Phenolic profile of a Parma violet unveiled by chemical and fluorescence imaging.

The ability of phenolic compounds to autofluoresce upon illumination by UV or blue light was exploited to explore the nature and distribution of these metabolites within the flower petals, leaves and roots of the violet, Viola alba subsp. dehnhardtii. This was achieved through a dual complementary approach that combined fluorescence microscopy imaging of living intact tissues and chemical extraction of pulverized material. The blue to red fluorescence displayed by living tissues upon illumination was indicative of their richness in phenolic compounds. Phenolic acids were found in all tissues, while flavonoids characterized the aerial part of the plant, anthocyanidins being restricted to the petals. The chemical quantification of phenolics in plant extracts confirmed their tissue-specific distribution and abundance. A key finding was that the spectral signatures obtained through confocal microscopy of endogenous fluorophores in living tissues and their counterpart extracts share the same fluorescence patterns, pointing out the potential of fluorescence imaging of intact organs for a proper estimation of their phenolic content. In addition, this study highlighted a few distinct morphology cell types, in particular foliar-glandular-like structures, and jagged petal cell walls. Altogether, these data provide a comprehensive histochemical localization of phenolics in living tissues of a violet. Converting fluorescence imaging into a chemical imprint indicated that one can rely on fluorescence microscopy of intact living tissues as a rapid, non-destructive means to follow their phenolic imprint under various environmental conditions.

A Low-Noise Direct Incremental A/D Converter for FET-Based THz Imaging Detectors.

This paper presents the design, implementation and characterization results of a pixel-level readout chain integrated with a FET-based terahertz (THz) detector for imaging applications. The readout chain is fabricated in a standard 150-nm CMOS technology and contains a cascade of a preamplification and noise reduction stage based on a parametric chopper amplifier and a direct analog-to-digital conversion by means of an incremental ΣΔ converter, performing a lock-in operation with modulated sources. The FET detector is integrated with an on-chip antenna operating in the frequency range of 325⁻375 GHz and compliant with all process design rules. The cascade of the FET THz detector and readout chain is evaluated in terms of responsivity and Noise Equivalent Power (NEP) measurements. The measured readout input-referred noise of 1.6 µ V r m s allows preserving the FET detector sensitivity by achieving a minimum NEP of 376 pW/ Hz in the optimum bias condition, while directly providing a digital output. The integrated readout chain features 65-dB peak-SNR and 80-µ W power consumption from a 1.8-V supply. The area of the antenna-coupled FET detector and the readout chain fits a pixel pitch of 455 µm, which is suitable for pixel array implementation. The proposed THz pixel has been successfully applied for imaging of concealed objects in a paper envelope under continuous-wave illumination.

Risk factors for postoperative hematoma after inguinal hernia repair: an update.

BACKGROUND: We recently sensed an increase in the frequency of groin hematoma after inguinal hernia repair (IHR) at our institution. The aim of this study was to provide a more updated assessment of the risk factors inherent to this complication. METHODS: We performed a case-control study of all adult patients (age ≥ 18 y) who developed a groin hematoma after IHR at our institution between 2003 and 2015. Univariate and multivariable analyses were performed to assess for independent predictors for groin hematoma. RESULTS: A total of 96 patients (among 6608 IHR) developed a groin hematoma, (60 were observed, 36 required intervention). The hematoma frequency increased from our previous study (1.4 % versus 0.9%, P < 0.01). Mean age was 64.6 y (range: 18-92), and 84.3% were men. There was no significant difference in the laterality, type, or technique of IHR between cases and controls. Univariate analysis (odds ratio [95% confidence interval], P) identified warfarin usage (3.5, [1.6-6.4], P < 0.01), valvular heart disease (11.6, [2.6-51.3], P < 0.01), atrial fibrillation (2.6, [1.2-5.5], P = 0.01), hypertension (2.03, [1.1-3.6], P = 0.02), recurrent hernia (3.7, [1.4-9.7], P < 0.01), and coronary artery disease (2.1, [1.0-4.4 ], P = 0.05) as significant preoperative factors. The proportion of patients on warfarin decreased since our prior report (31% versus 42%, P = 0.20). On multivariable regression, warfarin and recurrent hernia were independent predictors of hematoma development. CONCLUSIONS: Independent risk factors for the development of groin hematoma after IHR included warfarin use and recurrent hernia. Careful consideration for anticoagulation and surgical hypervigilance remains prudent in all patients undergoing IHR and especially those with recurrence.

