Clinical chemistry of serotonin and metabolites

Analyses of serotonin and other 5-hydroxyindoles, such as its precursor 5-hydroxytryptophan and major metabolite 5-hydroxyindoleacetic acid (5-HIAA), are indispensable for the elucidation of their (patho)physiological roles. In clinical chemistry attention is mainly focused on the diagnosis and follow-up of carcinoid tumours. For this most laboratories routinely measure urinary 5-HIAA. More recently, measurements of serotonin in platelets and urine have been advocated. Platelet serotonin may be the most sensitive indole marker for the detection of carcinoid tumours that secrete only small amounts of serotonin and/or its precursor 5-hydroxytryptophan. Although several chromatographic techniques have emerged for the analysis of tryptophan-related indoles, HPLC with either electrochemical or fluorometric detection have become the methods of choice for their quantification. HPLC-based methods combine selectivity, sensitivity and high precision, and enable the simultaneous investigation of several metabolically related indoles. This review aims to place the analysis of indoles in biological matrices in a biochemical, physiological and clinical perspective and highlights several important steps in their chromatographic analysis and quantification.     

Leptin Responses to Weight Loss in Postmenopausal Women: Relationship to Sex‐Hormone Binding Globulin and Visceral Obesity

Objective: Leptin concentrations increase with obesity and tend to decrease with weight loss. However, there is large variation in the response of serum leptin levels to decreases in body weight. This study examines which endocrine and body composition factors are related to changes in leptin concentrations following weight loss in obese, postmenopausal women. Research Methods and Procedures: Body composition (DXA), visceral obesity (computed tomography), leptin, cortisol, insulin, and sex hormone‐binding globulin (SHBG) concentrations were measured in 54 obese (body mass index [BMI] = 32.0 ± 4.5 kg/m²; mean ± SD), women (60 ± 6 years) before and after a 6‐month hypocaloric diet (250 to 350 kcal/day deficit). Results: Body weight decreased by 5.8 ± 3.4 kg (7.1%) and leptin levels decreased by 6.6 ± 11.9 ng/mL (14.5%) after the 6‐month treatment. Insulin levels decreased 10% (p < 0.05), but mean SHBG and cortisol levels did not change significantly. Relative changes in leptin with weight loss correlated positively with relative changes in body weight (r = 0.50, p < 0.0001), fat mass (r = 0.38, p < 0.01), subcutaneous fat area (r = 0.52, p < 0.0001), and with baseline values of SHBG (r = 0.38, p < 0.01) and baseline intra‐abdominal fat area (r = ?0.27, p < 0.06). Stepwise multiple regression analysis showed that baseline SHBG levels (r² = 0.24, p < 0.01), relative changes in body weight (cumulative r² = 0.40, p < 0.05), and baseline intra‐abdominal fat area (cumulative r² = 0.48, p < 0.05) were the only independent predictors of the relative change in leptin, accounting for 48% of the variance. Discussion: These results suggest that obese, postmenopausal women with a lower initial SHBG and more visceral obesity have a greater decrease in leptin with weight loss, independent of the amount of weight lost.     

PI Kinase-EhGEF2-EhRho5 axis contributes to LPA stimulated macropinocytosis in Entamoeba histolytica

Entamoeba histolytica is a protozoan responsible for several pathologies in humans. Trophozoites breach the intestinal site to enter the bloodstream and thus traverse to a secondary site. Macropinocytosis and phagocytosis, collectively accounting for heterophagy, are the two major processes responsible for sustenance of Entamoeba histolytica within the host. Both of these processes require significant rearrangements in the structure to entrap the target. Rho GTPases play an indispensable role in mustering proteins that regulate cytoskeletal remodelling. Unlike phagocytosis which has been studied in extensive detail, information on machinery of macropinocytosis in E. histolytica is still limited. In the current study, using site directed mutagenesis and RNAi based silencing, coupled with functional studies, we have demonstrated the involvement of EhRho5 in constitutive and LPA stimulated macropinocytosis. We also report that LPA, a bioactive phospholipid present in the bloodstream of the host, activates EhRho5 and translocates it from cytosol to plasma membrane and endomembrane compartments. Using biochemical and FRAP studies, we established that a PI Kinase acts upstream of EhRho5 in LPA mediated signalling. We further identified EhGEF2 as a guanine nucleotide exchange factor of EhRho5. In the amoebic trophozoites, EhGEF2 depletion leads to reduced macropinocytic efficiency of trophozoites, thus phenocopying its substrate. Upon LPA stimulation, EhGEF2 is found to sequester near the plasma membrane in a wortmannin sensitive fashion, explaining a possible mode for activation of EhRho5 in the amoebic trophozoites. Collectively, we propose that LPA stimulated macropinocytosis in E. histolytica is driven by the PI Kinase-EhGEF2-EhRho5 axis.     

