State of art and limitations in genetic engineering to induce stable chondrogenic phenotype

Current protocols for chondrocyte expansion and chondrogenic differentiation of stem cells fail to reduce phenotypic loss and to mitigate hypertrophic tendency. To this end, cell genetic manipulation is gaining pace as a means of generating cells with stable chondrocyte phenotype. Herein, we provide an overview of candidate genes that either induce cartilage regeneration or inhibit cartilage degeneration. We further discuss in vitro, ex vivo and in vivo viral transduction and non-viral transfection strategies for targeted cells (chondrocytes, mesenchymal stem cells, induced pluripotent stem cells and synovial cells), along with the most representative results obtained in pre-clinical models and in clinical trials. We highlight current challenges and associated risks that slowdown clinical acceptance and commercialisation of gene transfer technologies.     

Genome annotation and antimicrobial properties of Bacillus toyonensis VU‐DES13, isolated from the Folsomia candida gut

Antibiotic resistance necessitates the search for new bioactive compounds with novel mechanisms of action. Natural products derived from bacteria and fungi are widely used in the field of medicine and new environments can be explored as sources of antimicrobials. Bacteria associated with springtails have shown high inhibitory activity against pathogens. Here, we characterized a bacterial strain with high potential for antimicrobial activity, isolated from the gut of the springtail Folsomia candida Willem (Collembola: Isotomidae). The strain was characterized using the ‘analytical profile index’ and the ‘minimal inhibitory concentration’ assay to test for antibiotic resistance. Agar overlay and agar disk diffusion assays were used to test the inhibitory activity of the strain and its extract against a variety of pathogens, and reporter assays were used to investigate the mode of action. High‐performance liquid chromatography was used to analyze and fractionate the extract of bacterial culture, followed by additional assays on the fractions. The genome of the strain was screened for presence of antibiotic resistance genes and secondary metabolite gene clusters. The isolate was identified as Bacillus toyonensis Jiménez et al., but it displayed differences in metabolic profile when compared to the type species. The isolate was highly resistant to penicillin and inhibited the growth of a variety of pathogenic microorganisms. Genome analysis revealed an enrichment of resistance genes for β‐lactam antibiotics compared to the type isolate. Also, secondary metabolite clusters involved in the production of siderophores, bacteriocins, and nonribosomal peptide synthetases were identified. In conclusion, a unique Bacillus strain was isolated from the gut of F. candida, for which we provide evidence of inhibitory activity against an array of pathogens. This, coupled with high resistance to penicillin as substantiated by the presence of resistance genes, points to the potential of B. toyonensis VU‐DES13 to provide a new source of antimicrobial compounds.     

Structure-activity relationship study of position 4 in the urotensin-II receptor ligand U-II(4-11)

In the present study we describe the synthesis and biological evaluation of 24 analogues of the urotensin II (U-II) fragment U-II(4-11) substituted in position 4 with coded and non-coded aromatic amino acids. All of the new analogues behaved as full U-II receptor (UT) agonists. Our results indicated that aromaticity is well tolerated, size, length and chirality of the side chain are not important, while substituents with a nitrogen atom are preferred. Thus acylation of U-II(5-11) with small groups bearing nitrogen atoms could be instrumental in future studies for the identification of novel potent UT receptor ligands.     

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.     

Evaluation in rabbits of different anti-SHIV vaccine strategies based on DNA/fowlpox priming and virus-like particle boosting

Two different prime-boost immunization protocols were tested in rabbits and their immune response was evaluated and compared with the final aim of defining a vaccine strategy that might be able to protect non-human primates from infection with the pathogenic simian/human immunodeficiency virus, SHIV(89.6P). The two regimens were based on three priming immunizations with either an expression plasmid plus a fowlpox (FP) recombinant vector or with two FP recombinant vectors, each one expressing either the SIV(mac239) gag/pol or the HIV-1env(89.6P) genes. In both protocols, priming immunizations were followed by two boosts with SHIV-mimicking virus-like particles (VLP). A complete SHIV-specific response was observed in all animals. Interestingly, the DNA vaccine was three to 10 times more efficient than the FP recombinant in inducing an anti-gag humoral response. Real-time PCR confirmed the memory effect on T-cell subsets secreting interleukin-4 and interferon-gamma, as a consequence of stimulation of both arms of the immune system. Although both protocols were almost equally effective in eliciting homologous neutralizing antibodies and highlighted the efficacy of VLP administration for boosting, protocol A seemed to be more effective in promoting a balanced T-cell memory immune response and appears more promising for vaccine purposes.     

