Metabolomics- and proteomics-assisted genome annotation and analysis of the draft metabolic network of Chlamydomonas reinhardtii

We present an integrated analysis of the molecular repertoire of Chlamydomonas reinhardtii under reference conditions. Bioinformatics annotation methods combined with GCxGC/MS-based metabolomics and LC/MS-based shotgun proteomics profiling technologies have been applied to characterize abundant proteins and metabolites, resulting in the detection of 1069 proteins and 159 metabolites. Of the measured proteins, 204 currently do not have EST sequence support; thus a significant portion of the proteomics-detected proteins provide evidence for the validity of in silico gene models. Furthermore, the generated peptide data lend support to the validity of a number of proteins currently in the proposed model stage. By integrating genomic annotation information with experimentally identified metabolites and proteins, we constructed a draft metabolic network for Chlamydomonas. Computational metabolic modeling allowed an identification of missing enzymatic links. Some experimentally detected metabolites are not producible by the currently known and annotated enzyme set, thus suggesting entry points for further targeted gene discovery or biochemical pathway research. All data sets are made available as supplementary material as well as web-accessible databases and within the functional context via the Chlamydomonas-adapted MapMan annotation platform. Information of identified peptides is also available directly via the JGI-Chlamydomonas genomic resource database (http://genome.jgi-psf.org/Chlre3/Chlre3.home.html).     

The complex roles of neurosteroids in depression and anxiety disorders

The role of neurosteroids in neuropsychiatric disorders has been thoroughly investigated in many research studies that have stressed their significant pathophysiological function in neuropsychiatry. In this review, we will focus mainly on the steroids active on the GABA(A) receptors studied in anxiety and depression. The aim is to discuss the controversial results reported in research on anxiety and depressive disorders. We suggest the combined use of biological parameters linked to psychopathological dimensions to make more homogeneous diagnoses and to develop more precise therapies for the treatment of depression and anxiety disorders. We discuss the role of neurosteroids in the pathophysiology and therapy of anxiety and depression. Finally, we consider the possibility of using quantification of mRNA expression of steroidogenic enzymes from peripheral sources in neuropsychiatry.     

Identification and Validation of Urinary Biomarkers for Differential Diagnosis and Evaluation of Therapeutic Intervention in Anti-neutrophil Cytoplasmic Antibody-associated Vasculitis

Renal activity and smoldering disease is difficult to assess in anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) because of renal scarring. Even repeated biopsies suffer from sampling errors in this focal disease especially in patients with chronic renal insufficiency. We applied capillary electrophoresis coupled to mass spectrometry toward urine samples from patients with active renal AAV to identify and validate urinary biomarkers that enable differential diagnosis of disease and assessment of disease activity. The data were compared with healthy individuals, patients with other renal and non-renal diseases, and patients with AAV in remission. 113 potential biomarkers were identified that differed significantly between active renal AAV and healthy individuals and patients with other chronic renal diseases. Of these, 58 could be sequenced. Sensitivity and specificity of models based on 18 sequenced biomarkers were validated using blinded urine samples of 40 patients with different renal diseases. Discrimination of AAV from other renal diseases in blinded samples was possible with 90% sensitivity and 86.7–90% specificity depending on the model. 10 patients with active AAV were followed for 6 months after initiation of treatment. Immunosuppressive therapy led to a change of the proteome toward “remission.” 47 biomarkers could be sequenced that underwent significant changes during therapy together with regression of clinical symptoms, normalization of C-reactive protein, and improvement of renal function. Proteomics analysis with capillary electrophoresis-MS represents a promising tool for fast identification of patients with active AAV, indication of renal relapses, and monitoring for ongoing active renal disease and remission without renal biopsy.Systemic vasculitides are a heterogeneous group of disorders with inflammation of the blood vessel wall as their common hallmark. These disorders often pose difficulties with regard to diagnosis and monitoring of disease activity both at the initial presentation and during follow-up. In one subgroup of small vessel vasculitides, the advent of anti-neutrophil cytoplasmic antibodies (ANCAs)¹ in the 1980s not only provided a new pathogenetic concept but also a diagnostic marker (1, 2). In this group, the granulomatous ANCA-associated vasculitis (GAAV) (previously named Wegener granulomatosis) and the microscopic polyangiitis (MPA) share several common features, including pauci-immune focal crescentic necrotizing glomerulonephritis and often a pulmonary capillaritis (3). Because of the association with ANCA, these diseases (together with the Churg-Strauss syndrome) are sometimes collectively referred to as ANCA-associated vasculitis (AAV). Recently circulating endothelial cells have emerged as an important marker correlating with severity and activity of the systemic vasculitic disease, and their clinical use in small vessel AAV has been demonstrated (4, 5). Regarding renal involvement, which is found in up to 80–90% of the patients with AAV, activity is defined by kidney biopsy with pauci-immune necrotizing glomerulonephritis. Renal involvement may occur or recur at every point of the disease and the follow-up even if other organ involvement is controlled by immunosuppressive therapy. Early detection is important as renal prognosis depends on early administration of immunosuppressive treatment (6), and scarring and relapses increase the risk for terminal renal failure, which itself is a risk factor for patient survival. As kidney biopsy is invasive and the risk of bleeding increases with chronic renal damage, surrogate markers, such as rising creatinine, increasing proteinuria, and most importantly erythrocytes and erythrocyte cast in the urinary sediment, are used. However, these markers have limitations. Microhematuria might persist despite remission, proteinuria might increase despite improvement in renal function, and other renal diseases can also develop (7). Therefore, new markers for renal disease activity are eagerly awaited.Recently proteome analysis of urine has presented itself as a promising tool in the definition of chronic renal diseases (8, 9). We have developed an analytical platform for human urine analysis using capillary electrophoresis (CE) coupled on line to an ESI-TOF mass spectrometer (8, 10, 11). This approach permits the rapid analysis of the low molecular weight urinary proteome/peptidome in a single step and has enabled identification and validation of several urinary biomarkers in patients with different renal diseases (11–14).In this study, we aimed toward identification of biomarkers for active AAV and response to immunosuppressive treatment. The results indicate that urinary proteome analysis enables differential diagnosis and monitoring of renal disease activity of AAV.     

