Distal major pedicle of sartorius muscle flap: Anatomical study and its clinical implications.
Reconstruction of the popliteal region has limited option in terms of muscle flaps or myocutaneous flaps. Gastrocnemius muscle or the myocutaneous flaps are the option for majority of cases. However, reach of Gastrocnemius is limited if the wound is on the distal one-third thigh or the lateral aspect of knee region. Similarly, if the wound injures the muscle, then coverage becomes all the more difficult. Although inferiorly based fasciocutaneous flaps can cover the wound in case of bony injuries, muscle flaps are beneficial as they help in fracture healing. However, in cases with direct gastrocnemius muscle injury or if the wound on the distal one-third thigh or the lateral aspect then the options of muscle flaps is limited. An inferiorly based sartorius muscle can be one suitable alternative to cover this region. The aim is to devise an inferiorly based sartorius muscle flap for coverage of lower thigh, popliteal and upper one-third leg region. (1) To identify the location of distal major (largest diameter) pedicle in cadavers and its clinical application. (2) To determine the arc of rotation with distal major pedicle as pivot point. Ten Cadavers and 20 sartorius muscle dissected out. Prior silicone injection onto the femoral vessels was done to identify the location of the perforators for the sartorius muscle. The distance of perforators from anterior superior iliac spine (ASIS) measured and the diameter of each perforator by transverse cut measured using callipers. In clinical cases, the arc of rotation was measured by keeping the distal perforator intact as pedicle (detaching the muscle from the ASIS without detaching from the insertion and then rotating it). Out of the 10 cadavers analysed, 6 were male and 4 were female. The mean location of the distal major pedicle was at 35.25 cm from ASIS and range was between 30.4 cm to 38.3 cm. There was no significant variation between right and left limbs in individual cadaver (range 0.2 cm-1.6 cm). The mean diameter of the arterial component of distal major pedicle was 1.54 mm. In three clinical cases where this flap was harvested the arc of rotation were 95°, 110°, 125°. In one of the cases where flap was used to cover the tibial plateau (arc of rotation 155°), distal end of the muscle necrosed. This cadaver study supported by various other studies show that it has sizeable distal pedicle based on which whole muscle can be harvested as flap. In our study, the usual location of this pedicle is at 35 cm from ASIS. The mean diameter of the widest pedicle in distal one-third was 1.54 mm which along with other small diameter pedicle can support the entire muscle. This flap reached up to the infrapatellar region without any vascular compromise.


Assessing treatment readiness in violent offenders.
Although violent offenders are widely considered to be difficult to engage in therapeutic change, few methods of assessing treatment readiness currently exist. In this article the validation of a brief self-report measure designed to assess treatment readiness in offenders who have been referred to violent offender treatment programs is described. The measure, which is an adaptation of a general measure of treatment readiness developed in a previous work, displayed acceptable levels of convergent and discriminant validity and was able to successfully predict treatment engagement in violent offender treatment. These results suggest that the measure has utility in the assessment of treatment readiness in violent offenders.


Recombinant tumor necrosis factor enhances the locomotion of memory and naive B lymphocytes from human tonsils through the selective engagement of the type II receptor.
Recent studies performed in mice knocked out for the tumor necrosis factor (TNF ), the lymphotoxin-alpha, or the type I TNF receptor (R), genes have shown that these animals display gross defects in germinal center (GC) formation, suggesting that members of the TNF and TNFR superfamilies are involved in the control of B-cell migration. Based on these premises, we have here investigated the effects of human recombinant (r) TNF on the polarization and locomotion of tonsillar B cells. rTNF increased the spontaneous polarization and locomotion of unfractionated tonsillar B lymphocytes in a dose-dependent manner by inducing a true chemotactic response. Memory (IgD-, CD38(-)) and naive (IgD+, CD38(-)), but not GC (IgD-, CD38(+)) B cells purified from total tonsillar B lymphocytes, showed a significantly higher locomotion in the presence than in the absence of rTNF. Accordingly, type I and II TNF receptors (TNFRs) were detected by flow cytometry on the surface of memory and naive, but not GC, B lymphocytes. Blocking experiments with monoclonal antibodies to type I or II TNFR showed that rTNF enhanced the spontaneous chemotaxis of memory and naive B cells through the selective engagement of type II TNFR. Finally, the TNF gene was found to be expressed in memory, naive and GC B lymphocytes; the cytokine was released in culture supernatants from the three B-cell subsets after stimulation. These data may support the hypothesis that human TNF is involved in the paracrine and perhaps autocrine control of B-cell migration in secondary lymphoid tissues.


Macelignan inhibits melanosome transfer mediated by protease-activated receptor-2 in keratinocytes.
Skin pigmentation is the result of melanosome transfer from melanocytes to keratinocytes. Protease-activated receptor-2 (PAR-2) is a key mediator of melanosome transfer, which occurs as the melanocyte extends its dendrite toward surrounding keratinocytes that take up melanosomes by phagocytosis. We investigated the effects of macelignan isolated from Myristica fragrans HOUTT. (nutmeg) on melanosome transfer and the regulation of PAR-2 in human keratinocytes (HaCaT). HaCaT cells stimulated by the PAR-2-activating peptide Ser-Leu-Ile-Gly-Arg-Leu-NH₂ (SLIGRL) were treated with macelignan; PAR-2 expression was then determined by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. We evaluated the effects of macelignan on calcium mobilization and keratinocyte phagocytosis. In addition, B16F10 melanoma cells and keratinocytes were co-cultured to assess the effects of macelignan on prostaglandin E₂ (PGE₂) secretion and subsequent dendrite formation. Macelignan decreased HaCaT PAR-2 mRNA and protein levels in a dose-dependent manner. Furthermore, macelignan markedly reduced intracellular calcium mobilization and significantly downregulated keratinocyte phagocytosis, as shown by decreased ingestion of Escherichia coli bioparticles and fluorescent microspheres. In co-culture experiments, macelignan reduced keratinocyte PGE₂ secretion, thereby preventing dendrite formation in B16F10 melanoma cells compared with SLIGRL-treated controls. Macelignan inhibits melanosome transfer by downregulating PAR-2, thereby reducing keratinocyte phagocytosis and PGE₂ secretion, which in turn inhibits dendrite formation in B16F10 melanoma cells. Taken together, our findings suggest that macelignan could be used as a natural depigmenting agent to ameliorate hyperpigmentation.


