CRISPRi-based circuits to control gene expression in plants.

The construction of synthetic gene circuits in plants has been limited by a lack of orthogonal and modular parts. Here, we implement a CRISPR (clustered regularly interspaced short palindromic repeats) interference (CRISPRi)-based reversible gene circuit platform in plants. We create a toolkit of engineered repressible promoters of different strengths and construct NOT and NOR gates in Arabidopsis thaliana protoplasts. We determine the optimal processing system to express single guide RNAs from RNA Pol II promoters to introduce NOR gate programmability for interfacing with host regulatory sequences. The performance of a NOR gate in stably transformed Arabidopsis plants demonstrates the system's programmability and reversibility in a complex multicellular organism. Furthermore, cross-species activity of CRISPRi-based logic gates is shown in Physcomitrium patens, Triticum aestivum and Brassica napus protoplasts. Layering multiple NOR gates together creates OR, NIMPLY and AND logic functions, highlighting the modularity of our system. Our CRISPRi circuits are orthogonal, compact, reversible, programmable and modular and provide a platform for sophisticated spatiotemporal control of gene expression in plants.

TIEG1 Represses Smad7-Mediated Activation of TGF-ß1/Smad Signaling in Keloid Pathogenesis.

Transforming growth factor-ß (TGF-ß)/Smad signaling plays a key role in excessive fibrosis and keloid formations. Smad7 is a negative feedback regulator that prevents activation of TGF-ß/Smad signaling. However, the regulatory mechanism for Smad7 in the keloid pathogenic process remains elusive. Here, we show that expression of TIEG1 is markedly higher in keloid fibroblasts, whereas protein, mRNA, and promoter activity levels of Smad7 are decreased. When TIEG1 was knocked down with small interfering RNA, both the promoter activity and protein expression of Smad7 were increased, whereas collagen production and the proliferation, migration, and invasion of keloid fibroblasts were decreased. In contrast, TIEG1 overexpression led to a decrease in Smad7 expression and Smad7 promoter activity. Upon TGF-ß1 stimulation, TIEG1 promoted Smad2 phosphorylation by down-regulating Smad7. Luciferase reporter assays and chromatin immunoprecipitation assays further showed that TIEG1 can directly bind a GC-box/Sp1 site located between nucleotides -1392 and -1382 in the Smad7 promoter to repress Smad7 promoter activity. Taken together, these findings show that TIEG1 is highly expressed in human keloids and that it directly binds and represses Smad7 promoter-mediated activation of TGF-ß/Smad2 signaling, thus providing clues for development of TIEG1 blocking strategies for therapy or prophylaxis of keloids.

Effectiveness and safety of negative-pressure wound therapy for diabetic foot ulcers: a meta-analysis.

BACKGROUND: The authors conducted a meta-analysis to evaluate the effectiveness and safety of negative-pressure wound therapy for diabetic foot ulcers. METHODS: PubMed, EMBASE, and the Cochrane Library were searched to identify relevant studies published up to February of 2013. All randomized controlled trials comparing negative-pressure wound therapy and non-negative-pressure wound therapy (i.e., standard wound care) for diabetic foot ulcers were included. Results were pooled using meta-analysis to assess the efficacy and safety of negative pressure in managing diabetic foot ulcers. RESULTS: The databases were derived from eight qualified studies that included a total of 669 patients. Overall, compared with the non-negative-pressure wound therapy-treated diabetic foot ulcers, negative pressure resulted in a significantly higher proportion of healed ulcers (relative risk, 1.52; 95 percent CI, 1.23 to 1.89; p<0.001), more reduction of ulcer area (standardized mean difference, 0.89; 95 percent CI, 0.41 to 1.37; p=0.003), and shorter time to wound healing (standardized mean difference, -1.10; 95 percent CI, -1.83 to -0.37; p=0.003). Negative-pressure wound therapy patients also experienced significantly fewer major amputations (relative risk, 0.14; 95 percent CI, 0.04 to 0.51; p=0.003), but the rate of minor amputations was not affected (p=0.837). No significant difference was observed between negative-pressure wound therapy and non-negative-pressure wound therapy (p=0.683). No heterogeneity among studies was detected. CONCLUSION: Negative-pressure wound therapy appears to be more effective for diabetic foot ulcers compared with non-negative-pressure wound therapy, and has a similar safety profile. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.

