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Repeated single-point measurements of thoracic bioimpedance at a single (low) frequency are strongly related to fluid changes during hemodialysis. Extension to semi-continuous measurements may provide longitudinal details in the time pattern of the bioimpedance signal, and multi-frequency measurements may add in-depth information on the distribution between intra- and extracellular fluid. This study aimed to investigate the feasibility of semi-continuous multi-frequency thoracic bioimpedance measurements by a wearable device in hemodialysis patients. Therefore, thoracic bioimpedance was recorded semi-continuously (i.e., every ten minutes) at nine frequencies (8-160 kHz) in 68 patients during two consecutive hemodialysis sessions, complemented by a single-point measurement at home in-between both sessions. On average, the resistance signals increased during both hemodialysis sessions and decreased during the interdialytic interval. The increase during dialysis was larger at 8 kHz (∆ 32.6 Ω during session 1 and ∆ 10 Ω during session 2), compared to 160 kHz (∆ 29.5 Ω during session 1 and ∆ 5.1 Ω during session 2). Whereas the resistance at 8 kHz showed a linear time pattern, the evolution of the resistance at 160 kHz was significantly different ( p < 0.0001). Measuring bioimpedance semi-continuously and w i th a multi-frequency current is a major step forward in the understanding of fluid dynamics in hemodialysis patients. This study paves the road towards remote fluid monitoring.

In contrast to whole body bioimpedance, which estimates fluid status at a single point in time, thoracic bioimpedance applied by a wearable device could enable continuous measurements. However, clinical experience with thoracic bioimpedance in patients on dialysis is limited. To test the reproducibility of whole body and thoracic bioimpedance measurements and to compare their relationship with hemodynamic changes during hemodialysis, these parameters were measured pre- and end-dialysis in 54 patients during two sessions. The resistance from both bioimpedance techniques was moderately reproducible between two dialysis sessions (intraclass correlations of pre- to end-dialysis whole body and thoracic resistance between session 1 and 2 were 0.711 [0.58-0.8] and 0.723 [0.6-0.81], respectively). There was a very high to high correlation between changes in ultrafiltration volume and changes in whole body thoracic resistance. Changes in systolic blood pressure negatively correlated to both bioimpedance techniques. Although the relationship between changes in ultrafiltration volume and changes in resistance was stronger for whole body bioimpedance, the relationship with changes in blood pressure was at least comparable for thoracic measurements. These results suggest that thoracic bioimpedance, measured by a wearable device, may serve as an interesting alternative to whole body measurements for continuous hemodynamic monitoring during hemodialysis. NEW & NOTEWORTHY We examined the role of whole body and thoracic bioimpedance in hemodynamic changes during hemodialysis. Whole body and thoracic bioimpedance signals were strongly related to ultrafiltration volume and moderately, negatively, to changes in blood pressure. This work supports the further development of a wearable device measuring thoracic bioimpedance longitudinally in patients on hemodialysis. As such, it may serve as an innovative tool for continuous hemodynamic monitoring during hemodialysis in hospital or in a home-based setting.

Amyloid beta (Aβ) aggregation and tau hyper phosphorylation (p-tau) are key molecular factors in Alzheimer's disease (AD). The abnormal formation and accumulation of Aβ and p-tau lead to the formation of amyloid plaques and neurofibrillary tangles (NFTs) which ultimately leads to neuroinflammation and neurodegeneration. β- and γ-secretases produce Aβ peptides via the amyloidogenic pathway, and several kinases are involved in tau phosphorylation. Exosomes, a recently developed method of intercellular communication, derived from neuronal stem cells (NSC-exos), are intriguing therapeutic options for AD. Exosomes have ability to cross the BBB hence highly recommended for brain related diseases and disorders. In the current study, we examined how NSC-exos could protect human neuroblastoma cells SH-SY5Y (ATCC CRL-2266). NSC-exos were derived from Human neural stem cells (ATCC-BYS012) by ultracentrifugation and the therapeutic effects of the NSC-exos were then investigated in vitro. NSC-exos controlled the associated molecular processes to drastically lower Aβ and p-tau. A dose dependent reduction in β- and γ-secretase, acetylcholinesterase, GSK3β, CDK5, and activated α-secretase activities was also seen. We further showed that BACE1, PSEN1, CDK5, and GSK-3β mRNA expression was suppressed and downregulated, while ADAM10 mRNA was increased. NSC- Exos downregulate NF-B/ERK/JNK-related signaling pathways in activated glial cells HMC3 (ATCC-CRL-3304) and reduce inflammatory mediators such iNOS, IL-1β, TNF-α, and IL-6, which are associated with neuronal inflammation. The NSC-exos therapy ameliorated the neurodegeneration of human neuroblastoma cells SH-SY5Y by enhancing viability. Overall, these findings support that exosomes produced from stem cells can be a neuro-protective therapy to alleviate AD pathology., (© 2023. Springer Nature Limited.)