[Re-operation of a mitral porcine bioprosthesis 22-years after implantation].
A 49-year-old female suffered from increasing dyspnea from 2004. She had undergone mitral valve replacement with Carpentier-Edwards 1st-generation standard porcine bioprosthesis due to mitral stenosis 22-years ago. The echocardiography and catheterization showed mitral stenosis and, regurgitation. Re-replacement of the mitral prosthetic valve with ATS (27 mm) valve and tricuspid annuloplasty with Edwards MC3 Ring were successfully performed. There was no case who underwent so long post operative period after initial implantation of fragile prosthetic valve.


[MRI study of left ventricular function in patients with coronary disease and myocardial dysfunction before and after coronary revascularization].
To evaluate left ventricular (LV) myocardial function in ten patients with coronary artery disease (CAD) preoperatively and 6 months after coronary bypass grafting (CABG) by cardiac MRI. Ten patients (mean 65.2 +/- 5.9 years) with angiographically proven CAD and an indication for elective CABG underwent prospective evaluation of global LV function and regional wall motion by Cine-MRI at rest using a multiphase FLASH-2D sequence following regions of interest (ROI)-defined diagnostics of regional myocardial wall motion by means of levocardiography. Within the ROIs a total of 613 LV myocardial segments were analyzed preceding and following surgical revascularization. Results were compared with the data of 10 healthy volunteers. Preoperatively, patients showed reduced stroke volume and ejection fraction compared with volunteers (p < 0.01). Enddiastolic wall thickness (EDWT) and systolic wall thickening (SWT) were significantly lower in the patients (p < 0.01). Based on preoperative levocardiography ROI-defined myocardial segments showed a significantly lower preoperative EDWT in areas with wall motion abnormalities (7.4 +/- 2.5 mm; p < 0.01) than in normal myocardium (9.2 +/- 2.1 mm). Ejection fraction (p < 0.05), endsystolic wall thickness, and SWT (p < 0.01) improved significantly after bypass surgery. On ROI-defined analysis myocardial segments with impaired preoperative wall motion (n = 243) showed a significant increase of EDWT, ESWT and SWT (p < 0.01). In patients with CAD, cardiac MRI enables the non-invasive determination of postinfarctional LV remodeling with an increased EDWT of myocardial segments with normal regional wall motion and of the improvement in global and regional myocardial function following coronary bypass surgery.


Immunomodulation by antimicrobial drugs.
Immunomodulation aims at either enforcement of host defence mechanisms or dampening of the inflammatory response of the host. Thus, immunomodulatory drugs may enhance host defence by either stimulating the inflammatory response or inhibiting the counter-regulatory, anti-inflammatory response. On the other hand, the response of the host may be down-modulated through inhibition of the inflammatory response or induction of counter-regulatory, anti-inflammatory mechanisms. Antimicrobial drugs may have such immunomodulatory effects, but so far these effects have not been exploited clinically.


Taioba (Xanthosoma sagittifolium) leaves: nutrient composition and physiological effects on healthy rats.
Several studies have shown that fruits and vegetables contribute to protect against degenerative pathologies such as cardiovascular diseases, diabetes, and cancer, mainly due to the presence of dietary fiber (DF) and polyphenols. Taioba (Xanthosoma sagittifolium) is an edible aroid widely grown in many parts of Africa, America, and Asia. The tubers portions of taioba are widely consumed; however, the leafy portions are generally discarded, despite their high nutritive value. In this study, we have partly characterized the DF of lyophiized taioba leaf (LTL), and assessed the possible protective effects on biochemical parameters and on bile acid (BA) production in colon and cecum, when fed to healthy rats for 4 wk. Forty-five Wistar rats were assigned to either of 5 groups: group 1 received AIN 93G diet (CG: Control); group 2 received AIN 93G containing 2.5% of cellulose + 2.5% inulin (CEIN_5%); group 3 received AIN 93G containing 2.5% of cellulose + 2.5% taioba fiber (CETA_5%); group 4 received AIN 93G containing 5% cellulose + 2.5% taioba fiber (CETA_7.5%); group 5 received AIN 93G containing 5% cellulose + 2.5% of inulin (CEIN_7.5%). LTL showed high contents of total fiber, predominantly comprising insoluble DF with glucose as the major monomer. Rats receiving LTL had increased fecal mass and fat excretion, and improved BA profiles by diminishing the proportion of secondary acids, thus suggesting that consumption of taioba leaf may have the property of lowering the risk of colon cancer.


