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Colloidal gold nanoparticles (AuNPs) are important nanomaterials for chemical sensing and therapeutics. For their application, it is vital to develop a reliable and robust surface functionalization method that can be applied to diverse functional molecules and offer better stability under harsh biological conditions. Currently, thiol (SH) is the most commonly used functional group for forming stable covalent bonds with AuNPs. However, thiolated molecules typically require complicated preparation procedures, are susceptible to oxidation, and are not compatible with many electrophiles and reducing groups. In this study, we report that surface functionalization of AuNPs can be achieved using alkyne derivatives, which exhibit several advantages over classical thiolation and peptide-bond methods, including straightforward preparation of alkyne derivatives, rapid and simple conjugation in buffers and complex media, higher conjugation efficiency, long-term stability, and resistance to decomposition under harsh conditions. Several alkynylated biotin and fluorescein derivatives are prepared, and the alkynylated-AuNPs are characterized using a lateral flow assay, gel electrophoresis, and spectroscopy techniques to investigate the conjugation efficiencies, size distributions, protein interaction properties, and binding mode of the Au-alkyne bond. We also demonstrate that alkynylated-AuNPs can be used for the sensitive detection of hydrogen peroxide and streptavidin proteins.

The fish gill is a multifunctional organ that is important in multiple physiological processes such as gas transfer, ionoregulation, and chemoreception. This characteristic organ of fishes has received much attention, yet an often-overlooked point is that larval fishes in most cases do not have a fully developed gill, and thus larval gills do not function identically as adult gills. In addition, large changes associated with gas exchange and ionoregulation happen in gills during the larval phase, leading to the oxygen and ionoregulatory hypotheses examining the environmental constraint that resulted in the evolution of gills. This review thus focuses exclusively on the larval fish gill of teleosts, summarizing the development of teleost larval fish gills and its function in gas transfer, ionoregulation, and chemoreception, and comparing and contrasting it to adult gills where applicable, while providing some insight into the oxygen vs ionoregulatory hypotheses debate., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A fundamental requirement for all animals is to sense and respond to changes in environmental O 2 availability. Low O 2 (hypoxia) typically stimulates breathing, a universal and critical response termed the hypoxic ventilatory response (HVR). In this study, we test the hypothesis that taste-signaling pathways are used for O 2 sensing and activation of the HVR. We show that Merkel-like cells (MLCs), which are part of the taste-bud complex, function as O 2 chemoreceptor cells in larval zebrafish and that transduction of the O 2 signal uses taste-signaling pathways. Specifically, MLCs responded to hypoxia in vivo with an increase in Ca 2+ activity that can drive the HVR. In addition, MLCs transmit O 2 signals to afferent cranial nerves IX and X (nIX/X), which project into the area postrema within the hindbrain and synapse with interneurons that are in contact with vagal motor neurons. Hypoxia or chemo-activation of nIX/X caused Ca 2+ activity to increase within the area postrema and elicited hyperventilation. The results provide the first demonstration of an O 2 signaling pathway that commences with the activation of taste receptors (MLCs) to yield a critical physiological reflex, the HVR., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Zebrafish (Danio rerio) are unusual in having two paralogues of the serotonin re-uptake transporter (Sert), slc6a4a (serta) and slc6a4b (sertb), the transporter that serves in serotonin re-uptake from a synapse into the pre-synaptic cell or in serotonin uptake from the extracellular milieu into cells in the peripheral tissues. To address a knowledge gap concerning the specific roles of these paralogues, we used CRISPR/Cas9 technology to generate zebrafish knockout lines predicted to lack functional expression of Serta or Sertb. The consequences of loss-of-function of Serta or Sertb were assessed at the gene expression level, focusing on the serotonergic signalling pathway, and at the behaviour level, focusing on aggression. Whereas serta mRNA was expressed in all tissues examined, with high expression in the heart, gill and brain, only the brain displayed substantial sertb mRNA expression. In both serta -/- and sertb -/- fish, changes in transcript abundances of multiple components of the serotonin signalling pathway were detected, including proteins involved in serotonin synthesis (tph1a, tph1b, tph2, ddc), packaging (vmat2) and degradation (mao), and serotonin receptors (htr1aa, htr1ab). Using a mirror aggression test, serta -/- male but not female fish exhibited greater aggression than wildtype fish. However, both male and female sertb -/- fish displayed less aggression than their wildtype counterparts. These differences in behaviour between serta -/- and sertb -/- individuals hold promise for increasing our understanding of the neurophysiological basis of aggression in zebrafish., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Sarcopenia is characterized by the loss of muscle mass and function. It is well known that sarcopenia is often associated with aging, while in recent years, sarcopenia comorbid with chronic diseases such as cirrhosis has attracted widespread attention, whose underlying molecular mechanisms remain unclear. Since cirrhosis and sarcopenia are assumed to be closely interrelated in terms of pathogenesis, this review innovatively discussed the role of epigenetic modifications and microecological dysregulation in sarcopenia in the context of liver cirrhosis. Here we illustrated the relationship between sarcopenia and cirrhosis in the aspect of epigenetics, dysbiosis, and the crosstalk between gene modifications and intestinal microecology. Furthermore, the alterations in cirrhosis patients with sarcopenia, such as inflammatory response and oxidative stress, are found to present synergistic effects in the pathways of epigenetics and dysbiosis leading to sarcopenia. This review proposes that microbiome-based therapies are promising to break the vicious cycle between epigenetic modification and dysbiosis, providing strong support for the use of intestinal microecological interventions to prevent sarcopenia in cirrhotic patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xu, Pan, Zhang, Dai and Xu.)

