Altered Connectivity in Autistic Adults during Complex Facial Emotion Recognition: A Study of EEG Imaginary Coherence.

Difficulties in Facial Emotion Recognition (FER) are commonly associated with individuals diagnosed with Autism Spectrum Disorder (ASD). However, the mechanisms underlying these impairments remain inconclusive. While atypical cortical connectivity has been observed in autistic individuals, there is a paucity of investigation during cognitive tasks such as FER. It is possible that atypical cortical connectivity may underlie FER impairments in this population. Electroencephalography (EEG) Imaginary Coherence was examined in 22 autistic adults and 23 typically developing (TD) matched controls during a complex, dynamic FER task. Autistic adults demonstrated reduced coherence between both short and long range inter-hemispheric electrodes. By contrast, short range intra-hemispheric connectivity was increased in frontal and occipital regions during FER. These findings suggest altered network functioning in ASD.

Evaluation of DNA isolation procedures for detecting and quantifying environmental Legionella by real-time quantitative PCR.

Six methods, QiAamp DNA Mini Kit (Q), Q with Sepharose 4B gel column (Q/G), Q with low melting point agarose (Q/L), freeze-thaw/phenol-chloroform lysis (FT-PC), FT-PC/G, and FT-PC/L, were evaluated for their ability to isolate DNA of sufficient quality to quantify Legionella using qPCR. Samples of mixing Legionella pneumophila (ATCC33152) and humic acid (HA, 0-126.8 mg/l) were treated by the six methods. Q, Q/G, Q/L, FT-PC/G, and FT-PC/L removed HA from 1.9-126.8 to <1 mg/l determined by A260 with a spectrophotometer. Q obtained the highest DNA yield, followed by Q/G. Dilution (10- to 100-fold) of DNA arising from extraction using Q, Q/G, FT-PC, or FT-PC/G prevented qPCR inhibition. The highest recovery of cells was found in DNA extracted by Q and diluted 100-fold, and followed by Q/G. The applicability of Q and Q/G with dilution was further validated with cooling tower waters. Q or Q/G with 10-fold dilution increased L. pneumophila detection, whereas 100-fold dilution obtained the highest cell concentrations. Similar results were found for Legionella spp. except that both 10- and 100-fold dilutions increased cell concentrations. Thus, Q with 10-fold dilution is suggested to detect and quantify Legionella spp. and detect L. pneumophila. For L. pneumophila-positive samples, 100-fold diluted DNA must be re-analyzed to accurately quantify L. pneumophila.

DLC2 modulates angiogenic responses in vascular endothelial cells by regulating cell attachment and migration.

Deleted in liver cancer 1 (DLC1) is a RhoGTPase activation protein-containing tumor suppressor that associates with various types of cancer. Although DLC2 shares a similar domain structure with that of DLC1, the function of DLC2 is not well characterized. Here, we describe the expression and ablation of DLC2 in mice using a reporter-knockout approach. DLC2 is expressed in several tissues and in endothelial cells (ECs) of blood vessels. Although ECs and blood vessels show no histological abnormalities and mice appear overall healthy, DLC2-mutant mice display enhanced angiogenic responses induced by matrigel and by tumor cells. Silencing of DLC2 in human ECs has reduced cell attachment, increased migration, and tube formation. These changes are rescued by silencing of RhoA, suggesting that the process is RhoA pathway dependent. These results indicate that DLC2 is not required for mouse development and normal vessel formation, but may protect mouse from unwanted angiogenesis induced by, for example, tumor cells.

Rapid quantification of viable legionellae in water and biofilm using ethidium monoazide coupled with real-time quantitative PCR.

AIMS: To optimize ethidium monoazide (EMA) coupled with real-time quantitative PCR (qPCR) and to evaluate its environmental applicability on quantifying viable legionellae in water and biofilm of cooling towers and hot water systems. METHODS AND RESULTS: EMA (0.9-45.5 microg ml(-1)) and propidium monoazide (PMA, 0.9 and 2.3 microg ml(-1)) combined with qPCR (i.e. EMA-qPCR and PMA-qPCR, respectively) were applied to unheated and heated (70 degrees C for 30 min) Legionella pneumophila to quantify viable cells, which was also simultaneously determined by BacLight Bacterial Viability kit with epifluorogenic microscopic enumeration (BacLight-EM). The effects of nontarget microflora and sample matrix on the performance of EMA-qPCR were also evaluated. In comparison with BacLight-EM results, qPCR with EMA at 2.3 microg ml(-1) was determined as the optimal EMA-qPCR assay, which performed equally well as PMA-qPCR for unheated Leg. pneumophila but better than PMA-qPCR for heated Leg. pneumophila (P < 0.05). Moreover, qPCR with EMA at 2.3 microg ml(-1) accurately quantified viable Leg. pneumophila, Legionella anisa and Legionella-like amoebal pathogens 6 (LLAP 6) without interferences by heated legionellae, unheated nonlegionellae cells and cooling tower water matrix (P > 0.05). As for water and biofilm samples collected from cooling towers and hot water systems, the viable legionellae counts determined by EMA-qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR. CONCLUSIONS: The qPCR with EMA at 2.3 microg ml(-1) may accurately quantify viable legionellae (including fastidious LLAP 6) and Leg. pneumophila pretreated with superheating and is applicable for water and biofilm samples obtained from cooling towers and hot water systems. SIGNIFICANCE AND IMPACT OF THE STUDY: The EMA-qPCR assay may be useful in environmental surveillance for viable legionellae and in evaluation of superheating efficacy against legionellae.

