In vivo bioassay to test the pathogenicity of missense human AIP variants

Background Heterozygous germline loss-of-function mutations in the aryl hydrocarbon receptor-interacting protein gene (AIP) predispose to childhood-onset pituitary tumours. The pathogenicity of missense variants may pose difficulties for genetic counselling and family follow-up. Objective To develop an in vivo system to test the pathogenicity of human AIP mutations using the fruit fly Drosophila melanogaster. Methods We generated a null mutant of the Drosophila AIP orthologue, CG1847, a gene located on the Xchromosome, which displayed lethality at larval stage in hemizygous knockout male mutants (CG1847exon1_3 ). We tested human missense variants of ‘unknown significance’, with ‘pathogenic’ variants as positive control. Results We found that human AIP can functionally substitute for CG1847, as heterologous overexpression of human AIP rescued male CG1847exon1_3 lethality, while a truncated version of AIP did not restore viability. Flies harbouring patient-specific missense AIP variants (p.C238Y, p.I13N, p.W73R and p.G272D) failed to rescue CG1847exon1_3 mutants, while seven variants (p.R16H, p.Q164R, p.E293V, p.A299V, p.R304Q, p.R314W and p.R325Q) showed rescue, supporting a non-pathogenic role for these latter variants corresponding to prevalence and clinical data. Conclusion Our in vivo model represents a valuable tool to characterise putative disease-causing human AIP variants and assist the genetic counselling and management of families carrying AIP variants

Polymeric nanobiotics as a novel treatment for mycobacterial infections

Mycobacterium tuberculosis (Mtb) remains a major challenge to global health, made worse by the spread of multi-drug resistance. Currently, the efficacy and safety of treatment is limited by difficulties in achieving and sustaining adequate tissue antibiotic concentrations while limiting systemic drug exposure to tolerable levels. Here we show that nanoparticles generated from a polymer-antibiotic conjugate (‘nanobiotics’) deliver sustained release of active drug upon hydrolysis in acidic environments, found within Mtb-infected macrophages and granulomas, and can, by encapsulation of a second antibiotic, provide a mechanism of synchronous drug delivery. Nanobiotics are avidly taken up by infected macrophages, enhance killing of intracellular Mtb, and are efficiently delivered to granulomas and extracellular mycobacterial cords in vivo in an infected zebrafish model. We demonstrate that isoniazid (INH)-derived nanobiotics, alone or with additional encapsulation of clofazimine (CFZ), enhance killing of mycobacteria in vitro and in infected zebrafish, supporting the use of nanobiotics for Mtb therapy and indicating that nanoparticles generated from polymer-small molecule conjugates might provide a more general solution to delivering co-ordinated combination chemotherapy

The Smc5–Smc6 Complex Is Required to Remove Chromosome Junctions in Meiosis

Meiosis, a specialized cell division with a single cycle of DNA replication round and two consecutive rounds of nuclear segregation, allows for the exchange of genetic material between parental chromosomes and the formation of haploid gametes. The structural maintenance of chromosome (SMC) proteins aid manipulation of chromosome structures inside cells. Eukaryotic SMC complexes include cohesin, condensin and the Smc5–Smc6 complex. Meiotic roles have been discovered for cohesin and condensin. However, although Smc5–Smc6 is known to be required for successful meiotic divisions, the meiotic functions of the complex are not well understood. Here we show that the Smc5–Smc6 complex localizes to specific chromosome regions during meiotic prophase I. We report that meiotic cells lacking Smc5–Smc6 undergo catastrophic meiotic divisions as a consequence of unresolved linkages between chromosomes. Surprisingly, meiotic segregation defects are not rescued by abrogation of Spo11-induced meiotic recombination, indicating that at least some chromosome linkages in smc5–smc6 mutants originate from other cellular processes. These results demonstrate that, as in mitosis, Smc5-Smc6 is required to ensure proper chromosome segregation during meiosis by preventing aberrant recombination intermediates between homologous chromosomes

A Spaetzle-like role for Nerve Growth Factor β in vertebrate immunity to Staphylococcus aureus

Many key components of innate immunity to infection are shared between Drosophila and humans. However, the fly Toll ligand Spaetzle is not thought to have a vertebrate equivalent. We have found that the structurally related cystine-knot protein, nerve growth factor β (NGFβ), plays an unexpected Spaetzle-like role in immunity to Staphylococcus aureus infection in chordates. Deleterious mutations of either human NGFβ or its high-affinity receptor tropomyosin-related kinase receptor A (TRKA) were associated with severe S. aureus infections. NGFβ was released by macrophages in response to S. aureus exoproteins through activation of the NOD-like receptors NLRP3 and NLRC4 and enhanced phagocytosis and superoxide-dependent killing, stimulated proinflammatory cytokine production, and promoted calcium-dependent neutrophil recruitment. TrkA knockdown in zebrafish increased susceptibility to S. aureus infection, confirming an evolutionarily conserved role for NGFβ-TRKA signaling in pathogen-specific host immunity

