspring for neonatal outcomes and withdrawal symptoms. Female rats were implanted with a 28-day osmotic minipump delivering methadone (10 mg/kg per day), buprenorphine (1 mg/kg per day) or vehicle 5 days before mating. Continuous exposure to methadone or buprenorphine induced stable blood concentrations in the dams of 0.25 ± 0.02 µ M and 5.65 ± 0.16 nM, respectively. The fetal brain concentration of methadone (1.89 ± 0.35 nmol/g) was twice as high as that in the maternal brain, whereas the fetal brain concentration of buprenorphine (20.02 ± 4.97 pmol/g) was one-third the maternal brain concentration. The opioids remained in the offspring brain several days after the exposure ceased. Offspring prenatally exposed to methadone, but not buprenorphine, displayed reduced body weight and length and increased corticosterone levels. No significant changes in ultrasonic vocalizations were revealed. Our data in rat fetuses and neonates indicate that OMT with buprenorphine may be a better choice than methadone during pregnancy. SIGNIFICANCE STATEMENT: Concern has been raised about the use of opioid maintenance treatment during pregnancy because of the important role of the endogenous opioid system in brain development. Here, we show that the methadone concentration in the fetal rat brain was twice as high as that in the maternal brain, whereas the buprenorphine concentration was one-third the maternal concentration. Furthermore, buprenorphine allowed more favorable birth outcomes, suggesting that buprenorphine may be a better choice during pregnancy., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Streptococcus thermophilus is a lactic acid bacterium widely used in the syntrophic fermentation of milk into yogurt and cheese. Streptococcus thermophilus has adapted to ferment milk primarily through reductive genome evolution but also through acquisition of genes conferring proto-cooperation with Lactobacillus bulgaricus and efficient metabolism of milk macronutrients. Genomic analysis of Strep. thermophilus strains suggests that mobile genetic elements have contributed to genomic evolution through horizontal gene transfer and genomic plasticity. We previously used the endogenous type II CRISPR-Cas [clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated sequences (Cas)] system in Strep. thermophilus to isolate derivatives lacking the chromosomal mobile genetic element and expandable island that display decreased fitness under routine culturing conditions. Of note, the Lac operon and Leloir pathway genes were deleted in the largest expendable genomic island (102 kbp), rendering the strain incapable of acidifying milk. However, the removal of other open reading frames in the same island had unclear effects on the fitness and regulatory networks of Strep. thermophilus. To uncover the physiological basis for the observed phenotypic changes and underlying regulatory networks affected by deletion of the 102-kbp genomic island in Strep. thermophilus, we analyzed the transcriptome of the mutant that lacked ∼5% of its genome. In addition to the loss of transcripts encoded by the deleted material, we detected a total of 56 genes that were differentially expressed, primarily encompassing 10 select operons. Several predicted metabolic pathways were affected, including amino acid and purine metabolism, oligopeptide transport, and iron transport. Collectively, these results suggest that deletion of a 102-kb genomic island in Strep. thermophilus influences compensatory transcription of starvation stress response genes and metabolic pathways involved in important niche-related adaptation., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Advanced glycation end products (AGEs) have been implicated in the pathophysiology of type 2 diabetes and cardiovascular disease. We aimed to determine the associations of urinary carboxymethyl-lysine (CML) and methylglyoxal-hydroimidazolone (MG-H1) levels with cardiometabolic parameters in metabolically healthy obese women. Anthropometric, glycemic, cardiovascular, and urinary AGE parameters were measured in 58 metabolically healthy obese women (age: 39.98 ± 8.72 years; body mass index (BMI): 32.29 ± 4.05 kg/m 2 ). Urinary CML levels were positively associated with BMI ( r = 0.29, p = 0.02). After adjustment for age and BMI, there was a trend for positive associations between urinary CML levels and fasting ( p = 0.06) and 2 h insulin ( p = 0.05) levels, and insulin resistance measured by homeostatic model assessment (HOMA-IR) ( p = 0.06). Urinary MG-H1 levels were positively associated with systolic and diastolic blood pressure, pulse pressure, mean arterial pressure, and total and low-density lipoprotein cholesterol after adjustment for age, BMI, and HOMA-IR (all p ˂ 0.05). There were no associations between urinary CML levels and cardiovascular parameters, and between urinary MG-H1 levels and glycemic measurements. Our data support a role of urinary AGEs in the pathophysiology of insulin resistance and cardiovascular disease; however, future studies are highly warranted.