The study of breathing in fishes has featured prominently in Journal of Experimental Biology (JEB), particularly during the latter half of the past century. Indeed, many of the seminal discoveries in this important sub-field of comparative respiratory physiology were reported first in JEB. The period spanning 1960-1990 (the 'golden age of comparative respiratory physiology') witnessed intense innovation in the development of methods to study the control of breathing. Many of the guiding principles of piscine ventilatory control originated during this period, including our understanding of the dominance of O2 as the driver of ventilation in fish. However, a critical issue - the identity of the peripheral O2 chemoreceptors - remained unanswered until methods for cell isolation, culture and patch-clamp recording established that gill neuroepithelial cells (NECs) respond to hypoxia in vitro. Yet, the role of the NECs and other putative peripheral or central chemoreceptors in the control of ventilation in vivo remains poorly understood. Further progress will be driven by the implementation of genetic tools, most of which can be used in zebrafish (Danio rerio). These tools include CRISPR/Cas9 for selective gene knockout, and Tol2 systems for transgenesis, the latter of which enables optogenetic stimulation of cellular pathways, cellular ablation and in vivo cell-specific biosensing. Using these methods, the next period of discovery will see the identification of the peripheral sensory pathways that initiate ventilatory responses, and will elucidate the nature of their integration within the central nervous system and their link to the efferent motor neurons that control breathing., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Adjustments of ventilation in fishes to regulate the volume of water flowing over the gills are critically important responses to match branchial gas transfer with metabolic needs and to defend homeostasis during environmental fluctuations in O 2 and/or CO 2 levels. In this focused review, we discuss the control and consequences of ventilatory adjustments in fish, briefly summarizing ventilatory responses to hypoxia and hypercapnia before describing the current state of knowledge of the chemoreceptor cells and molecular mechanisms involved in sensing O 2 and CO 2 . We emphasize, where possible, insights gained from studies on early developmental stages. In particular, zebrafish ( Danio rerio ) larvae have emerged as an important model for investigating the molecular mechanisms of O 2 and CO 2 chemosensing as well as the central integration of chemosensory information. Their value stems, in part, from their amenability to genetic manipulation, which enables the creation of loss-of-function mutants, optogenetic manipulation, and the production of transgenic fish with specific genes linked to fluorescent reporters or biosensors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Perry, Pan and Gilmour.)

The sensing of environmental fluctuations and initiation of appropriate physiological responses is crucial to homeostasis. Neuroepithelial cells (NECs) in fishes are putative chemoreceptors, resembling mammalian Type I (glomus) cells, that respond in vitro to changes in O 2 , CO 2 , NH 3 , and pH. Cytosolic carbonic anhydrase (Ca17a) is thought to be involved in CO 2 sensing owing to its presence in NECs. Zebrafish ( Danio rerio ) lacking functional Ca17a were generated via CRISPR/Cas9 technology and used to assess the role of Ca17a in initiating the cardiorespiratory responses to elevated CO 2 (hypercapnia). Unfortunately, the homozygous knockout mutants ( ca17a -/- ) did not survive more than ∼12-14 days postfertilization (dpf), restricting experiments to early developmental stages (4-8 dpf). Changes in ventilation ( f V ) and cardiac ( f H ) frequency in response to hypercapnia (1% CO 2 ) in wild-type ( ca17a +/+ ), heterozygous ( ca17a +/- ) and ca17a -/- fish were used to investigate Ca17a-dependent CO 2 sensing and downstream signaling. Wild-type fish exhibited hyperventilation during hypercapnia as indicated by an increase in f V . In the ca17a -/- fish, the hyperventilatory response was attenuated markedly but only at 8 dpf. Hypercapnic tachycardia was observed for all genotypes and did not appear to be influenced by the absence of Ca17a. Interestingly, ca17a -/- fish exhibited a significantly lower resting f H that became more pronounced as the fish aged. The decrease in resting f H was prevented ("rescued") when ca17a -/- embryos were injected with ca17a mRNA. Collectively, the results of this study support a role for Ca17a in promoting hyperventilation during hypercapnia in larval zebrafish and suggest a previously unrecognized role for Ca17a in determining resting heart rate.

Aim: Although zebrafish are gaining popularity as biomedical models of cardiovascular disease, our understanding of their cardiac control mechanisms is fragmentary. Our goal was to clarify the controversial role of the ß1-adrenergic receptor (AR) in the regulation of heart rate in zebrafish., Methods: CRISPR-Cas9 was used to delete the adrb1 gene in zebrafish allowing us to generate a stable adrb1 -/- line. Larval heart rates were measured during pharmacological protocols and with exposure to hypercapnia. Expression of the five zebrafish adrb genes were measured in larval zebrafish hearts using qPCR., Results: Compared with genetically matched wild-types (adrb1 +/+ ), adrb1 -/- larvae exhibited ~20 beats min -1 lower heart rate, measured from 2 to 21 days post-fertilization (dpf). Nevertheless, adrb1 -/- larvae exhibited preserved positive chronotropic responses to pharmacological treatment with AR agonists (adrenaline, noradrenaline, isoproterenol), which were blocked by propranolol (general ß-AR antagonist). Regardless of genotype, larvae exhibited similar increases in heart rate in response to hypercapnia (1% CO 2 ) at 5 dpf, but tachycardia was blunted in adrb1 -/- larvae at 6 dpf. adrb1 gene expression was abolished in the hearts of adrb1 -/- larvae, confirming successful knockout. While gene expression of adrb2a and adrb3a was unchanged, adrb2b and adrb3b mRNA levels increased in adrb1 -/- larval hearts., Conclusion: Despite adrb1 contributing to the setting of resting heart rate in larvae, it is not strictly essential for zebrafish, as we generated a viable and breeding adrb1 -/- line. The chronotropic effects of adrenergic stimulation persist in adrb1 -/- zebrafish, likely due to the upregulation of other ß-AR subtypes., (© 2022 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Hypoxia-inducible factor 1-α (Hif-1α), an important transcription factor regulating cellular responses to reductions in O 2 , previously was shown to improve hypoxia tolerance in zebrafish ( Danio rerio ). Here, we examined the contribution of Hif-1α to hypoxic survival, focusing on the benefit of aquatic surface respiration (ASR). Wild-type and Hif-1α knockout lines of adult zebrafish were exposed to two levels (moderate or severe) of intermittent hypoxia. Survival was significantly compromised in Hif-1α knockout zebrafish prevented from accessing the surface during severe (16 mmHg) but not moderate (23 mmHg) hypoxia. When allowed access to the surface in severe hypoxia, survival times did not differ between wild-type and Hif-1α knockouts. Performing ASR mitigated the negative effects of the loss of Hif-1α with the knockouts initiating ASR at a higher P O 2 threshold and performing ASR for longer than wild-types. The loss of Hif-1α had little impact on survival in fish between 1 and 5 days post-fertilization, but as the larvae aged, their reliance on Hif-1α increased. Similar to adult fish, ASR compensated for the loss of Hif-1α on survival. Together, these results demonstrate that age, hypoxia severity and, in particular, the ability to perform ASR significantly modulate the impact of Hif-1α on survival in hypoxic zebrafish.

Purpose: It is difficult to distinguish cystic biliary atresia (CBA) from choledochal cyst (CC) before intraoperative cholangiography in neonates or young infants because of the similar ultrasonographic patterns and clinical manifestations. This study is to investigate the difference of clinical parameters between CBA and CC., Methods: 96 patients with cyst at hepatic hilum whose ages were less than 120 days during the period from Jan'2013 to Nov'2015 were retrospectively studied, they were divided into CBA group and CC group by intraoperative cholangiography, there were 29 cases of CBA and 67 cases of CC. It was compared and analyzed on laboratory data, preoperative ultrasonographic features, intraoperative cholangiography and histopathological results between the two groups. Data were quoted as means ± standard deviation (SD) or median (range), Student's t test, non-parametric test and χ2 test were used as appropriate. P < 0.05 was considered as significant statistical difference., Results: CBA group and CC group were comparable for the operative age (51.76 ± 23.99 days vs. 50.03 ± 26.38 days, P = 0.76), weight (4.60 ± 0.75 kg vs. 4.44 ± 1.03 kg, P = 0.46) and sex proportion (M/F: 9/20 vs. 20/47, P = 0.91). there were significant differences (CBA vs. CC) in ALT [59(3-375) vs. 30 (6-247) IU/L, P < 0.001], AST [140(30-694) vs. 44(18-410) IU/L, P < 0.001], T-Bil [190 (107-326) vs.87(5-310) μmol/L, P < 0.001], D-Bil [85 (31-174) vs.14(1-122) μmol/L, P < 0.001] and TBA [112 (23-269) vs.9(1-337) μmol/L, P < 0.001]. There was insignificant statistical difference in GGT [332(112-2154) vs. 226(21-1810) IU/L, P = 0.099] between CBA group and CC group. The cyst size in CBA group was obviously smaller than CC group (the maximum longitudinal diameter: 2.52 ± 1.16 cm vs. 4.71 ± 2.23 cm, P < 0.001; the maximum transverse diameter: 1.64 ± 0.75 cm vs. 3.41 ± 1.79 cm, P < 0.001). Significant differences presented in preoperative ultrasonographic features and histopathological results between the two groups., Conclusion: CBA should be suspected if ultrasonography indicates small cyst with characteristic manifestations of BA and elevated laboratory data. GGT was an insensitive indicator in distinguishing CBA from CC. Cholangiography should be done for the suspected CBA patients as soon as possible to confirm the diagnosis and have surgical intervention timely., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: With the advent of ultrasonography (US), perinatal diagnosis of biliary cystic malformation (BCM) has become more prevalent. BCM includes cystic biliary atresia (CBA) and choledochal cyst (CC). Both share similar ultrasonographic features and clinical manifestations, and yet the postnatal management is very different. This study was to assess whether CBA can be distinguished from CC by US., Methods: We retrospectively studied the clinical data of 98 BCM patients aged less than 130 days between January 2013 and November 2015. The patients were divided into the CBA group and the CC group based on intraoperative cholangiography. The ultrasonographic features in each group were then analyzed., Results: Thirty-one children had CBA (type I: 24, type III: 7) and 67 children had CC (all: type I). The cyst volume did not show a significant change in the CBA group while the cyst volume increased postnatally in the CC group. Preoperative cyst volume can be used as a sensitive indicator to distinguish choledochal cyst from cystic biliary atresia. Gallbladder size in the two groups differed significantly (P = 0.034); 23 of 31 patients in the CBA group had atrophic gallbladder, whereas none of the patients in the CC group had atrophic gallbladder (P < 0.001). The triangular cord sign was detected in ten patients in the CBA group and in none of the patients in the CC group (P < 0.001)., Conclusion: Serial prenatal and postnatal ultrasonographic studies may help differentiate CBA from CC in the majority of BCM, facilitating prenatal counseling and postnatal management.

