Methylmercury-induced hair cell loss requires hydrogen peroxide production and leukocytes in zebrafish embryos.

Hearing impairment and deafness is frequently observed as one of the neurological signs in patients with Minamata disease caused by methylmercury (MeHg) poisoning. Loss of hair cells in humans and animals is a consequence of MeHg poisoning. However, it is still not clear how MeHg causes hearing deficits. We employed the hair cells of the lateral line system of zebrafish embryos as a model to explore this question. We exposed transgenic zebrafish embryos to MeHg (30-360 µg/L) at the different stages, and scored the numbers of hair cells. We find that MeHg-induced reduction of hair cells is in a concentration dependent manner. By employing antisense morpholino against to pu.1, we confirm that loss of hair cells involves the action of leukocytes. Moreover, hair cell loss is attenuated by co-treating MeHg-exposed embryos with pharmacological inhibitors of NADPH oxidases named diphenyleneiodonium (DPI) and VAS2870. In situ gene expression analysis showed that genes encoding the SQSTM1-Keap1-Nrf2 systems involved in combating oxidative stress and immune responses are highly expressed in the lateral line organs of embryos exposed to MeHg. This suggests that induction of hydrogen peroxide (H2O2) is the primary effect of MeHg on the hair cells. Genes induced by MeHg are also involved in regeneration of the hair cells. These features are likely related to the capacity of the zebrafish to regenerate the lost hair cells.

Membrane fusion and drug delivery with carbon nanotube porins.

Drug delivery mitigates toxic side effects and poor pharmacokinetics of life-saving therapeutics and enhances treatment efficacy. However, direct cytoplasmic delivery of drugs and vaccines into cells has remained out of reach. We find that liposomes studded with 0.8-nm-wide carbon nanotube porins (CNTPs) function as efficient vehicles for direct cytoplasmic drug delivery by facilitating fusion of lipid membranes and complete mixing of the membrane material and vesicle interior content. Fusion kinetics data and coarse-grained molecular dynamics simulations reveal an unusual mechanism where CNTP dimers tether the vesicles, pull the membranes into proximity, and then fuse their outer and inner leaflets. Liposomes containing CNTPs in their membranes and loaded with an anticancer drug, doxorubicin, were effective in delivering the drug to cancer cells, killing up to 90% of them. Our results open an avenue for designing efficient drug delivery carriers compatible with a wide range of therapeutics.

Bilateral ultrasound-guided thoracic erector spinae plane blocks using a programmed intermittent bolus improve opioid-sparing postoperative analgesia in pediatric patients after open cardiac surgery: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Postoperative pain after pediatric cardiac surgery is usually treated with intravenous opioids. Recently, the focus has been on postoperative regional analgesia with the introduction of ultrasound-guided erector spinae plane blocks (ESPBs). We hypothesized that bilateral ESPB with a programmed intermittent bolus (PIB) regimen decreases postoperative morphine consumption at 48 hours and improves analgesia in children who undergo cardiac surgery. METHODS: This randomized, double-blind, placebo-controlled study comprised 50 children who underwent cardiac surgery through midline sternotomy. The patients were allocated randomly into two groups: ultrasound-guided bilateral ESPB at the level of T3-T4 transverse process then PIB with saline infusion (group 1, n=23) or PIB with 0.2% ropivacaine (group 2, n=27). Intravenous morphine at 30 µg/kg/hour was used as rescue analgesia. Postoperative pain was assessed using the COMFORT-B score for extubation, drain removal, and mobilization, and the FLACC (Face, Legs, Activity, Cry, Consolability) scale at 0, 2, 4, 6, 8, 12, 16, 20, 24, 36, and 48 hours after surgery. Adverse events were noted. RESULTS: The total dose of morphine in 48 hours was significantly decreased in patients receiving a bilateral ESPB with ropivacaine (120±320 µg/kg) compared with patients with saline infusion (512±560 µg/kg; p=0.03). Fourteen per cent of patients required rescue analgesia with morphine in group 2 compared with 41% in group 1 (p=0.05). The patients in group 2 demonstrated significantly reduced COMFORT-B scores at extubation, drain removal, and mobilization compared with those in group 1 and had reduced FLACC scale levels at 20 and 24 hours postoperatively (p=0.05 and p=0.001, respectively). No differences were reported for extubation and drain removal times or for length of hospital stay. In addition, vomiting episodes were decreased in group 2 (p=0.01). CONCLUSIONS: In pediatric cardiac surgery, the results of this study confirm our hypothesis that bilateral ESPB analgesia with ropivacaine decreases the postoperative morphine consumption at 48 hours and demonstrates better postoperative analgesia compared with a control group. Trial registration number NCT03593642.