Previous studies have suggested that at least some of the behavioral effects of heroin might be mediated by its active metabolite 6-acetylmorphine (6-AM). The aim of the present study was to investigate the reinforcing effects of 6-AM and its role in mediating those of heroin. We used an intravenous self-administration procedure in male Sprague-Dawley rats including four phases: acquisition, extinction, reinstatement of drug-seeking, and re-acquisition. Independent groups of rats readily learned to self-administer equimolar doses (0.135 μmol/kg) of either 6-AM (44.3 μg/kg) or heroin (50 μg/kg). Under a fixed ratio 1 (FR1) schedule of reinforcement, the rate of responding was the same for 6-AM and heroin, but it was significantly higher for 6-AM than for heroin under a FR2 schedule. A non-contingent infusion ('priming') of 0.068 μmol/kg of either 6-AM or heroin reinstated non-reinforced drug-seeking (relapse). The rats readily re-acquired self-administration behaviour when given access to one of two doses (0.068 and 0.135 μmol/kg) of 6-AM or heroin. Pretreatment with a specific monoclonal antibody (mAb) against 6-AM blocked the priming effect of 6-AM, and modified the rate of lever-pressing on re-acquisition of 6-AM self-administration in a manner compatible with a shift to the right of the dose-effect curve. The mAb did not affect heroin responding. The present results show that 6-AM possesses reinforcing effects similar to those of heroin. The lack of effect of 6-AM mAb on heroin priming and heroin self-administration calls for further studies to clarify the role of heroin and its metabolites in heroin reward. This article is part of the Special Issue entitled 'Opioid Neuropharmacology: Advances in treating pain and opioid addiction'., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Lactococcus lactis is globally used in food fermentation. Genomics is useful to investigate speciation and differential occurrence of (un)desired gene functions, often related to mobile DNA. This study investigates L. lactis for putative chromosomal mobile genetic elements through comparative genomics, and analyses how they contribute to chromosomal variation at strain level. Our work identified 95 loci that may range over 10% of the chromosome size when including prophages, and the loci display a marked differential occurrence in the analysed strains. Analysis of differential transcriptomics data revealed how mobile genetic elements may impact the host physiology in response to conditional changes. This insight in the genetic variation of mobile genetic elements in L. lactis holds potential to further identify important functions related to food and biotechnology applications within this important species., (© FEMS 2019.)

We demonstrate collinear phase-shifting holography for measuring complex optical modes of twisted light beams with orbital angular momentum (OAM) generated by passing a laser through a spatial light modulator (SLM). This technique measures the mode along the direction of propagation from the SLM and requires no additional optics, so it can be used to aid alignment of the SLM, to efficiently check for the effects of beam wander, and to fully characterize generated beams before use in other experiments. Optimized error analysis and careful SLM alignment allow us to generate and measure OAM with purity as high as 99.9%.

