To examine whether transplantation of human mesenchymal stem cells (MSCs) could reconstruct the corneal damage and also whether grafted MSCs could differentiate into corneal epithelial cells, we isolated MSCs from healthy donors. After growth and expansion on amniotic membrane, cells were transplanted into rat corneas 7 days after chemical burns. Reconstruction of the damaged cornea and the rat vision were measured once a week by slit lamp and by an optokinetic head-tracking instrument, respectively. Corneas were then cut out, fixed, and imbedded for immunofluorescent study of the expression of keratin 3 and keratin-pan as epithelial cell markers. Expression of CD45, interleukin 2, and metalloproteinase-2 was also investigated for inflammation and inflammation-related angiogenesis. The data showed that transplantation of MSCs, like limbal epithelial stem cells, successfully reconstructed damaged rat corneal surface. Interestingly, the therapeutic effect of the transplantation may be associated with the inhibition of inflammation and angiogenesis after transplantation of MSCs rather than the epithelial differentiation from MSCs. This study provides the first line of evidence that MSCs can be used for reconstruction of damaged corneas, presenting a new source for autotransplantation in the treatment of corneal disorders. STEM CELLS 2006;24:315-321

The binding and catalytic functions of proteins are generally mediated by a small number of functional residues held in place by the overall protein structure. Here, we describe deep learning approaches for scaffolding such functional sites without needing to prespecify the fold or secondary structure of the scaffold. The first approach, “constrained hallucination,” optimizes sequences such that their predicted structures contain the desired functional site. The second approach, “inpainting,” starts from the functional site and fills in additional sequence and structure to create a viable protein scaffold in a single forward pass through a specifically trained RoseTTAFold network. We use these two methods to design candidate immunogens, receptor traps, metalloproteins, enzymes, and protein-binding proteins and validate the designs using a combination of in silico and experimental tests.

The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains a challenge1–5. Here we describe a general solution to this problem that starts with a broad exploration of the vast space of possible binding modes to a selected region of a protein surface, and then intensifies the search in the vicinity of the most promising binding modes. We demonstrate the broad applicability of this approach through the de novo design of binding proteins to 12 diverse protein targets with different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and, following experimental optimization, bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of five of the binder–target complexes, and all five closely match the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein–protein interactions, and should guide improvements of both. Our approach enables the targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.

-The outcome of an udder infection is influenced by the pathogen species. We established a strictly defined infection model to better analyze the unknown molecular causes for these pathogen-specific effects, using Escherichia coli and Staphylococcus aureus strains previously asseverated from field cases of mastitis. Inoculation of quarters with 500 CFU of E. coli (n = 4) was performed 6 h, 12 h, and 24 h before culling. All animals showed signs of acute clinical mastitis 12 h after challenge: increased somatic cell count (SCC), decreased milk yield, leukopenia, fever, and udder swelling. Animals inoculated with 10 000 CFU of S. aureus for 24 h (n = 4) showed no or only modest clinical signs of mastitis. However, S. aureus caused clinical signs in animals, inoculated for 72 h-84 h. Real-time PCR proved that E. coli inoculation strongly and significantly upregulated the expression of β-defensins, TLR2 and TLR4 in the pathogen inoculated udder quarters as well as in mammary lymph nodes. TLR3 and TLR6 were not significantly regulated by the infections. Immuno-histochemistry identified mammary epithelial cells as sites for the upregulated TLR2 and β-defensin expression. S. aureus, in contrast, did not significantly regulate the expression of any of these genes during the first 24 h after pathogen inoculation. Only 84 h after inoculation, the expression of β-defensins, but not of TLRs was significantly (> 20 fold) upregulated in five out of six pathogen inoculated quarters. Using the established mastitis model, the data clearly demonstrate a pathogen-dependent difference in the time kinetics of induced pathogen receptors and defense molecules.toll-like receptor / mastitis / beta-defensin / mammary epithelial cell