Ultrasound-Guided Continuous Thoracic Erector Spinae Plane Block Within an Enhanced Recovery Program Is Associated with Decreased Opioid Consumption and Improved Patient Postoperative Rehabilitation After Open Cardiac Surgery-A Patient-Matched, Controlled Before-and-After Study.

OBJECTIVES: Open cardiac surgery may cause severe postoperative pain. The authors hypothesized that patients receiving a bundle of care using continuous erector spinae plane blocks (ESPB) would have decreased perioperative opioid consumption and improved early outcome parameters compared with standard perioperative management. DESIGN: A consecutive, patient-matched, controlled before-and-after study. SETTING: Two tertiary teaching hospitals. PARTICIPANTS: The study comprised 67 consecutive patients undergoing elective cardiac surgery with cardiopulmonary bypass. INTERVENTIONS: In a controlled before-and-after trial, this study compared a historical group of 20 consecutive open cardiac surgery patients matched with a prospective group of 47 consecutive patients receiving continuous bilateral ESPB (0.25 mL/kg/side of ropivacaine 0.5%) after general anesthesia induction. For postoperative analgesia, both groups received paracetamol. The control group received intravenous (IV) morphine, 0.5 mg/h, and IV nefopam, 100 mg/24 h. In the ESPB group, 8 hours after the loading dose, catheters were connected to a pump infusing intermittent automatic boluses of ropivacaine 0.2% every 6 hours. If needed, for both groups, rescue analgesia was provided with IV ketorolac, 30 mg, and IV morphine, 30 µg/kg. MEASUREMENTS AND MAIN RESULTS: Morphine consumption in the first 48 hours was significantly decreased in the ESPB group (40 [25-45] mg in the control group compared with 0 [0-0] mg in the ESPB group [p < 0.001]) as was intraoperative sufentanil (0.8 [0.6-0.9] µg/kg/h and 0.2 [0.16-0.3] µg/kg/h, respectively; p < 0.001). Times to chest tube removal, first mobilization, pain (Visual Analogue Scale) values 2 hours after chest tube removal, pain values at rest 1 month after surgery, and postoperative adverse events were significantly decreased in the ESPB group. There was no difference for extubation time and pain during first mobilization. CONCLUSION: The authors report for the first time that the use of a bundle of care including a continuous bilateral ESPB is associated with a significant decrease in intraoperative and postoperative opioid consumption, optimized rapid patient mobilization, and chest tube removal after open cardiac surgery.

N-Heterocyclic Carbene Analogues of Thiele and Chichibabin Hydrocarbons.

Stable N-heterocyclic carbene analogues of Thiele and Chichibabin hydrocarbons, [(IPr)(C6 H4 )(IPr)] and [(IPr)(C6 H4 )2 (IPr)] (4 and 5, respectively; IPr=C{N(2,6-iPr2 C6 H3 )}2 CHCH), are reported. In a nickel-catalyzed double carbenylation of 1,4-Br2 C6 H4 and 4,4'-Br2 (C6 H4 )2 with IPr (1), [(IPr)(C6 H4 )(IPr)](Br)2 (2) and [(IPr)(C6 H4 )2 (IPr)](Br)2 (3) were generated, which respectively afforded 4 and 5 as crystalline solids upon reduction with KC8 . Experimental and computational studies support the semiquinoidal nature of 5 with a small singlet-triplet energy gap ΔES-T of 10.7 kcal mol-1 , whereas 4 features more quinoidal character with a rather large ΔES-T of 25.6 kcal mol-1 . In view of the low ΔES-T , 4 and 5 may be described as biradicaloids. Moreover, 5 has considerable (41 %) diradical character.