Escalating opioid use among fertile women has increased the number of children being exposed to opioids during fetal life. Furthermore, accumulating evidence links prenatal opioid exposure, including opioid maintenance treatment, to long-term negative effects on cognition and behavior, and presses the need to explore novel treatment strategies for pregnant opioid users. The present study examined the potential of a monoclonal antibody (mAb) targeting heroin's first metabolite, 6-acetylmorphine (6-AM), in providing fetal protection against harmful effects of prenatal heroin exposure in mice. First, we examined anti-6-AM mAb's ability to block materno-fetal transfer of active metabolites after maternal heroin administration. Next, we studied whether maternal mAb pretreatment could prevent adverse effects in neonatal and adolescent offspring exposed to intrauterine heroin (3 × 1.05 mg/kg). Anti-6-AM mAb pretreatment of pregnant dams profoundly reduced the distribution of active heroin metabolites to the fetal brain. Furthermore, maternal mAb administration prevented hyperactivity and drug sensitization in adolescent female offspring prenatally exposed to heroin. Our findings demonstrate that passive immunization with a 6-AM-specific antibody during pregnancy provides fetal neuroprotection against heroin metabolites, and thereby prevents persistent adverse behavioral effects in the offspring. An immunotherapeutic approach to protect the fetus against long-term effects of prenatal drug exposure has not been reported previously, and should be further explored as prophylactic treatment of pregnant heroin users susceptible to relapse., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: In heroin-related deaths, it is often of interest to determine the approximate time span between intake of heroin and death, and to decide whether heroin or other opioids have been administered. In some autopsy cases, peripheral blood cannot be sampled due to decomposition, injuries or burns. The aim of the present study was to investigate whether measurements of heroin metabolites in matrices other than peripheral blood can be used to differentiate between rapid and delayed heroin deaths, and if morphine/codeine ratios measured in other matrices can separate heroin from codeine intakes., Methods: In this study, we included 51 forensic autopsy cases where morphine was detected in peripheral blood. Samples were collected from peripheral and cardiac blood, pericardial fluid, psoas and lateral vastus muscles, vitreous humor and urine. The opioid analysis included 6-acetylmorphine (6-AM), morphine, morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and codeine. Urine was only used for qualitative detection of 6-AM. 45 heroin-intake cases were divided into rapid deaths (n=24), based on the detection of 6-AM in blood, or delayed deaths (n=21), where 6-AM was detected in at least one other matrix but not in blood. An additional 6 cases were classified as codeine-intake cases, based on a morphine/codeine ratio below unity (<1) in peripheral blood, without detecting 6-AM in any matrix., Results: The median morphine concentrations were significantly higher in the rapid compared with the delayed heroin deaths in all matrices (p=0.004 for vitreous humor and p<0.001 for the other matrices). In the rapid heroin deaths, the M3G/morphine concentration ratios were significantly lower than in the delayed deaths both in peripheral and cardiac blood (p<0.001), as well as in pericardial fluid (p<0.001) and vitreous humor (p=0.006), but not in muscle. The morphine/codeine ratios measured in cardiac blood, pericardial fluid and the two muscle samples resembled the ratios in peripheral blood, although codeine was less often detected in other matrices than peripheral blood., Conclusions: Measurements of heroin-metabolites in cardiac blood, pericardial fluid and vitreous humor provide information comparable to that of peripheral blood regarding rapid and delayed heroin deaths, e.g. M3G/morphine ratios <2 indicate a rapid death while ratios >3 indicate a delayed death. However, considerable overlap in results from rapid and delayed deaths was observed, and measurements in muscle appeared less useful. Furthermore, matrices other than peripheral blood can be used to investigate morphine/codeine ratios, but vitreous humor seems less suited., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In some forensic autopsies blood is not available, and other matrices are sampled for toxicological analysis. The aims of the present study were to examine whether heroin metabolites can be detected in different post-mortem matrices, and investigate whether analyses in other matrices can give useful information about concentrations in peripheral blood. Effects of ethanol on the metabolism and distribution of heroin metabolites were also investigated. We included 45 forensic autopsies where morphine was detected in peripheral blood, concomitantly with 6-acetylmorphine (6-AM) detected in any matrix. Samples were collected from peripheral blood, cardiac blood, pericardial fluid, psoas muscle, lateral vastus muscle, vitreous humor and urine. Opioid analysis included 6-AM, morphine, codeine, and morphine glucuronides. The 6-AM was most often detected in urine (n = 39) and vitreous humor (n = 38). The median morphine concentration ratio relative to peripheral blood was 1.3 (range 0-3.6) for cardiac blood, 1.4 (range 0.07-5.3) for pericardial fluid, 1.2 (range 0-19.2) for psoas muscle, 1.1 (range 0-1.7) for lateral vastus muscle and 0.4 (range 0.2-3.2) for vitreous humor. The number of 6-AM positive cases was significantly higher (P = 0.03) in the ethanol positive group (n = 6; 86%) compared to the ethanol negative group (n = 14; 37%) in peripheral blood. The distribution of heroin metabolites to the different matrices was not significantly different between the ethanol positive and the ethanol negative group. This study shows that toxicological analyses of several matrices could be useful in heroin-related deaths. Urine and vitreous humor are superior for detection of 6-AM, while concentrations of morphine could be assessed from peripheral or cardiac blood, pericardial fluid, psoas muscle and lateral vastus muscle.

Environmental stressors inducing oxidative stress such as ionizing radiation may influence cognitive function and neuronal plasticity. Recent studies have shown that transgenic mice deficient of DNA glycosylases display unexpected cognitive deficiencies related to changes in gene expression in the hippocampus. The main objectives of the present study were to determine learning and memory performance in C57BL/6NTac 8-oxoguanine DNA glycosylase 1 (Ogg1) +/- (heterozygote) and Ogg1 +/+ (wild type, WT) mice, to study whether a single acute X-ray challenge (0.5 Gy, dose rate 0.457 Gy/min) influenced the cognitive performance in the Barnes maze, and if such differences were related to changes in gene expression levels in the hippocampus. We found that the Ogg1 +/- mice exhibited poorer early-phase learning performance compared to the WT mice. Surprisingly, X-ray exposure of the Ogg1 +/- animals improved their early-phase learning performance. No persistent effects on memory in the late-phase (6 weeks after irradiation) were observed. Our results further suggest that expression of 3 (Adrb1, Il1b, Prdx6) out of in total 35 genes investigated in the Ogg1 +/- hippocampus is correlated to spatial learning in the Barnes maze.

