Transplanted skin-derived precursor stem cells generate enteric ganglion-like structures in vivo

Introduction

Hirschsprung’s disease is characterized by a developmental arrest of neural crest cell migration, causing distal aganglionosis. Transplanted cells derived from the neural crest may regenerate enteric ganglia in this condition. We investigated the potential of skin-derived precursor cells (SKPs) to engraft and to differentiate into enteric ganglia in aganglionic rat intestine in vivo.

Methods

Adult Lewis rat jejunal segments were separated from intestinal continuity and treated with benzalkonium chloride to induce aganglionosis. Ganglia were identified via immunohistochemical stains for S100 and β-III tubulin (TUJ1). SKPs were procured from neonatal Lewis rats expressing enhanced green fluorescent protein (GFP) and cultured in neuroglial-selective media. SKP cell line expansion was quantified, and immunophenotypes were assessed by immunocytochemistry. Aganglionic segments underwent SKP transplantation 21–79 days after benzalkonium chloride treatment. The presence of GFP + cells, mature neurons, and mature glia was evaluated at posttransplant days 1, 6, and 9.

Results

Benzalkonium chloride-induced aganglionosis persisted for at least 85 days. Prior to differentiation, SKPs expressed S100, denoting neural crest lineage, and nestin, a marker of neuronal precursors. Differentiated SKPs in vitro expressed GFAP, a marker of glial differentiation, as well as TUJ1 and several enteric neurotransmitters. After transplantation, GFP + structures resembling ganglia were identified between longitudinal and circular smooth muscle layers.

Conclusion

SKPs are capable of engraftment, migration, and differentiation within aganglionic rodent intestine in vivo. Differentiated SKPs generate structures that resemble enteric ganglia. Our observations suggest that SKPs represent a potential gangliogenic therapeutic agent for Hirschsprung’s disease.     

Impact of preoperative immunonutrition on morbidity following cystectomy for bladder cancer: a case–control pilot study

Purpose

To compare postoperative complications in patients with or without preoperative immunonutrition before cystectomy.

Methods

A prospective, multicenter, pilot, case–control study was conducted during 6 months. Patients with 7-day preoperative immunonutrition were prospectively included and compared with a retrospective, matched control group without immunonutrition. Early complication rates and the length of hospital stay were analyzed. The bilateral type I error was <0.05; the power was 90 %. Thirty patients in each group were required.

Results

Thirty patients were included in each group, on a comparable basis. In the immunonutrition group, fewer postoperative complications (40 vs. 76.7 %; p = 0.008), less paralytic ileus at D7 (6.6 vs. 33.3 %; p = 0.02), fewer infections (23.3 vs. 60 %; p = 0.008), and in particular less pyelonephritis (16.7 vs. 46.7 %; p = 0.03) occurred. Clavien’s grades for complications were higher in the control group (p = 0.04). Mortality, pulmonary embolism, anastomotic fistulae, and wound dehiscence were similar between two groups. The length of stay was reduced by 3 days in the immunonutrition group.

Conclusions

In this pilot case–control study, immunonutrition is associated with a decrease in postoperative complications, urinary tract infections, Clavien’s grade for complications, and paralytic ileus in patients undergoing cystectomy for bladder cancer. Prospective randomized placebo control studies are needed to confirm these promising results.     

3D statistical modeling techniques to investigate the anatomy of the sacrum,its bone mass distribution,and the trans‐sacral corridors

The complex anatomy of the sacrum makes surgical fracture fixation challenging. We developed statistical models to investigate sacral anatomy with special regard to trans‐sacral implant fixation. We used computed tomographies of 20 intact adult pelves to establish 3D statistical models: a surface model of the sacrum and the trans‐sacral corridor S1, including principal component analysis (PCA), and an averaged gray value model of the sacrum given in Hounsfield Units. PCA demonstrated large variability in sacral anatomy markedly affecting the diameters of the trans‐sacral corridors. The configuration of the sacral alae and the vertical position of the auricular surfaces were important determinants of the trans‐sacral corridor dimension on level S1. The statistical model of trans‐sacral corridor S1 including the adjacent parts of the iliac bones showed main variation in length; however, the diameter was the main criterion for the surgically available corridor. The averaged gray value model revealed a distinct pattern of bone mass distribution with lower density particularly in the sacral alae. These advanced 3D statistical models provide a thorough anatomical understanding demonstrating the impact of sacral anatomy on positioning trans‐sacral implants. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1543–1548, 2014.     

