Bioprinting on sheet-based scaffolds applied to the creation of implantable tissue-engineered constructs with potentially diverse clinical applications: Tissue-Engineered Muscle Repair (TEMR) as a representative testbed.

Purpose: This report explores the overlooked potential of bioprinting to automate biomanufacturing of simple tissue structures, such as the uniform deposition of (mono)layers of progenitor cells on sheetlike decellularized extracellular matrices (dECM). In this scenario, dECM serves as a biodegradable celldelivery matrix to provide enhanced regenerative microenvironments for tissue repair. The Tissue-Engineered Muscle Repair (TEMR) technology-where muscle progenitor cells are seeded onto a porcine bladder acellular matrix (BAM), serves as a representative testbed for bioprinting applications. Previous work demonstrated that TEMR implantation improved functional outcomes following VML injury in biologically relevant rodent models.Materials and Methods: In the described bioprinting system, a cell-laden hydrogel bioink is used to deposit high cell densities (1.4 × 105-3.5 × 105 cells/cm2), onto both sides of the bladder acellular matrix as proof-of-concept.Results: These bioprinting methods achieve a reproducible and homogeneous distribution of cells, on both sides of the BAM scaffold, after just 24hrs, with cell viability as high as 98%. These preliminary results suggest bioprinting allows for improved dual-sided cell coverage compared to manual-seeding.Conclusions: Bioprinting can enable automated fabrication of TEMR constructs with high fidelity and scalability, while reducing biomanufacturing costs and timelines. Such bioprinting applications are underappreciated, yet critical, to expand the overall biomanufacturing paradigm for tissue engineered medical products. In addition, biofabrication of sheet-like implantable constructs, with cells deposited on both sides, is a process that is both scaffold and cell-type agnostic, and furthermore, is amenable to many geometries, and thus, additional tissue engineering applications beyond skeletal muscle.

Early outcomes of fluorescence angiography in the setting of endorectal mucosa advancement flaps.

BACKGROUND: Fistula-in-ano has a reported incidence of 31-34%. Besides fistulotomy, options for fistula repair are seton placement, endorectal advancement flap (ERAF), fibrin sealant, anal fistula plug and ligation of the intersphincteric fistula tract. Despite having a reported success rate as high as 75-98%, ERAF is not without complications, including flap breakdown, recurrence and fecal incontinence. Traditionally, maintaining a broad base to preserve blood supply has been advocated to reduce flap failure. And the aim of the present study was to evaluate outcomes of adult patients who underwent ERAF for complex fistula-in-ano with the use of intraoperative fluorescence angiography (FA) at our institution between July 2014 and July 2016. METHODS: We retrospectively reviewed consecutive cases of complex fistula-in-ano repair with ERAF and FA from a prospectively maintained dataset of adult patients with complex fistula-in-ano. Demographics, intraoperative data and 60-day outcomes were recorded and reviewed. RESULTS: Six patients [five males and one female with a mean age of 40 years (range 25-46 years)], with a total of seven fistulas, were identified. Six (85.7%) of these patients had undergone prior surgery for fistula-in-ano. No recurrences or complications of any type were noted at 2-week and 8-week follow-up. The majority of patients (71.4%) required flap revision based on intraoperative FA prior to flap fixation. CONCLUSIONS: FA is safe and offers real-time assessment of flap perfusion prior to and after fixation in anal fistula repair. The rate of flap ischemia may be underestimated, and therefore, to improve outcomes in ERAF, intraoperative FA should be included in the surgical armamentarium.

'Optimal' vortex rings and aquatic propulsion mechanisms.

Fishes swim by flapping their tail and other fins. Other sea creatures, such as squid and salps, eject fluid intermittently as a jet. We discuss the fluid mechanics behind these propulsion mechanisms and show that these animals produce optimal vortex rings, which give the maximum thrust for a given energy input. We show that fishes optimize both their steady swimming efficiency and their ability to accelerate and turn by producing an individual optimal ring with each flap of the tail or fin. Salps produce vortex rings directly by ejecting a volume of fluid through a rear orifice, and these are also optimal. An important implication of this paper is that the repetition of vortex production is not necessary for an individual vortex to have the 'optimal' characteristics.

On the mound of Macrotermes michaelseni as an organ of respiratory gas exchange.

Patterns and rates of air movements in the mounds and nests of Macrotermes michaelseni were studied using tracer methods. Wind is a significant source of energy for powering nest ventilation, despite the mound being a completely enclosed structure. Nests are ventilated by a tidal movement of air driven by temporal variation in wind speed and wind direction. Density gradients sufficiently steep to drive bulk flow by natural convection will be rare. However, metabolism-induced buoyant forces may interact with wind energy in a way that promotes homeostasis of the mound atmosphere.

Evaluation of the inactivation of infectious Herpes simplex virus by host-defense peptides.

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide microplate assay was adapted to screen for the ability of 20 host-defense peptides to inactivate herpes simplex virus type 1 and type 2. The procedure required minimal amounts of material, was reproducible, and was confirmed with standard antiviral testing techniques. In screening tests, with the exception of melittin, a highly cytotoxic and hemolytic peptide found in bee venom, the alpha-helical peptides in our test panel (magainins, cecropins, clavanins, and LL-37) caused little viral inactivation. Several beta-sheet peptides (defensins, tachyplesin, and protegrins) inactivated one or both viruses, sometimes with remarkable selectivity. Two peptides were identified as having antiviral activity against both viruses, indolicidin (a tryptophan-rich peptide from bovine neutrophils) and brevinin-1 (a peptide found in frog skin). The antiviral activity of these two peptides was confirmed with standard antiviral assays. Interestingly, the antiviral activity of brevinin-1 was maintained after reduction and carboxamidomethylation, procedures that abolished its otherwise prominent hemolytic and cytotoxic effects.

Molecular characterization of a globally distributed lineage of serotype 12F Streptococcus pneumoniae causing invasive disease.

These studies have identified a major genetic lineage of capsule serotype 12F Streptococcus pneumoniae, which has maintained two different types of the pneumococcal surface protein A (PspA) virulence factor and caused invasive disease in geographically disjoint locations. Twenty outbreak strains from a Texas jail and Maryland day care center and 16 reference strains from Texas, Maryland, Washington, Michigan, Oklahoma, Missouri, Alaska, and Australia were examined. Although the Texas and Maryland outbreak strains were indistinguishable by IS1167 and boxA genotyping procedures, all strains examined were members of a genetically similar lineage. The microevolutionary history of pspA differed from that of the overall genetic background of the strains. Taken together, these findings suggested that the Texas and Maryland outbreaks were caused by different clones of a major genetic lineage of serotype 12F pneumococci, within which at least one PspA has been acquired via localized genetic recombination.

Activity of protegrins against yeast-phase Candida albicans.

We used a two-stage radial diffusion assay to perform a structure-activity study of the antifungal effects of protegrin-1 (PG-1) on yeast-phase Candida albicans. While doing so, we computed MICs from the radial diffusion assay data by three methods and compared the respective values with results from colony count and broth microdilution assays. This allowed us to identify several technical modifications that improved the sensitivity and accuracy of radial diffusion assays. We found that both PG-1 and enantiomeric PG-1 (composed exclusively of D-amino acids) were potently fungicidal for yeast-phase C. albicans. The protegrins PG-2, -3, and -5, but not PG-4, were as effective as PG-1. At least one intramolecular disulfide bond was required to retain optimal candidacidal activity at physiological NaCl concentrations. Truncated variants of PG-1 that lacked its first four residues showed decreased candidacidal activity, although their activity against bacteria was substantially intact. Altering the beta-turn region (residues 9 to 12) of PG-1 or its variants further decreased candidacidal activity. These studies suggest that only 12 residues are needed to endow protegrin molecules with strong antibacterial activity and that at least 4 additional residues are needed to add potent antifungal properties. Thus, the 16-residue protegrin PG-2 likely represents the minimal structure needed for broad-spectrum antimicrobial activity encompassing bacteria and fungi.

On the thermal capacity of a bird's egg warmed by a brood patch.

Eggs of intermittently incubating birds are periodically rewarmed by a transient pulse of heat from the parent's brood patch. Estimating the energy cost of rewarming such an egg requires knowledge of the egg's thermal capacity, typically assumed to be the product of the egg's mass and its specific heat, designated here as the gravimetric thermal capacity. When chicken eggs are transiently warmed by an artificial brood patch, the energetic costs of the rewarming indicate that they have thermal capacities about one-third the gravimetric thermal capacity. In this article, I show that birds' eggs warmed locally by a brood patch have effective thermal capacities that differ substantially from the eggs' gravimetric thermal capacities, both in absolute magnitude and in response to varying the temporal properties of the transient pulse of heat. An effective thermal capacity exists because heat from a brood patch flows unevenly through an egg and because of thermal impedance effects on the unsteady component of heat flow into the egg. If these conditions in any way characterize the rewarming of eggs by intermittently incubating birds in nature, intermittent incubation may be considerably less costly in time and energy than has heretofore been assumed.

Zn2+-sensing by the cyanobacterial metallothionein repressor SmtB: different motifs mediate metal-induced protein-DNA dissociation.

SmtB is a member of a family of repressors which dissociate from DNA in the presence of metals; Zn2+ being the most potent inducer of metallothionein gene (smtA) transcription in vivo. In Synechococcus PCC 7942 cells devoid of chromosomal smtB, four plasmid-encoded mutants of SmtB (C61S, T11S/C14S, C121S and H105R/H106R) repressed lacZ expression driven by the smtA operator-promoter. Gel retardation assays with extracts from the complemented cells detected multiple SmtB-dependent complexes similar to those obtained with extracts from wild-type cells or with recombinant-SmtB. Elevated [Zn2+] alleviated repression in vivo by all of the mutants except H105R/H106R. These His residues (one or both) are therefore essential for Zn2+-sensing while, contrary to expectations, Cys residues are not. Hence different motifs facilitate metal-induced DNA-dissociation by SmtB and ArsR (the related oxyanion-sensing repressor), presumably generating variety in the spectra of metals sensed. Nucleotides and amino acids involved in DNA-SmtB interaction have been further defined/inferred and we also confirm that additional unknown factors form specific associations with the smt operator-promoter in elevated [Zn2+].

Evidence for the simultaneous expression of two PspAs by a clone of capsular serotype 6B Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) has been shown to be a serologically variable virulence factor of Streptococcus pneumoniae. In mice, PspA can elicit antibodies capable of protecting them against otherwise fatal infections with encapsulated pneumococci. In previous studies it has been reported that almost all isolates have two apparently unlinked genomic sequences that are highly homologous to the 5' and 3' halves of Rx1 pspA, although out MAbs to PspA have not detected more than one PspA in any given isolate of S. Pneumoniae. Recently, we have identified four isolates from a clone of capsular serotype 6B pneumococci (MC25-28) that simultaneously express two distinct PspAs. Each of the isolates (MC25-28) exhibited the same two Kpn I fragments (each containing a Hind III site) that hybridized with Rx1 pspA. MAbs specific for PspA detected two PspAs characterized by different molecular weights and different serologic patterns of reactivity (PspA type 6 detected by MAbs XiR278 and 2A4, and PspA type 34 detected only by MAb 7D2) in each of the four isolates. In previous studies XiR278 and 2A4 frequently have been observed to react with PspA epitopes of the same strain. Based on molecular weight data both epitopes were always present on the same molecule. Our present findings raise the possibility that pneumococci make a second serologically variable PspA which is generally not detected by currently available MAbs to PspA.

Limitation of life support: frequency and practice in a London and a Cape Town intensive care unit.

OBJECTIVES: To examine the frequency of limiting (withdrawing and withholding) therapy in the intensive care unit (ICU), the grounds for limiting therapy, the people involved in the decisions, the way the decisions are implemented and the patient outcome. DESIGN: Prospective survey. Ethical approval was obtained. SETTING: ICUs in tertiary centres in London and Cape Town. PATIENTS: All patients who died or had life support limited. INTERVENTIONS: Data collection only. RESULTS: There were 65 deaths out of 945 ICU discharges in London and 45 deaths out of 354 ICU discharges in Cape Town. Therapy was limited in 81.5% and 86.7% respectively (p = 0.6) of patients who died. The mean ages of patients whose therapy was limited were 60.2 years and 51.9 years (p = 0.014) and mean APACHE II scores 18.5 and 22.6 (p = 0.19) respectively. The most common reason for limiting therapy in both centres was multiple organ failure. Both medical and nursing staff were involved in most decisions, which were only implemented once wide consensus had been reached and the families had accepted the situation. Inotropes, ventilation, blood products, and antibiotics were most commonly withdrawn. The mean time from admission to the decision to limit therapy was 11.2 days in London and 9.6 days in Cape Town. The times to outcome (death in all patients) were 13.2 h and 8.1 h respectively. CONCLUSIONS: Withdrawal of therapy occurred commonly, most often because of multiple organ failure. Wide consensus was reached before a decision was made, and the time to death was generally short.

Expression of the type 2 metallothionein-like gene MT2 from Arabidopsis thaliana in Zn(2+)-metallothionein-deficient Synechococcus PCC 7942: putative role for MT2 in Zn2+ metabolism.

Zn2+ proteins pervade metabolism and are essential for gene expression. However, no proteins have been ascribed the central roles of Zn2+ donation to, or removal from, metalloproteins, or Zn2+ storage in vegetative plant tissue. In animals, such functions have been proposed for metallothioneins. Plants contain multiple metallothionein-like genes but their predicted products, which differ significantly from animal metallothioneins, remain to be isolated from vegetative tissue and their roles are uncertain. The type 2 metallothionein-like gene from Arabidopsis, MT2, was expressed under the control of Zn2+-responsive elements derived from the cyanobacterial metallothionein divergon, smt. Zn2+-dependent expression of MT2 transcripts in Synechococcus PCC 7942 was confirmed by northern analysis. The Arabidopsis MT2 gene partly complemented Zn2+ hypersensitivity in mutants of Synechococcus PCC 7942 which are functionally deficient in an endogenous Zn2+-metallothionein gene, smtA. MT2 was also expressed as a recombinant fusion protein in Escherichia coli, purified and shown to bind Zn2+ in vitro. The mean pH of half displacement of Zn2+ from MT2 was estimated to be 5.05. This suggests that MT2 has a greater affinity for Zn2+ than phytochelatins. The results presented here reveal that MT2 is capable of binding Zn2+ in vitro, conferring tolerance to elevated [Zn2+] in vivo within cyanobacteria and is likely to compete with other polypeptides for cellular Zn2+ in planta.

Difficulties in predicting outcome in cardiac surgery patients.

OBJECTIVE: To evaluate a novel combination of preoperative, intraoperative, and postoperative variables (including the Parsonnet, and the Acute Physiology and Chronic Health Evaluation II and III [APACHE II and III] scores) in cardiac surgery patients in order to predict hospital outcome, complications, and length of stay. DESIGN: Prospective survey. SETTING: Adult intensive care unit (ICU) at a tertiary care cardiothoracic surgery center. PATIENTS: All cardiac surgery patients admitted to the ICU over a 1-yr period. INTERVENTIONS: Medical history, Parsonnet score, intraoperative data (including bypass and ischemic times), APACHE II and III scores, complications, and outcome were collected for each patient. MEASUREMENTS AND MAIN RESULTS: One thousand eight patients were entered into the study. The mean Parsonnet score was 7.8 (range 0 to 33), mean APACHE II score 11.8 (range 2 to 33), and mean APACHE III score 42.5 (range 9 to 132). ICU mortality rate was 2.7% and hospital mortality rate was 3.8%. The mean APACHE II predicted risk of dying was 5.31%, which gave a standardized mortality ratio of 0.71. The above scores were all statistically well correlated with hospital mortality. Further, a logistic regression model was developed for the probability of hospital death. This model (which included bypass time, need for inotropes, mean arterial pressure, urea, and Glasgow Coma Scale) had an area under the receiver operating characteristic curve of 0.87, while the Parsonnet score had an area of 0.82 and the APACHE II risk of dying had an area of 0.84. CONCLUSIONS: Cardiac surgery remains a difficult area for outcome prediction. A combination of intraoperative and postoperative variables can improve predictive ability.

Construction of Zn2+/Cd(2+)-tolerant cyanobacteria with a modified metallothionein divergon: further analysis of the function and regulation of smt.

This paper reports the (de novo) construction of mutants of Synechococcus PCC 7942 lacking the repressor (SmtB) of the metallothionein gene, smtA. These smtA+/B- cells are more tolerant to elevated [Zn2+] and [Cd2+] than cells containing an intact metallothionein divergon (smt). Previously selected (by step-wise adaptation) Cd(2+)-tolerant mutants contain additional copies of smtA and possibly other undetected mutations. It is now confirmed that these cells also contain a deletion within 'all' copies of smtB and hence fail to revert to wild type following subculture in medium which has not been supplemented with Cd2+ or Zn2+. Northern analysis showed enhanced accumulation of smtA transcripts, even in the absence of added metal ions in these mutants. An increase in the accumulation of Zn2+ is reported in cells containing an intact metallothionein divergon compared to cells deficient in both smtA and smtB. This supports the assumption that SmtA binds Zn2+ within cyanobacterial cells. We also describe the use of the above mentioned mutants to identify additional factors involved in the regulation of transcription from the smtA operator-promoter.

Cyanobacterial metallothioneins: biochemistry and molecular genetics.

Metallothioneins have been extensively studied in many different eukaryotes where they sequester, and hence detoxify, excess amounts of certain metal ions. However, the precise functions of many of these molecules are not fully understood. This article reviews literature concerning their namesakes in prokaryotes.

Prone position for ventilation in patients with severe adult respiratory distress syndrome.

We report 3 cases of severe adult respiratory distress syndrome. The patients were very difficult to oxygenate, even with the most advanced conventional mechanical ventilation techniques. In all cases oxygenation improved dramatically when the patient was turned to the prone position. Reports of the use of the prone position for ventilation are scanty, and this is the first time it has been reported in this country. The proposed mechanisms for its effectiveness are reviewed.

