A Slc38a8 Mouse Model of FHONDA Syndrome Faithfully Recapitulates the Visual Deficits of Albinism Without Pigmentation Defects.

Purpose: We aimed to generate and phenotype a mouse model of foveal hypoplasia, optic nerve decussation defects, and anterior segment dysgenesis (FHONDA), a rare disease associated with mutations in Slc38a8 that causes severe visual alterations similar to albinism without affecting pigmentation. Methods: The FHONDA mouse model was generated with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology using an RNA guide targeting the Scl38a8 murine locus. The resulting mice were backcrossed to C57BL/6J. Melanin content was measured using spectrophotometry. Retinal cell architecture was analyzed through light and electron microscopy. Retinal projections to the brain were evaluated with anterograde labelling in embryos and adults. Visual function was assessed by electroretinography (ERG) and the optomotor test (OT). Results: From numerous Slc38a8 mouse mutant alleles generated, we selected one that encodes a truncated protein (p.196Pro*, equivalent to p.199Pro* in the human protein) closely resembling a mutant allele described in patients (p.200Gln*). Slc38a8 mutant mice exhibit wild-type eye and coat pigmentation with comparable melanin content. Subcellular abnormalities were observed in retinal pigment epithelium cells of Slc38a8 mutant mice. Anterograde labeling experiments of retinal projections in embryos and adults showed a reduction of ipsilateral fibers. Functional visual analyses revealed a decreased ERG response in scotopic conditions and a reduction of visual acuity in mutant mice measured by OT. Conclusions: Slc38a8 mutant mice recapitulate the phenotype of patients with FHONDA concerning their normal pigmentation and their abnormal visual system, in the latter being a hallmark of all types of albinism. These mice will be helpful in better understanding the pathophysiology of this genetic condition.

Embryonic disc formation following post-hatching bovine embryo development in vitro.

Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum- and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum- and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56% of the embryos and ~25% developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.

Trimeric heptad repeat synthetic peptides HR1 and HR2 efficiently inhibit HIV-1 entry.

The trimeric heptad repeat domains HR1 and HR2 of the human immunodeficiency virus 1 (HIV-1) gp41 play a key role in HIV-1-entry by membrane fusion. To develop efficient inhibitors against this step, the corresponding trimeric-N36 and C34 peptides were designed and synthesized. Analysis by circular dichroism of monomeric and trimeric N36 and C34 peptides showed their capacities to adopt α-helical structures and to establish physical interactions. At the virological level, while trimeric-C34 conserves the same high anti-fusion activity as monomeric-C34, trimerization of N36-peptide induced a significant increase, reaching 500-times higher in anti-fusion activity, against R5-tropic virus-mediated fusion. This result was associated with increased stability of the N36 trimer peptide with respect to the monomeric form, as demonstrated by the comparative kinetics of their antiviral activities during 6-day incubation in a physiological medium. Collectively, our findings demonstrate that while the trimerization of C34 peptide had no beneficial effect on its stability and antiviral activity, the trimerization of N36 peptide strengthened both stability and antiviral activity. This approach, promotes trimers as new promising HIV-1 inhibitors and point to future development aimed toward innovative peptide fusion inhibitors, microbicides or as immunogens.

Delivering wasp venom for cancer therapy.

Cytolytic peptides with potential therapeutic properties have appeared during the last three decades. However, the use of these natural weapons is relatively narrow due to their non-specific cytolytic activity as well as their rapid degradation and excretion when injected in blood. In order to rescue the use of these lytic peptides, we have designed pro-cytotoxic systems based on cytotoxic peptides conjugated to poly(l-glutamic acid) PGA polymer through specific cleavage sequences that are sensitive over-expressed tumor proteases, such as the metalloproteinase-2 (MMP-2) or cathepsin B. The potent cytotoxic peptide tested here, Mitoparan, is inactive when conjugated to the polymer and then become active again once released through the tumor proteases. Furthermore, this pro-cytotoxic system was decorated by a particular targeting peptide which binds to HER2 receptors over-expressed in some types of breast tumor cells, thereby increasing the selective release of cytolytic peptides inside tumor cell with exquisite spatiotemporal control. In this way, the system would improve the maximum tolerated dose and the pharmacokinetic parameters of cytotoxic peptides in vivo.

Dual system for the central nervous system targeting and blood-brain barrier transport of a selective prolyl oligopeptidase inhibitor.

