Parallel reverse genetic screening in mutant human cells using transcriptomics

Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease‐relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large‐scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.     

Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein

We examined the bactericidal activity of two proteins that are abundant in the cytoplasmic granules of human eosinophils, major basic protein (MBP) and eosinophil cationic protein (ECP). Unlike the human neutrophil's peptide defensins, both MBP and ECP killed stationary phase Staphylococcus aureus 502A in a simple nutrient-free buffer solution. Although MBP also killed Escherichia coli ML-35 with considerable efficacy under these experimental conditions, the in vitro activity of ECP against E. coli was considerably enhanced if mid-logarithmic phase bacteria replaced stationary phase organisms or if the assay medium was enriched with trypticase soy broth. The antibacterial activity of both eosinophil proteins was modulated by incubation time, protein concentration, temperature and pH. A pBR322-transformed derivative of E. coli ML-35 was used to examine the effects of ECP and MBP on integrity of the bacterial inner membrane (IM) and outer membrane. Although both MBP and ECP caused outer and inner membrane permeabilization when nutrients were present, only MBP was effective under nutrient-free conditions. Two proton ionophores (DNP and carbonyl cyanide m-chlorophenyl hydrazone) protected E. coli from the bactericidal effects of ECP but not from MBP. These findings establish that MBP and ECP have bactericidal properties and suggest that these proteins kill E. coli by similar but nonidentical mechanisms marked by an attack on the target cell's membranes. In view of evidence that high concentrations of ECP and MBP exist in cytoplasmic granules whose contents are translocated to phagocytic vacuoles, we suggest that MBP and ECP contribute to the eosinophil's ability to kill ingested bacteria.     

The structure of the rabbit macrophage defensin genes and their organ-specific expression

Defensins are a family of microbicidal and cytotoxic peptides abundant in the lysosomal granules of mammalian phagocytes. We present the cDNA and genomic sequences of two rabbit defensins, macrophage cationic peptides MCP-1 and MCP-2. Their cDNA and genomic sequences are highly homologous, reflecting the homology between the two defensins (32 of 33 amino acids). The MCP genes are closely linked (within 13 kb) suggesting that they evolved by a recent tandem gene duplication. Their cDNA sequences indicate that the peptides are synthesized as 95 amino acid prepro-MCPs, consistent with their lysosomal location. The MCP genes are separated into three exons encoding distinct domains: the 5' untranslated region, the prepropeptide domain, and the mature defensin sequence. Fully developed polymorphonuclear leukocytes, short-lived phagocytes with limited capacity for protein and nucleic acid synthesis, contained MCPs but lacked MCP mRNA. MCP mRNA was found in bone marrow and spleen, organs which contained immature polymorphonuclear leukocytes. MCP and MCP mRNA were detected in lung macrophages, but not in macrophages from other organs, nor in monocytes, the putative macrophage precursors. In macrophages, the expression of MCPs appears to be a marker of lung-specific differentiation.     

Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate

Lifelong neurogenesis in vertebrates relies on stem cells producing proliferation zones that contain neuronal precursors with distinct fates. Proliferation zones in the adult zebrafish brain are located in distinct regions along its entire anterior-posterior axis. We show a previously unappreciated degree of conservation of brain proliferation patterns among teleosts, suggestive of a teleost ground plan. Pulse chase labeling of proliferating populations reveals a centrifugal movement of cells away from their places of birth into the surrounding mantle zone. We observe tangential migration of cells born in the ventral telencephalon, but only a minor rostral migratory stream to the olfactory bulb. In contrast, the lateral telencephalic area, a domain considered homologous to the mammalian dentate gyrus, shows production of interneurons and migration as in mammals. After a 46-day chase, newborn highly mobile cells have moved into nuclear areas surrounding the proliferation zones. They often show HuC/D immunoreactivity but importantly also more specific neuronal identities as indicated by immunoreactivity for tyrosine hydroxylase, serotonin and parvalbumin. Application of a second proliferation marker allows us to recognize label-retaining, actively cycling cells that remain in the proliferation zones. The latter population meets two key criteria of neural stem cells: label retention and self renewal.     

Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens

A key target of many intracellular pathogens is the macrophage. Although macrophages can generate antimicrobial activity, neutrophils have been shown to have a key role in host defense, presumably by their preformed granules containing antimicrobial agents. Yet the mechanism by which neutrophils can mediate antimicrobial activity against intracellular pathogens such as Mycobacterium tuberculosis has been a long-standing enigma. We demonstrate that apoptotic neutrophils and purified granules inhibit the growth of extracellular mycobacteria. Phagocytosis of apoptotic neutrophils by macrophages results in decreased viability of intracellular M. tuberculosis. Concomitant with uptake of apoptotic neutrophils, granule contents traffic to early endosomes, and colocalize with mycobacteria. Uptake of purified granules alone decreased growth of intracellular mycobacteria. Therefore, the transfer of antimicrobial peptides from neutrophils to macrophages provides a cooperative defense strategy between innate immune cells against intracellular pathogens and may complement other pathways that involve delivery of antimicrobial peptides to macrophages.     

Despite having a common P1 Leu, eglin C inhibits alpha-lytic proteinase a million-fold more strongly than does turkey ovomucoid third domain

Results of the inhibition of alpha-lytic proteinase by two standard mechanism serine proteinase inhibitors, turkey ovomucoid third domain (OMTKY3) and eglin C, and many of their variants are presented. Despite similarities, including an identical P1 residue (Leu) in their primary contact regions, OMTKY3 and eglin C have vastly different association equilibrium constants toward alpha-lytic proteinase, with Ka values of 1.8 x 10(3) and 1.2 x 10(9) M(-1), respectively. Although 12 of the 13 serine proteinases tested in our laboratory for inhibition by OMTKY3 and eglin C are more strongly inhibited by the latter, the million-fold difference observed here with alpha-lytic proteinase is the largest we have seen. The million-fold stronger inhibition by eglin C is retained when the Ka values of the P1 Gly, Ala, Ser, and Ile variants of OMTKY3 and eglin C are compared. Despite the small size of the S1 pocket in alpha-lytic proteinase, interscaffolding additivity for OMTKY3 and eglin C holds well for the four P1 residues tested here. To better understand this difference, we measured Ka values for other OMTKY3 variants, including some that had residues elsewhere in their contact region that corresponded to those of eglin C. Assuming intrascaffolding additivity and using the Ka values obtained for OMTKY3 variants, we designed an OMTKY3-based inhibitor of alpha-lytic proteinase that was predicted to inhibit 10,000-fold more strongly than wild-type OMTKY3. This variant (K13A/P14E/L18A/R21T/N36D OMTKY3) was prepared, and its Ka value was measured against alpha-lytic proteinase. The measured Ka value was in excellent agreement with the predicted one (1.1 x 10(7) and 2.0 x 10(7) M(-1), respectively). Computational protein docking results are consistent with the view that the backbone conformation of eglin C is not significantly altered in the complex with alpha-lytic proteinase. They also show that the strong binding for eglin C correlates well with more favorable atomic contact energy and desolvation energy contributions as compared to OMTKY3.     

Regulation of iron acquisition and iron distribution in mammals

Both cellular iron deficiency and excess have adverse consequences. To maintain iron homeostasis, complex mechanisms have evolved to regulate cellular and extracellular iron concentrations. Extracellular iron concentrations are controlled by a peptide hormone hepcidin, which inhibits the supply of iron into plasma. Hepcidin acts by binding to and inducing the degradation of the cellular iron exporter, ferroportin, found in sites of major iron flows: duodenal enterocytes involved in iron absorption, macrophages that recycle iron from senescent erythrocytes, and hepatocytes that store iron. Hepcidin synthesis is in turn controlled by iron concentrations, hypoxia, anemia and inflammatory cytokines. The molecular mechanisms that regulate hepcidin production are only beginning to be understood, but its dysregulation is involved in the pathogenesis of a spectrum of iron disorders. Deficiency of hepcidin is the unifying cause of hereditary hemochromatoses, and excessive cytokine-stimulated hepcidin production causes hypoferremia and contributes to anemia of inflammation.