Background: This study aimed to evaluate the effects of different iterative reconstruction (IR) algorithms on coronary artery calcium (CAC) score quantification using the reduced radiation dose (RRD) protocol in an anthropomorphic phantom and in patients., Methods: A thorax phantom, containing 9 calcification inserts with varying hydroxyapatite (HA) densities, was scanned with the reference protocol [120 kv, 80 mAs, filtered back projection (FBP)] and RRD protocol (120 kV, 20-80 mAs, 5 mAs interval) using a 256-slice computed tomography (CT) scanner. Raw data were reconstructed with different reconstruction algorithms [iDose 4 levels 1-7 and iterative model reconstruction (IMR) levels 1-3]. Signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and Agatston score (AS) were calculated for each image series. The correction factor was derived from linear regression analysis between the reference image series and other image series with different parameters. Additionally, 40 patients were scanned with the RRD protocol (50 mAs) and reconstructed with FBP, iDose 4 level 4, and IMR level 2. AS was calculated for the 3-group image series, and was corrected by applying a correction factor for the IMR group. The agreement of risk stratification with different reconstruction algorithms was also analyzed., Results: For the phantom study, the iDose 4 and IMR groups had significantly higher SNR and CNR than the FBP group (all P<0.05). There were no significant differences in the total AS after comparing image series reconstructed with iDose 4 (level 1-7) and FBP (all P>0.05), while AS from the IMR (level 1-3) image series were lower than the FBP group (all P<0.05). The tube current of 50 mAs was determined for the clinical study, and the correction factor was 1.14. For the clinical study, the median AS from the iDose 4 and IMR groups were both significantly lower compared to the FBP image series [(112.89 (63.01, 314.09), 113.22 (64.78, 364.95) vs . 118.59 (65.05, 374.48), both P<0.05]. After applying the correction factor, the adjusted AS from the IMR group was not significantly different from that of the FBP group [126.48 (69.62, 355.85) vs . 118.59 (65.05, 374.48), P=0.145]. Moreover, the agreement in risk stratification between FBP and IMR improved from 0.81 to 0.85., Conclusions: The RRD CAC scoring scan using the IMR reconstruction algorithm is clinically feasible, and a correction factor can help reduce the AS underestimation effect., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/qims-20-437). The authors have no conflicts of interest to declare., (2021 Quantitative Imaging in Medicine and Surgery. All rights reserved.)

