Early identification of delayed wound healing in complex diabetic foot ulcers treated with a dermal regeneration template: a novel clinical target and its risk factors.

BACKGROUND: The dermal regeneration template (DRT), a tissue-engineered skin substitute composing a permanent dermal matrix and an upper temporary silicone layer that serves as the epidermis, has demonstrated efficacy in treating uncomplicated diabetic foot ulcers (DFUs). Our institution has obtained good outcomes with DRT in patients with more complicated DFUs. Because of its chronicity, the authors are working to identify a clinical target that anticipates delayed healing early in the treatment in addition to determining the risk factors linked to this endpoint to increase prevention. MATERIALS AND METHODS: This retrospective single-center study analyzed patients with DFUs who underwent wound reconstruction using DRT between 2016 and 2021. The patients were categorized into poor or good graft-take groups based on their DRT status on the 21st day after the application. Their relationship with complete healing (CH) rate at day 180 was analyzed. Variables were collected for risk factors for poor graft take at day 21. Independent risk factors were identified after multivariable analysis. The causes of poor graft take were also reported. RESULTS: This study examined 80 patients (38 and 42 patients in the poor and good graft-take groups, respectively). On day 180, the CH rate was 86.3% overall, but the poor graft-take group had a significantly lower CH rate (76.3 vs. 95.2%, P =0.021) than the good graft-take group. Our analysis identified four independent risk factors: transcutaneous oxygen pressure less than 30 mmHg (odds ratio, 154.14), off-loading device usage (0.03), diabetic neuropathy (6.51), and toe wound (0.20). The most frequent cause of poor graft take was infection (44.7%), followed by vascular compromise (21.1%) and hematoma (15.8%). CONCLUSION: Our study introduces the novel concept of poor graft take at day 21 associated with delayed wound healing. Four independent risk factors were identified, which allows physicians to arrange interventions to mitigate their effects or select patients more precisely. DRT represents a viable alternative to address DFUs, even in complicated wounds. A subsequent split-thickness skin graft is not always necessary to achieve CH.

Assessment of Intestinal Immunity and Permeability of Broilers on Partial Replacement Diets of Two-Stage Fermented Soybean Meal by Bacillus velezensis and Lactobacillus brevis ATCC 367.

The effect of soybean peptides from fermented soybean meal on the intestinal immunity and tight junction of broilers was assessed. Roughly, two-stage fermented soybean meal prepared with Bv and Lb (FSBMB+L), which has nearly three times higher soluble peptides than soybean meal (SBM), and reduced galacto-oligosaccharide (GOS) content and allergen protein. The one-stage fermented by Bv (FSBMB) has the highest soluble peptides, while commercial lactic acid bacteria (FSBML) has the highest Lactic acid bacteria count; these were used to compare the differences in the process. Ross308 broilers (n = 320) were divided into four groups: SBM diet and a diet replaced with 6% FSBMB+L, FSBMB, or FSBML. The growth performance was recorded during the experiment, and six birds (35-day-old) per group were euthanized. Analysis of their jejunum and ileum showed that the fermented soybean meal significantly improved the villus height in the jejunum (p < 0.05) and reduced the crypt hyperplasia. The FSBMB group had the highest reducing crypt depth; however, the FSBMB+L group had the highest villus height/crypt depth in the ileum (p < 0.05). In the jejunum, the relative mRNA of CLDN-1 and Occludin increased 2-fold in the treatments, and ZO-1 mRNA increased 1.5 times in FSBML and FSBMB+L (p < 0.05). Furthermore, the level of NF-κB and IL-6 mRNAs in FSBML increased, respectively, by 4 and 2.5 times. While FSBMB, along with FSBMB+L, had a 1.5-fold increase in the mRNA of IL-10, that of NF-κB increased 2-fold. FSBMB+L and FSBMB singly led to a 2- and 3-fold increase in IL-6 mRNA, respectively (p < 0.05). FSBMB and FSBMB+L can also upregulate MUC2 in the jejunum (p < 0.05). In short, using the soybean peptides from two-stage fermented soybean meal can ameliorate the negative factors of SBM and effectively regulate immune expression and intestinal repair, which will help broilers maintain intestinal integrity.

Crystal structure of the ligand-binding domain of a LysR-type transcriptional regulator: transcriptional activation via a rotary switch.