Response to fever and utilization of standby emergency treatment (SBET) for malaria in travellers to Southeast Asia: a questionnaire-based cohort study.
Guidelines in several European countries recommend standby emergency treatment (SBET) for travellers to regions with low or medium malaria transmission instead of continuous chemoprophylaxis: travellers are advised to seek medical assistance within 24 h in case of fever and to self-administer SBET only if they are not able to consult a doctor within the time period specified. Data on healthcare-seeking behaviour of febrile travellers and utilization of SBET is however scarce as only two studies were performed in the mid-1990s. Since tourism is constantly increasing and malaria epidemiology has dramatically changed in the meantime more knowledge is urgently needed. Some 876 travellers to destinations in South and Southeast Asia with low or medium malaria transmission were recruited in the travel clinic of the University Medical Center Hamburg-Eppendorf. Demographic and travel-related data were collected by using questionnaires. Pre-travel advice was carried out and SBET was prescribed in accordance to national guidelines. Post-travel phone interviews were performed to assess health incidents during travel and individual responses of travellers to febrile illness. Out of 714 patients who were monitored, 130 (18%) reported onset of fever during travel or 14 days after return. Of those travellers who reported fever, 100 (80%) carried SBET during travel. The vast majority of 79 (79%) febrile travellers who carried SBET did not seek medical assistance. Overall, 14 (14%) febrile patients who carried SBET and six (20%) patients who did not carry SBET took the correct measure (doctor visit or timely SBET administration) as a response to febrile illness, respectively. Only two travellers self-administered SBET, but both of them applied the wrong regimen. In view of declining malaria transmission and improving medical infrastructure in most countries of Southeast Asia and obvious obstacles concerning SBET as shown in this study the current strategy should be re-evaluated. Pre-travel advice concerning malaria in SEA should focus on appropriate mosquito bite protection and clearly emphasize the need to see a doctor within 24 h after onset of fever.


Adaptive behaviour of the spinal cord in the transition from quiet stance to walking.
Modulation of nociceptive withdrawal reflex (NWR) excitability was evaluated during gait initiation in 10 healthy subjects to investigate how load- and movement-related joint inputs activate lower spinal centres in the transition from quiet stance to walking. A motion analysis system integrated with a surface EMG device was used to acquire kinematic, kinetic and EMG variables. Starting from a quiet stance, subjects were asked to walk forward, at their natural speed. The sural nerve was stimulated and EMG responses were recorded from major hip, knee and ankle muscles. Gait initiation was divided into four subphases based on centre of pressure and centre of mass behaviours, while joint displacements were used to categorise joint motion as flexion or extension. The reflex parameters were measured and compared between subphases and in relation to the joint kinematics. The NWR was found to be subphase-dependent. NWR excitability was increased in the hip and knee flexor muscles of the starting leg, just prior to the occurrence of any movement, and in the knee flexor muscles of the same leg as soon as it was unloaded. The NWR was hip joint kinematics-dependent in a crossed manner. The excitability of the reflex was enhanced in the extensor muscles of the standing leg during the hip flexion of the starting leg, and in the hip flexors of the standing leg during the hip extension of the starting leg. No notable reflex modulation was observed in the ankle muscles. Our findings show that the NWR is modulated during the gait initiation phase. Leg unloading and hip joint motion are the main sources of the observed modulation and work in concert to prepare and assist the starting leg in the first step while supporting the contralateral leg, thereby possibly predisposing the lower limbs to the cyclical pattern of walking.


Diagnostic significance of IgG and albumin indices versus oligoclonal band types in demyelinating disorders.
The laboratory diagnosis of demyelinating inflammatory disorders (DIDs) relies on both intrathecal oligoclonal band (OCB) positivity and IgG index. Although OCB typing remains the gold-standard test for DIDs, it can be laborious and ambiguous, complicating diagnostics, and unduly increasing diagnostic time. We examined whether serum or cerebrospinal fluid (CSF) parameters can classify OCB types and, thus, be used as a replacement test to standard OCB typing. We retrospectively analysed >1000 prospectively collected samples of patients with DIDs and quantified albumin and IgG levels in the CSF and serum. We determined OCB types by isoelectric focusing combined with immunofixation and evaluated the diagnostic accuracies of IgG and albumin indices in discriminating OCB types by receiver operating characteristic curves and multinomial regression. An IgG index cut-off of 0.589 differentiated types 2/3 from types 1/4 (area under the curve 0.780, 95% CI 0.761 to 0.812, p<0.001; specificity: 71.10%, sensitivity: 73.45%). Albumin quotient cut-off values of 6.625 and of 6.707 discriminated type 1 from type 4 and type 2 from type 3, respectively (specificity: <55%, sensitivity: <75%). Female sex, age, IgG index, CSF IgG and serum albumin were associated with different OCB types. Our study reveals that IgG and albumin index can differentiate OCB types with adequate accuracy, especially if refined by age and gender.


Mid-infrared cavity resonator integrated grating filters.
Cavity Resonator Grating Filters (CRIGFs) working in the Mid-Infrared are reported, with narrow-band resonant reflectivity peaks around 2200 cm-1 (4.6 µm). They are fabricated in the GaAs/AlGaAs material system that can potentially cover the whole [1-12] µm spectral range. TE-polarized peak reflectivity is 30% with a 4 cm-1 full width at half maximum.


Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease.
Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries.


Benzylpenicillin-induced filament formation of Clostridium perfringens.
Growth of Clostridium perfringens with low concentrations of benzylpenicillin inhibited septum formation and division of the organisms. This resulted in continued growth of the organisms as aseptate filaments. The effect was reversed on removal of the antibiotic. The composition of walls isolated from organisms grown with the antibiotic was similar to that of walls from untreated bacteria. In addition, both contained non-N-acetylated glucosamine residues in their peptidoglycan. No differences were detected in the degree of cross-linkage of peptidoglycan. Clostridium perfringens contains six membrane-associated penicillin-binding proteins (PBPs) which have different affinities for [3H]benzylpenicillin. Concentrations of the antibiotic which were sufficient to cause filamentation of apparently all organisms in a culture caused almost complete saturation of PBPs 3, 4, 5 and 6. At these concentrations there was no measurable interaction with PBPs 1 and 2. Thus interaction of the antibiotic with the lower molecular weight PBPs is correlated with the inhibition of septum formation in C. perfringens.


