Robust Face Alignment via Deep Progressive Reinitialization and Adaptive Error-Driven Learning.

Regression-based face alignment involves learning a series of mapping functions to predict the true landmarks from an initial estimation of the alignment. Most existing approaches focus on learning efficacious mapping functions from some feature representations to improve performance. The issues related to the initial alignment estimation and the final learning objective, however, receive less attention. This work proposes a deep regression architecture with progressive reinitialization and a new error-driven learning loss function to explicitly address the above two issues. Given an image with a rough face detection result, the full face region is first mapped by a supervised spatial transformer network to a normalized form and trained to regress coarse positions of landmarks. Then, different face parts are further respectively reinitialized to their own normalized states, followed by another regression sub-network to refine the landmark positions. To deal with the inconsistent annotations in existing training datasets, we further propose an adaptive landmark-weighted loss function. It dynamically adjusts the importance of different landmarks according to their learning errors during training without depending on any hyper-parameters manually set by trial and error. A high level of robustness to annotation inconsistencies is thus achieved. The whole deep architecture permits training from end to end, and extensive experimental analyses and comparisons demonstrate its effectiveness and efficiency. The source code, trained models, and experimental results are made available at https://github.com/shaoxiaohu/Face_Alignment_DPR.git.

Activated factor X targeted stored in platelets as an effective gene therapy strategy for both hemophilia A and B.

BACKGROUND: Treatment of hemophiliacs with inhibitors remains challenging, and new treatments are in urgent need. Coagulation factor X plays a critical role in the downstream of blood coagulation cascade, which could serve as a bypassing agent for hemophilia therapy. Base on platelet-targeted gene therapy for hemophilia by our and other groups, we hypothesized that activated factor X (FXa) targeted stored in platelets might be effective in treating hemophilia A (HA) and B (HB) with or without inhibitors. METHODS: To achieve the storage of FXa in platelets, we constructed a FXa precursor and used the integrin αIIb promoter to control the targeted expression of FXa precursor in platelets. The expression cassette (2bFXa) was carried by lentivirus and introduced into mouse hematopoietic stem and progenitor cells (HSPCs), which were then transplanted into HA and HB mice. FXa expression and storage in platelets was examined in vitro and in vivo. We evaluated the therapeutic efficacy of platelet-stored FXa by tail bleeding assays and the thrombelastography. In addition, thrombotic risk was assessed in the recipient mice and the lipopolysaccharide induced inflammation mice. RESULTS: By transplanting 2bFXa lentivirus-transduced HSPCs into HA and HB mice, FXa was observed stably stored in platelet α-granules, the stored FXa is releasable and functional upon platelet activation. The platelet-stored FXa can significantly ameliorate bleeding phenotype in HA and HB mice as well as the mice with inhibitors. Meanwhile, no FXa leakage in plasma and no signs of increased risk of hypercoagulability were found in transplantation recipients and lipopolysaccharide induced septicemia recipients. CONCLUSIONS: Our proof-of-principle data indicated that target expression of the FXa precursor to platelets can generate a storage pool of FXa in platelet α-granules, the platelet-stored FXa is effective in treating HA and HB with inhibitors, suggesting that this could be a novel choice for hemophilia patients with inhibitors.

Preservation media, durations and cell concentrations of short-term storage affect key features of human adipose-derived mesenchymal stem cells for therapeutic application.