Nickel-catalysed direct C2-arylation of N-heterocyclic carbenes.

A highly efficient nickel catalysed method for the direct C2-arylation of N-heterocyclic carbenes (NHCs) is reported. This protocol enables a facile access to C2-arylated imidazolium salts (NHC-Ar)X (X = Cl, Br, I or OTf). Experimental and theoretical studies suggest the viability of a Ni(i)/Ni(ii) catalytic pathway in which the dinuclear Ni(i) species [(NHC)NiX]2 plays a key role.

Controlling uncertainty in aptamer selection.

The search for high-affinity aptamers for targets such as proteins, small molecules, or cancer cells remains a formidable endeavor. Systematic Evolution of Ligands by EXponential Enrichment (SELEX) offers an iterative process to discover these aptamers through evolutionary selection of high-affinity candidates from a highly diverse random pool. This randomness dictates an unknown population distribution of fitness parameters, encoded by the binding affinities, toward SELEX targets. Adding to this uncertainty, repeating SELEX under identical conditions may lead to variable outcomes. These uncertainties pose a challenge when tuning selection pressures to isolate high-affinity ligands. Here, we present a stochastic hybrid model that describes the evolutionary selection of aptamers to explore the impact of these unknowns. To our surprise, we find that even single copies of high-affinity ligands in a pool of billions can strongly influence population dynamics, yet their survival is highly dependent on chance. We perform Monte Carlo simulations to explore the impact of environmental parameters, such as the target concentration, on selection efficiency in SELEX and identify strategies to control these uncertainties to ultimately improve the outcome and speed of this time- and resource-intensive process.

Melanosomes in pigmented epithelia maintain eye lens transparency during zebrafish embryonic development.

Altered levels of trace elements are associated with increased oxidative stress that is eventually responsible for pathologic conditions. Oxidative stress has been proposed to be involved in eye diseases, including cataract formation. We visualized the distribution of metals and other trace elements in the eye of zebrafish embryos by micro X-ray fluorescence (µ-XRF) imaging. Many elements showed highest accumulation in the retinal pigment epithelium (RPE) of the zebrafish embryo. Knockdown of the zebrafish brown locus homologues tyrp1a/b eliminated accumulation of these elements in the RPE, indicating that they are bound by mature melanosomes. Furthermore, albino (slc45a2) mutants, which completely lack melanosomes, developed abnormal lens reflections similar to the congenital cataract caused by mutation of the myosin chaperon Unc45b, and an in situ spin trapping assay revealed increased oxidative stress in the lens of albino mutants. Finally transplanting a wildtype lens into an albino mutant background resulted in cataract formation. These data suggest that melanosomes in pigment epithelial cells protect the lens from oxidative stress during embryonic development, likely by buffering trace elements.

Rapid and specific drug quality testing assay for artemisinin and its derivatives using a luminescent reaction and novel microfluidic technology.

Globally, it is estimated that about 10-30% of pharmaceuticals are of poor quality. Poor-quality drugs lead to long-term drug resistance, create morbidity, and strain the financial structure of the health system. The current technologies for substandard drug detection either are too expensive for low-resource regions or only provide qualitative results. To address the current limitations with point-of-care technologies, we have developed an affordable and robust assay to quantify the amount of active pharmaceutical ingredients (APIs) to test product quality. Our novel assay consists of two parts: detection reagent (probe) and a microfluidic testing platform. As antimalarials are of high importance in the global fight against malaria and are often substandard, they are chosen as the model to validate our assay. As a proof-of-concept, we have tested the assay with artesunate pure and substandard samples (Arsuamoon tablets) from Africa and compared with the conventional 96-well plate with spectrophotometer to demonstrate the quantitative efficacy and performance of our system.

Gene responses in the central nervous system of zebrafish embryos exposed to the neurotoxicant methyl mercury.

Methyl mercury (MeHg) is a neurotoxicant with adverse effects on the development of the nervous system from fish to man. Despite a detailed understanding of the molecular mechanisms by which MeHg affects cellular homeostasis, it is still not clear how MeHg causes developmental neurotoxicity. We performed here a genome-wide transcriptional analysis of MeHg-exposed zebrafish embryos and combined this with a whole-mount in situ expression analysis of 88 MeHg-affected genes. The majority of the analyzed genes showed tissue- and region-restricted responses in various organs and tissues. The genes were linked to gene ontology terms like oxidative stress, transport and cell protection. Areas even within the central nervous system (CNS) are affected differently resulting in distinct cellular stress responses. Our study revealed an unexpected heterogeneity in gene responses to MeHg exposure in different tissues and neuronal subregions, even though the known molecular action of MeHg would predict a similar burden of exposed cells. The overall structure of the developing brain of MeHg-exposed embryos appeared normal, suggesting that the mechanism leading to differentiation of the CNS is not overtly affected by exposure to MeHg. We propose that MeHg disturbs the function of the CNS by disturbing the cellular homeostasis. As these cellular stress responses comprise genes that are also involved in normal neuronal activity and learning, MeHg may affect the developing CNS in a subtle manner that manifests itself in behavioral deficits.

Sample concentration and purification for point-of-care diagnostics.

The ability to increase the concentration of target analytes in a fixed sample volume can potentially lower the limit of detection for many biosensing techniques, and thus is key in sample preparation for infectious disease diagnosis. Concentration by evaporation is an effective method to achieve target enrichment. However, concentrating human samples, including blood and plasma, by evaporation-based methods is made challenging by high concentrations of proteins and electrolytes. Dehydration of the proteins causes the sample to turn into a gel, hindering further analysis. At the same time, decreasing the volume increases the overall concentration of electrolytes, causing bacterial or viral particle lysis, and making them more difficult to detect in affinity-based biosensors. Thus, we fabricated a microfluidic chip that incorporates both dialysis and concentration in a single design. The chip dialyzes the proteins from the plasma, while maintaining an appropriate concentration of electrolytes and concentrating the sample targets. The process to concentrate plasma or serum samples by a factor of 10 takes less than 30 minutes. As a proof-of-concept, we demonstrated the chip using a defective Human Immunodeficiency Virus (HIV). To distinguish patients on antiretroviral therapy who are failing therapy from those who are not, a diagnostic must be able to detect HIV in plasma down to at least 1000 particles per milliliter. For a number of technical reasons, it is difficult to get on-chip PCR reactions to reach this level of sensitivity, so concentration of HIV from lower viral load samples has the potential to improve the sensitivity of many types of molecular point-of-care viral load tests.

Methyl mercury suppresses the formation of the tail primordium in developing zebrafish embryos.

The objective of this study was to characterize the mechanisms of action of the model environmental toxicant methyl mercury (MeHg) in the zebrafish embryo. Zebrafish embryos were exposed to MeHg, and the effective concentration and window of exposure were determined in wild-type and fluorescent reporter transgenic zebrafish embryos. Genes were systematically assessed for altered expression in response to MeHg by in situ hybridization. MeHg impairs development of the fin fold and the tail fin primordium. Alterations in transgene expression were noted at 6 microg/l MeHg, making this shh:gfp line the most sensitive biosensor of MeHg exposure. The matrix metalloproteases mmp9 and mmp13 and eight other genes are induced in the embryonic tail in response to MeHg. Our data suggest that MeHg impairs tail development at least partially by activation of the tissue remodeling proteases Mmp9 and Mmp13.

Zebrafish embryos as models for embryotoxic and teratological effects of chemicals.

The experimental virtues of the zebrafish embryo such as small size, development outside of the mother, cheap maintenance of the adult made the zebrafish an excellent model for phenotypic genetic and more recently also chemical screens. The availability of a genome sequence and several thousand mutants and transgenic lines together with gene arrays and a broad spectrum of techniques to manipulate gene functions add further to the experimental strength of this model. Pioneering studies suggest that chemicals can have in many cases very similar toxicological and teratological effects in zebrafish embryos and humans. In certain areas such as cardiotoxicity, the zebrafish appears to outplay the traditional rodent models of toxicity testing. Several pilot projects used zebrafish embryos to identify new chemical entities with specific biological functions. In combination with the establishment of transgenic sensor lines and the further development of existing and new automated imaging systems, the zebrafish embryos could therefore be used as cost-effective and ethically acceptable animal models for drug screening as well as toxicity testing.

