Inoculation of Latilactobacillus sakei with Pichia kluyveri or Saccharomyces boulardii improves flavor compound profiles of salt-free fermented wheat-gluten: Effects from single strain inoculation.

Wheat-gluten, the protein-rich portion of wheat, can be processed to produce a highly savory sauce product after solid and liquid-state fermentation (SSF and LSF) with the inoculation of selected lactic acid bacteria (LAB) and yeast under salt-free condition. However, limited research has been done on the impact of different types of microbes in this process. This work studied the flavour impact on fermented wheat-gluten by the single inoculation of Latilactobacillus sakei or one yeast (Saccharomyces boulardii or Pichia kluyveri). Glucose was depleted during LSF in all treatments. Lactic acid production increased over time in L. sakei-fermented samples but not in yeast-fermented samples. Cysteine, serine and arginine remained low over LSF in L. sakei-fermented samples but increased in yeast-fermented samples. More fruity esters such as isoamyl acetate and isobutyl acetate were detected in samples fermented by P. kluyveri, while S. boulardii boosted the production of alcohols such as 3-methyl butanol and 2-phenylethyl alcohol. Principal component analysis revealed a clear difference in volatile profiles of the samples fermented with different strains. Therefore, the fermented sauce can potentially be processed into different flavor directions, and based on the flavor profile, be used in different food applications.

Delta Integration CRISPR-Cas (Di-CRISPR) in Saccharomyces cerevisiae.

Despite the advances made in genetic engineering of Saccharomyces cerevisiae, the multicopy genomic integration of large biochemical pathways remains a challenge. Here, we developed a Di-CRISPR (delta integration CRISPR-Cas) platform based on cleavage of the delta sites by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas) to enable unprecedented high-efficiency, multicopy, markerless integrations of large biochemical pathways into the S. cerevisiae genome. Detailed protocols are provided on the entire workflow which includes pDi-CRISPR plasmid and donor DNA construction, Di-CRISPR-mediated integration and analysis of integration efficiencies and copy numbers through flow cytometry and quantitative polymerase chain reaction (qPCR).

Enzyme-Induced Matrix Softening Regulates Hepatocarcinoma Cancer Cell Phenotypes.

The progression of cancer is often accompanied by changes in the mechanical properties of an extracellular matrix. However, limited efforts have been made to reproduce these biological events in vitro. To this end, this study demonstrates that matrix remodeling caused by matrix metalloproteinase (MMP)-1 regulates phenotypic activities and modulates radiosensitivity of cancer cells exclusively in a 3D matrix. In this study, hepatocarcinoma cells are cultured in a collagen-based gel tailored to present an elastic modulus of ≈4.0 kPa. The subsequent exposure of the gel to MMP-1 decreases the elastic modulus from 4.0 to 0.5 kPa. In response to MMP-1, liver cancer cells undergo active proliferation, downregulation of E-cadherin, and the loss of detoxification capacity. The resulting spheroids are more sensitive to radiation than the spheroids cultured in the stiffer gel not exposed to MMP-1. Overall, this study serves to better understand and control the effects of MMP-induced matrix remodeling.

A New Era of Genome Integration-Simply Cut and Paste!

Genome integration is a powerful tool in both basic and applied biological research. However, traditional genome integration, which is typically mediated by homologous recombination, has been constrained by low efficiencies and limited host range. In recent years, the emergence of homing endonucleases and programmable nucleases has greatly enhanced integration efficiencies and allowed alternative integration mechanisms such as nonhomologous end joining and microhomology-mediated end joining, enabling integration in hosts deficient in homologous recombination. In this review, we will highlight recent advances and breakthroughs in genome integration methods made possible by programmable nucleases, and their new applications in synthetic biology and metabolic engineering.

A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae.

Despite recent advances in genome editing capabilities for the model organism Saccharomyces cerevisiae, the chromosomal integration of large biochemical pathways for stable industrial production remains challenging. In this work, we developed a simple platform for high-efficiency, single-step, markerless, multi-copy chromosomal integration of full biochemical pathways in Saccharomyces cerevisiae. In this Di-CRISPR (delta integration CRISPR-Cas) platform based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas), we specifically designed guide RNA sequences to target multiple delta sites in the yeast genome. The generation of double stranded breaks at the delta sites allowed simultaneous integration of multiple copies of linearized donor DNA containing large biochemical pathways. With our newly developed Di-CRISPR platform, we were able to attain highly efficient and markerless integration of large biochemical pathways and achieve an unprecedented 18-copy genomic integration of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, thus generating a strain that was able to produce BDO directly from xylose. The simplicity and high efficiency of the Di-CRISPR platform could provide a superior alternative to high copy plasmids and would render this platform an invaluable tool for genome editing and metabolic engineering in S. cerevisiae.

Engineered pentafunctional minicellulosome for simultaneous saccharification and ethanol fermentation in Saccharomyces cerevisiae.

Several yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and ß-glucosidases in Saccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast.

Stiffness-modulated water retention and neovascularization of dermal fibroblast-encapsulating collagen gel.

