Radioimmunoassay of detomidine, a new benzylimidazole drug with analgesic sedation properties

A sensitive and specific radioimmunoassay was developed for detomidine, 4(5)-(2,3-dimethylbenzyl)imidazole. The antibodies were raised in rabbits against a conjugate of detomidine and bovine thyroglobulin prepared by diazo reaction. Detomidine was iodinated with chloramine-T and immunoreactive tracer was purified in cation exchange chromatography. The sensitivity of the RIA was 1.6 fmol/tube allowing direct detomidine measurements from minute serum and urine samples (0.1-0.2 microliter) as well as tissue homogenates (10 microliters). For concentrations below 16 pmol/ml chloroform extraction was used to extend the measurement range to 0.3 pmol/ml. Detomidine (80 micrograms/kg iv and im) was given to one horse and two calves and blood samples were taken and urine collected for 24 h whereafter the horse was slaughtered and tissue samples taken for RIA analyses. Serially diluted serum, urine and tissue samples produced a linear displacement curve parallel to synthetic detomidine in RIA. HPLC studies showed that serum and tissue immunoreactivity was unchanged detomidine whereas most immunoreactivity in the urine was due to an unknown metabolite.     

Effectiveness of a WHO self‐help psychological intervention for preventing mental disorders among Syrian refugees in Turkey: a randomized controlled trial

Refugees are at high risk of developing mental disorders. There is no evidence from randomized controlled trials (RCTs) that psychological interventions can prevent the onset of mental disorders in this group. We assessed the effectiveness of a self‐help psychological intervention developed by the World Health Organization, called Self‐Help Plus, in preventing the development of mental disorders among Syrian refugees experiencing psychological distress in Turkey. A two‐arm, assessor‐masked RCT was conducted in two Turkish areas. Eligible participants were adult Syrian refugees experiencing psychological distress (General Health Questionnaire ≥3), but without a diagnosis of mental disorder. They were randomly assigned either to the Self‐Help Plus arm (consisting of Self‐Help Plus combined with Enhanced Care as Usual, ECAU) or to ECAU only in a 1:1 ratio. Self‐Help Plus was delivered in a group format by two facilitators over five sessions. The primary outcome measure was the presence of any mental disorder assessed by the Mini International Neuropsychiatric Interview at six‐month follow‐up. Secondary outcome measures were the presence of mental disorders at post‐intervention, and psychological distress, symptoms of post‐traumatic stress disorder and depression, personally identified psychological outcomes, functional impairment, subjective well‐being, and quality of life at post‐intervention and six‐month follow‐up. Between October 1, 2018 and November 30, 2019, 1,186 refugees were assessed for inclusion. Five hundred forty‐four people were ineligible, and 642 participants were enrolled and randomly assigned to either Self‐Help Plus (N=322) or ECAU (N=320). Self‐Help Plus participants were significantly less likely to have any mental disorders at six‐month follow‐up compared to the ECAU group (21.69% vs. 40.73%; Cramer''s V = 0.205, p<0.001, risk ratio: 0.533, 95% CI: 0.408‐0.696). Analysis of secondary outcomes suggested that Self‐Help Plus was not effective immediately post‐intervention, but was associated with beneficial effects at six‐month follow‐up in terms of symptoms of depression, personally identified psychological outcomes, and quality of life. This is the first prevention RCT ever conducted among refugees experiencing psychological distress but without a mental disorder. Self‐Help Plus was found to be an effective strategy for preventing the onset of mental disorders. Based on these findings, this low‐intensity self‐help psychological intervention could be scaled up as a public health strategy to prevent mental disorders in refugee populations exposed to ongoing adversities.     

Membrane targeting and activation of the Lowe syndrome protein OCRL1 by rab GTPases

The X-linked disorder oculocerebrorenal syndrome of Lowe is caused by mutation of the OCRL1 protein, an inositol polyphosphate 5-phosphatase. OCRL1 is localised to the Golgi apparatus and early endosomes, and can translocate to lamellipodia upon growth factor stimulation. We show here that OCRL1 interacts with several members of the rab family of small GTPases. Strongest interaction is seen with Golgi-associated rab1 and rab6 and endosomal rab5. Point mutants defective in rab binding fail to target to the Golgi apparatus and endosomes, strongly suggesting rab interaction is required for targeting of OCRL1 to these compartments. Membrane recruitment via rab binding is required for changes in Golgi and endosomal dynamics induced by overexpression of catalytically inactive OCRL1. In vitro experiments demonstrate that rab5 and rab6 directly stimulate the 5-phosphatase activity of OCRL1. We conclude that rabs play a dual role in regulation of OCRL1, firstly targeting it to the Golgi apparatus and endosomes, and secondly, directly stimulating the 5-phosphatase activity of OCRL1 after membrane recruitment.     