Application of the design of experiments in optimization of drug layering of pellets with an insight into drug polymer interactions.
This study consists of two experimental designs. Within the first one, suitable technique for application of model drug onto inactive pellets was evaluated and formulation and process parameters with greatest impact to process efficency and useful yield were determined. Results of experiments showed that formulation characteristics were the ones with the greatest impact on coating efficiency and that suspension layering technique was significantly better for drug application onto inactive pellets in comparison to solution layering during which pronounced agglomeration of pellets occurred. Analysis of drug-polymer interactions by differential scanning calorimetry was performed to explain the results of experiments. The reason for agglomeration of pellets during solution layering was formation of low Tg amorphous form of model drug. The second set of experiments was performed according to central composite design experimental plan in order to optimize level of binder and concentration of solids in the coating liquid which were found to have greatest positive impact on process efficiency and useful yield in the screening study. Statistically significant models were obtained by response surface methodology and it was possible to use them to define optimal levels of excipients in the formulation.


Evaluation of the Continuous Positive Airway Pressure Effect on Neurotrophins' Gene Expression and Protein Levels.
Neurotrophins (NT) might be associated with the pathophysiology of obstructive sleep apnea (OSA) due to concurrent intermittent hypoxia and sleep fragmentation. Such a relationship could have implications for the health and overall well-being of patients; however, the literature on this subject is sparse. This study investigated the alterations in the serum protein concentration and the mRNA expression of the brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NTF3), and neurotrophin-4 (NTF4) proteins following a single night of continuous positive airway pressure (CPAP) therapy. This study group consisted of 30 patients with OSA. Venous blood was collected twice after a diagnostic polysomnography (PSG) and PSG with CPAP treatment. Gene expression was assessed with a quantitative real-time polymerase chain reaction. An enzyme-linked immunosorbent assay was used to determine the protein concentrations. After CPAP treatment, BDNF, proBDNF, GDNF, and NTF4 protein levels decreased (p = 0.002, p = 0.003, p = 0.047, and p = 0.009, respectively), while NTF3 increased (p = 0.001). Sleep latency was correlated with ΔPSG + CPAP/PSG gene expression for BDNF (R = 0.387, p = 0.038), NTF3 (R = 0.440, p = 0.019), and NTF4 (R = 0.424, p = 0.025). OSA severity parameters were not associated with protein levels or gene expressions. CPAP therapy could have an impact on the posttranscriptional stages of NT synthesis. The expression of different NTs appears to be connected with sleep architecture but not with OSA severity.


An autoencoder-based snow drought index.
In several regions across the globe, snow has a significant impact on hydrology. The amounts of water that infiltrate the ground and flow as runoff are driven by the melting of snow. Therefore, it is crucial to study the magnitude and effect of snowmelt. Snow droughts, resulting from reduced snow storage, can drastically impact the water supplies in basins where snow predominates, such as in the western United States. Hence, it is important to detect the time and severity of snow droughts efficiently. We propose the Snow Drought Response Index or SnoDRI, a novel indicator that could be used to identify and quantify snow drought occurrences. Our index is calculated using cutting-edge ML algorithms from various snow-related variables. The self-supervised learning of an autoencoder is combined with mutual information in the model. In this study, we use Random Forests for feature extraction for SnoDRI and assess the importance of each variable. We use reanalysis data (NLDAS-2) from 1981 to 2021 for the Pacific United States to study the efficacy of the new snow drought index. We evaluate the index by confirming the coincidence of its interpretation and the actual snow drought incidents.


Comparison of the galactopoietic response to pituitary-derived and recombinant-derived variants of bovine growth hormone.
Two studies were designed to examine the differences in galactopoietic potency of molecular variants of pituitary- and recombinant-derived bovine GH (bGH). The recombinant bGH molecules included amino-terminal and position-127 amino acid substitutions which are representative of two of the four natural pituitary variants or of partially degraded bGH molecules. Amino-terminal variants of bGH included methionine (Met1), alanine (Ala1), serine (Ser1) or deletion of four amino acids (delta 1-4). The delta 1-4 variants were representative of degradation products previously isolated in pituitary bGH preparations. In the first study, 54 lactating Holstein cows received i.m. injections of a buffer solution (control), pituitary-derived bGH, or recombinant-derived [Met1,Leu127]-bGH, [Met1,Val127]-bGH, [Ala1,Leu127]-bGH, or [Ala1,Val127]-bGH. Cows received 25 mg bGH/day for 21 days. Substitution of the amino-terminal alanyl residue with methionine did not affect milk response. GH variants with Val127 elicited a greater milk response (8.5 kg/day) than Leu127 bGH variants (6.5 kg/day). The average milk response to the four recombinant bGH variants was 7.5 kg/day greater than controls compared with 4.4 kg/day for pituitary-derived bGH. In contrast, blood bGH concentrations were equivalent for pituitary and recombinant bGH treatments, approximately 20 micrograms/l more than control levels at 3 h after injection. Blood free fatty acid concentrations were increased, but insulin and glucose levels were unaffected by bGH treatment. In the second study, 54 lactating Holstein cows received i.m. injections of a buffer control solution or recombinant-derived [Met1,Leu127]-bGH, [Ser1,Leu127]-bGH, [Ser1,Val127]-bGH, [delta 1-4,Leu127]-bGH or [delta 1-4,Val127]-bGH. Cows received 25 mg bGH/day for 28 days. The milk response to full-length bGH variants was 6.6 kg/day greater than the response to the amino-terminal deletion variants (P less than 0.05). Substitution of valine for leucine did not affect milk response to either the deletion (delta 1-4) or full-length (Met1 or Ser1) bGH molecules. In conclusion, the lowered galactopoietic potency of pituitary bGH preparations was demonstrated, at least in part, to be due to the presence of amino-terminal amino acid deletions rather than differences in amino acid sequences of recombinant bGH. Ala1 bGH variants with valine at position 127 elicited a greater milk response than Leu127 variants.