Clostridiodes difficile strains from the NAPCR1/ST54 and NAP1/ST01 types have caused outbreaks despite of their notable differences in genome diversity. By comparing whole genome sequences of 32 NAPCR1/ST54 isolates and 17 NAP1/ST01 recovered from patients infected with C. difficile we assessed whether mutation, homologous recombination (r) or nonhomologous recombination (NHR) through lateral gene transfer (LGT) have differentially shaped the microdiversification of these strains. The average number of single nucleotide polymorphisms (SNPs) in coding sequences (NAPCR1/ST54 = 24; NAP1/ST01 = 19) and SNP densities (NAPCR1/ST54 = 0.54/kb; NAP1/ST01 = 0.46/kb) in the NAPCR1/ST54 and NAP1/ST01 isolates was comparable. However, the NAP1/ST01 isolates showed 3× higher average dN/dS rates (8.35) that the NAPCR1/ST54 isolates (2.62). Regarding r, whereas 31 of the NAPCR1/ST54 isolates showed 1 recombination block (3,301-8,226 bp), the NAP1/ST01 isolates showed no bases in recombination. As to NHR, the pangenome of the NAPCR1/ST54 isolates was larger (4,802 gene clusters, 26% noncore genes) and more heterogeneous (644 ± 33 gene content changes) than that of the NAP1/ST01 isolates (3,829 gene clusters, ca. 6% noncore genes, 129 ± 37 gene content changes). Nearly 55% of the gene content changes seen among the NAPCR1/ST54 isolates (355 ± 31) were traced back to MGEs with putative genes for antimicrobial resistance and virulence factors that were only detected in single isolates or isolate clusters. Congruently, the LGT/SNP rate calculated for the NAPCR1/ST54 isolates (26.8 ± 2.8) was 4× higher than the one obtained for the NAP1/ST1 isolates (6.8 ± 2.0). We conclude that NHR-LGT has had a greater role in the microdiversification of the NAPCR1/ST54 strains, opposite to the NAP1/ST01 strains, where mutation is known to play a more prominent role.

A novel ion pair reversed phase ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of the stress hormones adrenaline, noradrenaline and corticosterone in rodent blood was developed and fully validated. Separations were performed on an Acquity HSS T3 column (2.1mm i.d.×100mm, 1.8μm) with gradient elution and a runtime of 5.5min. The retention of adrenaline and noradrenaline was substantially increased by employing the ion pair reagent heptafluorobutyric acid (HFBA). Ion pair reagents are usually added to the mobile phase only, but we demonstrate for the first time that including HFBA to the sample reconstitution solvent as well, has a major impact on the chromatography of these compounds. The stability of adrenaline and corticosterone in rodent blood was investigated using the surrogate analytes adrenaline-d 3 and corticosterone-d 8 . The applicability of the described method was demonstrated by measuring the concentration of stress hormones in rodent blood samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mechanochemistry continues to reveal new possibilities in chemistry including the opportunity for "greening" reactions. Nevertheless, a clear understanding of the energetic transformations within mechanochemical systems remains elusive. We employed a uniquely modified ball mill and strategically chosen Diels-Alder reactions to evaluate the role of several ball-milling variables. This revealed three different energetic regions that we believe are defining characteristics of most, if not all, mechanochemical reactors. Relative to the locations of a given ball mill's regions, activation energy determines whether a reaction is energetically easy (Region I), challenging (Region II), or unreasonable (Region III) in a given timeframe. It is in Region II, that great sensitivity to mechanochemical conditions such as vial material and oscillation frequency emerge. Our unique modifications granted control of reaction vessel temperature, which in turn allowed control of the locations of Regions I, II, and III for our mill. Taken together, these results suggest envisioning vibratory mills (and likely other mechanochemical methodologies) as molecular-collision facilitating devices that act upon molecules occupying a thermally-derived energy distribution. This unifies ball-milling energetics with solution-reaction energetics via a common tie to the Arrhenius equation, but gives mechanochemistry the unique opportunity to influence either half of the equation. In light of this, we discuss a strategy for translating solvent-based reaction conditions to ball milling conditions. Lastly, we posit that the extra control via frequency factor grants mechanochemistry the potential for greater selectivity than conventional solution reactions.

