Molecular Signatures of Hemagglutinin Stem-Directed Heterosubtypic Human Neutralizing Antibodies against Influenza A Viruses

Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs). Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.     

Polyandry in the medfly - shifts in paternity mediated by sperm stratification and mixing

Background

In the Mediterranean fruit fly (medfly), Ceratitis capitata, a highly invasive agricultural pest species, polyandry, associated with sperm precedence, is a recurrent behaviour in the wild. The absence of tools for the unambiguous discrimination between competing sperm from different males in the complex female reproductive tract has strongly limited the understanding of mechanisms controlling sperm dynamics and use.

Results

Here we use transgenic medfly lines expressing green or red fluorescent proteins in the spermatozoa, which can be easily observed and unambiguously differentiated within the female fertilization chamber. In twice-mated females, one day after the second mating, sperm from the first male appeared to be homogenously distributed all over the distal portion of each alveolus within the fertilization chamber, whereas sperm from the second male were clearly concentrated in the central portion of each alveolus. This distinct stratified sperm distribution was not maintained over time, as green and red sperm appeared homogeneously mixed seven days after the second mating. This dynamic sperm storage pattern is mirrored by the paternal contribution in the progeny of twice-mated females.

Conclusions

Polyandrous medfly females, unlike Drosophila, conserve sperm from two different mates to fertilize their eggs. From an evolutionary point of view, the storage of sperm in a stratified pattern by medfly females may initially favour the fresher ejaculate from the second male. However, as the second male's sperm gradually becomes depleted, the sperm from the first male becomes increasingly available for fertilization. The accumulation of sperm from different males will increase the overall genetic variability of the offspring and will ultimately affect the effective population size. From an applicative point of view, the dynamics of sperm storage and their temporal use by a polyandrous female may have an impact on the Sterile Insect Technique (SIT). Indeed, even if the female's last mate is sterile, an increasing proportion of sperm from a previous mating with a fertile male may contribute to sire viable progeny.

     

Development of a Nuclear Transformation System for Oleaginous Green Alga Lobosphaera (Parietochloris) incisa and Genetic Complementation of a Mutant Strain,Deficient in Arachidonic Acid Biosynthesis

Microalgae are considered a promising source for various high value products, such as carotenoids, ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The unicellular green alga Lobosphaera (Parietochloris) incisa is an outstanding candidate for the efficient phototrophic production of arachidonic acid (AA), an essential ω-6 PUFA for infant brain development and a widely used ingredient in the baby formula industry. Although phototrophic production of such algal products has not yet been established, estimated costs are considered to be 2–5 times higher than competing heterotrophic production costs. This alga accumulates unprecedented amounts of AA within triacylglycerols and the molecular pathway of AA biosynthesis in L. incisa has been previously elucidated. Thus, progress in transformation and metabolic engineering of this high value alga could be exploited for increasing the efficient production of AA at competitive prices. We describe here the first successful transformation of L. incisa using the ble gene as a selection marker, under the control of the endogenous RBCS promoter. Furthermore, we have succeeded in the functional complementation of the L. incisa mutant strain P127, containing a mutated, inactive version of the delta-5 (Δ5) fatty acid desaturase gene. A copy of the functional Δ5 desaturase gene, linked to the ble selection marker, was transformed into the P127 mutant. The resulting transformants selected for zeocine resistant, had AA biosynthesis partially restored, indicating the functional complementation of the mutant strain with the wild-type gene. The results of this study present a platform for the successful genetic engineering of L. incisa and its long-chain PUFA metabolism.     

Combinatorial and Computational Approaches to Identify Interactions of Macrophage Colony-stimulating Factor (M-CSF) and Its Receptor c-FMS

The molecular interactions between macrophage colony-stimulating factor (M-CSF) and the tyrosine kinase receptor c-FMS play a key role in the immune response, bone metabolism, and the development of some cancers. Because no x-ray structure is available for the human M-CSF·c-FMS complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-FMS. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSF_M) single mutants and screened this library to isolate variants with reduced affinity for c-FMS using FACS. Sequencing yielded a number of single M-CSF_M variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSF_M structure and to classify the mutations into three groups as follows: those that significantly decrease protein stability; those that destroy favorable intermolecular interactions; and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSF_M and three variants were produced as soluble proteins; their affinity and structure were analyzed; and very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSF_M residues critical for M-CSF·c-FMS interactions, we have laid down the basis for a deeper understanding of the M-CSF·c-FMS signaling mechanism and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF·c-FMS binding interface.     

Differences in inhibition by IDF45 (an inhibitory diffusible factor) of early RNA synthesis stimulation induced by pp60 v-src and various mitogens.