[Proteomic analysis between keloid and normal skin].

OBJECTIVE: To investigate and search correlative proteins of keloid by comparing the results of differential proteomic analysis between keloid and normal skin. METHODS: From January 2010 to June 2010 two-dimensional gel electrophoresis was used to define patterns of protein expression in keloid skin from 8 patients and matched normal skin from 3 patients. Differential expression protein spots were showed and analyzed by matrix-assisted laser desorption ionization-time of flying/time of flying (MALDI-TOF/TOF) mass spectrometry. RESULTS: This study succeeded to provide a two-dimensional protein profiling comparison between normal skin and keloid. Gel-analysis software identified an average of 2978 spots in keloid while 3053 spots in normal skin and statistical filtering yielded 40 spots of a 4-fold change, 32 of which were identified by using mass spectrometry, 20 were up-regulated and 12 were down-regulated. Functional analysis revealed that these proteins could be fractionated to carrier proteins (3 proteins), signal transduction proteins (4 proteins), proliferation and apoptosis related proteins (2 proteins), cytoskeleton proteins (6 proteins), extracellular matrix proteins (8 proteins), immunity related proteins (3 proteins), tumor related proteins (2 proteins), and function unknown protein (4 proteins). CONCLUSIONS: Proteomic analysis can identify the proteins with variance of keloid versus normal skin. The further research to these differential proteins may help reveal the pathogenesis of keloid and provide new treatments for keloid.

Early escharectomy and concurrent composite skin grafting over human acellular dermal matrix scaffold for covering deep facial burns.

BACKGROUND: Although escharectomy and full-thickness skin autografting have been widely used to treat deep facial burns, the clinical outcomes remain unacceptable. Composite razor-thin skin grafting over acellular dermal matrix scaffold has been used successfully in repairing burns of the trunk and limbs, but its use in covering deep facial burns has rarely been reported. In this study, the authors investigated the clinical outcomes of early escharectomy and concurrent composite razor-thin skin autografting and acellular dermal matrix scaffold for treating deep facial burns. METHODS: Patients with deep facial burns (n = 16) involving 8 to 30 percent of the total body surface area received early escharectomy by postburn day 3 and concurrent, one-stage, large, razor-thin skin autografting on top of human acellular dermal matrix scaffold. Wound dressings were changed on postoperative days 7, 9, and 12 to examine the survival of skin autografts. Patients were followed up for 12 months to evaluate their facial profiles. RESULTS: The take rate of composite skin autografts was 97.3 percent at postoperative day 12. At the follow-up visit, the skin autografts appeared normal in color, with soft texture and good elasticity. The skin junctures showed little scarring. The patients exhibited a chubby facial appearance and abundant expression, except for one patient with microstomia and two patients with ectropion who required further plastic surgical interventions. CONCLUSION: Early escharectomy and concurrent composite razor-thin skin autografting on top of acellular dermal matrix scaffold constitute an effective and favorable option for covering deep facial burns, especially for patients with limited donor sites.

[Expression of Smads in keloid scarring].

OBJECTIVE: To investigate the differential expression of different types of Smads in keloids, normal scars and normal skins and its possible clinicopathological significance. METHODS: RT-PCR and Western blot methods were used to examine the expression of Smads mRNA and proteins level in 10 cases of keloid, in 10 cases of normal scar and in 10 cases of normal skin tissues and fibroblasts. Fibroblasts of keloid, normal scar and normal skin were cultured in vitro. The expression difference were compared and analyzed by t-test, there was statistical difference when P < 0.05. RESULTS: The mRNA and protein expression of inhibitory Smad7 were significantly down regulated in keloid compared with normal scar (P < 0.05) and normal skin (P < 0.05). However, no significant difference of the mRNA and protein expression of Smad2, 3 and the protein expression of phosphorylation of Smad2, 3 in keloid, normal scar, normal skin tissues and fibroblasts. CONCLUSIONS: The decreased expression of Smad7 in keloid might play a significant role in the increased TGF-beta1/Smads signal transduction, which can not be terminated by autologous negative feedback cycle.

[Observation of the effect of the mixed composite skin graft on deep partial thickness burn wounds].