The chromatoid body: a germ-cell-specific RNA-processing centre

The chromatoid body, a unique cloud-like structure of male germ cells, moves dynamically in the cytoplasm of haploid spermatids, but its function has remained elusive for decades. Recent findings indicate that microRNA and RNA-decay pathways converge to the chromatoid body. This highly specialized structure might function as an intracellular focal domain that organizes and controls RNA processing in male germ cells.     

Harmonization is more important than experience—results of the first Nordic–Baltic diatom intercalibration exercise 2007 (stream monitoring)

The goal of this study was a harmonization of diatom identification and counting among diatomists from the Scandinavian and Baltic countries to improve the comparison of diatom studies in this geographical area. An analysis of the results of 25 diatomists following the European standard EN 14407 during an intercalibration exercise showed that a high similarity was achieved by harmonization and not because of a long experience with diatoms. Sources of error were wrong calibration scales, overlooking of small taxa, especially small Navicula s.l., misidentifications (Eunotia rhomboidea was mistaken for Eunotia incisa) and unclear separation between certain taxa in the identification literature. The latter was discussed during a workshop with focus on the Achnanthes minutissima group, the separation of Fragilaria capucina var. gracilis from F. capucina var. rumpens, and Nitzschia palea var. palea from N. palea var. debilis. The exercise showed also that the Swedish standard diatom method tested here worked fine with acceptable error for the indices IPS (Indice de Polluo-sensibilité Spécifique) and ACID (ACidity Index for Diatoms) when diatomists with a low similarity (Bray–Curtis <60%) with the auditor in at least one of the samples are excluded.     

Role of two sequence motifs of mesencephalic astrocyte-derived neurotrophic factor in its survival-promoting activity