The Ross-Macdonald model has exerted enormous influence over the study of malaria transmission dynamics and control, but it lacked features to describe parasite dispersal, travel, and other important aspects of heterogeneous transmission. Here, we present a patch-based differential equation modeling framework that extends the Ross-Macdonald model with sufficient skill and complexity to support planning, monitoring and evaluation for Plasmodium falciparum malaria control. We designed a generic interface for building structured, spatial models of malaria transmission based on a new algorithm for mosquito blood feeding. We developed new algorithms to simulate adult mosquito demography, dispersal, and egg laying in response to resource availability. The core dynamical components describing mosquito ecology and malaria transmission were decomposed, redesigned and reassembled into a modular framework. Structural elements in the framework-human population strata, patches, and aquatic habitats-interact through a flexible design that facilitates construction of ensembles of models with scalable complexity to support robust analytics for malaria policy and adaptive malaria control. We propose updated definitions for the human biting rate and entomological inoculation rates. We present new formulas to describe parasite dispersal and spatial dynamics under steady state conditions, including the human biting rates, parasite dispersal, the "vectorial capacity matrix," a human transmitting capacity distribution matrix, and threshold conditions. An [Formula: see text] package that implements the framework, solves the differential equations, and computes spatial metrics for models developed in this framework has been developed. Development of the model and metrics have focused on malaria, but since the framework is modular, the same ideas and software can be applied to other mosquito-borne pathogen systems., Competing Interests: The authors declare no competing interests., (Copyright: © 2023 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Germin (GER) and germin-like proteins (GLPs) play an important role in various plant processes. Zea mays contains 26 germin-like protein genes ( ZmGLPs ) located on chromosomes 2, 4, and 10; most of which are functionally unexplored. The present study aimed to characterize all ZmGLPs using the latest computational tools. All of them were studied at a physicochemical, subcellular, structural, and functional level, and their expression was predicted in plant development, against biotic and abiotic stresses using various in silico approaches. Overall, ZmGLPs showed greater similarity in their physicochemical properties, domain architecture, and structure, mostly localized in the cytoplasmic or extracellular regions. Phylogenetically, they have a narrow genetic background with a recent history of gene duplication events on chromosome 4. Functional analysis revealed novel enzymatic activities of phosphoglycolate phosphatase, adenosylhomocysteinase, phosphoglycolate phosphatase-like, osmotin/thaumatin-like, and acetohydroxy acid isomeroreductase largely mediated by disulfide bonding. Expression analysis revealed their crucial role in the root, root tips, crown root, elongation and maturation zones, radicle, and cortex with the highest expression being observed during germination and at the maturity levels. Further, ZmGLPs showed strong expression against biotic ( Aspergillus flavus , Colletotrichum graminicola , Cercospora zeina , Fusarium verticillioides , and Fusarium virguliforme ) while limited expression was noted against abiotic stresses. Concisely, our results provide a platform for additional functional exploration of the ZmGLP genes against various environmental stresses., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Saponins are natural compounds found in plants and have a diverse range of applications. However, the therapeutic potential of saponins in regulating cytotoxicity, angiogenesis, and inflammation in mammalian cells is yet to be explored. Here, we investigated the therapeutic effects of saponins from green tea by exploring the cytotoxic effects of saponins by inducing apoptosis in the human cancer cell lines hepatocellular carcinoma (HEPG2) and colorectal adenocarcinoma (HT29). The anti-angiogenesis effect of saponins was also investigated in human umbilical vein endothelial cells (HUVEC). We explored the ability of saponins to attenuate inflammation in a dose-dependent manner in normal human cells. It was found that saponins exhibit cytotoxic effects in cancer cells and not in normal cells at the same concentration. Cytotoxicity was measured by inducing apoptosis by enhancing caspase-3 (cas-3) activation and B-cell lymphoma-2 ( Bcl-2 )-associated X protein ( BAX ) gene expression and suppressing the antiapoptotic protein, Bcl-2. The inhibition of HUVEC proliferation was due to the suppression of the phosphoinositide 3-kinase (PI3K), protein kinase B ( AKT ), vascular endothelial growth factor receptor-2 ( VEGFR-2 ), and nuclear factor kappa B ( NF-κB ). We also observed the antioxidant potential of green tea-derived saponins against free radicals in reactive oxygen species (ROS)-induced cells. Here we observed that the saponins exhibited free radical scavenging activities and activated nuclear factorerythroid 2-related factor 2 (NRF-2) leading to the upregulation of antioxidant-related genes in human embryonic kidney 293 (HEK293) cells. Furthermore, we demonstrated that the anti-inflammatory effects were due to the suppression of pro-inflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor-alpha (TNF-α), and inducible nitric oxide synthase (iNOS) in HEK293 cells. The significance of the work is we are the first to report on the anti-cancer effects of saponins based on the anti-inflammatory, antioxidant, anti-angiogenesis, and apoptosis induction properties. In conclusion, green tea-derived saponins could be effective therapeutics for the treatment of cancer.

A limited number of studies using differing imaging approaches suggest that there are regional variation in the cerebrovascular response to hypercapnia and hypoxia. However there are limitations to these studies. In particular, it is not clear if existing studies of hypoxia have fully accounted for the confounding effects of the changes in arterial PCO2 on cerebral perfusion that, if uncontrolled, will accompany the hypoxic stimulus. We determined quantitative maps of grey matter cerebral blood flow using a multi-slice pulsed arterial spin labelling MRI method at 3 T at rest, during conditions of isocapnic euoxia, hypercapnia, and mild isocapnic hypoxia. From these data, we determined grey matter cerebrovascular reactivity maps which show the spatial distribution of the responses to these interventions. Whilst, overall, cerebral perfusion increased with hypercapnia and hypoxia, hypoxia cerebrovascular reactivity maps showed very high variation both within and between individuals: most grey matter regions exhibiting a positive cerebrovascular reactivity, but some exhibiting a negative reactivity. The physiological explanation for this variation remains unclear and it is not known if these local differences will vary with state or with regional brain activity. The potential interaction between hypoxic or hypercapnic cerebrovascular changes and neurally related changes in brain perfusion is of particular interest for functional imaging studies of brain activation in which arterial blood gases are altered. We have determined the interaction between global hypoxia and hypercapnia-induced blood oxygen level-dependent (BOLD) MRI signal and local neurally related BOLD signal. Although statistically significant interactions were present, physiologically the effects were weak and, in practice, they did not change the statistical outcome related to the analysis of the neurally related signals. These data suggest that such respiratory-related confounds can be successfully accounted for in functional imaging studies.