Thermal ecology of two sympatric saxicolous lizards of the genus Phymaturus from the Payunia region (Argentina)

We evaluated the thermal biology of two sympatric saxicolous species of the genus Phymaturus, endemic from the Argentine Payunia region. Taking into account that the distributional range of Phymaturus roigorum (the largest species) is greater than the range of P. payuniae, we evaluated the habitat (type of rocks) used by these species. We recorded body temperature and operative temperatures in different habitats, and we determined the preferred body temperature in the laboratory. We compared the thermal quality of habitats occupied and not occupied by Phymaturus payuniae, and the accuracy and effectiveness of thermoregulation between species.     

Evaluation of Porcine Intestinal Epitheliocytes as an In vitro Immunoassay System for the Selection of Probiotic Bifidobacteria to Alleviate Inflammatory Bowel Disease

Probiotics and Antimicrobial Proteins - The use of in vitro systems that allow efficient selection of probiotic candidates with immunomodulatory properties could significantly minimize the use of...     

Metabotropic glutamate receptors in neurodegeneration/neuroprotection: Still a hot topic?

Moving from early studies, we here review the most recent evidence linking metabotropic glutamate (mGlu) receptors to processes of neurodegeneration/neuroprotection. The use of knockout mice and subtype-selective drugs has increased our knowledge of the precise role played by individual mGlu receptor subtypes in these processes. Activation of mGlu1 and mGlu5 receptors may either amplify or reduce neuronal damage depending on the context and the nature of the toxic insults. In contrast, mGlu1 and mGlu5 receptors antagonists are consistently protective in in vitro and in vivo models of neuronal death. A series of studies suggest that mGlu1 receptor antagonists or negative allosteric modulators (NAMs) are promising candidates for the treatment of ischemic brain damage, whereas mGlu5 receptor NAMs, which have been clinically developed for the treatment of Parkinson's disease (PD) and l-DOPA-induced dyskinesias, protect nigro-striatal dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity in mice and monkeys. Activation of glial mGlu3 receptors promotes the formation of various neurotrophic factors, such as transforming growth factor-β (TGF-β), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF). Hence, selective mGlu3 receptor agonists or positive allosteric modulators (PAMs) (not yet available) are potentially helpful in the treatment of chronic neurodegenerative disorders such as PD, Alzheimer's disease (AD), and amyotrophic lateral sclerosis. Selective mGlu2 receptor PAMs should be used with caution in AD patients because these drugs are shown to amplify β-amyloid neurotoxicity. Finally, mGlu4 receptor agonists/PAMs share with mGlu5 receptor NAMs the ability to improve motor symptoms associated with PD and attenuate nigro-striatal degeneration at the same time. No data are yet available on the role of mGlu7 and mGlu8 receptors in neurodegeneration/neuroprotection.     

Influence of antimicrobial feed additives on broiler commensal posthatch gut microbiota development and performance

The effects of avilamycin, zinc bacitracin, and flavophospholipol on broiler gut microbial community colonization and bird performance in the first 17 days posthatch were investigated. Significant differences in gut microbiota associated with gut section, dietary treatment, and age were identified by terminal restriction fragment length polymorphism (T-RFLP), although no performance-related differences between dietary treatments were detected. Similar age-related shifts in the gut microbiota were identified regardless of diet but varied between the ilea and ceca. Interbird variabilities in ileal bacterial communities were reduced (3 to 7 days posthatch) in chicks fed with feed containing antimicrobial agents. Avilamycin and flavophospholipol had the most consistent effect on gut microbial communities. Operational taxonomic units (OTU) linked to changes in gut microbiota in birds on antimicrobial-supplemented diets were characterized and identified. Some OTUs could be identified to the species level; however, the majority could be only tentatively classified to the genus, family, order, or domain level. OTUs 140 to 146 (Lachnospiraceae), OTU 186/188 (Lactobacillus johnsonii), OTU 220 (Lachnospiraceae), OTUs 284 to 288 (unclassified bacterial spp. or Ruminococcaceae), OTU 296/298 (unclassified bacterium or Clostridiales), and OTU 480/482 (Oxalobacteraceae) were less prevalent in the guts of chicks fed antimicrobial-supplemented diets. OTU 178/180 (Lactobacillus crispatus), OTU 152 (Lactobacillus reuteri or unclassified Clostridiales), OTU 198/200 (Subdoligranulum spp.), and OTU 490/492 (unclassified bacterium or Enterobacteriaceae) were less prevalent in the gut of chicks raised on the antimicrobial-free diet. The identification of key bacterial species influenced by antimicrobial-supplemented feed immediately posthatch may assist in the formulation of diets that facilitate beneficial gut microbial colonization and, hence, the development of alternatives to current antimicrobial agents in feed for sustainable poultry production.