Paired source testing of optical systems.
A technique is described for obtaining interferometric data on an optical system without modification of the system, and using the detector intrinsic to the system. A pair of coherent point sources is imaged by the system and examined in an out-of-focus position. The period of the resulting fringe pattern is used to determine the focus position unambiguously. The structure of the fringe pattern indicates other system errors. An experiment to verify the practicality of the technique is described.


Abnormal sex-duct development in female moles: the role of anti-Müllerian hormone and testosterone.
We have performed a morphological, hormonal and molecular study of the development of the sex ducts in the mole Talpa occidentalis. Females develop bilateral ovotestes with a functional ovarian portion and disgenic testicular tissue. The Müllerian ducts develop normally in females and their regression is very fast in males, suggesting a powerful action of the anti-Müllerian hormone in the mole. RT-PCR demonstrated that the gene governing this hormone begins to be expressed in males coinciding with testis differentiation, and expression continues until shortly after birth. Immunohistochemical studies showed that expression occurs in the Sertoli cells of testes. No expression was detected in females. Wolffian duct development was normal in males and degenerate in prenatal females, but developmental recovery after birth gave rise to the formation of rudimentary epididymides. This event coincides in time with increasing serum testosterone levels and Leydig cell differentiation in the female gonad, thus suggesting that testosterone produced by the ovotestes is responsible for masculinisation of female moles. During postnatal development, serum testosterone concentrations decreased in males but increased in females, thus approaching the levels that adult males and females have during the non-breeding season.


Characteristics of laser Doppler perfusion imaging in vitro and in vivo.
Traditional laser Doppler flowmetry (LDF) employs continuous recording of perfusion at one point with time. In order to eliminate the large spatial and temporal fluctuations that occur in the microcirculation, laser Doppler perfusion imaging (LDI) integrates flow readings over a large area. This paper describes a number of experiments to identify some of the characteristics of the LDI, its relationship to flow and no-flow conditions and to compare it with LDF. We undertook experiments to establish the effect of scanner head height, avascular skin thickness and haematocrit on LDI output. We also investigated the contribution of the biological zero signal (the signal obtained from skin when flow is arrested) to the LDI output. LDI output increased with scanner height in vitro and in vivo. Increasing avascular skin thickness reduced the LDI output although linear output characteristics with flow were maintained over the flow range studied. Increasing the haematocrit resulted in a loss of linearity of output with flow at lower velocities. The biological zero signal contributes a similar proportion of the output signal in LDF and LDI. We have presented a series of experiments that will contribute to the understanding of the characteristics of laser Doppler perfusion imaging, its comparison to laser Doppler flowmetry and its relationship to flow and no flow situations. However, our experiments were restricted to one machine, and may not necessarily be applicable to other instruments.


Purkinje cell vulnerability to developmental ethanol exposure in the rat cerebellum.
Ethanol exposure is a consistent and reliable producer of neuronal toxicity, especially during periods of enhanced neuronal vulnerability. For rat cerebellar Purkinje cells, the postnatal period during the time of the brain growth spurt exhibits the greatest vulnerability to ethanol. Analyses of studies completed over more than 20 years provides sufficient detail to allow for the determination of the specific vulnerable window for ethanol-induced loss of Purkinje cells. Data reporting Purkinje cell counts after ethanol exposure were compiled from 18 studies published since 1975. We conducted linear regression analysis between peak blood ethanol concentration (BEC) and percent reduction in Purkinje cells for the following individual postnatal (PN) days: PN4, PN5. PN6, PN7, and PN8 or beyond (+). The slope of the regression and the coefficients of determination (r2) were the primary factors of interest. Analysis of variance of the regressions was conducted to identify whether the slopes were significantly different from zero, or from each other. Exposures involving the PN4-6 period demonstrated the greatest significance in the relationship between BEC and reduction of Purkinje cell number. No significant differences were identified between different ethanol exposure techniques or for different Purkinje cell counting techniques. In addition, the initial day of exposure and the duration of exposure were not identified as critical variables. The literature database, developed over the past 20 years is clear in its direction that studies designed to identify the ethanol-specific mechanisms of Purkinje cell death are best designed to involve ethanol exposure during the vulnerable window of postnatal days 4-6.