Background/objectives: High blood pressure (HBP) is one of the most important risk factors for cardiovascular diseases and it has a high prevalence in pediatric populations. However, the determinants of the incidence of Pre-HBP and HBP in children are not well known. i) To describe the incidence of HBP in European children; and ii) to evaluate the effect of physical activity (PA) and sedentary behavior (SB) on the Pre-HBP and HBP., Methods: The IDEFICS cohort study. A total of 16,228 children 2-9years at baseline were recruited by complex sampling population-based survey in eight European countries. At baseline (T0), 5221 children were selected for accelerometer measurements; 5061 children were re-examined 2years later (T1). We estimated the incidence of Pre-HBP and HBP and evaluate the effect of PA and SB on the Pre-HBP and HBP, by computing relative risks and the corresponding 95% confidence intervals (RR, 95% CI)., Results: Incidences of Pre-HBP and HBP per year were: 121/1000 children and 110/1000 children, respectively. We found that children maintaining SB>2h/d during the two year follow-up showed a RR of having HBP of 1.28 (1.03-1.60). Children in T1 not performing the recommended amount of PA (<60min/d) have a RR of HBP of 1.53 (1.12 to 2.09). We found no association between pre-HBP and the behaviors., Conclusion: The incidence of pre-HBP and HBP is high in European children. Maintaining sedentary behaviors during childhood increases the risk of developing HBP after two years of follow-up., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: Non-invasive quantification of lower limb alignment using navigation technology is now possible throughout knee flexion owing to software developments. We report the precision and accuracy of a non-invasive system measuring mechanical alignment of the lower limb including coronal stress testing of the knee., Methods: Twelve cadaveric limbs were tested with a commercial invasive navigation system against the non-invasive system. Coronal mechanical femorotibial (MFT) alignment was measured with no stress, then 15 Nm varus and valgus applied moments. Measurements were recorded at 10° intervals from extension to 90° flexion. At each flexion interval, coefficient of repeatability (CR) tested precision within each system, and limits of agreement (LOA) tested agreement between the two systems. Limits for CR & LOA were set at 3° based on requirements for surgical planning and evaluation., Results: Precision was acceptable throughout flexion in all conditions of stress using the invasive system (CR ≤ 1.9°). Precision was acceptable using the non-invasive system from extension to 50° flexion (CR ≤ 2.4°), beyond which precision was unacceptable (> 3.4°). With no coronal stress applied, agreement remained acceptable from extension to 40° (LOA ≤ 2.4°), and when 15 Nm varus or valgus stress was applied agreement was acceptable from extension to 30° (LOA ≤ 2.9°). Higher angles of knee flexion had a negative impact on precision and accuracy., Conclusion & Clinical Relevance: The non-invasive system provides reliable quantitative data in-vitro on coronal MFT alignment and laxity in the range relevant to assessment of collateral ligament injury, pre-operative planning of arthroplasty and flexion instability following arthroplasty. In-vivo validation should be performed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Two operative approaches are commonly used for isolated talonavicular arthrodesis: the medial and the dorsal approach. It is recognized that access to the lateral aspect of the talonavicular joint can be limited when using the medial approach, and it is our experience that using the dorsal approach addresses this issue. We performed an anatomical study using cadaver specimens, to compare the amount of articular surface that can be accessed by each operative approach., Methods: Medial and dorsal approaches to the talonavicular joint were performed on each of 11 cadaveric specimens (10 fresh frozen, 1 embalmed). Distraction of the joint was performed as used intraoperatively and the accessible area of articular surfaces was marked for each of the 2 approaches using a previously reported technique. Disarticulation was performed and the marked surface area was quantified using an immersion digital microscribe, allowing a 3-dimensional virtual model of the articular surfaces to be assessed., Results: The median percentage of total accessible talonavicular articular surface area for the medial and dorsal approaches was 71% and 92%, respectively (Wilcoxon signed-rank test, P < .001)., Conclusion: This study provides quantifiable measurements of the articular surface accessible by the medial and dorsal approaches to the talonavicular joint., Clinical Relevance: These data support for the use of the dorsal approach for talonavicular arthrodesis, particularly in cases where access to the lateral half of the joint is necessary., (© The Author(s) 2014.)

Purpose: The ability to quantify rotational laxity of the knee would increase understanding of functional rotatory instability, identify the best treatment methods for soft tissue injury, and have a role in diagnosis of soft tissue injury. This study aimed to report the reliability, repeatability and precision of a non-invasive adaptation of image-free navigation technology by comparing with a validated invasive system used for computer-assisted surgery., Methods: Twelve cadaveric lower limbs were tested with a commercial image-free navigation system using passive trackers secured by bone screws. They were then tested a non-invasive fabric-strap system. Manual application of torque was used consistent with clinical examination to rotate the tibia to the end of internal rotation and external rotation range. Measurements were taken at 10° intervals from full extension to 90° flexion, and protocol was repeated twice using each system. Intraclass correlation coefficient (ICC) was used to reflect reliability of measurements. At each flexion interval, coefficient of repeatability (CR) was calculated for each system, and limits of agreement (LOA) were used to reflect agreement between the systems., Results: The results for internal and external rotation were combined throughout flexion: ICC invasive; 0.94 (0.86-0.99), non-invasive; 0.92 (0.7-0.99), CR invasive; 2.4° (1.3-4.8°), non-invasive; 3.5° (1.8-6.6), LOA; 8.2° (4.3-13.5)., Conclusion: Non-invasive optical tracker fixation gives improved agreement with a validated method of measurement compared with devices measuring tibial rotation by foot position. This system gives the added possibility of dynamic, weight-bearing testing in the clinically important range of 0°-30° knee flexion without the need for any limb restraint.