LysR-type transcriptional regulators (LTTRs) generally bind to target promoters in two conformations, depending on the availability of inducing ligands. OccR is an LTTR that regulates the octopine catabolism operon of Agrobacterium tumefaciens. OccR binds to a site located between the divergent occQ and occR promoters. Octopine triggers a conformational change that activates the occQ promoter, and does not affect autorepression. This change shortens the length of bound DNA and relaxes a high-angle DNA bend. Here, we describe the crystal structure of the ligand-binding domain (LBD) of OccR apoprotein and holoprotein. Pairs of LBDs form dimers with extensive hydrogen bonding, while pairs of dimers interact via a single helix, creating a tetramer interface. Octopine causes a 70° rotation of each dimer with respect to the opposite dimer, precisely at the tetramer interface. We modeled the DNA binding domain (DBD), linker helix and bound DNA onto the apoprotein and holoprotein. The two DBDs of the modeled apoprotein lie far apart and the bound DNA between them has a high-angle DNA bend. In contrast, the two DBDs of the holoprotein lie closer to each other, with a low DNA bend angle. This inter-dimer pivot fully explains earlier studies of this LTTR.

Glucose repression can be alleviated by reducing glucose phosphorylation rate in Saccharomyces cerevisiae.

Microorganisms commonly exhibit preferential glucose consumption and diauxic growth when cultured in mixtures of glucose and other sugars. Although various genetic perturbations have alleviated the effects of glucose repression on consumption of specific sugars, a broadly applicable mechanism remains unknown. Here, we report that a reduction in the rate of glucose phosphorylation alleviates the effects of glucose repression in Saccharomyces cerevisiae. Through adaptive evolution under a mixture of xylose and the glucose analog 2-deoxyglucose, we isolated a mutant strain capable of simultaneously consuming glucose and xylose. Genome sequencing of the evolved mutant followed by CRISPR/Cas9-based reverse engineering revealed that mutations in the glucose phosphorylating enzymes (Hxk1, Hxk2, Glk1) were sufficient to confer simultaneous glucose and xylose utilization. We then found that varying hexokinase expression with an inducible promoter led to the simultaneous utilization of glucose and xylose. Interestingly, no mutations in sugar transporters occurred during the evolution, and no specific transporter played an indispensable role in simultaneous sugar utilization. Additionally, we demonstrated that slowing glucose consumption also enabled simultaneous utilization of glucose and galactose. These results suggest that the rate of intracellular glucose phosphorylation is a decisive factor for metabolic regulations of mixed sugars.

X-ray crystal structures of the pheromone-binding domains of two quorum-hindered transcription factors, YenR of Yersinia enterocolitica and CepR2 of Burkholderia cenocepacia.

The ability of LuxR-type proteins to regulate transcription is controlled by bacterial pheromones, N-acylhomoserine lactones (AHLs). Most LuxR-family proteins require their cognate AHLs for activity, and some of them require AHLs for folding and stability, and for protease-resistance. However, a few members of this family are able to fold, dimerize, bind DNA, and regulate transcription in the absence of AHLs; moreover, these proteins are antagonized by their cognate AHLs. One such protein is YenR of Yersinia enterocolitica, which is antagonized by N-3-oxohexanoyl-l-homoserine lactone (OHHL). This pheromone is produced by the OHHL synthase, a product of the adjacent yenI gene. Another example is CepR2 of Burkholderia cenocepacia, which is antagonized by N-octanoyl-l-homoserine lactone (OHL), whose synthesis is directed by the cepI gene of the same bacterium. Here, we describe the high-resolution crystal structures of the AHL binding domains of YenR and CepR2. YenR was crystallized in the presence and absence of OHHL. While this ligand does not cause large scale changes in the YenR structure, it does alter the orientation of several highly conserved YenR residues within and near the pheromone-binding pocket, which in turn caused a significant movement of a surface-exposed loop.

GroE chaperonins assisted functional expression of bacterial enzymes in Saccharomyces cerevisiae.

Rapid advances in the capabilities of reading and writing DNA along with increasing understanding of microbial metabolism at the systems-level have paved an incredible path for metabolic engineering. Despite these advances, post-translational tools facilitating functional expression of heterologous enzymes in model hosts have not been developed well. Some bacterial enzymes, such as Escherichia coli xylose isomerase (XI) and arabinose isomerase (AI) which are essential for utilizing cellulosic sugars, cannot be functionally expressed in Saccharomyces cerevisiae. We hypothesized and demonstrated that the mismatching of the HSP60 chaperone systems between bacterial and eukaryotic cells might be the reason these bacterial enzymes cannot be functionally expressed in yeast. The results showed that the co-expression of E. coli GroE can facilitate the functional expression of E. coli XI and AI, as well as the Agrobacterium tumefaciens D-psicose epimerase in S. cerevisiae. The co-expression of bacterial chaperonins in S. cerevisiae is a promising post-translational strategy for the functional expression of bacterial enzymes in yeast. Biotechnol. Bioeng. 2016;113: 2149-2155. © 2016 Wiley Periodicals, Inc.