Adult respiratory distress syndrome: has there been a change in outcome predictive measures?

BACKGROUND: Studies suggest that the mortality in adults with acute respiratory distress (ARDS) has not changed over the past two decades, despite the introduction of new therapeutic techniques and sophisticated ventilatory support devices. Mortality and physiological variables that might predict outcome in patients with ARDS were therefore assessed. METHODS: A retrospective survey was undertaken in 41 patients with ARDS. RESULTS: Mortality was 66%. Only the presence of sepsis predicted death. CONCLUSION: Mortality from ARDS is unchanged. Currently available severity scoring systems are not helpful in predicting outcome.

Fiberoptic bronchoscopy in the intensive care unit--a prospective study of 147 procedures in 107 patients.

OBJECTIVE: To determine the value and safety of fiberoptic bronchoscopy in an intensive care unit (ICU). DESIGN: Prospective survey. SETTING: ICUs at a tertiary care hospital (except for seven procedures that were performed at a peripheral hospital ICU). PATIENTS: A total of 107 patients with a mean age of 43.9 yrs (range 15 to 84). INTERVENTIONS: One hundred forty-seven fiberoptic bronchoscopy procedures (116 performed on patients who were undergoing mechanical ventilation) were performed on 107 patients. Ninety-four procedures were for diagnostic reasons (upper and lower airway inspection, focal and diffuse pulmonary infiltrates), 37 for therapeutic reasons (bronchial toilet, pulmonary hemorrhage, endotracheal intubation), and 16 for both reasons. Topical anaesthesia was used for fiberoptic bronchoscopy; sedation was rarely needed. Appropriate diagnostic and therapeutic procedures were performed. MEASUREMENTS AND MAIN RESULTS: Oxygen saturation, electrocardiogram, and blood pressure were monitored. Transbronchial biopsies (all on mechanical ventilation) for diffuse pulmonary infiltrates were diagnostic in five of seven cases, and were suggestive of the diagnosis in a further case. Endobronchial biopsies were not diagnostic in any of three cases. Bronchial brushings for microbiology were positive in nine of 50 procedures and for cytology in one of nine procedures. Protected specimen brushes for pulmonary infiltrates gave positive microbiology findings in five of 23 procedures. In pulmonary hemorrhage, focal bleeding was found in five cases, diffuse bleeding in four, and no bleeding source in three. In lobar atelectasis, bronchial toilet led to full reexpansion (n = 20 procedures), partial reexpansion (n = 5), and no change (n = 3). Intubation with fiberoptic bronchoscopy was successful in four of five patients. Hypoxemia (oxygen saturation < 90%) occurred in 29 procedures; it caused no problems. Complications included hemorrhage (n = 2), supraventricular tachycardia (n = 1), pneumothorax (n = 1), pneumatocele (n = 1), and bronchospasm (n = 1). No deaths were attributable to fiberoptic bronchoscopy. CONCLUSIONS: Fiberoptic bronchoscopy in the ICU is safe, contributes valuable diagnostic information, and is useful for therapeutic purposes.

Immobilization of a lysine-terminated heparin to polyvinyl alcohol.

Lysine terminated heparin, prepared by the nitrous acid partial depolymerization and reductive amination of heparin, failed to increase the active heparin content of a heparin-polyvinyl alcohol (heparin-PVA) hydrogel relative to the unmodified commercial heparin. The depolymerization of heparin resulted in a loss of biological activity which outweighed the increase in the terminal amine groups (produced by reductive amination), that were used for glutaraldehyde immobilization to the PVA. The loss in anti-thrombin activity (thrombin time or chromogenic substrate) paralleled the increase in anhydromannose end groups due to depolymerization making it necessary to optimize the loss of activity against the increase in terminal amine groups after amination. For example, depolymerization at a high sodium nitrite concentration (81 g/l) at pH4 and 25 degrees C for 20 min, resulted in a loss of 22-40% of the biological activity but achieved an anhydromannose content of 600 nmoles/mg (approximately 7 cleavage sites/molecule). After the anhydromannose groups were reductively aminated by lysine, the anhydromannose content was reduced to 190 nmol/mg indicating a terminal lysine content of 410 nmol/mg. This resulted in an increase in heparin content of the final hydrogel by 53% on mass terms. However, given the reduction in biological activity, it was not surprising that the modified heparin-PVA hydrogel coated on a polyethylene tube was no better than the hydrogel with unmodified heparin in inactivating thrombin in a flow circuit. These results point out the need for care in interpreting heparin immobilization results and for new strategies to increase the active heparin content of this hydrogel.