OBJECTIVE: To evaluate the effect of mixed composite skin graft on the deep partial thickness burn wounds after tangential excision in burn patients. METHODS: Tangential excision was performed in 30 extremities of 23 burn patients within 3 postburn days (PBDs). Then large pieces of homologous acellular dermal matrix were grafted onto the superficial fascia with razor thin autoskin on top of them. The survival rate of skin grafts, the appearance and the functional recovery of the extremities were observed on 10 to 12 post operative day (POD). Skin samples from a healed wound of a patient were harvested three months after the injury for pathologic examination. RESULTS: The survival rate of the composite skin grafts was 93%. Necrosis was encountered in 7% of the grafts in the lower extremities due to the poor fixation of the grafts leading to separation of autologous skin and the dermal template, and also due to infection resulting in lysis of the grafts. The grafted skin was excellent in the appearance and elasticity, and function of the injured extremities recovered well after grafting after 3 - 6 months of follow-up. Epidermal and dermal texture was also good as shown by pathologic examination. CONCLUSION: Mixed composite skin grafting after early tangential excision might be an ideal and effective method in the management of deep partial thickness burn wounds.

[In vitro cleavage activity of the ribozymes constructed specifically for the second exons of pro alpha I and III collagen genes].

OBJECTIVE: To study the in vitro cleavage activity and the reaction conditions of the hammerhead ribozymes for the second exons of pro alpha 1I and III collagen genes, and observe the effect of the two ribozymes on collagen synthesis by the fibroblasts in scar tissue. METHODS: The fragments of the second exons of pro alpha 1I and III collagen genes were cloned into the plasmids pT-I and pT-III and labeled with (32)P during transcription to obtain the target RNA. The transcription products of the plasmids pT-gI and pT-gIII containing specific ribozymes were incubated with the label target RNAs, respectively, under various conditions, and the results observed by electrophoresis autoradiography. The constructed ribozymes were transduced into cultured fibroblasts via liposomes for investigation of their effects on mRNA synthesis of type I and III collagen protein by image analysis. RESULTS: The two ribozymes (rgI and rg III) could efficiently cleave their target RNAs at both 37 degrees Celsius and 42 degrees Celsius, in the presence of Mg(2+) within a relatively wide concentration range form 10 to 20 mmol/L. When the temperature was lowered from 65 degrees Celsius to 37 degrees Celsius and maintained at 37 degrees Celsius, the cleavage activity of the ribozymes reached the maximum. The expression of mRNA of I and III collagen decreased accompanied by reduced collagen synthesis. CONCLUSION: Hammerhead ribozymes for the fragments of the second exons of pro alpha 1I and III collagen genes can cleave its target RNAs in vitro and effectively inhibit the collagen synthesis by the scar-derived fibroblasts.

[A study of the dynamic relationship between AVP mRNA expression in PVN and immuno-responsiveness in rats].

OBJECTIVE: To investigate the proper dynamic relationship between AVP mRNA expression in PVN and immune response. METHODS: Through the establishment of the immuno-enhancement and immunosuppression rat models, the in situ hybridization histochemical methods and the images analytical techniques were used to detect the dynamic variations of AVP mRNA content of PVN during different periods of immune response. RESULTS: 1. The average area of AVP mRNA positive signal in single cell of PVN in the immune suppression group was significantly smaller than that in the immune enhancement group and control group (P < 0.05). 2. The average gray degree of AVP mRNA positive signal in single cell of PVN in the maximum period of immune response was lower than that in the incipient enhancement period of immune response (P < 0.05). On the other hand, the average gray degree was clearly higher in the minimum period of immune response than that in the incipient suppression period of immune response (P < 0.05). 3. The total area that the AVP mRNA-positive signal occupied in the whole PVN in the maximum enhancement period of immune response was significantly larger than that in the incipient enhancement period response and the control group (P < 0.05). Conversely, it was evidently smaller in the minimum suppression period than in the incipient suppression period of immune response (P < 0.05). 4. The average gray degree of AVP mRNA positive signal in the whole PVN in the maximum enhancement period of immune response was significantly lower than that in the incipient enhancement period and the control group (P < 0.05). In turn, it was obviously higher in the minimum suppression period than in the incipient suppression period of immune response and the control group (P < 0.05). CONCLUSION: There is a positive relationship between the AVP mRNA content of PVN and the intensity of immune responsiveness, indicating that the situation of AVP gene transcription varies with the variation of immune responsiveness.