Acute elevations of plasma asymmetric dimethylarginine and impaired endothelial function in response to a high-fat meal in patients with type 2 diabetes.

Asymmetric dimethylarginine (ADMA), a compound detectable in human plasma, is an endogenous inhibitor of NO synthase. Endothelial dysfunction is an early event in atherogenesis, and large-vessel atherosclerosis is a major cause of morbidity and mortality in patients with type 2 diabetes mellitus. Fifty patients with type 2 diabetes mellitus were studied at baseline and 5 hours after ingestion of a high-fat meal. Plasma ADMA measured by using high-performance liquid chromatography increased from 1.04+/-0.99 to 2.51+/-2.27 micromol/L (P:<0.0005). Brachial arterial vasodilation after reactive hyperemia, a NO-dependent function, measured by high-resolution ultrasound, decreased from 6.9+/-3.9% at baseline to 1.3+/-4.5% (P:<0.0001). These changes occurred in association with increased plasma levels of triglycerides and very low density lipoprotein triglycerides, with reduced low density lipoprotein cholesterol and high density lipoprotein cholesterol, and with no changes in total cholesterol. The increase in plasma ADMA in response to a high-fat meal was significantly and inversely related to the decrease in percent vasodilation. In 10 of the subjects studied with a similar protocol on another day, no significant changes in the brachial artery flow responses or in plasma ADMA were observed 5 hours after ingestion of a nonfat isocaloric meal. The data suggest that ADMA may contribute to abnormal blood flow responses and to atherogenesis in type 2 diabetics.

Bacterial growth in saline implants: in vitro and in vivo studies.

The survival of bacteria was evaluated in custom-made saline breast implants with integral injection ports in vitro and in 10 New Zealand White rabbits for Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Serratia marcescens. Pseudomonas and Serratia survived in vitro in saline-filled implants and multiplied 24-fold and 22-fold, respectively, from the initial inocula of 300 colony-forming units per cubic centimeter in 21 days. Serratia alone survived in saline implants placed on the dorsum of rabbits, proliferated 80-fold in 7 days, and tapered to 10-fold at the end of 3 weeks. Chemical analysis revealed the presence of glucose in fluid from the implants in the animal study (mean, 1.2 mg per deciliter; standard error of mean [SEM], 0.6) after 21 days and from human subjects (mean, 3.8 mg per deciliter; SEM, 1.0) after 8 months to 10 years. Serratia incubated in human breast implant fluid samples proliferated 7-fold to 30-fold greater than in the saline control in a nonaerated environment. We conclude that some bacteria are able to proliferate in saline in breast implants. Furthermore, their survival may be facilitated by a substance (i.e., glucose) that diffuses across the implant outer shell.

The roles of revascularization and resorption on endurance of craniofacial onlay bone grafts in the rabbit.

A total of 32 New Zealand white rabbits underwent subperiosteal implantation of fresh autogenous unicortical calvarial and iliac crest grafts on their snouts with microscrew rigid fixation. After 3 and 10 days, vascularity was assessed by latex casting, and osteoclastic activity was determined by histochemical staining for tartrate-resistant acid phosphatase. After 70 days, volumetric analysis and tartrate-resistant acid phosphatase staining were performed on six animals. The calvarial grafts demonstrated greater volume maintenance than the iliac bone (72 percent versus 32 percent, p < 0.025). There were significantly greater osteoclastic activity and revascularization in the cancellous portion of calvarial and iliac crest bone grafts by the 10th day of onlay grafting. Minimal activities were present at the cortical bone. Because calvarial grafts contain more cortical bone, its superior volume maintenance can be understood by the architectural influence on revascularization and resorption.

The effect of systemic antibiotic and antibiotic-impregnated polymethylmethacrylate beads on the bacterial clearance in wounds containing contaminated dead bone.

