Attenuative effects of collagen peptide from milkfish (Chanos chanos) scales on ovariectomy-induced osteoporosis.

Osteoporosis is characterized by low bone mass, bone microarchitecture disruption, and collagen loss, leading to increased fracture risk. In the current study, collagen peptides were extracted from milkfish scales (MS) to develop potential therapeutic candidates for osteoporosis. MS was used to synthesize a crude extract of fish scales (FS), collagen liquid (COL), and hydroxyapatite powder (HA). COL samples were further categorized according to the peptide size of total COL (0.1 mg/mL), COL < 1 kDa (0.1 mg/mL), COL: 1-10 kDa (0.1 mg/mL), and COL > 10 kDa (0.1 mg/mL) to determine it. Semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR) and immunofluorescence labeling were used to assess the expression levels of specific mRNA and proteins in vitro. For in vivo studies, mice ovariectomy (OVX)-induced postmenopausal osteoporosis were developed, while the sham surgery (Sham) group was treated as a control. Collagen peptides (CP) from MS inhibited osteoclast differentiation in RAW264.7 cells following an insult with nuclear factor kappa-B ligand (RANKL). CP also enhanced osteoblast proliferation in MG-63 cells, possibly through downregulating NFATc1 and TRAP mRNA expression and upregulating ALP and OPG mRNA levels. Furthermore, COL1 kDa also inhibited bone density loss in osteoporotic mice. Taken together, CP may reduce RANKL-induced osteoclast activity while promoting osteoblast synthesis, and therefore may act as a potential therapeutic agent for the prevention and control of osteoporosis.

An optimized electrotransformation protocol for Lactobacillus jensenii.

Engineered bacteria are promising candidates for in situ detection and treatment of diseases. The female uro-genital tract presents several pathologies, such as sexually transmitted diseases or genital cancer, that could benefit from such technology. While bacteria from the gut microbiome are increasingly engineered, the use of chassis isolated from the female uro-genital resident flora has been limited. A major hurdle to implement the experimental throughput required for efficient engineering in these non-model bacteria is their low transformability. Here we report an optimized electrotransformation protocol for Lactobacillus jensenii, one the most widespread species across vaginal microflora. Starting from classical conditions, we optimized buffers, electric field parameters, cuvette type and DNA quantity to achieve an 80-fold improvement in transformation efficiency, with up to 3.5·103 CFUs/µg of DNA in L. jensenii ATCC 25258. We also identify several plasmids that are maintained and support reporter gene expression in L. jensenii. Finally, we demonstrate that our protocol provides increased transformability in three independent clinical isolates of L. jensenii. This work will facilitate the genetic engineering of L. jensenii and enable its use for addressing challenges in gynecological healthcare.

Engineered l-Lactate Responding Promoter System Operating in Glucose-Rich and Anoxic Environments.

Bacteria equipped with genetically encoded lactate biosensors are promising tools for biopharmaceutical production, diagnostics, and cellular therapies. However, many applications involve glucose-rich and anoxic environments, in which current whole-cell lactate biosensors show low performance. Here we engineer an optimized, synthetic lactate biosensor system by repurposing the natural LldPRD promoter regulated by the LldR transcriptional regulator. We removed glucose catabolite and anoxic repression by designing a hybrid promoter, containing LldR operators and tuned both regulator and reporter gene expressions to optimize biosensor signal-to-noise ratio. The resulting lactate biosensor, termed ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia), can operate in glucose-rich, aerobic and anoxic conditions. We show that ALPaGA works reliably in the probiotic chassisEscherichia coliNissle 1917 and can detect endogenous l-lactate produced by 3D tumor spheroids with an improved dynamic range. In the future, the ALPaGA system could be used to monitor bioproduction processes and improve the specificity of engineered bacterial cancer therapies by restricting their activity to the lactate-rich microenvironment of solid tumors.

Antibody variable domain interface and framework sequence requirements for stability and function by high-throughput experiments.