Locomotory behaviour of epitheliocytes and fibroblasts on metallic grids.
Behaviour of epitheliocytes and fibroblasts on special discontinuous substrata (metallic grids with square openings of 45x45 microm2) was examined in order to compare the ability of these cells to spread in two mutually perpendicular directions and to stretch over the void spaces. Two cell types with typical fibroblastic morphology, the AGO 1523 line of human foreskin fibroblasts and secondary cultures of mouse embryo fibroblasts, and three cell types with typical epithelial morphology, primary mouse hepatocytes, the IAR-2 line of rat liver cells and the MDCK line of canine kidney epithelial cells (clone 20) were used. We also examined the epitheliocytes (MDCK cells, clone 20) transformed to fibroblast-like morphology by treatment with hepatocyte growth factor/scatter factor (HGF/SF). Time-lapse video microscopy, scanning electron microscopy and immunofluorescence microscopy were used to examine cell reorganizations at various stages of spreading. It was found that early stages of spreading of fibroblasts and epitheliocytes were similar: the cell spread along two bars, perpendicular to each other (bar and crossbar), with the formation of a small triangular lamellar cytoplasm stretched over the opening. Later central parts of the bodies of the fibroblasts retracted from the bars so that the cells remained attached only by their polar lamellae. Successive expansions and partial retractions of these lamellae led to elongation of the cell body crossing several openings of the grid. Epitheliocytes, in contrast to fibroblasts, at the late stages of spreading did not retract their bodies and did not contract polar lamellae. As a result, their central lamellae stretched progressively over the openings. As a result of the treatment of MDCK epitheliocytes with HGF/SF the behaviour of the cells on the grids became similar to that of fibroblasts. It is suggested that these distinct spreading patterns of epitheliocytes and fibroblasts are due to the type-specific differences in the actin-myosin cortex. Experiments with microtubule-specific drugs, colcemid and taxol, indicate that the organization of this cortex is under microtubular control.


Injuries, Violence, and Bullying Among Middle School Students in Oman.
Injuries account for a substantial proportion of the burden of disease in adolescents globally. This paper describes injury rates and associated exposures, and risk behaviors in Oman's 2010 Global School-based Student Health Survey (GSHS). This study used complex samples analysis to examine nationally-representative data from 1 606 students in grades eight, nine, and 10 who participated in the 2010 Oman GSHS. In total, 34.0% of the students reported having at least one injury in the past year that caused at least one full day abscence from usual activities or required medical treatment. The most common injury type reported was a broken bone or dislocated joint. The most common injury cause was falling. Additionally, 38.4% of the students reported being bullied in the past month, 38.8% reported being physically attacked in the past year, and 47.6% reported being in physical fights. Both injured boys and girls reported experiencing significantly more bullying, fights, and attacks (odds ratio > 2) than their non-injured classmates, even though only 9.6% of injured students reported that their most serious injury in the past year was the result of an assault, and students reporting assaults did not have significantly higher odds of exposure to these types of peer violence. More than half of the bullied students reported that the most frequent type of peer victimization they experienced was being made fun of with sexual jokes, comments, or gestures. Sexual bullying was the most common type of bullying reported by girls and boys. Promoting healthier peer relationships may help to reduce injuries in this age group as well as reducing the harmful effects of bullying.


Overview of the Anticancer Potential of the "King of Spices" Piper nigrum and Its Main Constituent Piperine.
The main limits of current anticancer therapy are relapses, chemoresistance, and toxic effects resulting from its poor selectivity towards cancer cells that severely impair a patient's quality of life. Therefore, the discovery of new anticancer drugs remains an urgent challenge. Natural products represent an excellent opportunity due to their ability to target heterogenous populations of cancer cells and regulate several key pathways involved in cancer development, and their favorable toxicological profile. Piper nigrum is one of the most popular spices in the world, with growing fame as a source of bioactive molecules with pharmacological properties. The present review aims to provide a comprehensive overview of the anticancer potential of Piper nigrum and its major active constituents-not limited to the well-known piperine-whose undeniable anticancer properties have been reported for different cancer cell lines and animal models. Moreover, the chemosensitizing effects of Piper nigrum in association with traditional anticancer drugs are depicted and its toxicological profile is outlined. Despite the promising results, human studies are missing, which are crucial for supporting the efficacy and safety of Piper nigrum and its single components in cancer patients.


Appendicular robusticity and the paleobiology of modern human emergence.
The emergence of modern humans in the Late Pleistocene, whatever its phylogenetic history, was characterized by a series of behaviorally important shifts reflected in aspects of human hard tissue biology and the archeological record. To elucidate these shifts further, diaphyseal cross-sectional morphology was analyzed by using cross-sectional areas and second moments of area of the mid-distal humerus and midshaft femur. The humeral diaphysis indicates a gradual reduction in habitual load levels from Eurasian late archaic, to Early Upper Paleolithic early modern, to Middle Upper Paleolithic early modern hominids, with the Levantine Middle Paleolithic early modern humans being a gracile anomalous outlier. The femoral diaphysis, once variation in ecogeographically patterned body proportions is taken into account, indicates no changes across the pre-30,000 years B.P. samples in habitual locomotor load levels, followed by a modest decrease through the Middle Upper Paleolithic.


Metagenomic sequencing complements routine diagnostics in identifying viral pathogens in lung transplant recipients with unknown etiology of respiratory infection.
Lung transplant patients are a vulnerable group of immunosuppressed patients that are prone to frequent respiratory infections. We studied 60 episodes of respiratory symptoms in 71 lung transplant patients. Almost half of these episodes were of unknown infectious etiology despite extensive routine diagnostic testing. We re-analyzed respiratory samples of all episodes with undetermined etiology in order to detect potential viral pathogens missed/not accounted for in routine diagnostics. Respiratory samples were enriched for viruses by filtration and nuclease digestion, whole nucleic acids extracted and randomly amplified before high throughput metagenomic virus sequencing. Viruses were identified by a bioinformatic pipeline and confirmed and quantified using specific real-time PCR. In completion of routine diagnostics, we identified and confirmed a viral etiology of infection by our metagenomic approach in four patients (three Rhinovirus A, one Rhinovirus B infection) despite initial negative results in specific multiplex PCR. Notably, the majority of samples were also positive for Torque teno virus (TTV) and Human Herpesvirus 7 (HHV-7). While TTV viral loads increased with immunosuppression in both throat swabs and blood samples, HHV-7 remained at low levels throughout the observation period and was restricted to the respiratory tract. This study highlights the potential of metagenomic sequencing for virus diagnostics in cases with previously unknown etiology of infection and in complex diagnostic situations such as in immunocompromised hosts.