Background: The arterial supply to the talus has been extensively studied previously but never to specifically examine the subchondral region of the talar dome, a frequent site of localised pathology. This study aims to analyse and quantify the subchondral vascularity of the talar dome., Methods: We performed cadaveric arterial injection studies. After processing, the vascularity to the subchondral region of the talar dome was visualised and mapped using three-dimensional computer technology, then quantified and reported using a nine-section anatomical grid., Results: The areas of relative poor perfusion across the talar dome are the posterior/medial, posterior/lateral and middle/medial sections of a nine-section grid. The rest of the subchondral region shows more richly vascularised bone., Conclusions: The vascularity of the subchondral surface of the talar dome is not uniformly distributed. This may be relevant to the aetiology and management of osteochondral lesions and shows some correlation with their more frequent locations., (Copyright © 2013 European Foot and Ankle Society. Published by Elsevier Ltd. All rights reserved.)

Objective: Non-invasive navigation techniques have recently been developed to determine mechanical femorotibial alignment (MFTA) in extension. The primary aim of this study was to evaluate the precision and accuracy of an image-free navigation system with new software designed to provide multiple kinematic measurements of the knee. The secondary aim was to test two types of strap material used to attach optical trackers to the lower limb., Methods: Seventy-two registrations were carried out on 6 intact embalmed cadaveric specimens (mean age: 77.8 ± 12 years). A validated fabric strap, bone screws and novel rubber strap were used to secure the passive tracker baseplate for four full experiments with each knee. The MFTA angle was measured under the conditions of no applied stress, valgus stress, and varus stress. These measurements were carried out at full extension and at 30°, 40°, 50° and 60° of flexion. Intraclass correlation coefficients, repeatability coefficients, and limits of agreement (LOA) were used to convey precision and agreement in measuring MFTA with respect to each of the independent variables, i.e., degree of flexion, applied coronal stress, and method of tracker fixation. Based on the current literature, a repeatability coefficient and LOA of ≤ 3° were deemed acceptable., Results: The mean fixed flexion for the 6 specimens was 12.8° (range: 6-20°). The mean repeatability coefficient measuring MFTA in extension with screws or fabric strapping of the baseplate was ≤ 2°, compared to 2.3° using rubber strapping. When flexing the knee, MFTA measurements taken using screws or fabric straps remained precise (repeatability coefficient ≤ 3°) throughout the tested range of flexion (12.8-60°); however, using rubber straps, the repeatability coefficient was >3° beyond 50° flexion. In general, applying a varus/valgus stress while measuring MFTA decreased precision beyond 40° flexion. Using fabric strapping, excellent repeatability (coefficient ≤ 2°) was observed until 40° flexion; however, beyond 50° flexion, the repeatability coefficient was >3°. As was the case with precision, agreement between the invasive and non-invasive systems was satisfactory in extension and worsened with flexion. Mean limits of agreement between the invasive and non-invasive system using fabric strapping to assess MFTA were 3° (range: 2.3-3.8°) with no stress applied and 3.9° (range: 2.8-5.2°) with varus and valgus stress. Using rubber strapping, the corresponding values were 4.4° (range: 2.8-8.5°) with no stress applied, 5.5° (range: 3.3-9.0°) with varus stress, and 5.6° (range: 3.3-11.9°) with valgus stress., Discussion: Acceptable precision and accuracy may be possible when measuring knee kinematics in early flexion using a non-invasive system; however, we do not believe passive trackers should be mounted with rubber strapping such as was used in this study. Flexing the knee appears to decrease the precision and accuracy of the system. The functions of this new software using image-free navigation technology have many potential clinical applications, including assessment of bony and soft tissue deformity, pre-operative planning, and post-operative evaluation, as well as in further pure research comparing kinematics of the normal and pathological knee.

Control programmes for vaccine preventable diseases typically operate under logistic constraints such as limited resources and in spatially structured populations where the assumption of homogeneous mixing is invalid. It is unclear, therefore, how to maximise the effectiveness of campaigns in such populations. We investigate how to deploy vaccine in metapopulations by comparing the effectiveness of alternative vaccination strategies on reducing disease occurrence (presence/absence), using canine rabies as a model system, and a domestic dog population within a Tanzanian district divided into sub-populations corresponding to villages. We use patch-occupancy models to quantify the contribution of sub-populations to disease occurrence ("risk") and model allocation strategies for a limited number of vaccine doses that prioritize villages based on their size, risk, or the reduction in risk for the entire population that would result from vaccination. We assume that a maximum of 70% of susceptible individuals in a village could be vaccinated, and that only susceptible dogs are vaccinated. The most effective strategy maximised the reduction in risk of the entire population, and was up to 62% more effective than the other strategies. Large, single-pulse campaigns provided the greatest short-term protection, but higher frequencies of smaller pulses were more effective at reducing long-term disease occurrence. Vaccine allocation on a per-dose basis was substantially more effective than a per-village strategy, indicating that operational constraints can reduce control effectiveness. The spatial distribution and abundance of hosts have an important influence on disease dynamics and these results demonstrate that metapopulation models can be used to substantially improve the effectiveness of vaccination campaigns and optimize the allocation of limited control resources., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Urbanization affects lifestyles in the developing world but no studies have assessed the impact on objectively measured physical activity in children and adolescents from sub-Saharan Africa., Purpose: To compare objectively measured habitual physical activity, sedentary time, and indices of adiposity in adolescents from rural and urban areas of Kenya., Methods: Physical activity and sedentary time were assessed by accelerometry for 5 consecutive days in 97 (50 female and 47 male) rural and 103 (52 female and 51 male) urban adolescents (mean age 13 ± 1 years). Body Mass Index (BMI) and BMI z-scores were used to assess adiposity., Results: Rural males spent more time in moderate-to-vigorous intensity physical activity (MVPA) compared with urban males (68 ± 22 vs. 50 ± 17 min, respectively; P < .001). Similarly, Rural females spent more time in MVPA compared with urban females (62 ± 20 vs. 37 ± 20 min, respectively; P < .001). Furthermore, there were significant differences in daily sedentary time between rural and urban subjects. Residence (rural vs. urban) significantly (P < .001) influenced BMI z-score (R(2) = .46)., Conclusion: Rural Kenyan adolescents are significantly more physically active (and less sedentary) and have lower indices of adiposity compared with urban adolescents and this is a likely reflection of the impact of urbanization on lifestyle in Kenya.