Rabies epidemiology,prevention and control in Nigeria: Scoping progress towards elimination

BackgroundHuman rabies remains a significant public health problem in Africa with outbreaks reported in most countries. In Nigeria–the most populous country in Africa–rabies causes a significant public health burden partly due to perennial obstacles to implementing a national prevention and control program.MethodsWe conducted a scoping review using standard Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to identify and select published articles from Nigeria during 1978–2020 reporting on rabies virus infections (human, canine, livestock, and wildlife), canine bites, knowledge, attitudes and practices (KAP) surveys on rabies and canine ecology studies. We extracted information on study location, year and additional details of each study such as rabies prevalence, general characteristics of offending dogs, dog vaccination status and health-seeking behaviours.FindingsBetween 1978 and 2020, 90 published articles met our inclusion criteria. The prevalence of rabies virus antigen detection varied between 3% and 28%, with more studies in the north. Most bites were unprovoked from dog bite studies (36.4%-97%), by dogs with low vaccination rates (12–38%). A more significant proportion of biting dogs were owned (31–90%). Laboratory confirmation for biting was available for only a small proportion of studies (6%; n = 2/32). Of the dogs surveyed during ecology studies, indigenous dogs accounted for the majority (62–98%), used mostly for security purposes (52–98%), with the vaccination rate between 15% and 38% in most states. Studies conducted in areas distant from rabies diagnostic facilities accounted for more human rabies cases and fewer dog rabies cases.ConclusionSignificant improvements are necessary to achieve the elimination of human rabies mediated via dogs by 2030.     

The pathogenesis of lyme neuroborreliosis: from infection to inflammation

This review describes the current knowledge of the pathogenesis of acute Lyme neuroborreliosis (LNB), from invasion to inflammation of the central nervous system. Borrelia burgdorferi (B.b.) enters the host through a tick bite on the skin and may disseminate from there to secondary organs, including the central nervous system. To achieve this, B.b. first has to evade the hostile immune system. In a second step, the borrelia have to reach the central nervous system and cross the blood-brain barrier. Once in the cerebrospinal fluid (CSF), the spirochetes elicit an inflammatory response. We describe current knowledge about the infiltration of leukocytes into the CSF in LNB. In the final section, we discuss the mechanisms by which the spirochetal infection leads to the observed neural dysfunction. To conclude, we construct a stringent concept of the pathogenesis of LNB.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

ConA的抗着床效应

本文用凝集素为探针,探索糖复合物在胚泡着床中的作用,报道了与甘露糖苷有专一结合的伴刀豆凝集素(ConA)有明显的抗小鼠胚泡着床作用。妊娠4d的小鼠,每只子宫角中注入Con A 25μg,22只子宫角中只有4只子宫角有胚泡着床,着床率为18.2％,与生理盐水对照组的着床率87.5％相比有明显差异。将相同剂量的Con A先与0.4mol/L α-甲基-D-甘露糖苷温育1—2h后再注入子宫,20只子宫角中有15只子宫角有胚泡着床,着床率提高到75％。用辣根过氧化物酶直接标记法证明,着床前子宫内膜细胞表面有Con A受体存在,并随着妊娠天数而增加,尤其是间质细胞,发情期时时为阴性反应,到着床期蜕膜细胞膜表面呈现出大量Con A受体。提示精复合物在着床中的重要作用。与甘露糖苷同样专一结合的,但寡糖结构专一性与Con A不同的豌豆凝集素注入子宫则无抗着床效应,着床率为85.7％。由此可以推测,N-连接的包含二个未被取代的或只在C-2位被取代的α-甘露糖苷的寡糖参于胚泡与子宫内膜相互作用的着床过程。     

Description and quantification of pteropod shell dissolution: a sensitive bioindicator of ocean acidification

Anthropogenic ocean acidification is likely to have negative effects on marine calcifying organisms, such as shelled pteropods, by promoting dissolution of aragonite shells. Study of shell dissolution requires an accurate and sensitive method for assessing shell damage. Shell dissolution was induced through incubations in CO₂‐enriched seawater for 4 and 14 days. We describe a procedure that allows the level of dissolution to be assessed and classified into three main types: Type I with partial dissolution of the prismatic layer; Type II with exposure of underlying crossed‐lamellar layer, and Type III, where crossed‐lamellar layer shows signs of dissolution. Levels of dissolution showed a good correspondence to the incubation conditions, with the most severe damage found in specimens held for 14 days in undersaturated condition (Ω ~ 0.8). This methodology enables the response of small pelagic calcifiers to acidified conditions to be detected at an early stage, thus making pteropods a valuable bioindicator of future ocean acidification.