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a prosurvival protein that protects the cells when applied intracellularly in vitro or extracellularly in vivo. Its protective mechanisms are poorly known. Here we studied the role of two short sequence motifs within the carboxy-(C) terminal domain of MANF in its neuroprotective activity: the CKGC sequence (a CXXC motif) that could be involved in redox reactions, and the C-terminal RTDL sequence, an endoplasmic reticulum (ER) retention signal. We mutated these motifs and analyzed the antiapoptotic effect and intracellular localization of these mutants of MANF when overexpressed in cultured sympathetic or sensory neurons. As an in vivo model for studying the effect of these mutants after their extracellular application, we used the rat model of cerebral ischemia. Even though we found no evidence for oxidoreductase activity of MANF, the mutation of CXXC motif completely abolished its protective effect, showing that this motif is crucial for both MANF''s intracellular and extracellular activity. The RTDL motif was not needed for the neuroprotective activity of MANF after its extracellular application in the stroke model in vivo. However, in vitro the deletion of RTDL motif inactivated MANF in the sympathetic neurons where the mutant protein localized to Golgi, but not in the sensory neurons where the mutant localized to the ER, showing that intracellular MANF protects these peripheral neurons in vitro only when localized to the ER.The prosurvival proteins that actively keep the cells alive function as a counterbalance to the prodeath programs of the cell and are thereby essential players in morphogenesis and adult tissue homeostasis. Such survival-promoting proteins are also potential candidates for the treatment of pathological conditions, especially in the nervous system where the lost neurons are only rarely replaced by new ones. Some prosurvival proteins act extracellularly. For example, neurotrophic factors (NTFs) are secreted proteins that bind the cognate receptors on the surface of the cells, thereby triggering prosurvival signaling cascades.^{1, 2, 3} Other prosurvival proteins, such as Akt kinase, antiapoptotic Bcl2 family members, inhibitor of apoptosis (IAP) proteins and so on are not secreted and protect the cells intracellularly. A new family of survival-promoting proteins has recently been described⁴ that can act in both ways. This family consists of two proteins, mesencephalic astrocyte-derived neurotrophic factor (MANF)⁵ and cerebral dopamine neurotrophic factor (CDNF).⁶ Both factors, when delivered into the extracellular space of the brain or applied via viral vectors potently antagonize neurological damage in the rodent models of Parkinson''s disease. MANF is also protective against cerebral ischemia^{6, 7, 8, 9, 10} and prevents degeneration of Purkinje cells in spinocerebellar ataxia.¹¹ In these experiments, MANF and CDNF halted the death of the neurons and also stimulated regrowth of the dopaminergic fibers, acting thus as typical NTFs. On the other hand in non-neuronal cells, MANF was also shown to be a resident protein of the endoplasmic reticulum (ER) protecting the cells intracellularly against ER stress.^{12, 13, 14, 15, 16, 17}In line with its role in counteracting cell death, MANF promotes pancreatic β-cell proliferation and survival in vivo, and lack of MANF leads to chronic unfolded protein response (UPR) activation in pancreatic islets.¹⁸ We have also shown that microinjected intracellular MANF protects cultured sympathetic neurons of the superior cervical ganglion (SCG) against apoptosis-inducing toxins, whereas it does not protect or bind these neurons when applied to the culture medium.¹⁹ Thus, MANF can protect the neurons when applied extracellularly (at least in vivo) and intracellularly. Of note, MANF also has both intra- and extracellular activities on the pancreatic β cells.¹⁸ However, the mechanisms of intra- and extracellular action of MANF are currently not well defined.Structurally MANF consists of amino- (N) terminal saposin-like domain and carboxy-(C) terminal SAP domain-like domain that are connected by a flexible linker.^{19, 20} From the amino-acid sequence and structure, two potentially functional motifs can be distinguished in the carboxy-terminal domain of MANF (C-MANF).^{19, 20} First, a conserved^{21, 22} CXXC motif (CKGC) was found in the loop connecting the helices α7 and α8 where two cysteines were connected with a disulfide bond. The CXXC motif is found in the active center of the enzymes of thiol-disulfide oxidoreductase superfamily.²³ Second, a conserved RTDL sequence belonging to the class of KDEL-like ER retention signal²⁴ is found at the very end of C terminus of MANF^{19, 20} and is shown to be required for its retrieval from the Golgi to ER.^{15, 16, 25} The role of the RTDL motif in the survival-promoting activity of MANF has not been studied.In this study, we set up to investigate the importance of these motifs of MANF for its survival-promoting activity both in the intracellular in vitro as well as extracellular in vivo paradigms. We mutated the CXXC and RTDL motifs and tested the activity of the mutants by microinjecting plasmid DNAs encoding the mutant proteins into apoptotic sympathetic SCG neurons, our validated model for testing the neuroprotective bioactivity of MANF,^{19, 21} and sensory dorsal root ganglion (DRG) neurons. Subcellular localization of the MANF mutants was also studied in the same neurons. We show that the CXXC motif is critically required for the survival-promoting activity of MANF, as mutation of this motif prevents MANF from being neuroprotective in both types of neurons in vitro and also in the in vivo rat model of cerebral ischemia. In addition, we show, using the same in vivo model, that the RTDL motif is not required for the neuroprotective activity of extracellularly applied MANF. However, intracellularly the mutant without RTDL motif was inactive only when it was not retrieved to the ER.     

A heart to heart on temperature: Impaired temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of cardiac arrhythmia

Triploid (3N) salmonids are of interest to aquaculture and sport fishing industries, however it has been shown that 3N fish have impaired tolerance to high temperatures. To test the hypothesis that poor high temperature tolerance in 3N salmonids is related to impaired O₂ delivery to the body, maximum heart rate (fH) was measured in 2N (diploid) and 3N rainbow trout (Oncorhynchus mykiss) during an incremental temperature challenge. fH of both ploidies was similar at 10 °C. However, a significant effect of ploidy on the response of fH to temperature from 10 to 22 °C was reflected in a lower Q₁₀ for 3N individuals. Additionally, all 3N trout developed a cardiac arrhythmia by 22 °C, where 30% of 2N trout continued to maintain a rhythmic heartbeat. These findings suggest that reduced 3N high temperature tolerance could be due to early collapse of the cardiovascular system's ability to deliver O₂ to the body during warming.