Rapid and marker-free refactoring of xylose-fermenting yeast strains with Cas9/CRISPR.

Genomic integration of expression cassettes containing heterologous genes into yeast with traditional methods inevitably deposits undesirable genetic elements into yeast chromosomes, such as plasmid-borne multiple cloning sites, antibiotic resistance genes, Escherichia coli origins, and yeast auxotrophic markers. Specifically, drug resistance genes for selecting transformants could hamper further industrial usage of the resulting strains because of public health concerns. While we constructed an efficient and rapid xylose-fermenting Saccharomyces cerevisiae, the engineered strain (SR8) might not be readily used for a large-scale fermentation because the SR8 strain contained multiple copies of drug resistance genes. We utilized the Cas9/CRISPR-based technique to refactor an efficient xylose-fermenting yeast strain without depositing any undesirable genetic elements in resulting strains. In order to integrate genes (XYL1, XYL2, and XYL3) coding for xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis, and delete both PHO13 and ALD6, a double-strand break formation by Cas9 and its repair by homologous recombination were exploited. Specifically, plasmids containing guide RNAs targeting PHO13 and ALD6 were sequentially co-transformed with linearized DNA fragments containing XYL1, XYL2, and XYL3 into S. cerevisiae expressing Cas9. As a result, two copies of XYL1, XYL2, and XYL3 were integrated into the loci of PHO13 and ALD6 for achieving overexpression of heterologous genes and knockout of endogenous genes simultaneously. With further prototrophic complementation, we were able to construct an engineered strain exhibiting comparable xylose fermentation capabilities with SR8 within 3 weeks. We report a detailed procedure for refactoring xylose-fermenting yeast using any host strains. The refactored strains using our procedure could be readily used for large-scale fermentations since they have no antibiotic resistant markers.

Yeast synthetic biology toolbox and applications for biofuel production.

Yeasts are efficient biofuel producers with numerous advantages outcompeting bacterial counterparts. While most synthetic biology tools have been developed and customized for bacteria especially for Escherichia coli, yeast synthetic biological tools have been exploited for improving yeast to produce fuels and chemicals from renewable biomass. Here we review the current status of synthetic biological tools and their applications for biofuel production, focusing on the model strain Saccharomyces cerevisiae We describe assembly techniques that have been developed for constructing genes, pathways, and genomes in yeast. Moreover, we discuss synthetic parts for allowing precise control of gene expression at both transcriptional and translational levels. Applications of these synthetic biological approaches have led to identification of effective gene targets that are responsible for desirable traits, such as cellulosic sugar utilization, advanced biofuel production, and enhanced tolerance against toxic products for biofuel production from renewable biomass. Although an array of synthetic biology tools and devices are available, we observed some gaps existing in tool development to achieve industrial utilization. Looking forward, future tool development should focus on industrial cultivation conditions utilizing industrial strains.

Most mutant OccR proteins that are defective in positive control hold operator DNA in a locked high-angle bend.

OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid. Positive control of the occ genes occurs in response to octopine, a nutrient released from crown gall tumors. OccR also functions as an autorepressor in the presence or absence of octopine. OccR binds to a site between occQ and occR in the presence or absence of octopine, although octopine triggers a conformational change that shortens the DNA footprint and relaxes a DNA bend. In order to determine the roles of this conformational change in transcriptional activation, we isolated 11 OccR mutants that were defective in activation of the occQ promoter but were still capable of autorepression. The mutations in these mutants spanned most of the length of the protein. Two additional positive-control mutants were isolated using site-directed mutagenesis. Twelve mutant proteins displayed a high-angle DNA bend in the presence or absence of octopine. One mutant, the L26A mutant, showed ligand-responsive DNA binding similar to that of wild-type OccR and therefore must be impaired in a subsequent step in activation.

The quorum-hindered transcription factor YenR of Yersinia enterocolitica inhibits pheromone production and promotes motility via a small non-coding RNA.