Prevalence of hereditary prosopagnosia (HPA) in Hong Kong Chinese population.

Prosopagnosia (PA), or the inability to recognize a familiar person by the face alone, had been considered to be a rare dysfunction mainly acquired by trauma to the brain. Recently we have shown that the congenital form of PA, which was considered to be even rarer, is common in Caucasians, with a prevalence of 2.5%. As these cases were familial we coined the term Hereditary Prosopagnosia (HPA). The present study is the first systematic screening for HPA in a defined population of ethnic Chinese. In 2004-2005, 533 out of around 750 medical students of The University of Hong Kong took part in a questionnaire-based screening. The responses of 133 students indicated that they were likely to be candidates for PA. One hundred twenty agreed for diagnostic interview. Finally we made the clinical diagnosis of PA in 10 subjects. A prevalence of 1.88% (95% CI, 1.05-2.71) is established which is in the same range as in Caucasians. We took a detailed family history of four index prosopagnosic persons and were able to further investigate the families of four probands. Each had other first-degree relatives with the same visual cognitive dysfunction. Thus, as in the Caucasians, regular autosomal dominant inheritance might best explain the segregation pattern.

Cloning and developmental expression of kinesin superfamily7 (kif7) in the brackish medaka (Oryzias melastigma), a close relative of the Japanese medaka (Oryzias latipes).

kif7 is a member of the kinesin superfamily members which are molecular motor proteins that move along microtubules in a highly regulated manner through ATP hydrolysis. In this paper, we report on the cloning of the Oryziasmelastigmakif7 (omkif7) using primers designed according to the Japanese medaka (Oryziaslatipes) database. The cloned omkif7 has an open reading frame of 3762bp and is deduced to encode a polypeptide of 1254 amino acids that possesses the putative ATP-binding and microtubule-binding motifs in its motor domain at the N-terminal region. We characterized the cloned omkif7 by comparison with the zebrafish kif7. Both omkif7 and zebrafish kif7 are shown to be expressed in all embryonic stages and adult tissues examined with higher expression level in the testis and ovary. Whole-mount in situ hybridization revealed that the expression of omkif7 is ubiquitous during the early stages of embryonic development, but became more restrictive and localized to the brain, fin bud and eye at later development. This study suggested that the brackish O.melastigma can serve as a good seawater model organism for developmental studies by utilizing the resources developed from its close relative of the Japanese medaka.

Targeted inactivation of the IL-4 receptor alpha chain I4R motif promotes allergic airway inflammation.

The insulin/interleukin-4 (IL-4) receptor (I4R) motif mediates the association of insulin receptor substrate (IRS)-2 with the interleukin-4 (IL-4)Ralpha chain and transduces mitogenic signals in response to IL-4. Its physiological functions were analyzed in mice with a germline point mutation that changed the motif's effector tyrosine residue into phenylalanine (Y500F). The Y500F mutation abrogated IRS-2 phosphorylation and impaired IL-4-induced CD4+ T lymphocyte proliferation but left unperturbed Stat6 activation, up-regulation of IL-4-responsive gene products, and Th cell differentiation under Th2 polarizing conditions. However, in vivo the Y500F mutation was associated with increased allergen-induced IgE production, airway responsiveness, tissue eosinophilia, and mucus production. These results define an important role for the I4R motif in regulating allergic inflammation.

Calcium calmodulin-dependent protein kinase IV is required for fear memory.

The ability to remember potential dangers in an environment is necessary to the survival of animals and humans. The cyclic AMP responsive element binding protein (CREB) is a key transcription factor in synaptic plasticity and memory consolidation. We have found that in CaMKIV(-/-) mice--which are deficient in a component of the calcium calmodulin-dependent protein kinase (CaMK) pathway, a major pathway of CREB activation--fear memory, but not persistent pain, was significantly reduced. CREB activation by fear conditioning and synaptic potentiation in the amygdala and cortical areas was reduced or blocked. We propose that cognitive memory related to a noxious shock can be disassociated from behavioral responses to tissue injury and inflammation.