BACKGROUND: Adipose-derived mesenchymal stem cells (ADSCs) have shown great potential in the treatment of various diseases. However, the optimum short-term storage condition of ADSCs in 2∼8 °C is rarely reported. This study aimed at optimizing a short-term storage condition to ensure the viability and function of ADSCs before transplantation. METHODS: Preservation media and durations of storage were evaluated by cell viability, apoptosis, adhesion ability and colony-forming unit (CFU) capacity of ADSCs. The abilities of cell proliferation and differentiation were used to optimize cell concentrations. Optimized preservation condition was evaluated by cell surface markers, cell cycle and immunosuppressive capacity. RESULTS: A total of 5% human serum albumin in multiple electrolytes (ME + HSA) was the optimized medium with high cell viability, low cluster rate, good adhesion ability and high CFU capacity of ADSCs. Duration of storage should be limited to 24 h to ensure the quality of ADSCs before transplantation. A concentration of 5 × 106 cells/ml was the most suitable cell concentration with low late stage apoptosis, rapid proliferation and good osteogenic and adipogenic differentiation ability. This selected condition did not change surface markers, cell cycle, indoleamine 2, 3-dioxygenase 1 (IDO1) gene expression and kynurenine (Kyn) concentration significantly. DISCUSSION: In this study, ME + HSA was found to be the best medium, most likely due to the supplement of HSA which could protect cells, the physiological pH (7.4) of ME and sodium gluconate ingredient in ME which could provide energy for cells. Duration should be limited to 24 h because of reduced nutrient supply and increased waste and lactic acid accumulation during prolonged storage. To keep cell proliferation and limit lactic acid accumulation, the proper cell concentration is 5× 106 cells/ml. Surface markers, cell cycle and immunosuppressive capacity did not change significantly after storage using the optimized condition, which confirmed our results that this optimized short-term storage condition of MSCs has a great potential for the application of cell therapy.

An improved system for the surface immobilisation of proteins on Bacillus thuringiensis vegetative cells and spores through a new spore cortex-lytic enzyme anchor.

An improved surface-immobilisation system was engineered to target heterologous proteins onto vegetative cells and spores of Bacillus thuringiensis plasmid-free recipient strain BMB171. The sporulation-dependent spore cortex-lytic enzyme from B. thuringiensis YBT-1520, SceA, was expressed in vegetative cells and used as the surface anchoring motif. Green fluorescent protein (GFP) and a Bacillus endo-ß-1,3-1,4-glucanase (BglS) were used as the fusion partners to test the binding efficiency and the functional activities of immobilised surface proteins. The surface localisation of the SceA-GFP fusion protein on vegetative cells and spores was confirmed by Western blot, immunofluorescence microscopy and flow cytometry. The GFP fluorescence intensity from both vegetative cells and spores was measured and compared to a previously characterised surface display system using a peptidoglycan hydrolase anchor (Mbg). Results demonstrated comparable efficiency of SceA- and Mbg-mediated immobilisation on vegetative cells but a more efficient immobilisation on spores using the SceA anchor, suggesting SceA has greater potential for spore-based applications. The SceA protein was then applied to target BglS onto vegetative cells and spores, and the surface immobilisation was verified by the substantial whole-cell enzymatic activity and enhanced whole-spore enzymatic activity compared to vegetative cells. A dually active B. thuringiensis vegetative cell and spore display system could prove especially valuable for the development of regenerable and heat-stable biocatalysts that function under adverse environmental conditions, for example, an effective feed additive for improved digestion and nutrient absorption by livestock.

Complete genome sequence of Bacillus thuringiensis subsp. chinensis strain CT-43.

Bacillus thuringiensis has been widely used as an agricultural biopesticide for a long time. As a producing strain, B. thuringiensis subsp. chinensis strain CT-43 is highly toxic to lepidopterous and dipterous insects. It can form various parasporal crystals consisting of Cry1Aa3, Cry1Ba1, Cry1Ia14, Cry2Aa9, and Cry2Ab1. During fermentation, it simultaneously generates vegetative insecticidal protein Vip3Aa10 and the insecticidal nucleotide analogue thuringiensin. Here, we report the finished, annotated genome sequence of B. thuringiensis strain CT-43.

Complete genome sequence of Bacillus thuringiensis mutant strain BMB171.

Bacillus thuringiensis has been widely used as a biopesticide for a long time. Here we report the finished and annotated genome sequence of B. thuringiensis mutant strain BMB171, an acrystalliferous mutant strain with a high transformation frequency obtained and stocked in our laboratory.

Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor.