Aims: The aim of this study was to update and extend our knowledge of the bacterial load and microbial composition in Norway lobster (Nephrops norvegicus) under commercially relevant storage conditions to optimize handling procedures., Methods and Results: Total viable counts were performed at different storage temperatures (0, 4, 8, 10, 12 or 16°C) and after different storage times (1-7 days). Storage at 16°C was found to be most detrimental, and storage at 0°C was found to be optimal. 16S-rRNA sequencing was utilized to determine the composition of the bacteria within the microflora. In this way, Photobacterium isolates, especially Photobacterium phosphoreum, were identified as the main specific spoilage organisms. The abilities to reduce trimethylamineoxide (TMAO) and to produce H(2)S were analysed in a selection of bacterial isolates. The higher the incubation temperature during storage, the more isolates were found to reduce TMAO and produce H(2)S., Conclusions: Nephrops norvegicus possesses an unusually high initial microbial load when fresh. Storage temperature is the most crucial factor affecting microbial growth, microbial activity and spoilage potential in N. norvegicus produce. Spoilage can be attributed mainly to P. phosphoreum., Significance and Impact of the Study: This study presents significant new findings with regard to the progression and causative agents of spoilage in N. norvegicus. Based on the results, we can recommend that N. norvegicus tails should be stored in a 0°C environment immediately after catch. Stored this way, the growth and spoilage activity of the microflora may be reduced significantly and an extension of shelf life might be attained., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Laboratory-based studies have demonstrated that adding protein (PRO) to a carbohydrate (CHO) supplement can improve thermoregulatory capacity, exercise performance and recovery. However, no study has investigated these effects in a competitive sporting context. This study assessed the effects of combined CHO-PRO supplementation on physiological responses and exercise performance during 8 days of strenuous competition in a hot environment. Twenty-eight cyclists participating in the TransAlp mountain bike race were randomly assigned to fitness-matched placebo (PLA 76 g L(-1) CHO) or CHO-PRO (18 g L(-1) PRO, 72 g L(-1) CHO) groups. Participants were given enough supplements to allow ad libitum consumption. Physiological and anthropometric variables were recorded pre- and post-exercise. Body mass decreased significantly from race stage 1 to 8 in the PLA group (-0.75 ± 0.22 kg, P = 0.01) but did not change in the CHO-PRO group (0.42 ± 0.42 kg, P = 0.35). Creatine kinase concentration and muscle soreness were substantially elevated during the race, but were not different between groups (P = 0.82, P = 0.44, respectively). Urine osmolality was significantly higher in the CHO-PRO versus the PLA group (P = 0.04) and the rise in tympanic temperature from pre- to post-exercise was significantly less in CHO-PRO versus PLA (P = 0.01). The CHO-PRO group also completed the 8 stages significantly quicker than the PLA group (2,277 ± 127 vs. 2,592 ± 68 min, respectively, P = 0.02). CHO-PRO supplementation therefore appears to prevent body mass loss, enhance thermoregulatory capacity and improve competitive exercise performance despite no effect on muscle damage.