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We examined the repertoire and extent of inflammation dependent gene regulation in a bovine mammary epithelial cell (MEC) model, to better understand the contribution of the MEC in the immune defence of the udder. We challenged primary cultures of MEC from cows with heat inactivated Escherichia coli pathogens and used Affymetrix DNA-microarrays to profile challenge related alterations in their transcriptome. Compared to acute mastitis, the most prominently activated genes comprise those encoding chemokines, interleukins, beta-defensins, serum amyloid A and haptoglobin. Hence, the MEC exert sentinel as well as effector functions of innate immune defence. E. coli stimulated a larger fraction of genes (30%) in the MEC belonging to the functional category Inflammatory Response than we recorded with the same microarrays during acute mastitis in the udder (17%). This observation underscores the exquisite immune capacity of MEC. To more closely examine the adequacy of immunological regulation in MEC, we compared the inflammation dependent regulation of factors contributing to the complement system between the udder versus the MEC. In the MEC we observed only up regulation of several complement factor-encoding genes. Mastitis, in contrast, in the udder strongly down regulates such genes encoding factors contributing to both, the classical pathway of complement activation and the Membrane Attack Complex, while the expression of factors contributing to the alternative pathway may be enhanced. This functionally polarized regulation of the complex complement pathway is not reflected in the MEC models.

Computational studies were performed in an effort to understand the relative reactivity of oxoesters and thioesters in nucleophilic acyl transfer reactions. Transition state models were developed for the reactions of methyl acetate and methyl thioacetate with hydroxide, ammonia, and methylcyanoacetate carbanion. Quantum mechanical calculations based on these models reproduced experimental observations that oxoesters and thioesters have similar reactivity toward hydroxide while thioesters are about 100-fold and at least 2000-fold more reactive than oxoesters toward amine and carbanion nucleophiles, respectively. NBO analysis was performed to elucidate the role of electron delocalization in reactant and transition state stabilization. These calculations indicate similar losses of delocalization energy for the oxoester and thioester in going from the reactants to the transition state in reaction with hydroxide while the loss of delocalization energy is significantly greater for the oxoester in reactions with the other nucleophiles. Bond rotational analysis of the transition states for the reactions with hydroxide and ammonia provide support for an important role of the p(X) --> sigma(C-Nu) interaction (X = O or S of the oxoester or thioester respectively, Nu = nucleophile) in governing the reactivity of oxoesters and thioesters in nucleophilic acyl substitution.

Prolactin stimulates the expression of milk genes during lactation through the activation of STAT5 transcription factors, which subsequently bind to their cognate target sequence on the promoters. Demethylation of 5methylCpG dinucleotides permits the tissue-specific accessibility of transcription factor-binding sites during development, but remethylation has not been shown to contribute to acute suppression of gene expression. We characterize functionally a novel STAT5-binding lactational enhancer in the far upstream promoter (wK10 kbp) of the bovine aS1-casein-encoding gene. This promoter area is hypomethylated in the lactating udder only. Remethylation of this area accompanies an experimentally elicited acute shutdown of casein synthesis in fully lactating cows, whose udder quarters have experimentally been infected with a pathogenic E. coli strain. Within 24 h after infection, the relevant promoter area was remethylated from 10% of the DNA molecules in the uninfected control quarters to w50% in the infected quarters, the typical values for fully lactating and not lactating udders respectively. Increased methylation resulted in tighter chromatin packing. Concomitantly, the aS1-casein mRNA concentration dropped to w50% while the protein synthesis was shut down to w2 . 5% in the infected quarters, alone. The methylation status of the promoter from a not lactationally regulated gene was unaltered, and the distal aS1-casein promoter was not remethylated in udder quarters with subclinical Staphylococcus aureus infections featuring sustained casein synthesis. Hence, infection-related remethylation of the aS1-casein promoter and chromatin remodelling serves as an acute, spatially restricted regulatory mechanism, which might insulate the promoter against the systemically unchanged high levels of circulating prolactin. This provides a rare example for an acute regulatory significance of CpG methylation.