Serotonergic neuroepithelial cells (NECs) in larval zebrafish are believed to be O 2 chemoreceptors. Serotonin (5-HT) within these NECs has been implicated as a neurotransmitter mediating the hypoxic ventilatory response (HVR). Here, we use knockout approaches to discern the role of 5-HT in regulating the HVR by targeting the rate limiting enzyme for 5-HT synthesis, tryptophan hydroxylase (Tph). Using transgenic lines, we determined that Tph1a is expressed in skin and pharyngeal arch NECs, as well as in pharyngeal arch Merkel-like cells (MLCs), whereas Tph1b is expressed predominately in MLCs. Knocking out the two tph1 paralogs resulted in similar changes in detectable serotonergic cell density between the two mutants, yet their responses to hypoxia (35 mmHg) were different. Larvae lacking Tph1a (tph1a -/- mutants) displayed a higher ventilation rate when exposed to hypoxia compared to wild-types, whereas tph1b -/- mutants exhibited a lower ventilation rate suggesting that 5-HT located in locations other than NECs, may play a dominant role in regulating the HVR., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In fishes, branchial cytosolic carbonic anhydrase (CA) plays an important role in ion and acid-base regulation. The Ca17a isoform in zebrafish ( Danio rerio ) is expressed abundantly in Na + -absorbing/H + -secreting H + -ATPase-rich (HR) cells. The present study aimed to identify the role of Ca17a in ion and acid-base regulation across life stages using CRISPR/Cas9 gene editing. However, in preliminary experiments, we established that ca17a knockout is lethal with ca17a -/- mutants exhibiting a significant decrease in survival beginning at ∼12 days postfertilization (dpf) and with no individuals surviving past 19 dpf. Based on these findings, we hypothesized that ca17a -/- mutants would display alterations in ion and acid-base balance and that these physiological disturbances might underlie their early demise. Na + uptake rates were significantly increased by up to 300% in homozygous mutants compared with wild-type individuals at 4 and 9 dpf; however, whole body Na + content remained constant. While Cl - uptake was significantly reduced in ca17a -/- mutants, Cl - content was unaffected. Reduction of CA activity by Ca17a morpholino knockdown or ethoxzolamide treatments similarly reduced Cl - uptake, implicating Ca17a in the mechanism of Cl - uptake by larval zebrafish. H + secretion, O 2 consumption, CO 2 excretion, and ammonia excretion were generally unaltered in ca17a -/- mutants. In conclusion, while the loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-dependent Cl - uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.

Beginning with the discovery more than 35 years ago that oxygen chemoreceptors of the fish gill are enriched with serotonin, numerous studies have examined the importance of this, and other neuroendocrine factors in piscine chemoreceptor function, and in particular on the chemoreceptor-mediated reflex control of breathing. However, despite these studies, there is continued debate as to the role of neuroendocrine factors in the initiation or modulation of breathing during environmental disturbances or physical activity. In this review, we summarize the state-of-knowledge surrounding the neuroendocrine control of oxygen chemoreception in fish and the associated reflex adjustments to ventilation. We focus on neurohumoral substances that either are present in chemosensory cells or those that are localised elsewhere but have also been implicated in the direct control of breathing. These substances include serotonin, catecholamines (adrenaline and noradrenaline), acetylcholine, purines and gaseous neurotransmitters. Despite the growing indirect evidence for an involvement of these neuroendocrine factors in chemoreception and ventilatory control, direct evidence awaits the incorporation of novel methods currently under development., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Fish increase ventilation during hypoxia, a reflex termed the hypoxic ventilatory response (HVR). The HVR is an effective mechanism to increase O 2 uptake, but at a high metabolic cost. Therefore, when hypoxia becomes severe enough, ventilation declines, as its benefit is diminished. The water oxygen partial pressure ( P w O 2 ) at which this decline occurs is expected to be near the critical P w O 2  ( P crit ), the P w O 2  at which O 2 consumption begins to decline. Our results indicate that in zebrafish ( Danio rerio ), the relationship between peak HVR and P crit is dependent on developmental stage. Peak ventilation occurred at P w O 2  values higher than P crit in larvae, but at a P w O 2  significantly lower than P crit in adults. Larval zebrafish use cutaneous respiration to a greater extent than branchial respiration and the cost of sustaining the HVR may outweigh the benefit, whereas adult zebrafish, which rely on branchial respiration, may benefit from using HVR at P w O 2  below P crit ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Weightlessness-induced cardiovascular dysfunction can lead to physiological and pathological consequences. It has been shown that spaceflight or simulated microgravity can alter expression profiles of some microRNAs (miRNAs). Here, we attempt to identify the role of miRNAs in human umbilical vein endothelial cells (HUVECs) apoptosis under simulated microgravity. RNA-sequencing and quantitative real-time PCR (qRT-PCR) assays were used to identify differentially expressed miRNAs in HUVECs under simulated microgravity. Then we obtained the target genes of these miRNAs through target analysis software. Moreover, GO and KEGG enrichment analysis were performed. The effects of these miRNAs on HUVECs apoptosis were evaluated by flow cytometry, Western blot and Hoechst staining. Furthermore, we obtained the target gene of miR-27b-5p by luciferase assay, qRT-PCR and Western blot. Finally, we investigated the relationship between this target gene and miR-27b-5p in HUVECs apoptosis under normal gravity or simulated microgravity. We found 29 differentially expressed miRNAs in HUVECs under simulated microgravity. Of them, the expressions of 3 miRNAs were validated by qRT-PCR. We demonstrated that miR-27b-5p affected HUVECs apoptosis by inhibiting zinc fingers and homeoboxes 1 (ZHX1). Our results reported here demonstrate for the first time that simulated microgravity can alter the expression of some miRNAs in HUVECs and miR-27b-5p may protect HUVECs from apoptosis under simulated microgravity by targeting ZHX1.