Epidemiology of yellow fever virus in humans,arthropods, and non-human primates in sub-Saharan Africa: A systematic review and meta-analysis

Yellow fever (YF) has re-emerged in the last two decades causing several outbreaks in endemic countries and spreading to new receptive regions. This changing epidemiology of YF creates new challenges for global public health efforts. Yellow fever is caused by the yellow fever virus (YFV) that circulates between humans, the mosquito vector, and non-human primates (NHP). In this systematic review and meta-analysis, we review and analyse data on the case fatality rate (CFR) and prevalence of YFV in humans, and on the prevalence of YFV in arthropods, and NHP in sub-Saharan Africa (SSA). We performed a comprehensive literature search in PubMed, Web of Science, African Journal Online, and African Index Medicus databases. We included studies reporting data on the CFR and/or prevalence of YFV. Extracted data was verified and analysed using the random effect meta-analysis. We conducted subgroup, sensitivity analysis, and publication bias analyses using the random effect meta-analysis while I² statistic was employed to determine heterogeneity. This review was registered with PROSPERO under the identification CRD42021242444. The final meta-analysis included 55 studies. The overall case fatality rate due to YFV was 31.1% (18.3–45.4) in humans and pooled prevalence of YFV infection was 9.4% (6.9–12.2) in humans. Only five studies in West and East Africa detected the YFV in mosquito species of the genus Aedes and in Anopheles funestus. In NHP, YFV antibodies were found only in members of the Cercopithecidae family. Our analysis provides evidence on the ongoing circulation of the YFV in humans, Aedes mosquitoes and NHP in SSA. These observations highlight the ongoing transmission of the YFV and its potential to cause large outbreaks in SSA. As such, strategies such as those proposed by the WHO’s Eliminate Yellow Fever Epidemics (EYE) initiative are urgently needed to control and prevent yellow fever outbreaks in SSA.     

Abundance,distribution, and territory areas of rock-dwelling Lake Tanganyika cichlid fish species

Lake Tanganyika, the second-oldest and second-deepest lake in the world, harbors an impressive cichlid fish fauna counting about 250 endemic species that are characterized by a great level of ecological, morphological, and behavioral specialization. This study describes and compares cichlid fish communities at two rocky shores with differential human impact in the south of Lake Tanganyika. Species inventories and depth-dependent abundances were elaborated. About 41 and 46 sympatric cichlid species were recorded in the two study sites, respectively. Variabilichromis moorii was the most abundant species (29–60% of total number of fishes), followed by Aulonocranus dewindti (3–19%), Tropheus moorii (12%), Ophthalmotilapia ventralis (4–10%), Eretmodus cyanostictus (6–11%), and Cyathopharynx furcifer (0.01–9%). All other species had abundances below 5%. It further emerged that large cichlids such as Petrochromis species, Cyathopharynx furcifer, and Lobochilotes labiatus were very rare at one location, with frequencies of 0.55% or less. Territorial sizes of three particularly abundant species, Variabilichromis moorii, Aulonocranus dewindti, and Tropheus moorii, were assessed by behavioral observations. We distinguished between territorial core areas and total defended area, yielding average core areas between 0.4 (V. moorii) and 1.6 m² (T. moorii), and total defended areas averaging for each species between 1.6 (V. moorii) and 5.0 m² (A. dewindti) with no significant differences between the two study sites. The data on individual densities are also relevant for evolutionary studies, in that they allow more accurate calculations of effective population sizes.     

A high degree of aneuploidy in frozen-thawed human preimplantation embryos

We have studied the chromosomal content in 68 normally fertilised freeze-thawed human embryos of good morphology from 34 patients with an average maternal age of 32,6 years. Forty embryos showed post-thaw cellular division and twenty-eight post-thaw cleavage arrest. After spreading of the embryos on microscope slides, analysis of chromosomes X, Y, 15, 16, 17 and 18 was performed using two rounds of fluorescent in situ hybridisation (FISH). According to the results, the embryos were divided into four groups: (I) normal, all nuclei uniformly diploid, (II) diploid mosaics, normal diploid blastomeres in combination with abnormal blastomeres, (III) abnormal, all nuclei abnormal, (IV) chaotic, the chromosome constitution varies randomly from cell to cell. Approximately 25% of the embryos had normal number of the chromosomes tested, while the majority of the embryos were abnormal. Most of the abnormal embryos were diploid mosaics (57%). This was true for the embryos showing cleavage division as well as the embryos showing cleavage arrest. Our data show a slightly higher incidence of abnormal embryos compared to those obtained with FISH in non-cryopreserved embryos and confirm that the majority of preimplantation embryos fertilised in vitro contain abnormal blastomeres. The results, mechanisms, significance and implications are discussed. Received: 19 November 1998 / Accepted: 4 March 1999     

Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER*

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.     

Reconfiguration of a Multi-oscillator Network by Light in the Drosophila Circadian Clock

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Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation.