Next-generation sequencing to generate interactome datasets

Next-generation sequencing has not been applied to protein-protein interactome network mapping so far because the association between the members of each interacting pair would not be maintained in en masse sequencing. We describe a massively parallel interactome-mapping pipeline, Stitch-seq, that combines PCR stitching with next-generation sequencing and used it to generate a new human interactome dataset. Stitch-seq is applicable to various interaction assays and should help expand interactome network mapping.     

Activation of hypothalamic RIP‐Cre neurons promotes beiging of WAT via sympathetic nervous system

Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat‐insulin‐promoter‐Cre (RIP‐Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT. Genetic ablation of APPL2 in RIP‐Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP‐Cre neurons, inactivation of VMH AMPK, or treatment with a β3‐adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP‐Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP‐Cre neurons, in which the APPL2–AMPK signaling axis is crucial for this defending mechanism to cold and obesity.     

Visualization and interpretation of birth defects data using linked micromap plots

BACKGROUND: Many states have implemented birth defects surveillance systems to monitor and disseminate information regarding birth defects. However, many of these states rely on tabular methods to disseminate statistical birth defects summaries. An innovative presentation technique for birth defect data that portrays the information in a joint geographical and statistical context is the linked micromap (LM) plot. METHODS: LM plots were generated for oral cleft data at two geographical resolutions—USA states and counties of Utah. The LM plots also included demographic and behavioral risk data. RESULTS: A LM plot for the USA reveals spatial patterns indicating higher oral cleft occurrence in the southwest and the midwest and lower occurrence in the east. The LM plot also indicates relationships between oral cleft occurrence and maternal smoking rates and the proportion of American Indians and Alaskan Natives. In particular, the five states with the highest oral cleft occurrence had a higher proportion of American Indians and Alaskan Natives. Among the 15 states with the highest oral cleft occurrence, nine had a smoking rate of 16% or higher while among the 15 states with the lowest oral cleft occurrence only one state had a smoking rate greater than 16%. The LM plot for the state of Utah shows no clear geographic pattern, due perhaps to a relatively small number of cases in a limited geographic area. CONCLUSIONS: LM plots are effective in representing complex and large volume birth defects data. Integration to birth defects surveillance systems will improve both presentation and interpretation. Birth Defects Research (Part A), 2008. © 2007 Wiley‐Liss, Inc.     

Morphology and diagnosis of some fourth-stage larvae of cyathostomines (Nematoda: Strongyloidea) in donkeys Equus asinus L. from Ethiopia

Fourth-stage larvae of four species of the Cyathostominae Nicoll, 1927 parasitic in donkeys Equus asinus L. from Ethiopia were identified mainly using moulting specimens. They are Cylicocyclus asini Matthee, Krecek & Gibbons, 2001, C. auriculatus (Looss, 1900) Chaves, 1930, Cyathostomum tetracanthum (Mehlis, 1831) Molin, 1861 (sensu Looss, 1900) and Cylindropharynx brevicauda Leiper, 1911. The larva of Cylicocyclus asini is similar to those of C. nassatus (Looss, 1900) Chaves, 1930 and C. leptostomum Kotlán, 1920, but differs from the former by the shape of the dorsal tooth in the oesophageal funnel, which is notably smaller than in C. asini but similar to that of C. leptostomum. However, the larva of C. asini differs from that of C. leptostomum in the size of the buccal capsule, which is notably larger than in C. leptostomum but similar to that of C. nassatus. The larva of C. auriculatus is very similar to that of C. insigne (Boulenger, 1917) Chaves, 1930. The larva of Cyathostomum tetracanthum is similar to those of Cylicostephanus bidentatus (Ihle, 1925) Lichtenfels, 1975, C. goldi (Boulenger, 1917) Lichtenfels, 1975 and Cyathostomum catinatum Looss, 1900. The larva of Cylindropharynx brevicauda is similar to that of Petrovinema skrjabini (Ershov, 1930) Ershov, 1943 but smaller in size. Including the four cyathostomine species identified in the present study, 36 species belonging to 13 genera have so far been described and identified of a total of 64 recognised and confirmed species of equine strongyles.     

Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa

Background  

While the larval-juvenile transition (metamorphosis) in the spionid polychaete Pseudopolydora vexillosa involves gradual morphological changes and does not require substantial development of juvenile organs, the opposite occurs in the barnacle Balanus amphitrite. We hypothesized that the proteome changes during metamorphosis in the spionids are less drastic than that in the barnacles. To test this, proteomes of pre-competent larvae, competent larvae (ready to metamorphose), and juveniles of P. vexillosa were compared using 2-dimensional gel electrophoresis (2-DE), and they were then compared to those of the barnacle.     