Freshwater (FW) fishes inhabit dilute environments and must actively absorb ions in order to counteract diffusive salt loss. Neuroendocrine control of ion uptake in FW fishes is an important feature of ion homeostasis and several important neuroendocrine factors have been identified. The role of serotonin (5-HT), however, has received less attention despite several studies pointing to a role for 5-HT in the control of ion balance. Here, we used a gene knockout approach to elucidate the role of 5-HT in regulating Na + and Ca 2+ uptake rates in larval zebrafish. Tryptophan hydroxylase (TPH) is the rate-limiting step in 5-HT synthesis and we therefore hypothesized that ion uptake rates would be altered in zebrafish larvae carrying knockout mutations in tph genes. We first examined the effect of tph1b knockout (KO) and found that tph1bKO larvae, obtained from Harvard University, had reduced rates of Na + and Ca 2+ uptake compared to wild-type (WT) larvae from our institution (uOttawa WT), lending support to our hypothesis. However, further experiments controlling for differences in genetic background demonstrated that WT larvae from Harvard University (Harvard WT) had lower ion uptake rates than those of uOttawa WT, and that ion uptake rate between Harvard WT and tph1bKO larvae were not significantly different. Therefore, our initial observation that tph1bKO larvae (Harvard source) had reduced ion uptake rates relative to uOttawa WT was a function of genetic background and not of knockout itself. These data provide a cautionary tale of the importance of controlling for genetic background in gene knockout experiments., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Astronauts exposed to a gravity-free environment experience cardiovascular deconditioning that causes post-spaceflight orthostatic intolerance and other pathological conditions. Endothelial dysfunction is an important factor responsible for this alteration. Our previous study showed enhanced autophagy in endothelial cells under simulated microgravity. The present study explored the cytoprotective role of autophagy under microgravity in human umbilical vein endothelial cells (HUVECs). We found that clinorotation for 48 h induced apoptosis and endoplasmic reticulum (ER) stress in HUVECs. ER stress and the unfolded protein response (UPR) partially contributed to apoptosis under clinorotation. Autophagy partially reduced ER stress and restored UPR signaling by autophagic clearance of ubiquitin-protein aggregates, thereby reducing apoptosis. In addition, the ER stress antagonist 4-phenylbutyric acid upregulated autophagy in HUVECs. Taken together, these findings indicate that autophagy plays a protective role against apoptosis under clinorotation by clearing protein aggregates and partially restoring the UPR.

In water-breathing fishes, the hypoxic ventilatory response (HVR) represents an increase in water flow over the gills during exposure to lowered ambient O 2 levels. The HVR is a critical defense mechanism that serves to delay the negative consequences of hypoxia on aerobic respiration. However, the physiological significance of the HVR in larval fishes is unclear as they do not have a fully developed gill and rely primarily on cutaneous gas transfer. Using larval zebrafish (4, 7, 10 and 15 days post-fertilization; dpf), we examined HVR under three levels of hypoxia (25, 45 and 60 mmHg). The larvae exhibited widely different HVRs as a function of developmental age and level of the hypoxia. Yet, critical O 2 tensions ( P crit ) remained constant (30-34 mmHg) over the same period of development. Micro-optrode O 2 sensors were used to measure a significant decrease in buccal cavity water O 2 tensions in 4 and 7 dpf larvae compared with the water they inspired, demonstrating significant extraction of O 2 from the buccal cavity. To assess the physiological significance of the HVR, ventilatory water flow was prevented in larvae at 4 and 7 dpf by embedding their heads in agar. An increase in P crit was observed in larvae at 7 dpf but not 4 dpf, suggesting that buccal ventilation is important for O 2 extraction by 7 dpf. Combined, these data indicate that branchial/buccal gas transfer plays a significant role in O 2 uptake during hypoxia, and supports a physiological benefit of the HVR in early life stages of zebrafish., (© 2019. Published by The Company of Biologists Ltd.)