Background and Purpose: Many G protein-coupled receptors internalize following agonist binding. The studies were designed to identify novel means to effectively quantify this process using the orexin OX(1) receptor and the cannabinoid CB(1) receptor as exemplars., Experimental Approach: The human OX(1) and CB(1) receptors were modified to incorporate both epitope tags and variants (SNAP and CLIP) of the enzyme O(6)-alkylguanine-DNA-alkyltransferase within their extracellular, N-terminal domain. Cells able to regulate expression of differing amounts of these constructs upon addition of an antibiotic were developed and analysed., Key Results: Cell surface forms of each receptor construct were detected by both antibody recognition of the epitope tags and covalent binding of fluorophores to the O(6)-alkylguanine-DNA-alkyltransferase variants. Receptor internalization in response to agonists but not antagonists could be monitored by each approach but sensitivity was up to six- to 10-fold greater than other approaches when employing a novel, time-resolved fluorescence probe for the SNAP tag. Sensitivity was not enhanced, however, for the CLIP tag, possibly due to higher levels of nonspecific binding., Conclusions and Implications: These studies demonstrate that highly sensitive and quantitative assays that monitor cell surface CB(1) and OX(1) receptors and their internalization by agonists can be developed based on introduction of variants of O(6)-alkylguanine-DNA-alkyltransferase into the N-terminal domain of the receptor. This should be equally suitable for other G protein-coupled receptors., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Background and Purpose: GPR35 is a poorly characterized G protein-coupled receptor at which kynurenic acid has been suggested to be the endogenous ligand. We wished to test this and develop assays appropriate for the study of this receptor., Experimental Approach: Human and rat orthologues of GPR35 were engineered and expressed and assays developed to assess interaction with β-arrestin-2, activation of Gα₁₃ and agonist-induced internalization., Key Results: GPR35-β-arrestin-2 interaction assays confirmed that both the endogenous tryptophan metabolite kynurenic acid and the synthetic ligand zaprinast had agonist action at each orthologue. Zaprinast was substantially more potent than kynurenic acid at each and both agonists displayed substantially greater potency at rat GPR35. Two novel thiazolidinediones also displayed agonism and displayed similar potency at each GPR35 orthologue. The three ligand classes acted orthosterically with respect to each other, suggesting overlapping binding sites and, consistent with this, mutation to alanine of the conserved arginine at position 3.36 or tyrosine 3.32 in transmembrane domain III abolished β-arrestin-2 recruitment in response to each ligand at each orthologue., Conclusions and Implications: These studies indicate that β-arrestin-2 interaction assays are highly appropriate to explore the pharmacology of GPR35 and that Gα₁₃ activation is an alternative avenue of signal generation from GPR35. Arginine and tyrosine residues in transmembrane domain III are integral to agonist recognition and function of this receptor. The potency of kynurenic acid at human GPR35 is sufficiently low, however, to question whether it is likely to be the true endogenous ligand for this receptor., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Daughters of diabetes patients have lower insulin sensitivity than women with no diabetes family history, but increase insulin sensitivity to a greater extent with exercise training. This study aimed to determine whether differences in circulating concentrations of adiponectin and leptin, and adipose tissue expression of their genes and receptors played a role. Women offspring of patients with type 2 diabetes mellitus (n = 34; age, 35.6 ± 7.0 years; body mass index, 28.1 ± 5.1 kg/m²) and matched controls with no diabetes family history (n = 36; age, 33.6 ± 6.1 years; body mass index, 27.3 ± 4.7 kg/m²) participated. Blood and abdominal subcutaneous adipose tissue samples were obtained at baseline and after a controlled 7-week endurance-type exercise intervention (sessions were performed at 65%-80% of maximum heart rate). At baseline, no significant differences were observed between groups in circulating leptin or adiponectin concentrations, or expression of their genes or receptors. In response to exercise, plasma leptin decreased more in offspring than controls (-32.2% vs -7.3%, P = .005 for interaction); and the long isoform of the leptin receptor messenger RNA (mRNA) increased significantly only in the offspring (+39.4%, P = .026 vs +7.7%, P = .892). Leptin mRNA decreased similarly in both groups (-24.7% vs -25.0%, P < .05 for both). Furthermore, changes in plasma leptin (r = -0.432, P < .001) and leptin mRNA (r = -0.298, P = .019) correlated significantly with changes in insulin sensitivity. Plasma adiponectin decreased similarly in both groups (-12.1% vs -15.2%, P < .01 for both), but no significant changes were observed in adiponectin-related gene expression. This work shows that exercise training has differing effects on leptin-related variables between women with and without a diabetes family history and suggests that these molecular differences may contribute to the differential effects of exercise training on insulin sensitivity between these 2 groups., (© 2011 Elsevier Inc. All rights reserved.)

The simple diamine diaminoethane (ethylenediamine, EDA) has been shown to activate GABA receptors in the central and peripheral nervous systems, partly by a direct action and partly by releasing endogenous GABA. These effects have been shown to be produced by the complexation of EDA with bicarbonate to form a carbamate. The present work has compared EDA, GABA and β-alanine responses in rat CA1 neurons using extracellular and intracellular recordings, as well as neocortical evoked potentials in vivo. Superfusion of GABA onto hippocampal slices produced depolarisation and a decrease of field epsps, both effects fading rapidly, but showing sensitivity to blockade by bicuculline. EDA produced an initial hyperpolarisation and increase of extracellular field epsp size with no fade and only partial sensitivity to bicuculline, with subsequent depolarisation, while β-alanine produces a much larger underlying hyperpolarisation and increase in fepsps, followed by depolarisation and inhibition of fepsps. The responses to β-alanine, but not GABA or EDA, were blocked by strychnine. In vivo experiments, recording somatosensory evoked potentials, confirmed that EDA produced an initial increase followed by depression, and that this effect was not fully blocked by bicuculline. Overall the results indicate that EDA has actions in addition to the activation of GABA receptors. These actions are not attributable to activation of β-alanine-sensitive glycine receptors, but may involve the activation of sites sensitive to adipic acid, which is structurally equivalent to the dicarbamate of EDA. The results emphasise the complex pharmacology of simple amines in bicarbonate-containing solutions., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Recent advances in G protein-coupled receptors have challenged traditional definitions of agonism, antagonism, affinity and efficacy. The discovery of agonist functional selectivity and receptor allosterism has meant researchers have an expanded canvas for designing and discovering novel drugs. Here we describe modes of agonism emerging from the discovery of functional selectivity and allosterism. We discuss the concept of ago-allosterism, where ligands can initiate signaling by themselves and influence the actions of another ligand at the same receptor. We introduce the concept of dualsteric ligands that consist of distinct elements which bind to each of the orthosteric and an allosteric domain on a single receptor to enhance subtype selectivity. Finally, the concept that efficacy should be defined by the activity of an endogenous ligand will be challenged by the discovery that some ligands act as 'super-agonists' in specific pathways or at certain receptor mutations., (Crown Copyright © 2010. Published by Elsevier Ireland Ltd. All rights reserved.)