NHS-A isoform of the NHS gene is a novel interactor of ZO-1

Mutations in the NHS (Nance-Horan Syndrome) gene lead to severe congenital cataracts, dental defects and sometimes mental retardation. NHS encodes two protein isoforms, NHS-A and -1A that display cell-type dependent differential expression and localization. Here we demonstrate that of these two isoforms, the NHS-A isoform associates with the cell membrane in the presence of intercellular contacts and it immunoprecipitates with the tight junction protein ZO-1 in MDCK (Madin Darby Canine Kidney) epithelial cells and in neonatal rat lens. The NHS-1A isoform however is a cytoplasmic protein. Both Nhs isoforms are expressed during mouse development. Immunolabelling of developing mouse with the anti-NHS antibody that detects both isoforms revealed the protein in the developing head including the eye and brain. It was primarily expressed in epithelium including neural epithelium and certain vascular endothelium but only weakly expressed in mesenchymal cells. In the epithelium and vascular endothelium the protein associated with the cell membrane and co-localized with ZO-1, which indirectly indicates expression of the Nhs-A isoform in these structures. Membrane localization of the protein in the lens vesicle similarly supports Nhs-A expression. In conclusion, the NHS-A isoform of NHS is a novel interactor of ZO-1 and may have a role at tight junctions. This isoform is important in mammalian development especially of the organs in the head.     

Genomic analysis of SARS-CoV-2 reveals local viral evolution in Ghana

The confirmed case fatality rate for the coronavirus disease 2019 (COVID-19) in Ghana has dropped from a peak of 2% in March to be consistently below 1% since May 2020. Globally, case fatality rates have been linked to the strains/clades of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a specific country. Here we present 46 whole genomes of SARS-CoV-2 circulating in Ghana, from two separate sequencing batches: 15 isolates from the early epidemic (March 12–April 1 2020) and 31 from later time-points ( 25–27 May 2020). Sequencing was carried out on an Illumina MiSeq system following an amplicon-based enrichment for SARS-CoV-2 cDNA. After genome assembly and quality control processes, phylogenetic analysis showed that the first batch of 15 genomes clustered into five clades: 19A, 19B, 20A, 20B, and 20C, whereas the second batch of 31 genomes clustered to only three clades 19B, 20A, and 20B. The imported cases (6/46) mapped to circulating viruses in their countries of origin, namely, India, Hungary, Norway, the United Kingdom, and the United States of America. All genomes mapped to the original Wuhan strain with high similarity (99.5–99.8%). All imported strains mapped to the European superclade A, whereas 5/9 locally infected individuals harbored the B4 clade, from the East Asian superclade B. Ghana appears to have 19B and 20B as the two largest circulating clades based on our sequence analyses. In line with global reports, the D614G linked viruses seem to be predominating. Comparison of Ghanaian SARS-CoV-2 genomes with global genomes indicates that Ghanaian strains have not diverged significantly from circulating strains commonly imported into Africa. The low level of diversity in our genomes may indicate lower levels of transmission, even for D614G viruses, which is consistent with the relatively low levels of infection reported in Ghana.     

Involvement of claudin-7 in HIV infection of CD4(-) cells

Background

The antibacterial activity of host defense peptides (HDP) is largely mediated by permeabilization of bacterial membranes. The lipid membrane of enveloped viruses might also be a target of antimicrobial peptides. Therefore, we screened a panel of naturally occurring HDPs representing different classes for inhibition of early, Env-independent steps in the HIV replication cycle. A lentiviral vector-based screening assay was used to determine the inhibitory effect of HDPs on early steps in the replication cycle and on cell metabolism.

Results

Human LL37 and porcine Protegrin-1 specifically reduced lentiviral vector infectivity, whereas the reduction of luciferase activities observed at high concentrations of the other HDPs is primarily due to modulation of cellular activity and/or cytotoxicity rather than antiviral activity. A retroviral vector was inhibited by LL37 and Protegrin-1 to similar extent, while no specific inhibition of adenoviral vector mediated gene transfer was observed. Specific inhibitory effects of Protegrin-1 were confirmed for wild type HIV-1.

Conclusion

Although Protegrin-1 apparently inhibits an early step in the HIV-replication cycle, cytotoxic effects might limit its use as an antiviral agent unless the specificity for the virus can be improved.