Loss-of-function technologies, such as morpholino- and RNAi-mediated gene knockdown, and TALEN- and CRISPR/Cas9-mediated gene knockout, are widely used to investigate gene function and its physiological significance. Here, we provide a general overview of the various knockdown and knockout technologies commonly used in comparative physiology and discuss the merits and drawbacks of these technologies with a particular focus on research conducted in zebrafish. Despite their widespread use, there is an ongoing debate surrounding the use of knockdown versus knockout approaches and their potential off-target effects. This debate is primarily fueled by the observations that, in some studies, knockout mutants exhibit phenotypes different from those observed in response to knockdown using morpholinos or RNAi. We discuss the current debate and focus on the discrepancies between knockdown and knockout phenotypes, providing literature and primary data to show that the different phenotypes are not necessarily a direct result of the off-target effects of the knockdown agents used. Nevertheless, given the recent evidence of some knockdown phenotypes being recapitulated in knockout mutants lacking the morpholino or RNAi target, we stress that results of knockdown experiments need to be interpreted with caution. We ultimately argue that knockdown experiments should not be discontinued if proper control experiments are performed, and that with careful interpretation, knockdown approaches remain useful to complement the limitations of knockout studies (e.g. lethality of knockout and compensatory responses)., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The Gulf of Mexico was home to the Deepwater Horizon oil spill, and is also known to exhibit seasonal declines in oxygen availability. Oil exposure in fish is known to impact oxygen uptake through cardiac impairment, which raises questions about the additive effects of these two stressors. Here we explore this question on the Atlantic croaker using two measures of hypoxia tolerance: critical oxygen threshold (P crit ), and time to loss of equilibrium (LOE). We first demonstrated that 24 h exposure to 10.1 and 23.2 μg l -1 ΣPAH 50 significantly impaired oxygen uptake. There was no effect of exposure on P crit or LOE. Exposure did result in significantly different repeatability between pre- and post-exposure P crit , suggesting that hypoxia tolerant individual may see greater impacts following exposure. These results suggest oil exposure does not have wide scale detrimental outcomes for hypoxia tolerance in fish, yet there may be fine scale impairments of ecological significance., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Individuals exposed to long-term spaceflight often experience cardiovascular dysfunctions characterized by orthostatic intolerance, disability on physical exercise, and even frank syncope. Recent studies have showed that the alterations of cardiovascular system are closely related to the functional changes of endothelial cells. We have shown previously that autophagy can be induced by simulated microgravity in human umbilical vein endothelial cells (HUVECs). However, the mechanism of enhanced autophagy induced by simulated microgravity and its role in the regulation of endothelial function still remain unclear. We report here that 48 h clinorotation promoted cell migration in HUVECs by induction of autophagy. Furthermore, clinorotation enhanced autophagy by the mechanism of human murine double minute 2 (HDM2)-dependent degradation of cytoplasmic p53 at 26S proteasome, which results in the suppression of mechanistic target of rapamycin (mTOR), but not via activation of AMPK in HUVECs. These results support the key role of HDM2-p53 in direct downregulation of mTOR, but not through AMPK in microgravity-induced autophagy in HUVECs.

Hemoglobin (Hb) multiplicity is common in fish, yet despite its ubiquitous nature, the functional significance is unclear. Here we explore the hypothesis that Hb multiplicity plays a role in hypoxia tolerance using the red drum (Sciaenops ocellatus). Red drum is an economically and ecologically important species native to coastal regions and estuaries of the Gulf of Mexico - habitats that routinely experience pronounced hypoxic events. Using a transcriptomic approach, we demonstrate that red drum red blood cells express 7 and 5 Hbα and Hbβ isoforms, respectively. Phylogenetic analysis grouped these isoforms into distinct isoHb clades, and provided evidence of lineage specific expression of particular isoHbs. In normoxia, three isoHbs predominated (Hbα-3.1, -3.2, and Hbβ-3.1). A three-week hypoxia acclimation (48 mmHg) resulted in significant up-regulation of Hbα-2, Hbα-3.2, and Hbβ-3.1, effectively switching the predominantly expressed isoforms. Changes in subunit expression were correlated with a decrease in non-stripped hemolysate P 50 . Similarly, hypoxia acclimation resulted in a 20% reduction in whole animal critical oxygen threshold (P crit ). Hypoxia acclimation was not associated with changes in gill morphology, hematocrit, or relative ventricular mass. Overall, these data provide support for the hypothesis that Hb isoform switching can provide a physiological benefit to counteract environmental stress in fishes.

Mass-specific oxygen consumption rate, i.e. standard metabolic rate (Rs ) and critical oxygen tension (Pcrit ) of red drum Sciaenops ocellatus were measured and scaled over a 2500-fold range in mass (MF ; 0·26-686 g). Rs conformed to well established models (Rs  = 3·73·91 MF (-0·21) ; r(2)  = 0·86) while Pcrit increased over the size range (Pcrit  = 3·15 log10 MF  + 16·19; r(2)  = 0·44). This relationship may be ecologically advantageous as it would allow smaller S. ocellatus to better utilize hypoxic zones as habitat and refuge from predators., (© 2016 The Fisheries Society of the British Isles.)