A System-Wide Investigation of the Dynamics of Wnt Signaling Reveals Novel Phases of Transcriptional Regulation

Aberrant Wnt signaling has been implicated in a wide variety of cancers and many components of the Wnt signaling network have now been identified. Much less is known, however, about how these proteins are coordinately regulated. Here, a broad, quantitative, and dynamic study of Wnt3a-mediated stimulation of HEK 293 cells revealed two phases of transcriptional regulation: an early phase in which signaling antagonists were downregulated, providing positive feedback, and a later phase in which many of these same antagonists were upregulated, attenuating signaling. The dynamic expression profiles of several response genes, including MYC and CTBP1, correlated significantly with proliferation and migration (P<0.05). Additionally, their levels tracked with the tumorigenicity of colon cancer cell lines and they were significantly overexpressed in colorectal adenocarcinomas (P<0.05). Our data highlight CtBP1 as a transcription factor that contributes to positive feedback during the early phases of Wnt signaling and serves as a novel marker for colorectal cancer progression

A Spaetzle-like role for nerve growth factor beta in vertebrate immunity to Staphylococcus aureus

Many key components of innate immunity to infection are shared between Drosophila and humans. However, the fly Toll ligand Spaetzle is not thought to have a vertebrate equivalent. We have found that the structurally related cystine-knot protein, nerve growth factor β (NGFβ), plays an unexpected Spaetzle-like role in immunity to Staphylococcus aureus infection in chordates. Deleterious mutations of either human NGFβ or its high-affinity receptor tropomyosin-related kinase receptor A (TRKA) were associated with severe S. aureus infections. NGFβ was released by macrophages in response to S. aureus exoproteins through activation of the NOD-like receptors NLRP3 and NLRC4 and enhanced phagocytosis and superoxide-dependent killing, stimulated proinflammatory cytokine production, and promoted calcium-dependent neutrophil recruitment. TrkA knockdown in zebrafish increased susceptibility to S. aureus infection, confirming an evolutionarily conserved role for NGFβ-TRKA signaling in pathogen-specific host immunity

Frequent and Efficient Use of the Sister Chromatid for DNA Double-Strand Break Repair during Budding Yeast Meiosis

Studies of DNA double-strand break repair during meiosis reveal that a substantial fraction of recombination occurs between sister chromatids

Sudden cardiac death and pump failure death prediction in chronic heart failure by combining ECG and clinical markers in an integrated risk model

BACKGROUND: Sudden cardiac death (SCD) and pump failure death (PFD) are common endpoints in chronic heart failure (CHF) patients, but prevention strategies are different. Currently used tools to specifically predict these endpoints are limited. We developed risk models to specifically assess SCD and PFD risk in CHF by combining ECG markers and clinical variables. METHODS: The relation of clinical and ECG markers with SCD and PFD risk was assessed in 597 patients enrolled in the MUSIC (MUerte Súbita en Insuficiencia Cardiaca) study. ECG indices included: turbulence slope (TS), reflecting autonomic dysfunction; T-wave alternans (TWA), reflecting ventricular repolarization instability; and T-peak-to-end restitution (ΔαTpe) and T-wave morphology restitution (TMR), both reflecting changes in dispersion of repolarization due to heart rate changes. Standard clinical indices were also included. RESULTS: The indices with the greatest SCD prognostic impact were gender, New York Heart Association (NYHA) class, left ventricular ejection fraction, TWA, ΔαTpe and TMR. For PFD, the indices were diabetes, NYHA class, ΔαTpe and TS. Using a model with only clinical variables, the hazard ratios (HRs) for SCD and PFD for patients in the high-risk group (fifth quintile of risk score) with respect to patients in the low-risk group (first and second quintiles of risk score) were both greater than 4. HRs for SCD and PFD increased to 9 and 11 when using a model including only ECG markers, and to 14 and 13, when combining clinical and ECG markers. CONCLUSION: The inclusion of ECG markers capturing complementary pro-arrhythmic and pump failure mechanisms into risk models based only on standard clinical variables substantially improves prediction of SCD and PFD in CHF patients