Tumor-Free Transplantation of Patient-Derived Induced Pluripotent Stem Cell Progeny for Customized Islet Regeneration.

UNLABELLED: Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms, yet their clinical translation has been compromised by their biosafety concern. Here, we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Moreover, removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable, depending on the oncogenic load, with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus, transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. SIGNIFICANCE: Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products, especially when reprogrammed with integrating vectors. Two major underlying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in testing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature ß cell phenotype would lead to safe islet replacement therapy for diabetes.

Catch me if you can early simulation efforts affect fundamental surgical skill assessment scores.

BACKGROUND: We evaluated whether early exposure to a simulation curriculum enhances acquired surgical skills. METHODS: The "Surgical Olympics" evaluates interns on basic surgical skills and knowledge. After the Summer Olympics (July), interns were randomly divided into groups: "A" participated in a 7-week curriculum once a week, whereas "B" attended 7 weeks of lectures once a week. All interns then participated in the October Olympics. The 2 groups then switched. Finally, all interns completed a January Olympics. RESULTS: Scores were tabulated for the July, October, and January Olympics. Mean scores (A = 182 ± 42, Group B = 188 ± 34; P = .70) were similar in July; in October, group A (mean score = 237 ± 31) outperformed group B (mean score = 200 ± 32; P = .01). Mean total scores in January (A = 290 ± 34, B = 276 ± 34; P = .32) were similar. CONCLUSIONS: Early exposure to a surgical simulation curriculum enhances surgical intern performance in our Surgical Olympics. Subsequent simulation experience helps learners close this gap.

Needle thoracostomy: Clinical effectiveness is improved using a longer angiocatheter.

BACKGROUND: Decompression of tension physiology may be lifesaving, but significant doubts remain regarding ideal needle thoracostomy (NT) catheter length in the treatment of tension physiology. We aimed to demonstrate increased clinical effectiveness of longer NT angiocatheter (8 cm) compared with current Advanced Trauma Life Support recommendations of 5-cm NT length. METHODS: This is a retrospective review of all adult trauma patients from 2003 to 2013 (age > 15 years) transported to a Level I trauma center. Patients underwent NT at the second intercostal space midclavicular line, either at the scene of injury, during transport (prehospital), or during initial hospital trauma resuscitation. Before March 2011, both prehospital and hospital trauma team NT equipment routinely had a 5-cm angiocatheter available. After March 2011, prehospital providers were provided an 8-cm angiocatheter. Effectiveness was defined as documented clinical improvement in respiratory, cardiovascular, or general clinical condition. RESULTS: There were 91 NTs performed on 70 patients (21 bilateral placements) either in the field (prehospital, n = 41) or as part of resuscitation in the hospital (hospital, n = 29). Effectiveness of NT was 48% until March 2011 (n = 24). NT effectiveness was significantly higher in the prehospital setting than in the hospital (68.3% success rate vs. 20.7%, p < 0.01). Patients who underwent NT using 8 cm compared with 5 cm were significantly more effective (83% vs. 41%, respectively, p = 0.01). No complications of NT were identified in either group. CONCLUSION: Eight-centimeter angiocatheters are more effective at chest decompression compared with currently recommended 5 cm at the second intercostal space midclavicular line. LEVEL OF EVIDENCE: Therapeutic study, level IV.

Do You See What I See? How We Use Video as an Adjunct to General Surgery Resident Education.

OBJECTIVE: Preparation of learners for surgical operations varies by institution, surgeon staff, and the trainees themselves. Often the operative environment is overwhelming for surgical trainees and the educational experience is substandard due to inadequate preparation. We sought to develop a simple, quick, and interactive tool that might assess each individual trainee's knowledge baseline before participating in minimally invasive surgery (MIS). DESIGN: A 4-minute video with 5 separate muted clips from laparoscopic procedures (splenectomy, gastric band removal, cholecystectomy, adrenalectomy, and inguinal hernia repair) was created and shown to medical students (MS), general surgery residents, and staff surgeons. Participants were asked to watch the video and commentate (provide facts) on the operation, body region, instruments, anatomy, pathology, and surgical technique. Comments were scored using a 100-point grading scale (100 facts agreed upon by 8 surgical staff and trainees) with points deducted for incorrect answers. All participants were video recorded. Performance was scored by 2 separate raters. SETTING: An academic medical center. PARTICIPANTS: MS = 10, interns (n = 8), postgraduate year 2 residents (PGY)2s (n = 11), PGY3s (n = 10), PGY4s (n = 9), PGY5s (n = 7), and general surgery staff surgeons (n = 5). RESULTS: Scores ranged from -5 to 76 total facts offered during the 4-minute video examination. MS scored the lowest (mean, range; 5, -5 to 8); interns were better (17, 4-29), followed by PGY2s (31, 21-34), PGY3s (33, 10-44), PGY4s (44, 19-47), PGY5s (48, 28-49), and staff (48, 17-76), p < 0.001. Rater concordance was 0.98-measured using a concordance correlation coefficient (95% CI: 0.96-0.99). Only 2 of 8 interns acknowledged the critical view during the laparoscopic cholecystectomy video clip vs 10 of 11 PGY2 residents (p < 0.003). Of 8 interns, 7 misperceived the spleen as the liver in the splenectomy clip vs 2 of 7 chief residents (p = 0.02). CONCLUSIONS: Not surprisingly, more experienced surgeons were able to relay a larger number of laparoscopic facts during a 4-minute video clip of 5 MIS operations than inexperienced trainees. However, even tenured staff surgeons relayed very few facts on procedures they were unfamiliar with. The potential differentiating capabilities of such a quick and inexpensive effort has pushed us to generate better online learning tools (operative modules) and hands-on simulation resources for our learners. We aim to repeat this and other studies to see if our learners are better prepared for video assessment and ultimately, MIS operations.

Nuclear reprogramming with a non-integrating human RNA virus.

INTRODUCTION: Advances in the field of stem cells have led to novel avenues for generating induced pluripotent stem cells (iPSCs) from differentiated somatic cells. iPSCs are typically obtained by the introduction of four factors--OCT4, SOX2, KLF4, and cMYC--via integrating vectors. Here, we report the feasibility of a novel reprogramming process based on vectors derived from the non-integrating vaccine strain of measles virus (MV). METHODS: We produced a one-cycle MV vector by substituting the viral attachment protein gene with the green fluorescent protein (GFP) gene. This vector was further engineered to encode for OCT4 in an additional transcription unit. RESULTS: After verification of OCT4 expression, we assessed the ability of iPSC reprogramming. The reprogramming vector cocktail with the OCT4-expressing MV vector and SOX2-, KLF4-, and cMYC-expressing lentiviral vectors efficiently transduced human skin fibroblasts and formed iPSC colonies. Reverse transcription-polymerase chain reaction and immunostaining confirmed induction of endogenous pluripotency-associated marker genes, such as SSEA-4, TRA-1-60, and Nanog. Pluripotency of derived clones was confirmed by spontaneous differentiation into three germ layers, teratoma formation, and guided differentiation into beating cardiomyocytes. CONCLUSIONS: MV vectors can induce efficient nuclear reprogramming. Given the excellent safety record of MV vaccines and the translational capabilities recently developed to produce MV-based vectors now used for cancer clinical trials, our MV vector system provides an RNA-based, non-integrating gene transfer platform for nuclear reprogramming that is amenable for immediate clinical translation.

Beta cell regeneration after single-round immunological destruction in a mouse model.