There is increasing evidence that matrix stiffness modulates various phenotypic activities of cells surrounded by a three-dimensional (3D) matrix. These findings suggest that matrix stiffness can also regulate dermal fibroblasts activities to remodel, repair, and recreate skin dermis, but this has not yet been systematically demonstrated to date. This study examines the effects of matrix rigidity on the morphology, growth rates, and glycosaminoglycan (GAG) production of dermal fibroblasts cultured in collagen-based hydrogels with controlled elastic moduli. The elastic moduli (E) of collagen hydrogels were increased from 0.7 to 1.6 and 2.2 kPa by chemically cross-linking collagen fibrils with poly(ethylene glycol) disuccinimidylester. Increasing E of the hydrogel led to decreases in cellular spreading, nuclear aspect ratio, and growth rate. In contrast, the cellular GAG production level was elevated by increasing E from 0.7 to 1.6 kPa. The larger accumulation of GAG in the stiffer hydrogel led to increased water retention during exposure to air, as confirmed with magnetic resonance imaging. Additionally, in a chicken chorioallantoic membrane, a cell-encapsulating hydrogel with E of 1.6 kPa created dermis-like tissue with larger amount of GAG and density of blood vessels, while a cell-hydrogel construct with E of 0.7 kPa generated scar-like tissue. Overall, the results of this study will be highly useful for designing advanced tissue engineering scaffolds that can enhance the quality of a wide array of regenerated tissues including skin.

Generation of Cell-Instructive Collagen Gels through Thermodynamic Control.

Recent studies have demonstrated the usefulness of three-dimensional hydrogel scaffolds for cell instruction. However, the control of gel architectures in cell-friendly conditions remains a challenge. Here, we report a novel method to generate unique three-dimensional collagen gel structures for the modulation of cell phenotypes. This was achieved by directing collagen self-assembly with unreactive hydrophilic polyethylene glycol (PEG) chains. Our approach allowed the fiber sizes and mechanics of three-dimensional collagen gels to be readily controlled. It also enabled the recapitulation of distinctive structures such as large perimysial collagen cables. Through different experiments, we elucidated the underlying mechanism for this PEG-mediated thermodynamic regulation of gel structure. We further used these cell-instructive three-dimensional gels to bring about pronounced morphological changes in encapsulated fibroblasts and their activation to form contractile bundles. Overall, our platform fills a gap in the existing array of collagen scaffolds and can potentially be adapted to a variety of self-assembling systems.

A cell-instructive hydrogel to regulate malignancy of 3D tumor spheroids with matrix rigidity.

Three dimensional (3D) tumor spheroid models are becoming important biomedical tools for both fundamental and applied cancer studies, but current models do not account for different levels of cancer malignancy. Several studies have reported that the mechanical rigidity of a hydrogel plays a significant role in regulating the phenotypes of cancer cells adhered to the gel surface. This finding suggests that matrix rigidity should also modulate the malignancy of 3D tumor spheroids. However, the role of matrix stiffness is often confounded by concurrent changes in 3D matrix permeability. This study reports an advanced strategy to assemble 3D liver tumor spheroids with controlled intercellular organization, phenotypes, and angiogenic activities using hydrogels with controlled stiffness and minimal differences in molecular diffusivity. The elastic moduli of cell-encapsulated collagen gels were increased by stiffening interconnected collagen fibers with varied amounts of poly(ethylene glycol) di-(succinic acid N-hydroxysuccinimidyl ester). Interestingly, hepatocellular carcinoma cells encapsulated in a fat-like, softer hydrogel formed malignant cancer spheroids, while cells cultured in a liver-like, stiffer gel formed compact hepatoids with suppressed malignancy. Overall, both the hydrogel and the 3D tumor spheroids developed in this study will be greatly useful to better understand and regulate the emergent behaviors of various cancer cells.

[A case report of neuromyelitis optica with 37 years' interval between optic neuropathy and spinal cord lesion and literature review].

OBJECTIVE: To improve the recolonization of long term interval between optic neuropathy and spinal cord injury of neuromyelitis optica. METHODS: One 51-year old male patient with 37 years' interval between optic neuropathy and spinal cord injury of neuromyelitis optica underwent the examination of plasma and cerebrospinal fluid and head and spinal MRI examinations, who was also followed up. His clinical data were analyzed and related literature was reviewed. RESULTS: The myelin basic protein and IgG index in his cerebrospinal fluid was high, his oligoclonal band of cerebrospinal fluid was positive, and abnormal finding in visual evoked potential. Abnormal intramedullary long T(2) signals was showed in spinal cord MRI at T(1-7) segment. When diagnosed as neuromyelitis optica, the clinical symptom and signs was improved with corticosteroid and gamma globulin therapy. The patient was in stable condition at present. CONCLUSIONS: There could be a long term interval between optic neuritis and myelitis. One should pay attention to clinical features and imaging examination of subclinical lesion in spinal cord and brain and conoid the possibility of developing neuromyelitis optica or multiple sclerosis.

Tuning the non-equilibrium state of a drug-encapsulated poly(ethylene glycol) hydrogel for stem and progenitor cell mobilization.

Injectable and biodegradable hydrogels have been increasingly studied for sustained drug delivery in various molecular therapies. However, it remains a challenge to attain desired delivery rate at injection sites due to local tissue pressures exerted on the soft hydrogels. Furthermore, there is often limited controllability of stiffness and degradation rates, which are key factors required for achieving desired drug release rate and therapeutic efficacy. This study presents a stiff and metastable poly(ethylene glycol) diacrylate (PEGDA)-poly(ethylene imine) (PEI) hydrogel which exhibits an elastic modulus equivalent to bulk plastic materials, and controllable degradation rate independent of its initial elastic modulus. Such unique stiffness was attained from the highly branched architecture of PEI, and the decoupled controllability of degradation rate was achieved by tuning the non-equilibrium swelling of the hydrogel. Furthermore, a single intramuscular administration of granulocyte colony stimulating factor (GCSF)-encapsulated PEGDA-PEI hydrogel extended the mobilization of mononuclear cells to four days. A larger yield of expanded CD34+ and CD31+ endothelial progenitor cells (EPCs) was also obtained as compared to the daily bolus administration. Overall, the hydrogel created in this study will be useful for the controlled and sustained delivery of a wide array of drug molecules.