Less than 2% of all potential neurotherapeutics cross the blood-brain barrier (BBB). Here, we sought to build a construct with the capacity to cross this barrier, to behave as a chemical delivery system, and, once inside the central nervous system, to be transformed and then act as an enzyme inhibitor. With all this in mind, here, we describe the entire process to obtain such a compound, from the initial candidate selection to preparation of the compound library and posterior evaluation and final selection of the most promising candidates in terms of selectivity, serum stability, and BBB-transport.

Delivery of gold nanoparticles to the brain by conjugation with a peptide that recognizes the transferrin receptor.

The treatment of Alzheimer's disease and many other brain-related disorders is limited because of the presence of the blood-brain barrier, which highly regulate the crossing of drugs. Metal nanoparticles have unique features that could contribute to the development of new therapies for these diseases. Nanoparticles have the capacity to carry several molecules of a drug; furthermore, their unique physico-chemical properties allow, for example, photothermal therapy to produce molecular surgery to destroy tumor cells and toxic structures. Recently, we demonstrated that gold nanoparticles conjugated to the peptide CLPFFD are useful to destroy the toxic aggregates of ß-amyloid, similar to the ones found in the brains of patients with Alzheimer's disease. However, nanoparticles, like many other compounds, have null or very low capacity to cross the blood-brain barrier. In order to devise a strategy to improve drug delivery to the brain, here we introduced the peptide sequence THRPPMWSPVWP into the gold nanoparticle-CLPFFD conjugate. This peptide sequence interacts with the transferrin receptor present in the microvascular endothelial cells of the blood-brain barrier, thus causing an increase in the permeability of the conjugate in brain, as shown by experiments in vitro and in vivo. Our results are highly relevant for the therapeutic applications of gold nanoparticles for molecular surgery in the treatment of neurodegenerative diseases such as Alzheimer's disease.

Selenomethionine incorporation into amyloid sequences regulates fibrillogenesis and toxicity.

BACKGROUND: The capacity of a polypeptide chain to engage in an amyloid formation process and cause a conformational disease is contained in its sequence. Some of the sequences undergoing fibrillation contain critical methionine (Met) residues which in vivo can be synthetically substituted by selenomethionine (SeM) and alter their properties. METHODOLOGY/PRINCIPAL FINDINGS: Using peptide synthesis, biophysical techniques and cell viability determinations we have studied the effect of the substitution of methionine (Met) by selenomethionine (SeM) on the fibrillogenesis and toxic properties of Aß40 and HuPrP(106-140). We have found that the effects display site-specificity and vary from inhibition of fibrillation and decreased toxicity ([SeM(35)]Aß40, [SeM(129)]HuPrP(106-140) and [SeM(134)]HuPrP(106-140)), retarded assembly, modulation of polymer shape and retention of toxicity ([SeM(112)]HuPrP(106-140) to absence of effects ([SeM(109)]HuPrP(106-140)). CONCLUSIONS/SIGNIFICANCE: This work provides direct evidence that the substitution of Met by SeM in proamyloid sequences has a major impact on their self-assembly and toxic properties, suggesting that the SeM pool can play a major role in dictating the allowance and efficiency of a polypeptide chain to undergo toxic polymerization.

Genetic polymorphisms among C57BL/6 mouse inbred strains.

Mice from the inbred C57BL/6 strain have been commonly used for the generation and analysis of transgenic and knockout animal models. However, several C57BL/6 substrains exist, and these are genetically and phenotypically different. In addition, each of these substrains can be purchased from different animal providers and, in some cases, they have maintained their breeding stocks separated for a long time, allowing genetic differences to accumulate due to individual variability and genetic drift. With the aim of describing the differences in the genotype of several C57BL/6 substrains, we applied the Illumina(®) Mouse Medium Density Linkage Mapping panel, with 1,449 single nucleotide polymorphisms (SNPs), to individuals from ten C57BL/6-related strains: C57BL/6JArc, C57BL/6J from The Jackson Lab, C57BL/6J from Crl, C57BL6/JRccHsd, C57BL/6JOlaHsd, C57BL/6JBomTac, B6(Cg)-Tyr ( c-2j )/J, C57BL/6NCrl, C57BL/6NHsd and C57BL/6NTac. Twelve SNPs were found informative to discriminate among the mouse strains considered. Mice derived from the original C57BL/6J: C57BL/6JArc, C57BL/6J from The Jackson Lab and C57BL/6J from Crl, were indistinguishable. Similarly, all C57BL/6N substrains displayed the same genotype, whereas the additional substrains showed intermediate cases with substrain-specific polymorphisms. These results will be instrumental for the correct genetic monitoring and appropriate mouse colony handling of different transgenic and knockout mice produced in distinct C57BL/6 inbred substrains.