Non-destructive methods of collecting DNA from small fish species can be problematic, as fin clips can potentially affect behaviour or survivorship in the wild. Swabbing body mucus may provide a less invasive method of DNA collection. However, risk of contamination from other individuals in high density groups could give erroneous genotyping results. We compared multilocus microsatellite genotypes from the same individuals when collected at low and high density and compared this with fin clips. We found no differences between these categories, with a genotyping error rate of 0.42%, validating the use of body mucus swabbing for DNA collection in fish., (© 2010 Blackwell Publishing Ltd.)

hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

The control of gene expression at certain times during the mitotic cell division cycle is a common feature in eukaryotes. In fission yeast, at least five waves of gene expression have been described, with one transcribed at the M-G1 interval under the control of the PBF transcription factor complex. PBF consists of at least three transcription factors, two forkhead-like proteins Sep1p and Fkh2p, and a MADS box-like protein Mbx1p, and binds to PCB motifs found in the gene promoters. Mbx1p is under the direct control of the polo-like kinase Plo1p and the Cdc14p-like phosphatase Clp1p (Flp1p). Here, we show that M-G1 gene expression in fission yeast is also regulated by the anillin-like protein, Mid1p (Dmf1p). Mid1p binds in vivo to both Fkh2p and Sep1p, and to the promoter regions of M-G1 transcribed genes. Mid1p promoter binding is dependent on Fkh2p, Plo1p and Clp1p. The absence of mid1(+) in cells results in partial loss of M-G1 specific gene expression, suggesting that it has a negative role in controlling gene expression. This phenotype is exacerbated by also removing clp1(+), suggesting that Mid1p and Clp1p have overlapping functions in controlling transcription. As mid1(+) is itself expressed at M-G1, these observations offer a new mechanism whereby Mid1p contributes to controlling cell cycle-specific gene expression as part of a feedback loop.

Regulated gene expression makes an important contribution to cell cycle control mechanisms. In fission yeast, a group of genes is coordinately expressed during a late stage of the cell cycle (M phase and cytokinesis) that is controlled by common cis-acting promoter motifs named pombe cell cycle boxes (PCBs), which are bound by a trans-acting transcription factor complex, PCB binding factor (PBF). PBF contains at least three transcription factors, a MADS box protein Mbx1p and two forkhead transcription factors, Sep1p and Fkh2p. Here we show that the fission yeast Cdc14p-like phosphatase Clp1p (Flp1p) controls M-G1 specific gene expression through PBF. Clp1p binds in vivo both to Mbx1p, a MADS box-like transcription factor, and to the promoters of genes transcribed at this cell cycle time. Because Clp1p dephosphorylates Mbx1p in vitro, and is required for Mbx1p cell cycle-specific dephosphorylation in vivo, our observations suggest that Clp1p controls cell cycle-specific gene expression through binding to and dephosphorylating Mbx1p.

Studies of the fat mass and obesity-associated (FTO) gene provide compelling evidence of genetic variation in the general population that influences fat levels and obesity risk. Studies of the interaction between genetic and environmental factors such as physical activity (PA) will promote the understanding of how lifestyle can modulate genetic contributions to obesity. In this study, we investigated the effect of FTO genotype, and interactions with PA or energy intake, in young children and adolescents. In all, 1-5-year-old children from the Growth, Exercise and Nutrition Epidemiological Study in preSchoolers (GENESIS) study (N=1980) and 11-18-year-old Greek adolescents (N=949) were measured for adiposity-related phenotypes and genotyped at the FTO single-nucleotide polymorphism (SNP) marker, rs17817449. Adolescents were classified as physically active or inactive based on self-reported levels of PA. In adolescents, FTO genotype influenced weight (P=0.001) and BMI (P=0.007). There was also a significant SNP(*)PA(*)gender interaction (P=0.028) on BMI, which reflected the association between FTO genotype and BMI in males (P=0.016), but not females (P=0.15), and significant SNP(*)PA interaction in males (P=0.007), but not females (P=0.74). The FTO genotype effect was more pronounced in inactive than active males. Inactive males homozygous for the G allele had a mean BMI 3 kg/m(2) higher than T carriers (P=0.008). In the GENESIS study, no significant association between FTO genotype and adiposity was found. The present findings highlight PA as an important factor modifying the effect of FTO genotype.

Reconstructive surgery of the midface using facial artery perforator (FAP) flaps is being used more frequently now as it has been reported to provide better aesthetic results and reduce a traditional two-stage procedure to a one-stage technique. However, the wide acceptance of this approach is limited by a poor understanding of the anatomy associated with this technique. This was investigated through a cadaveric study. The facial artery (FA) of 16 cadaveric half-faces were each identified, cannulated with coloured latex and then dissected to give an accurate and quantified description of FA perforating branches. A lateral-view picture of each specimen was taken and analysed using ImageJ 1.42q. Cadaveric dissections showed that each hemiface could be regarded as a single entity. The values of the means were as follows: FA length=116±22 mm, FA diameter=2.62±0.74 mm, number of FAPs=4±2, FAP length=14.12±3.46 mm and FAP diameter=0.94±0.29 mm. A reference point, A, where FAPs were consistently found to originate, was also identified. Therefore, the FAP flap is a viable and valuable addition to plastic reconstructive techniques. The localisation of point A with precise measurements can facilitate the design and use of such FAP flaps for the reconstruction of nasal, as well as perinasal and perioral defects., (Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Drug therapies currently used for second stage Human African Trypanosomiasis (HAT) exhibit problems with toxicity, difficulty of administration, and resistance linked to the loss of transporter function. Key to the development of new drugs for HAT is a better understanding of the transport properties of candidate compounds. Standard methods for studying transport utilize radio-labelled permeant or HPLC-MS, however the natural fluorescence of many trypanocidal compounds can be exploited. Here we present a fluorescence-based assay for measuring uptake, by trypanosomes, of CPD0801, a drug candidate for second stage HAT. Sample fluorescence is measured in a 96-well format using a benchtop fluorimeter. Our method is directly applicable to the study of other diamidines with similar fluorescent properties and readily adapted for use with other cell types or fluorescent molecules as we demonstrate for the veterinary trypanocide ethidium., (Copyright © 2010 Elsevier B.V. All rights reserved.)