Anatomical feasibility of the anterior obturator nerve transfer to restore bowel and bladder function

Total sacrectomies are radical procedures required to treat tumorigenic processes involving the sacrum. The purpose of our anatomical study was to assess the feasibility of a novel nerve transfer involving the anterior obturator nerve to the pudendal and pelvic nerves to the rectum and bladder. Anterior dissection of the obturator nerve was performed in eight hemipelvis cadaver specimens. The common obturator nerve branched into the anterior and posterior at the level of the obturator foramen. The anterior branch then divided into two separate branches (adductor longus and gracilis). The branch to the gracilis was on average longer and also larger than the branch to the adductor longus (8.7 ± 2.1 cm vs. 6.7 ± 2.6 cm in length and 2.6 ± 0.2 mm vs 1.8 ± 0.4 mm in diameter). Each branch of the anterior obturator was long enough to reach the pelvic nerves. The novel transfer of the anterior branch of the obturator nerve to reinnervate the bladder and bowel is anatomically feasible. This represents a promising option with minimal donor site deficit. © 2014 Wiley Periodicals, Inc. Microsurgery 34:459–463, 2014.     

Effects of Purines and Pyrimidines on the Fungistatic Activity of 5-Fluorocytosine in Aspergillus Species

The ability of some exogenously supplied purines, pyrimidines, and their nucleosides to antagonize the in vitro fungistatic activity of 5-fluorocytosine (5-FC) in Aspergillus species was examined. All compounds tested except thymidine were capable of altering the minimal inhibitory concentration of 5-FC for seven strains of aspergilli tested. In most instances, there was a reciprocal correlation between the ability of a compound to antagonize the fungistatic activity of 5-FC and the quantity of 5-FC taken up by cells in the presence of the compound over a 72-h period.     