A dorsal muscular wound model was used in 40 New Zealand White rabbits to study the effect of systemic and local antibiotics on the bacterial clearance of contaminated dead bone. Devitalized iliac crest bone preincubated with Staphylococcus aureus was implanted in each deep muscular wound with or without tobramycin-impregnated polymethylmethacrylate beads. Either systemic tobramycin or cefazolin was administered for 7 days. Animals were sacrificed at 7 and 14 days. The wounds containing tobramycin beads had significantly fewer bacteria than those without antibiotic beads (2.0 x 10(2) versus 1.3 x 10(6); p < 0.008). The reduction in bacteria due to the tobramycin beads did not differ significantly with respect to the concurrent systemic antibiotics or to the duration of incubation. We conclude that tobramycin-impregnated beads are effective in reducing bacterial count in contaminated bony wounds treated with systemic antibiotics. Furthermore, the bactericidal effect of the antibiotic beads is independent of and additive to the systemic antibiotic delivered to the wounds by well-perfused muscles.

Potentiation of angiostatic steroids by a synthetic inhibitor of arylsulfatase.

Sodium 2-hydroxy-5-nitro-alpha-toluenesulfonate (HNT, compound II, Fig. 7) is a synthetic inhibitor of arylsulfatase (E.C. 3.1.6.1.). At 1 to 10 mM, HNT increased the clotting time of heparinized rabbit blood by 7-fold. In the 6-day chick embryo, mixtures of heparin and hydrocortisone applied to the chorioallantoic membrane, are known to inhibit specifically the growth of capillary blood vessels. HNT potentiated this antiangiogenic activity in a dose-dependent manner when the concentration of heparin was suboptimal. Potentiation of the antiangiogenic activity of steroids by HNT correlated inversely with the concentration of exogenous heparin. In the absence of exogenous heparin, hydrocortisone did not inhibit angiogenesis. Hydrocortisone and HNT, however, inhibited angiogenesis to the same extent as hydrocortisone and heparin. An arylsulfatase with a Km of 1.5 mM for nitrocatechol sulfate as substrate and a Ki of 10.0 microM for the inhibition of HNT, was identified in the chick embryo chorioallantoic membrane. Preincubation of heparin with a commercially available arylsulfatase caused a 50% reduction in antiangiogenic activity of heparin-steroid mixtures applied to the chorioallantoic membrane. This loss of activity was prevented completely by addition of HNT to the arylsulfatase-heparin incubation mixture. These results suggest that HNT (a) potentiates the anticoagulant function of heparin, (b) prevents the inactivation of antiangiogenic activity of heparin by an endogenous arylsulfatase in the chorioallantoic membrane and by a commercial arylsulfatase, and (c) in the presence of angiostatic steroids can inhibit angiogenesis in the chick embryo without the addition of exogenous heparin. On the basis of these data, we propose that this inhibitor of arylsulfatase acts to potentiate angiostatic steroids by suppressing the desulfation of tissue heparin.

Porphobilinogen synthase modification with methylmethanethiosulfonate. A protocol for the investigation of metalloproteins.

A stable, reversibly sulfhydryl-modified, Zn2+-free porphobilinogen synthase (mod-apo-PBG synthase) has been prepared using methylmethanethiosulfonate. Mod-apo-PBG synthase prepared from holo-PBG synthase using [methyl-14C]methanethiosulfonate incorporated three thiomethyl groups/subunit. When apo-PBG synthase was prepared using EDTA alone, subsequent reaction with [methyl-14C]methanethiosulfonate resulted in incorporation of only two thiomethyl groups/subunit. Mod-apo-PBG synthase was catalytically inactive and contained less than 0.1 mol of Zn/mol of octameric enzyme; it could be reconstituted to full activity using 2-mercaptoethanol and Zn2+. A variety of metal ions were screened for their ability to reconstitute and/or reactivate mod-apo-PBG synthase. Only Zn2+ and Cd2+ reconstitute mod-apo-PBG synthase to full activity. When comparing mod-apo-PBG synthase prepared from holo-PBG synthase in the presence of EDTA with mod-apo-PBG synthase prepared from holo-PBG synthase in the absence of EDTA, no difference was detected in either Zn content, stoichiometry of 14C-labeling, or kinetic behavior. We have confirmed both the observations that four Zn2+/mol of octameric apoenzyme are necessary for full catalytic activity and that holoenzyme, isolated in the presence of 10 microM ZnCl2, contains eight Zn2+/octamer. The additional four binding sites are not catalytically important. Methylmethanethiosulfonate modification is presented as a generally useful method for the investigation of metalloproteins because it provides a route for the preparation of stable apoproteins and a direct method for metal ion replacement.