Physical inactivity and percent breast density among Hispanic women.
Results of epidemiologic studies suggest an inverse association between breast cancer risk and physical activity; this is one of the few modifiable breast cancer risk factors identified to date. However, only 2 previous studies assessed the association between physical activity and the extent of mammographically-detected fibroglandular breast density, a marker of breast cancer risk. Moreover, there has been no study of physical inactivity and percent breast density, nor a study of this relationship in Hispanic women, who are less physically active than non-Hispanic whites. In the Chicago Breast Health Project, we collected information on sociodemographic, reproductive, medical and lifestyle factors and percent breast density, assessed quantitatively using full-field digital mammography, from 294 Hispanic women. In our study, we examined the independent associations of hours per day of physical inactivity with percent breast density using multivariate linear regression analysis adjusting for age, education, body mass index, parity, menopausal status, use of hormone replacement therapy and smoking status. Overall, the mean percent breast density was low (i.e., 17.7%) and ranged from 1.9% to 54.6%. There was no difference in percent breast density for women who reported 1.5-3.0 hr of physical inactivity per day compared to women who reported 0-1 hr per day (beta = -0.08, p = 0.95), but percent density was marginally significantly higher for women who were reported at least 3.5 hr per day of physical inactivity (beta = 3.18, p = 0.056). Results were similar, albeit less statistically significant, in analyses of pre/perimenopausal and postmenopausal women separately. These results support the need for further research investigating the effect of physical activity on breast cancer risk.


Transformation and expression of a cloned fimA gene in Porphyromonas gingivalis.
The Porphyromonas gingivalis fimbria is an important virulence factor involved in the adherence and colonization of the organism in the oral cavity. In this study, we transformed this organism with a gene, fimA381, encoding the fimbrial subunit of P. gingivalis 381 (fimbrillin) by using the host-vector system that we developed previously and examined expression of the cloned fimA381 gene. The recombinant plasmid pYHF2 was constructed by ligating a fragment containing the fimA381 gene into the plasmid vector pYH420 and transformed into the restriction-deficient P. gingivalis host YH522. pYHF2 was autonomously maintained in YH522 cells, and the fimbrillin polypeptide (recombinant fimbrillin) was fully expressed. The molecular mass of the recombinant fimbrillin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 41 kDa, which was identical to that of the native fimbrillin of strain 381. The amino acid sequences of the 20 amino-terminal residues of the recombinant fimbrillin and the native fimbrillin of the strain 381 were identical. In addition, characteristic long and thin fimbrial structures (recombinant fimbriae) that were distinguishable from the host's native fimbriae when examined by immunogold electron microscopy were observed around the cell surface of the transformants containing the fimA381 gene. These results suggested that transformation of fimA gene from a different strain of P. gingivalis followed by accumulation of the mature fimbrial subunit protein was sufficient for production of fimbrial structures that were observable by electron microscopy.


Improved transient silencing of gene expression in the mosquito female Aedes aegypti.
Gene silencing using RNA interference (RNAi) has become a widely used genetic technique to study gene function in many organisms. In insects, this technique is often applied through the delivery of dsRNA. In the adult female Aedes aegypti, a main vector of human-infecting arboviruses, efficiency of gene silencing following dsRNA injection varies greatly according to targeted genes. Difficult knockdowns using dsRNA can thus hamper gene function analysis. Here, by analysing silencing of three different genes in female Ae. aegypti (p400, ago2 and E75), we show that gene silencing can indeed be dsRNA sequence dependent but different efficiencies do not correlate with dsRNA length. Our findings suggest that silencing is likely also gene dependent, probably due to gene-specific tissue expression and/or feedback mechanisms. We demonstrate that use of high doses of dsRNA can improve knockdown efficiency, and injection of a transfection reagent along with dsRNA reduces the variability in efficiency between replicates. Finally, we show that gene silencing cannot be achieved using siRNA injection in Ae. aegypti adult females. Overall, this work should help future gene function analyses using RNAi in adult females Ae. aegypti, leading toward a better understanding of physiological and infectious processes.


Retrorectal cystic hamartoma: report of 5 cases with malignancy arising in 2.
Retrorectal cystic hamartomas, or tailgut cysts, are rare congenital lesions that typically present as presacral masses. These lesions are frequently clinically unrecognized and misdiagnosed. Malignant change is extremely rare. Only 10 additional cases with associated malignancy were recovered from the literature. We describe the clinicopathologic features of 5 cases, including 2 cases with malignant transformation. All patients were women (age range, 36-69 years). The most common symptoms were pain with defecation and rectal bleeding. One patient was asymptomatic. All lesions presented as multicystic presacral masses and all were surgically resected. The lesions varied in size from approximately 2 to 12 cm (average, 9.5 cm) and overall had similar histology composed of a variety of epithelial linings (stratified squamous, transitional, and simple or ciliated pseudostratified columnar). Skin adnexa, neural elements, and heterologous mesenchymal tissue, discriminators between retrorectal cystic hamartoma and teratoma, were not identified. Arising in association with the cysts was a focus of adenocarcinoma in one case and a neuroendocrine carcinoma in another. The clinical diagnoses in our cases were often delayed, which in part may be due to unfamiliarity with this entity. The main diagnostic difficulty is distinction from presacral mature cystic teratomas and rectal duplication cysts. Tailgut cysts require complete surgical excisions to prevent future recurrences and to preclude possible malignant transformation. Meticulous gross examination and adequate sampling are important to document the exact nature of these cysts and to rule out possible coexisting malignancies, which may be focal.