Corrosion and biological properties of microarc oxidized calcium phosphate (CaP) coatings on Mg-Zn-Zr alloy were improved by optimizing negative power density parameters. Scanning electron microscope (SEM) and X-ray diffractometer (XRD) were employed to characterize the coating morphology and phase composition. The in vitro cytotoxicity and systemic toxicity tests were carried out to evaluate the coating biocompatibility. The degradability and bioactivity of the coatings were determined by in vitro simulated body fluid (SBF) immersion test. The coating microstructure, thickness and growth rate can be influenced by negative power density through changing direction of ions movements, rate of ions exchanges and affecting formation of plasma. The CaP coatings reduced the substrate degradation rate. Calcium phosphates, such as hydroxyapatite (Ca10(PO4)6(OH)2, HA) and calcium pyrophosphate (Ca2P2O7, CPP), etc., were induced after 30 days SBF immersion, indicating that the coatings have bioactivity. The CaP coatings have no toxicity to cell and living mice, indicating that the coatings are safe to serve as implants., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Calcium phosphate (CaP) coatings were prepared on Mg-Zn-Zr magnesium alloy by micro-arc oxidation (MAO) in electrolyte containing calcium acetate monohydrate (CH3COO)2Ca·H2O) and different phosphates (i.e. disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), sodium phosphate (Na3PO4·H2O) and sodium hexametaphosphate((NaPO3)6)). Scanning electron microscope (SEM), energy-dispersive X-ray spectrometry (EDS) and X-ray diffractometer (XRD) were employed to characterize the microstructure, elemental distribution and phase composition of the CaP coatings. Simulated body fluid (SBF) immersion test was used to evaluate the coating bioactivity and degradability. Systemic toxicity test was used to evaluate the coating biocompatibility. Fluoride ion selective electrode (ISE) was used to measure F(-) ions concentration during 30 days SBF immersion. The CaP coatings effectively reduced the corrosion rate and the surfaces of CaP coatings were covered by a new layer formed of numerous needle-like and scale-like apatites. The formation of these calcium phosphate apatites indicates that the coatings have excellent bioactivity. The coatings formed in (NaPO3)6-containging electrolyte exhibit thicker thickness, higher adhesive strength, slower degradation rate, better apatite-inducing ability and biocompatibility., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Ceramic coatings were prepared on ZK60 magnesium alloy in electrolyte with different concentration ratio of calcium and phosphorus (Ca/P) by micro-arc oxidation (MAO) technique at constant voltage. The microstructure, phase composition, elemental distribution, corrosion resistance, and adhesion of the coatings were investigated by scanning electron microscope (SEM), X-ray diffractometer (XRD), energy-dispersive X-ray spectrometry (EDS), electrochemical workstation, and scratch spectrometer, respectively. The coating biocompatibility was evaluated by in vitro cytotoxicity tests and systemic toxicity tests, and the bioactivity and degradability were evaluated by simulation body fluid (SBF) immersion tests. SEM shows that pores with different shapes distribute all over the coating surface. The adhesion and thickness of the coatings increases with increasing Ca/P ratio of electrolyte. The in vitro cytotoxicity tests and systemic toxicity texts demonstrate that the coatings have no toxicity to cell and living animal, which show that the coatings have excellent biocompatibility. XRD analysis shows that bioactive calciumphosphate (CaP) phases such as hydroxyapatite (HA, Ca(10)(PO(4))(6)(OH)(2)) and calcium pyrophosphate (CPP, Ca(2)P(2)O(7)) are induced in the immersed coatings, indicating that the MAO coatings have excellent bioactivity., (Copyright © 2012 Wiley Periodicals, Inc.)

Objective: To investigate the therapeutical effect and side-effect of docetaxel combined with cisplatin (DDP) on the treatment of local advanced esophageal cancer with concomitant radiation therapy., Methods: Ninety patients with LOCAL advanced esophageal squamous cell carcinoma were divided into two groups: (DDP + 5-Fu) group and (docetaxel + DDP) group. Chemotherapy was carried out every 4 weeks for a total of 4 courses. The radiation dose was 50.4 Gy/28FX., Results: The median survival time of patients in the (DDP + 5-Fu) group was 16 months and that in (docetaxel + DDP) group was 21 months (P = 0.0278). The 3-year survival rate in the (docetaxel + DDP) group was obviously higher than that in the (DDP + 5-Fu) group (23.9% vs. 12.1%). The ORR in (docetaxel + DDP) group (84.5%) was significantly higher than that in the (DDP + 5-Fu) group (71.1%) (P = 0.025). No significant differences were observed in the incidence of side-effects in the two groups., Conclusions: The conventional dose chemotherapy of docetaxel + DDP with concomitant radiation therapy showed a better partial remission rate and long-term survival rate for the treatment of local advanced esophageal cancer than the traditional chemotherapy (DDP + 5-Fu) with concomitant radiation therapy and the side-effects are not increased.

PML nuclear body (PML NB) is an important macromolecular nuclear structure that is involved in many essential aspects of cellular function. Tens of proteins have been found in PML NBs, and promyelocytic leukemia protein (PML) has been proven to be essential for the formation of this structure. Here, we showed that TRAF and TNF receptor-associated protein (TTRAP) was a novel PML NBs-associated protein. TTRAP colocalized with three important PML NBs-associated proteins, PML, DAXX and Sp100 in the typical fashion of PML NBs. By yeast mating assay, TTRAP was identified to interact with these PML NBs-associated proteins. The transcription and expression of TTRAP could be induced by IFN-gamma, representing another common feature of PML NBs-associated proteins. These results would not only be important for understanding PML NBs but also be helpful in studying the TTRAP function in the future.