Peptidoglycan-binding protein TsaP functions in surface assembly of type IV pili

Type IV pili (T4P) are ubiquitous and versatile bacterial cell surface structures involved in adhesion to host cells, biofilm formation, motility, and DNA uptake. In Gram-negative bacteria, T4P pass the outer membrane (OM) through the large, oligomeric, ring-shaped secretin complex. In the β-proteobacterium Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which we name T4P secretin-associated protein (TsaP), was identified as a phylogenetically widely conserved component of the secretin complex that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the OM in a PilQ-dependent manner. In the δ-proteobacterium Myxococcus xanthus, TsaP is also important for surface assembly of T4P, and it accumulates and localizes in a PilQ-dependent manner to the cell poles. Our results show that TsaP is a novel protein associated with T4P function and suggest that TsaP functions to anchor the secretin complex to the peptidoglycan.Type IV pili systems (T4PSs) are involved in the assembly of long, thin fibers, which are found on the surfaces of many bacteria and archaea (1). Type IV pili (T4P) function in host cell adhesion, twitching motility, virulence, DNA uptake, and biofilm formation and are evolutionary related to type II secretion systems (T2SSs), bacterial transformation systems, and the archaellum (2–4). T4PSs can be divided into T4aPSs and T4bPSs that are distinguished based on pilin size and assembly systems (5, 6). T4aPSs form the most abundant class, and the T4P formed by these systems can undergo cycles of extension, adhesion, and retraction, which is a feature that distinguishes them from the other bacterial surface structures (7, 8). T4aP retract at rates up to 1 μm/s and can generate forces up to 150 pN (9, 10). Generally, T4bPSs are not associated with retraction. Here, we focus on T4aPSs and refer to these as T4PSs unless specifically indicated. T4PSs have been studied extensively in many bacteria but are especially well characterized in Neisseria and Pseudomonas spp. and in Myxococcus xanthus. Different nomenclature is used for different T4PSs (Table S1). Here, the Neisseria gonorrhoeae nomenclature is used.T4P are composed of major (e.g., PilE) and minor (in N. gonorrhoeae; e.g., PilV, PilX, ComP) pilins that are synthesized as preproteins with a type III signal peptide. After cleavage of the signal peptide by the prepilin peptidase PilD (11, 12), the T4P are assembled by a multiprotein complex (13). In Gram-negative bacteria, the proteins of T4PSs can be divided into three subcomplexes: the inner membrane (IM) motor complex, the alignment complex, and the outer membrane (OM) pore complex (6). The IM motor complex drives both the assembly and the retraction of T4P. Pilin subunits are extruded from the IM by the platform protein PilG (14) and the hexameric ATPase PilF (15). Disassembly of T4P with retraction occurs when PilF is replaced by the hexameric ATPase PilT (7, 16). PilU, a PilT paralog, is involved in retraction to a lesser extent (17). The alignment complex consisting of PilM, PilN, PilO, and PilP is proposed to connect the IM motor complex and the OM pore complex, and it is also thought to be involved in the stability and/or gating of the OM complex (18–20). In the OM, PilQ forms a homooligomeric ring that serves as a conduit for T4P (21–23).PilQ is a member of the secretin protein family. Proteins belonging to this family are present in many Gram-negative bacteria and are components of T4PSs, T2SSs, type III secretion systems (T3SSs), and extrusion systems of filamentous phages (24). Secretins are multidomain proteins with a signal sequence and a conserved C-terminal OM-spanning domain. Most secretins contain multiple copies of an N-terminal α/β domain (the N domains). PilQ proteins are integral OM proteins and form large gated channels. Oligomeric secretin complexes with different symmetries have been identified. Structural characterization by EM of purified PilQ from Neisseria meningitidis showed a dodecameric structure with a chamber sealed at both ends (25, 26), whereas the T2SS secretins PulD (27) and GspD (28) of the Klebsiella oxytoca pullanase and Vibrio cholerae toxin secretion systems, respectively, showed dodecameric structures with a chamber open at the periplasmic side and closed at the OM side. The structure of the InvG secretin complex of the T3SS of the Salmonella typhimurium needle complex showed 15-fold symmetry and is open at both ends (29), and the phage pIV secretin showed 14-fold symmetry (30). The structure of the C-terminal OM-spanning domain involved in multimer formation is currently not known. Crystal structures of the periplasmic N domains of GspD of the T2SS of enterotoxigenic Escherichia coli (31), of EscC of the T3SS of S. typhimurium (32), and of N. meningitidis PilQ (25) showed that these domains consist of α-helices packed against three-stranded β-sheets. Secretins of T4P systems also contain B domains, which are not present in other secretins and are located N-terminal to the N domains. The structure of the B2 domain of N. meningitidis PilQ consists of several β-strands (25). Remarkably, when the sequence conservation of the B2 domain was mapped to the structure of the B2 domain of N. meningitidis PilQ, a highly conserved patch was identified that was proposed to form the binding site for a currently unidentified T4PS protein (25).Secretins interact with several other proteins. Pilotin proteins are small lipoproteins that interact with the extreme C terminus of secretins and are responsible for OM targeting and oligomerization of secretins (33–38). Secretins of T4PSs also interact with the alignment complex. For N. meningitidis, Pseudomonas aeruginosa, and M. xanthus PilQ, a direct interaction was demonstrated between the respective PilPs and the N0 domains of the PilQs (25, 39, 40). Recently, ExeA of the T2SS of Aeromonas hydrophila (41) and FimV of the T4PS of P. aeruginosa (42) were also implicated in secretin assembly. They contain, respectively, PF01471 and LysM peptidoglycan (PG)-binding domains that might attach them to the PG. However, neither of these two proteins is ubiquitously conserved in bacteria assembling T4P.We have previously shown that the PilQ secretin of N. gonorrhoeae embedded in OM sheets is surrounded by a peripheral structure, which is formed by an additional peripheral ring as well as spikes (43). The proteins that make up these structures are not known. Here, we identify a widely conserved protein, which we name T4P secretin-associated protein (TsaP), that is important for the formation of the peripheral structure. Phylogenomic analysis of 450 genomes of Proteobacteria showed that the presence of the tsaP gene is strongly linked to the presence of genes for T4aPSs. We characterize the TsaP protein and demonstrate the importance of TsaP for T4aP assembly in the two phylogenetically widely separated model organisms N. gonorrhoeae and M. xanthus.     

Comparison of signal quality between EASI and Mason-Likar 12-lead electrocardiograms during physical activity.

BACKGROUND: Myoelectric noise and baseline wander, artifacts that appear when patients move during electrocardiographic monitoring, can cause false alarms. This problem can be addressed by using a reduced lead set and placing electrodes on the anterior part of the torso only. The Mason-Likar modification of the standard 12-lead electrocardiogram and the EASI lead system are 2 alternative systems for lead placement. OBJECTIVES: To test the hypothesis that the EASI lead system is less susceptible to artifacts than is the Mason-Likar modification of the standard 12-lead electrocardiogram. METHODS: Baseline wander and myoelectric noise amplitudes of EASI and Mason-Likar 12-lead electrocardiograms were compared. Twenty healthy volunteers participated. Both lead systems were recorded simultaneously for different types of physical activities. For each lead in each subject, baseline wander and myoelectric noise were measured for both systems, at rest and during each physical activity. RESULTS: The outcome for baseline wander was mixed. For myoelectric noise content, the EASI system performed better for the limb leads in the different physical activities. In the precordial leads, the differences were minimal or mixed. However, for supine-to-right turning, EASI performed worse than the Mason-Likar system. CONCLUSIONS: The 2 systems have similar susceptibilities to baseline wander. The EASI system is, however, less susceptible to myoelectric noise than is the Mason-Likar system. EASI performed worse than Mason-Likar for turning supine to right, because only the EASI system uses an electrode in the right-midaxillary line.