There is increasing evidence that some non-essential substances or environmental stressors can have stimulatory or beneficial effects at low exposure levels while being toxic at higher levels, and that environmental 'priming' of certain physiological processes can result in their improved functioning in later life. These kinds of nonlinear dose-response relationships are referred to as hormetic responses and have been described across a wide range of organisms (from bacteria to vertebrates), in response to exposure to at least 1000 different chemical and environmental stressors. Although most work in this area has been in the fields of toxicology and human health, the concept of hormesis also has general applicability in ecology and evolutionary biology as it provides an important conceptual link between environmental conditions and organism function - both at the time of initial exposure to stressors and later in life. In this review, we discuss and clarify the different ways in which the term hormesis is used and provide a framework that we hope will be useful for ecologists interested in the fitness consequences of exposure to stressors. By using ecologically relevant examples from the existing literature, we show that hormesis is connected with both acclimation and phenotypic plasticity, and may play an important role in allowing animals to adjust to changing environments., (© 2010 Blackwell Publishing Ltd/CNRS.)

Internalisation of the mu opioid receptor from the surface of cells is generally achieved by receptor occupancy with agonist ligands of high efficacy. However, in many situations the potent analgesic morphine fails to promote internalisation effectively and whether there is a direct link between this and the propensity for the sustained use of morphine to result in both tolerance and dependence has been studied intensely. Although frequently described as a partial agonist, this characteristic appears insufficient to explain the poor capacity of morphine to promote internalisation of the mu opioid receptor. Experiments performed using both transfected cell systems and ex vivo/in vivo models have provided evidence that when morphine can promote internalisation of the mu receptor there is a decrease in the development of tolerance and dependence. Although aspects of this model are controversial, such observations suggest a number of approaches to further enhance the use of morphine as an analgesic.

Central to the pathogenesis of many bacterial pathogens is the ability to deliver effector proteins directly into the cells of their eukaryotic host. EspF is one of many effector proteins exclusive to the attaching and effacing pathogen family that includes enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli. Work in recent years has revealed EspF to be one of the most multifunctional effector proteins known, with defined roles in several host cellular processes, including disruption of the epithelial barrier, antiphagocytosis, microvillus effacement, host membrane remodelling, modulation of the cytoskeleton, targeting and disruption of the nucleolus, intermediate filament disruption, cell invasion, mitochondrial dysfunction, apoptosis, and inhibition of several important epithelial transporters. Surprisingly, despite this high number of functions, EspF is a relatively small effector protein, and recent work has begun to decipher the molecular events that underlie its multifunctionality. This review focuses on the activities of EspF within the host cell and discusses recent findings and molecular insights relating to the virulence functions of this fascinating bacterial effector.

Lamina II contains a large number of interneurons involved in modulation and transmission of somatosensory (including nociceptive) information. However, its neuronal circuitry is poorly understood due to the difficulty of identifying functional populations of interneurons. This information is important for understanding nociceptive processing and for identifying changes that underlie chronic pain. In this study, we compared morphology, neurotransmitter content, electrophysiological and pharmacological properties for 61 lamina II neurons recorded in slices from adult rat spinal cord. Morphology was related to transmitter content, since islet cells were GABAergic, while radial and most vertical cells were glutamatergic. However, there was considerable diversity among the remaining cells, some of which could not be classified morphologically. Transmitter phenotype was related to firing pattern, since most (18/22) excitatory cells, but few (2/23) inhibitory cells had delayed, gap or reluctant patterns, which are associated with A-type potassium (I(A)) currents. Somatostatin was identified in axons of 14/24 excitatory neurons. These had variable morphology, but most of those tested showed delayed-firing. Excitatory interneurons are therefore likely to contribute to pain states associated with synaptic plasticity involving I(A) currents. Although noradrenaline and serotonin evoked outward currents in both inhibitory and excitatory cells, somatostatin produced these currents only in inhibitory neurons, suggesting that its pro-nociceptive effects are mediated by disinhibition. Our results demonstrate that certain distinctive populations of inhibitory and excitatory interneuron can be recognised in lamina II. Combining this approach with identification of other neurochemical markers should allow further clarification of neuronal circuitry in the superficial dorsal horn., (Copyright © 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Masquerading animals have evolved striking visual resemblances to inanimate objects. These animals gain protection from their predators not simply by avoiding detection, but by causing their predators to misclassify them as the 'models' that they appear to resemble. Using domestic chicks as predators and twig-mimicking caterpillars as prey, we demonstrated that masquerading prey were more likely to be misclassified as their models when viewed in isolation from their models than when viewed alongside examples of their model, although they benefitted from masquerade to some extent in both conditions. From this, we predict a selection pressure on masqueraders to use microhabitats that reduce the risk of them being viewed simultaneously with examples of their model, and/or to more closely resemble their model in situations where simultaneous viewing is commonplace.