BACKGROUND: Previous studies have revealed that the lysin motif (LysM) domains of bacterial cell wall-degrading enzymes are able to bind to peptidoglycan moieties of the cell wall. This suggests an approach for a cell surface display system in Gram-positive bacteria using a LysM-containing protein as the anchoring motif. In this study, we developed a new surface display system in B. thuringiensis using a LysM-containing peptidoglycan hydrolase, endo-beta-N-acetylglucosaminidase (Mbg), as the anchor protein. RESULTS: Homology searching in the B. thuringiensis YBT-1520 genome revealed a putative peptidoglycan hydrolase gene. The encoded protein, Mbg, exhibited substantial cell-wall binding capacity. The deduced amino acid sequence of Mbg was structurally distinguished as an N-terminal domain with two tandemly aligned LysMs and a C-terminal catalytic domain. A GFP-fusion protein was expressed and used to verify the surface localization by Western blot, flow cytometry, protease accessibility, SDS sensitivity, immunofluorescence, and electron microscopy assays. Low-level constitutive expression of Mbg was elevated by introducing a sporulation-independent promoter of cry3Aa. Truncated Mbg domains with separate N-terminus (Mbgn), C-terminus (Mbgc), LysM1, or LysM2 were further compared for their cell-wall displaying efficiencies. The Mbgn moiety contributed to cell-wall anchoring, while LysM1 was the active domain. Two tandemly repeated Mbgns exhibited the highest display activity, while the activity of three repeated Mbgns was decreased. A heterologous bacterial multicopper oxidase (WlacD) was successfully displayed onto the surface of B. thuringiensis target cells using the optimum (Mbgn)2 anchor, without radically altering its catalytic activity. CONCLUSION: Mbg can be a functional anchor protein to target different heterologous proteins onto the surface of B. thuringiensis cells. Since the LysM domain appears to be universal in Gram-positive bacteria, the strategy presented here could be applicable in other bacteria for developing this type of system.

Improved phosphate biosorption by bacterial surface display of phosphate-binding protein utilizing ice nucleation protein.

The conventional enhanced biological phosphorus removal (EBPR) system often deteriorates at low chemical oxygen demand (COD) or under aeration conditions. A new approach that incorporates phosphate-eutrophic wastewater remediation was introduced through immobilization of an intracellular phosphate-binding protein (PBP) onto the surface of Pseudomonas putida or Escherichia coli, using the N-terminal anchor (InaQ-N) of a newly identified ice nucleation protein from Pseudomonas syringae. A green fluorescent protein-fusion protein was expressed and used to confirm surface localization. The PBP was then targeted to the surface of E. coli JM109 and P. putida AB92019. The engineered P. putida and E. coli microorganisms were capable of absolute biosorption of total phosphates at rates of 60 and 80 mg L(-1), respectively, over 5 h. In the recombinant P. putida cells, a surface-immobilized PBP fusion that had three tandemly repeated InaQ-Ns exhibited the maximum increment in phosphate biosorption, at sixfold compared with the control strain. Even heat-killed recombinant cells of either P. putida or E. coli retained substantial biosorptive activities. The current study demonstrates that the bacterial surface display of PBP should be considered as a strong contender for generating organisms capable of functioning in EBPR systems under low COD, resulting in improved removal of eutrophic phosphorus from wastewaters.

[Functional cell surface display of endo-beta-1, 3-1, 4-glucanase in Lactococcus lactis using N-acetylmuraminidase as the anchoring motif].

In this report, we utilized N-Acetylmuraminidase (AcmA) to develop a whole-cell catalyst of endo-beta-1, 3-1, 4-glucanase in Lactococcus lactis. The PCR-amplified full-length acmA gene from L. lactis MB191 was fused with the green fluorescent gene (gfp), followed by ligating the chimeric acmA-gfp into the Escherichia coli-L. lactis shuttle expression vector pMG36k, yielding the recombinant plasmid pMB137. SDS-PAGE analysis showed that the constitutive expression of AcmA-GFP fusion protein in the L. lactis AS1.2829 construct harboring pMB137 (named MB137), with the predicted Mr of 74 kD. Western blotting, GFP specific fluorescence intensity assays and flow cytometry analysis confirmed that AcmA-GFP was immobilized on the outer membrane, which constituted approx. 35% of the total intracellular fusion protein. Furthermore, acmA was fused with a PCR-amplified encoding fragment of the endo-beta-1, 3-1, 4-glucanase gene (gls) from Bacillus sublitis BF7658, resulting in the recombinant plasmid pMB138. By transferring pMB138 into L. lactis AS1.2829, the derived L. lactis MB138 expressing the AcmA-GLS fusion enzyme exhibited a distinct whole-cell glucanase activity (by 12 U/mL) compared to the control strain, indicating AcmA had served as a functional anchoring motif to immobilize the heterologous enzyme on the cell surface of L. lactis.