The YenR and YenI proteins of Yersinia enterocolitica resemble the quorum sensing proteins LuxR and LuxI of Vibrio fischeri. Apo-YenR activated a gene, designated yenS, that lies adjacent to and divergent from yenR. YenR-dependent expression of yenS was inhibited by endogenous or exogenous 3-oxohexanoylhomoserine lactone (OHHL) a pheromone made by YenI. Purified apo-YenR bound non-cooperatively to two 20-nucleotide sites that lie upstream of yenS. Binding occurred in the absence of (OHHL), and YenR was largely released from the DNA by this pheromone. yenS encoded two non-translated RNAs 169 and 105 nucleotides long that share the same 5' end but have different 3' ends. One or both RNAs inhibited the translation and accumulation of the yenI mRNA by binding to a region that overlaps the YenI start codon. A mutation in yenI strongly stimulated swarming motility on the surface of semi-solid agar, while exogenous OHHL completely suppressed this phenotype. Hypermotility in yenI mutants was also suppressed by mutations in yenR or yenS, suggesting that YenS plays a direct, stimulatory role in swarming motility.

LuxR-type quorum-sensing regulators that are detached from common scents.

The ability of LuxR-type proteins to regulate transcription is controlled by bacterial pheromones, N-acylhomoserine lactones (AHLs). Most LuxR-family proteins require their cognate AHLs for activity, and at least some of them require AHLs for folding and protease resistance. However, a few members of this family are able to fold, dimerize, bind DNA, and regulate transcription in the absence of AHLs; moreover, these proteins are antagonized by their cognate AHLs. Complexes between some of these proteins and their DNA binding sites are disrupted by AHLs in vitro. All such proteins are fairly closely related within the larger LuxR family, indicating that they share a relatively recent common ancestor. The 3' ends of the genes encoding these receptors invariably overlap with the 3' ends of the cognate AHL synthase genes, suggesting additional antagonism at the level of mRNA synthesis, stability or translation.

Reconstitution of the biochemical activities of the AttJ repressor and the AttK, AttL, and AttM catabolic enzymes of Agrobacterium tumefaciens.

The attKLM operon encodes a lactonase (AttM) that hydrolyzes acylhomoserine lactone autoinducers, as well as two putative dehydrogenases (AttK and AttL). Here we show that AttK, AttL, and AttM collectively covert gamma-butyrolactone to succinate. Two metabolic intermediates, gamma-hydroxybutyrate and succinic semialdehyde, inactivated the AttJ repressor in vitro and induced attKLM transcription in vivo.

Direct binding of the quorum sensing regulator CepR of Burkholderia cenocepacia to two target promoters in vitro.

Burkholderia cenocepacia is an opportunistic human pathogen that can aggressively colonize the cystic fibrosis lung. This organism has a LuxR/LuxI-type quorum sensing system that enables cell-cell communication via exchange of acyl homoserine lactones (AHLs). The CepR and CepI proteins constitute a global regulatory system, controlling expression of at least 40 genes, including those controlling swarming motility and biofilm formation. In this study, we isolated seven lacZ fusions in a clinical isolate of B. cenocepacia that are inducible by octanoyl-HSL. Induction of all of these genes requires CepR. The cepI promoter was tested for induction by a set of 33 synthetic autoinducers and analogues, and was most strongly induced by long-chain AHLs lacking 3-oxo substitutions. Expression of this promoter was inhibited by high concentrations of three different autoinducers, each having six-carbon acyl chains. When CepR protein was overproduced in Escherichia coli, it accumulated in a soluble form in the presence of octanoyl-HSL, but accumulated only as insoluble inclusion bodies in its absence. Purified CepR-OHL complexes bound to specific DNA sequences at the cepI and aidA promoters with high specificity. These binding sites included a 16-nucleotide imperfect dyad symmetry. Both CepR binding sites are centred approximately 44 nucleotides upstream of the respective transcription start sites.

Regulation of the Bacillus subtilis extracytoplasmic function protein sigma(Y) and its target promoters.

The Bacillus subtilis extracytoplasmic function sigma factor sigma(Y) is of unknown function. We demonstrate that the sigY operon is expressed from an autoregulatory promoter site, P(Y). We selected for transposon-induced mutations that upregulate P(Y) transcription in an attempt to identify genes involved in sigma(Y) regulation. The resulting insertions disrupted yxlC, the gene immediately downstream of sigY. However, the phenotype of the yxlC::Tn10 insertion was due to polarity on the downstream genes of the sigY operon; a nonpolar insertion in yxlC did not lead to derepression of P(Y). Further analyses revealed that both yxlD and yxlE encoded proteins important for the negative regulation of sigma(Y) activity. A comparison of the transcriptomes of wild-type and yxlC::Tn10 mutant strains revealed elevated expression of several operons. However, only one additional gene, ybgB, was unambiguously identified as a direct target for sigma(Y). This was supported by analysis of direct targets for sigma(Y) transcription with whole-genome runoff transcription followed by macroarray analysis.