Protein structural stability and biological functionality are dictated by the formation of intradomain cores and interdomain interfaces, but the intricate sequence-structure-function interrelationships in the packing of protein cores and interfaces remain difficult to elucidate due to the intractability of enumerating all packing possibilities and assessing the consequences of all the variations. In this work, groups of ß strand residues of model antibody variable domains were randomized with saturated mutagenesis and the functional variants were selected for high-throughput sequencing and high-throughput thermal stability measurements. The results show that the sequence preferences of the intradomain hydrophobic core residues are strikingly flexible among hydrophobic residues, implying that these residues are coupled indirectly with antigen binding through energetic stabilization of the protein structures. By contrast, the interdomain interface residues are directly coupled with antigen binding. The interdomain interface should be treated as an integral part of the antigen-binding site.

Loop-sequence features and stability determinants in antibody variable domains by high-throughput experiments.

Protein loops are frequently considered as critical determinants in protein structure and function. Recent advances in high-throughput methods for DNA sequencing and thermal stability measurement have enabled effective exploration of sequence-structure-function relationships in local protein regions. Using these data-intensive technologies, we investigated the sequence-structure-function relationships of six complementarity-determining regions (CDRs) and ten non-CDR loops in the variable domains of a model vascular endothelial growth factor (VEGF)-binding single-chain antibody variable fragment (scFv) whose sequence had been optimized via a consensus-sequence approach. The results show that only a handful of residues involving long-range tertiary interactions distant from the antigen-binding site are strongly coupled with antigen binding. This implies that the loops are passive regions in protein folding; the essential sequences of these regions are dictated by conserved tertiary interactions and the consensus local loop-sequence features contribute little to protein stability and function.

Design of phage-displayed cystine-stabilized mini-protein libraries for proteinaceous binder engineering.

Cystine-stabilized mini-proteins are important scaffolds in the combinatorial search of binders for molecular recognition. The structural determinants of a cystine-stabilized scaffold are the critical residues determining the formation of the native disulfide-bonding configuration, and thus should remain unchanged in the combinatorial libraries so as to allow a large portion of the library sequences to be compatible with the scaffold structure. A high-throughput molecular evolution procedure has been developed to select and screen for the polypeptide sequences folding into a specific cystine-stabilized structure. Patterns of sequence preference that emerge from the resultant sequence profiles provide structural determinant information, which facilitates the designs of combinatorial libraries for combinatorial approaches as in phage display. This methodology enables artificial cystine-stabilized proteins to be engineered with enhanced folding and binding properties.

Rationalization and design of the complementarity determining region sequences in an antibody-antigen recognition interface.

Protein-protein interactions are critical determinants in biological systems. Engineered proteins binding to specific areas on protein surfaces could lead to therapeutics or diagnostics for treating diseases in humans. But designing epitope-specific protein-protein interactions with computational atomistic interaction free energy remains a difficult challenge. Here we show that, with the antibody-VEGF (vascular endothelial growth factor) interaction as a model system, the experimentally observed amino acid preferences in the antibody-antigen interface can be rationalized with 3-dimensional distributions of interacting atoms derived from the database of protein structures. Machine learning models established on the rationalization can be generalized to design amino acid preferences in antibody-antigen interfaces, for which the experimental validations are tractable with current high throughput synthetic antibody display technologies. Leave-one-out cross validation on the benchmark system yielded the accuracy, precision, recall (sensitivity) and specificity of the overall binary predictions to be 0.69, 0.45, 0.63, and 0.71 respectively, and the overall Matthews correlation coefficient of the 20 amino acid types in the 24 interface CDR positions was 0.312. The structure-based computational antibody design methodology was further tested with other antibodies binding to VEGF. The results indicate that the methodology could provide alternatives to the current antibody technologies based on animal immune systems in engineering therapeutic and diagnostic antibodies against predetermined antigen epitopes.

Probiotic Bio-Three induces Th1 and anti-inflammatory effects in PBMC and dendritic cells.