Factors inhibiting cell growth have been isolated from different cell types. However, little information is available concerning their mode of action. A novel growth inhibitory factor of 45 kDa (IDF45) was recently purified to homogeneity from medium conditioned by 3T3 cells. This molecule was able to inhibit DNA synthesis and the growth of chick embryo fibroblasts (CEF) in a reversible manner. By contrast, DNA synthesis stimulated by v-src expression in CEF was poorly inhibited by IDF45. In order to gain further insight into the IDF45 mode of action in normal and transformed CEF, we compared the effects of IDF45 on early stimulation of RNA synthesis induced in CEF by different mitogenic factors and by v-src gene expression. Stimulation, by serum, of RNA synthesis was inhibited by IDF45; however, inhibition increased when cells were preincubated with IDF45 before addition of serum and cell labeling for 2 h. IDF45 was also able to inhibit partially the stimulation of RNA synthesis induced by PMA and PDGF but was unable to inhibit stimulation of RNA synthesis induced by insulin and v-src expression. By contrast, stimulation of RNA synthesis induced by IGF-I was rapidly 100% inhibited by IDF45. The effect of IDF45 on DNA synthesis stimulated by the different mitogens was also determined and was correlated with the effect of IDF45 on RNA synthesis. These results suggest that the modes of action of IDF45 on stimulation of RNA synthesis by v-src and by insulin are similar. Our present results agree with others showing the bifunctional activity of IDF45 as an IGF-binding protein and as an inhibitory molecule in DNA stimulation induced by serum.     

IGFBP-1, an insulin like growth factor binding protein, is a cell growth inhibitor

A novel cell growth inhibitor, IDF45 (inhibitory diffusible factor), was recently purified to apparent homogeneity. It is a bifunctional molecule: able to bind Insulin like growth factor (IGF) and to 100% inhibit DNA synthesis stimulated by serum in fibroblasts. It was of interest to verify whether other members of the IGF-binding protein (IGFBP) family show the same bifunctional growth inhibitory properties. In this paper we show that purified IGFBP-1 derived from amniotic fluid is a cell growth inhibitor. In chick embryo fibroblasts, it inhibited DNA synthesis stimulated by serum. However the stimulation was maximally 60% inhibited and half of the inhibition was observed with 100ng/ml IGFBP-1. So the specific activity of IGFBP-1 is lower than that of IDF45. IGFBP-1 also reversibly prevented the CEF growth. In the same cells IGFBP-1 inhibited DNA synthesis stimulated by IGF-I. We demonstrated that the same protein IGFBP-1 is able to inhibit DNA synthesis stimulated by serum and by IGF-I. The possibility that IGFBP-1 is a bifunctional molecule is discussed.     

Presence of IDF45 (mlGFBP-3) binding sites on chick embryo fibroblasts.

IDF45 (inhibitory diffusible factor) a mouse insulin-like growth factor binding protein (mlGFBP-3) has been shown to 100 percent inhibit DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF). Our previous results suggested that this large inhibition by IDF45 of serum stimulation was not just the result of its inhibitory activity toward IGF present in serum. The addition of Mn2+ (10(-3)M) in the incubation medium enables us to show the presence of numerous binding sites per cells (about 60,000) of mlGFBP-3. However the dissociation constant (10(-8)M) indicated that this mouse IGFBP-3 bound to the membrane with low affinity. These findings lend new support to the assumption of the bifunctional property of IGFBP-3, which would have an effect outside the cell (binding of IGF in the medium) and another effect within cells or on the surface.     

Pollinator‐mediated selection on floral size and tube color in Linum pubescens: Can differential behavior and preference in different times of the day maintain dimorphism?

Diversity of flower traits is often proposed as the outcome of selection exerted by pollinators. Positive directional pollinator‐mediated selection on floral size has been widely shown to reduce phenotypic variance. However, the underlying mechanism of maintaining within‐population floral color polymorphism is poorly understood. Divergent selection, mediated by different pollinators or by both mutualists and antagonists, may create and maintain such polymorphism, but it has rarely been shown to result from differential behavior of one pollinator. We tested whether different behaviors of the same pollinators in morning and evening are associated with dimorphic floral trait in Linum pubescens, a Mediterranean annual plant that exhibits variable within‐population frequencies of dark‐ and light‐colored flower tubes. Usia bicolor bee‐flies, the major pollinators of L. pubescens, are mostly feeding in the flower in the morning, while in the evening they are mostly visiting the flowers for mating. In 2 years of studying L. pubescens in a single large population in the Carmel, Israel, we found in one year that dark‐centered flowers received significantly higher fraction of visits in the morning. Fitness was positively affected by number of visits, but no fitness differences were found between tube‐color morphs, suggesting that both morphs have similar pollination success. Using mediation analysis, we found that flower size was under positive directional pollinator‐mediated selection in both years, but pollinator behavior did not explain entirely this selection, which was possibly mediated also by other agents, such as florivores or a‐biotic stresses. While most pollinator‐mediated selection studies show that flower size signals food reward, in L. pubescens, it may also signal for mating place, which may drive positive selection. While flower size found to be under pollinator‐mediated selection in L. pubescens, differential behavior of the pollinators in morning and evening did not seem to explain flower color polymorphism.