Melanin precursors prevent premature age-related and noise-induced hearing loss in albino mice.

Strial melanocytes are required for normal development and correct functioning of the cochlea. Hearing deficits have been reported in albino individuals from different species, although melanin appears to be not essential for normal auditory function. We have analyzed the auditory brainstem responses (ABR) of two transgenic mice: YRT2, carrying the entire mouse tyrosinase (Tyr) gene expression-domain and undistinguishable from wild-type pigmented animals; and TyrTH, non-pigmented but ectopically expressing tyrosine hydroxylase (Th) in melanocytes, which generate the precursor metabolite, L-DOPA, but not melanin. We show that young albino mice present a higher prevalence of profound sensorineural deafness and a poorer recovery of auditory thresholds after noise-exposure than transgenic mice. Hearing loss was associated with absence of cochlear melanin or its precursor metabolites and latencies of the central auditory pathway were unaltered. In summary, albino mice show impaired hearing responses during ageing and after noise damage when compared to YRT2 and TyrTH transgenic mice, which do not show the albino-associated ABR alterations. These results demonstrate that melanin precursors, such as L-DOPA, have a protective role in the mammalian cochlea in age-related and noise-induced hearing loss.

Diketopiperazines as a tool for the study of transport across the blood-brain barrier (BBB) and their potential use as BBB-shuttles.

Here we prepared and evaluated two libraries of mono-N-methylated and di-N-methylated diketopiperazines (DKPs) by parallel artificial membrane permeability assay and immobilized artificial membrane chromatography in order to obtain information on the features that govern the passage of peptidic molecules across the blood-brain barrier (BBB) by passive diffusion. On the basis of the results from these two libraries, we prepared and evaluated several DKP-baicalin and DKP-dopamine constructs. The DKPs or cyclic dipeptide scaffolds can be considered a novel family of brain delivery systems (BBB-shuttles) to transport to the brain drugs and other cargos that cannot cross the BBB unaided.

Evolutionary combinatorial chemistry, a novel tool for SAR studies on peptide transport across the blood-brain barrier. Part 2. Design, synthesis and evaluation of a first generation of peptides.

The use of high-throughput methods in drug discovery allows the generation and testing of a large number of compounds, but at the price of providing redundant information. Evolutionary combinatorial chemistry combines the selection and synthesis of biologically active compounds with artificial intelligence optimization methods, such as genetic algorithms (GA). Drug candidates for the treatment of central nervous system (CNS) disorders must overcome the blood-brain barrier (BBB). This paper reports a new genetic algorithm that searches for the optimal physicochemical properties for peptide transport across the blood-brain barrier. A first generation of peptides has been generated and synthesized. Due to the high content of N-methyl amino acids present in most of these peptides, their syntheses were especially challenging due to over-incorporations, deletions and DKP formations. Distinct fragmentation patterns during peptide cleavage have been identified. The first generation of peptides has been studied by evaluation techniques such as immobilized artificial membrane chromatography (IAMC), a cell-based assay, log Poctanol/water calculations, etc. Finally, a second generation has been proposed.

Arylboronic acids and arylpinacolboronate esters in Suzuki coupling reactions involving indoles. Partner role swapping and heterocycle protection.

Yields of Suzuki couplings involving indoles depended upon (i) whether arylboronic acids or arylpinacolboronate esters were used, (ii) whether the heterocycle was the aryl halide or the arylboron coupling partner, and (iii) whether the heterocycle was protected or not. Highest yields, which were unaffected by incorporating Boc or Tos protection at the heterocyclic nitrogen, were obtained when indole bromides were reacted with phenylboronic acids. When indolylboronic acids were reacted with phenyl bromides, yields were somewhat lower and depended on the nitrogen substituent, being highest in the absence of protection, lower in the presence of the Boc group, and lowest of all with the Tos group. Arylpinacolboronate esters were less reactive than arylboronic acids. They required considerably longer reaction times and furnished generally lower yields of biaryl. Furthermore, irrespective of whether the heterocycle was the aryl bromide or the arylpinacolboronate ester, these yields were highest when it was protected with the Tos group. Yields were lower with the Boc group, and unprotected heterocycles gave only traces of biaryl. Careful selection of arylboron reagent, of coupling partner roles, and of protecting groups are essential to ensuring optimum results in these Suzuki couplings. These results may also be relevant to couplings involving other substrates.