AIMS/HYPOTHESIS: Achieving a better understanding of beta cell regeneration after immunological destruction is crucial for the development of immunotherapy approaches for type 1 diabetes. In previous type 1 diabetes models, sustained immune activation eliminates regenerating beta cells, thus limiting the study of the regenerative capacity of beta cells upon immunological destruction. Here, we employed an adeno-associated virus 8 (AAV8) vector for beta cell-targeted overexpression of a foreign antigen to induce single-round immunological destruction of existing beta cells. METHODS: Young and aged C57BL/6J mice were treated with AAV8 vectors expressing the foreign antigen luciferase. Islet inflammation and regeneration was observed at 3, 6, 10 and 22 weeks post-AAV delivery. RESULTS: In young C57BL/6J mice, robust humoral and cellular immune responses were developed towards antigen-expressing beta cells, leading to decreased beta cell mass. This was followed by beta cell mass replenishment, along with enhanced proliferation of insulin-positive cells, recruitment of nestin/CD34-positive endothelial cells, displacement of alpha cells and mobilisation of cytoplasmic neurogenin 3-positive cells. Mice with recovering beta cells showed normal or reduced fasting blood glucose levels and faster glucose clearance than controls. Although aged mice demonstrated similar responses to the treatment, they initially exhibited notable islet scarring and fluctuations in blood glucose levels, indicating that beta cell regeneration is slower in aged mice. CONCLUSIONS/INTERPRETATION: Our hit-and-run, beta cell-targeted antigen expression system provides an opportunity to monitor the impact of single-round immunological beta cell destruction in animals with diverse genetic backgrounds or ageing status.

Transgenic sequences are frequently lost in Phytophthora parasitica transformants without reversion of the transgene-induced silenced state.

Little data exist on the mechanism and stability of transformation in Phytophthora parasitica, a major oomycete parasite of plants. Here, we studied the stability of drug-resistant protoplast transformants by analyzing single-zoospore derivatives. We show that the transgenic sequences are not stably integrated into the chromosomes, resulting in the loss of drug resistance in single-zoospore derivatives. However, in strains where the P. parasitica gene encoding the CBEL elicitor was silenced by transformation with sense or antisense constructs, silencing is not reversed when the transgenic sequences are lost. This suggests that instability of P. parasitica transformants is not an obstacle for loss-of-function studies in this organism.

Cellulose binding domains of a Phytophthora cell wall protein are novel pathogen-associated molecular patterns.

The cellulose binding elicitor lectin (CBEL) from Phytophthora parasitica nicotianae contains two cellulose binding domains (CBDs) belonging to the Carbohydrate Binding Module1 family, which is found almost exclusively in fungi. The mechanism by which CBEL is perceived by the host plant remains unknown. The role of CBDs in eliciting activity was investigated using modified versions of the protein produced in Escherichia coli or synthesized in planta through the potato virus X expression system. Recombinant CBEL produced by E. coli elicited necrotic lesions and defense gene expression when injected into tobacco (Nicotiana tabacum) leaves. CBEL production in planta induced necrosis. Site-directed mutagenesis on aromatic amino acid residues located within the CBDs as well as leaf infiltration assays using mutated and truncated recombinant proteins confirmed the importance of intact CBDs to induce defense responses. Tobacco and Arabidopsis thaliana leaf infiltration assays using synthetic peptides showed that the CBDs of CBEL are essential and sufficient to stimulate defense responses. Moreover, CBEL elicits a transient variation of cytosolic calcium levels in tobacco cells but not in protoplasts. These results define CBDs as a novel class of molecular patterns in oomycetes that are targeted by the innate immune system of plants and might act through interaction with the cell wall.

A novel Arabidopsis-Colletotrichum pathosystem for the molecular dissection of plant-fungal interactions.

The ability of a Colletotrichum sp., originally isolated from Brassica campestris, to infect Arabidopsis thaliana was examined. Sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal (r)DNA showed the pathogen to be Colletotrichum destructivum. The host range was broad, including many cruciferous plants and some legumes. At 25 degrees C, all A. thaliana accessions tested were susceptible to the Brassica isolates of C. destructivum; however, at 15 degrees C, the accession Ws-2 showed a temperature-dependant resistance, in which single epidermal cells underwent a rapid hypersensitive response. Legume isolates of C. destructivum were unable to infect A. thaliana and induced deposition of callose papillae at sites of attempted penetration. In compatible interactions, C. destructivum showed a two-stage, hemibiotrophic infection process. The initial biotrophic phase was associated with large, intracellular primary hyphae and was confined to one epidermal cell; whereas, in the subsequent necrotrophic phase, narrow secondary hyphae extensively colonized the tissue and conidia were produced in acervuli. An efficient transformation system was established for C. destructivum, using Agrobacterium-mediated transfer of DNA. The ability to genetically manipulate both partners in the interaction is an important advantage, and the Arabidopsis-Colletotrichum pathosystem should provide a valuable new model for dissecting plant-fungal interactions.