Base excision repair (BER) is a major pathway for removal of DNA base lesions and maintenance of genomic stability, which is essential in cancer prevention. DNA glycosylases recognize and remove specific lesions in the first step of BER. The existence of a number of these enzymes with overlapping substrate specificities has been thought to be the reason why single knock-out models of individual DNA glycosylases are not cancer prone. In this work we have characterized DNA glycosylases NEIL1 and NEIL2 (Neil1 -/- /Neil2 -/- ) double and NEIL1, NEIL2 and NEIL3 (Neil1 -/- /Neil2 -/- /Neil3 -/- ) triple knock-out mouse models. Unexpectedly, our results show that these mice are not prone to cancer and have no elevated mutation frequencies under normal physiological conditions. Moreover, telomere length is not affected and there was no accumulation of oxidative DNA damage compared to wild-type mice. These results strengthen the hypothesis that the NEIL enzymes are not simply back-up enzymes for each other but enzymes that have distinct functions beyond canonical repair.

Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus . Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus , suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall.

Clinical decision support systems, when used effectively, can improve the quality of care. However, such systems can malfunction, and these malfunctions can be difficult to detect. In this poster, we describe four methods of detecting and resolving issues with clinical decision support: 1) statistical anomaly detection, 2) visual analytics and dashboards, 3) user feedback analysis, 4) taxonomization of failure modes/effects.

Objective: To illustrate ways in which clinical decision support systems (CDSSs) malfunction and identify patterns of such malfunctions., Materials and Methods: We identified and investigated several CDSS malfunctions at Brigham and Women's Hospital and present them as a case series. We also conducted a preliminary survey of Chief Medical Information Officers to assess the frequency of such malfunctions., Results: We identified four CDSS malfunctions at Brigham and Women's Hospital: (1) an alert for monitoring thyroid function in patients receiving amiodarone stopped working when an internal identifier for amiodarone was changed in another system; (2) an alert for lead screening for children stopped working when the rule was inadvertently edited; (3) a software upgrade of the electronic health record software caused numerous spurious alerts to fire; and (4) a malfunction in an external drug classification system caused an alert to inappropriately suggest antiplatelet drugs, such as aspirin, for patients already taking one. We found that 93% of the Chief Medical Information Officers who responded to our survey had experienced at least one CDSS malfunction, and two-thirds experienced malfunctions at least annually., Discussion: CDSS malfunctions are widespread and often persist for long periods. The failure of alerts to fire is particularly difficult to detect. A range of causes, including changes in codes and fields, software upgrades, inadvertent disabling or editing of rules, and malfunctions of external systems commonly contribute to CDSS malfunctions, and current approaches for preventing and detecting such malfunctions are inadequate., Conclusion: CDSS malfunctions occur commonly and often go undetected. Better methods are needed to prevent and detect these malfunctions., (© The Author 2016. Published by Oxford University Press on behalf of the American Medical Informatics Association.)

Virulent strains of Clostridium difficile have become a global health problem associated with morbidity and mortality. Traditional typing methods do not provide ideal resolution to track outbreak strains, ascertain genetic diversity between isolates, or monitor the phylogeny of this species on a global basis. Here, we investigate the occurrence and diversity of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) in C. difficile to assess the potential of CRISPR-based phylogeny and high-resolution genotyping. A single Type-IB CRISPR-Cas system was identified in 217 analyzed genomes with cas gene clusters present at conserved chromosomal locations, suggesting vertical evolution of the system, assessing a total of 1,865 CRISPR arrays. The CRISPR arrays, markedly enriched (8.5 arrays/genome) compared with other species, occur both at conserved and variable locations across strains, and thus provide a basis for typing based on locus occurrence and spacer polymorphism. Clustering of strains by array composition correlated with sequence type (ST) analysis. Spacer content and polymorphism within conserved CRISPR arrays revealed phylogenetic relationship across clades and within ST. Spacer polymorphisms of conserved arrays were instrumental for differentiating closely related strains, e.g., ST1/RT027/B1 strains and pathogenicity locus encoding ST3/RT001 strains. CRISPR spacers showed sequence similarity to phage sequences, which is consistent with the native role of CRISPR-Cas as adaptive immune systems in bacteria. Overall, CRISPR-Cas sequences constitute a valuable basis for genotyping of C. difficile isolates, provide insights into the micro-evolutionary events that occur between closely related strains, and reflect the evolutionary trajectory of these genomes., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)