AIM: To investigate the immune response of peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) that were stimulated by probiotic preparations. METHODS: PBMCs were isolated, cultured, and stimulated with Bio-Three (a mixture of Bacillus mesentericus, Clostridium butyricum and Enterococcus faecalis; 10(5), 10(6) and 10(7) CFU/mL for 24 h). Cytokine production of (1) circulating PBMCs; (2) PBMCs stimulated by probiotic preparation; (3) monocyte-derived DCs; and (4) DC and T cell co-culture was determined by enzyme-linked immunosorbent assay. Phenotypic analysis of circulating PBMCs was also investigated by flow cytometry. Blood was obtained from individuals who consumed Bio-Three (10(9) CFU/d B. mesentericus, C. butyricum and E. faecalis) for 2 wk, or those who did not take probiotics orally. RESULTS: In culture supernatants, interferon-gamma (IFN-gamma) and interleukin (IL)-10 production increased, but IL-4 and tumor necrosis factor-alpha (TNF-alpha) production by PBMCs decreased after 1 and 2 wk of probiotic treatment. Flow cytometry was also performed on day 14 and detected enhanced expression of CD11b, HLA-DR, CD4, CD45RA, CD25, CD44 and CD69 in response to Bio-Three. Furthermore, IL-10 and IL-12 were upregulated in supernatants of monocyte-derived DCs, and IFN-gamma and IL-10 were enhanced in supernatants of CD4(+) T cells co-cultured with DCs. CONCLUSION: Bio-Three appeared to stimulate the Th1 immune response, downregulate pro-inflammatory cytokines (TNF-alpha) and upregulate anti-inflammatory cytokine (IL-10). Probiotics could be effective in activation of PBMCs and DCs.

Engineering anti-vascular endothelial growth factor single chain disulfide-stabilized antibody variable fragments (sc-dsFv) with phage-displayed sc-dsFv libraries.

Phage display of antibody fragments from natural or synthetic antibody libraries with the single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. However, the nature of the artificial single chain constructs results in unstable proteins expressed on the phage surface or as soluble proteins secreted in the bacterial culture medium. The stability of the variable domain structures can be enhanced with interdomain disulfide bond, but the single chain disulfide-stabilized constructs (sc-dsFv) have yet to be established as a feasible format for bacterial phage display due to diminishing expression levels on the phage surface in known phage display systems. In this work, biological combinatorial searches were used to establish that the c-region of the signal sequence is critically responsible for effective expression and functional folding of the sc-dsFv on the phage surface. The optimum signal sequences increase the expression of functional sc-dsFv by 2 orders of magnitude compared with wild-type signal sequences, enabling the construction of phage-displayed synthetic antivascular endothelial growth factor sc-dsFv libraries. Comparison of the scFv and sc-dsFv variants selected from the phage-displayed libraries for vascular endothelial growth factor binding revealed the sequence preference differences resulting from the interdomain disulfide bond. These results underlie a new phage display format for antibody fragments with all the benefits from the scFv format but without the downside due to the instability of the dimeric interface in scFv.

Molecular evolution of cystine-stabilized miniproteins as stable proteinaceous binders.

Small cystine-stabilized proteins are desirable scaffolds for therapeutics and diagnostics. Specific folding and binding properties of the proteinaceous binders can be engineered with combinatorial protein libraries in connection with artificial molecular evolution. The combinatorial protein libraries are composed of scaffold variants with random sequence variation, which inevitably produces a portion of the library sequences incompatible with the parent structure. Here, we used artificial molecular evolution to elucidate structure-determining residues in a smallest cystine-stabilized scaffold. The structural determinant information was then applied to designing cystine-stabilized miniproteins binding to human vascular endothelial growth factor. This work demonstrated a general methodology on engineering artificial cystine-stabilized proteins as antibody mimetics with simultaneously enhanced folding and binding properties.

Factor Xa active site substrate specificity with substrate phage display and computational molecular modeling.

Structural origin of substrate-enzyme recognition remains incompletely understood. In the model enzyme system of serine protease, canonical anti-parallel beta-structure substrate-enzyme complex is the predominant hypothesis for the substrate-enzyme interaction at the atomic level. We used factor Xa (fXa), a key serine protease of the coagulation system, as a model enzyme to test the canonical conformation hypothesis. More than 160 fXa-cleavable substrate phage variants were experimentally selected from three designed substrate phage display libraries. These substrate phage variants were sequenced and their specificities to the model enzyme were quantified with quantitative enzyme-linked immunosorbent assay for substrate phage-enzyme reaction kinetics. At least three substrate-enzyme recognition modes emerged from the experimental data as necessary to account for the sequence-dependent specificity of the model enzyme. Computational molecular models were constructed, with both energetics and pharmacophore criteria, for the substrate-enzyme complexes of several of the representative substrate peptide sequences. In contrast to the canonical conformation hypothesis, the binding modes of the substrates to the model enzyme varied according to the substrate peptide sequence, indicating that an ensemble of binding modes underlay the observed specificity of the model serine protease.