Magnetic resonance imaging with hyperpolarized [1,4-(13)C2]fumarate allows detection of early renal acute tubular necrosis.
Acute kidney injury (AKI) is a common and important medical problem, affecting 10% of hospitalized patients, and it is associated with significant morbidity and mortality. The most frequent cause of AKI is acute tubular necrosis (ATN). Current imaging techniques and biomarkers do not allow ATN to be reliably differentiated from important differential diagnoses, such as acute glomerulonephritis (GN). We investigated whether (13)C magnetic resonance spectroscopic imaging (MRSI) might allow the noninvasive diagnosis of ATN. (13)C MRSI of hyperpolarized [1,4-(13)C(2)]fumarate and pyruvate was used in murine models of ATN and acute GN (NZM2410 mice with lupus nephritis). A significant increase in [1,4-(13)C(2)]malate signal was identified in the kidneys of mice with ATN early in the disease course before the onset of severe histological changes. No such increase in renal [1,4-(13)C(2)]malate was observed in mice with acute GN. The kidney [1-(13)C]pyruvate/[1-(13)C]lactate ratio showed substantial variability and was not significantly decreased in animals with ATN or increased in animals with GN. In conclusion, MRSI of hyperpolarized [1,4-(13)C(2)]fumarate allows the detection of early tubular necrosis and its distinction from glomerular inflammation in murine models. This technique may have the potential to identify a window of therapeutic opportunity in which emerging therapies might be applied to patients with ATN, reducing the need for acute dialysis with its attendant morbidity and cost.


TLR2-dependent pathway of heterologous down-modulation for the CC chemokine receptors 1, 2, and 5 in human blood monocytes.
During innate immune responses, the inflammatory CC chemokine receptors CCR1, CCR2, and CCR5 mediate the recruitment of blood monocytes to infected tissues by promoting cell migration in response to chemokines CCL2-5. Toll-like receptors also play an essential role, allowing pathogen recognition by the recruited monocytes. Here, we demonstrate that Toll-like receptor 2 (TLR2) stimulation by lipoteichoic acid (LTA) from Staphylococcus aureus leads to gradual down-modulation of CCR1, CCR2, and CCR5 from the plasma membrane of human blood-isolated monocytes and inhibits chemotaxis. Interestingly, LTA does not promote rapid desensitization of chemokine-mediated calcium responses. We found that the TLR2 crosstalk with chemokine receptors is not dependent on the Toll/interleukin-1 receptor domain-containing adaptor protein, but instead involves phospholipase C, the small G protein Rac1, and is phorbol ester sensitive. Activation of this pathway by LTA lead to β-arrestin-mediated endocytosis of Ser349-phosphorylated CCR5 into recycling endosomes, as does CCL5 treatment. However, LTA-induced internalization of CCR5 is a slower process associated with phospholipase C-mediated and phorbol ester-sensitive phosphorylation. Overall, our data indicate that TLR2 negatively regulates CCR1, CCR2, and CCR5 on human blood monocytes by activating the machinery used to support chemokine-dependent down-modulation and provide a molecular mechanism for inhibiting monocyte migration after pathogen recognition.


Theranostic nanoparticles for future personalized medicine.
The concept of personalized medicine has recently emerged as a promising way to address unmet medical needs. Due to the limitations of standard diagnostic and therapeutic strategies, the disease treatment is moving towards tailored treatment for individual patients, considering the inter-individual variability in therapeutic response. Theranostics, which involves the combination of therapy and diagnostic imaging into a single system, may fulfill the promise of personalized medicine. By integrating molecular imaging functionalities into therapy, theranostic approach could be advantageous in therapy selection, treatment planning, objective response monitoring and follow-up therapy planning based on the specific molecular characteristics of a disease. Although the field of therapy and imaging of its response have been independently developed thus far, developing imaging strategies can be fully exploited to revolutionize the theranostic systems in combination with the therapy modality. In this review, we describe the recent advances in molecular imaging technologies that have been specifically developed to evaluate the therapeutic efficacy for theranostic purposes.


Genetic, epigenetic and transcriptional comparison of esophagus tumor‑associated and adjacent normal myofibroblasts.
Myofibroblasts (MFs) are present in healthy tissues and are also key components of the tumor microenvironment. In the present study a comparative analysis of MFs obtained from various gastrointestinal tumor tissues and from tumor‑adjacent normal tissues of cancer patients was performed, with the aim to evaluate differences in MF morphology, gene expression profile and function. The goal was to correlate the observed morphological and functional variations with the underlying genetic and epigenetic backgrounds. The mutation frequency of MFs was assessed by next generation sequencing. The transcript levels of cancer‑specific genes were determined by TaqMan array and quantitative polymerase chain reaction. Epigenetic modifications were analyzed by immunocytochemistry and western blotting. The migratory capacity of MFs was assessed by scratch assay, whereas matrix metalloproteinase expression and activity were obtained by quantitative polymerase chain reaction and zymography. The results of the present study demonstrate that MFs were present in an increased number and with altered morphology in tumor samples compared with the healthy tissue. Although the detected number of mutations in tumor‑associated and normal tissue‑derived MFs did not differ markedly, shifts in the level of specific acetylated and methylated histone proteins, namely decreased levels of trimethylated H3K9 and acetylated H4K16 were demonstrated in tumor‑associated MFs. Transcript levels of several tumor‑specific genes involved in metastasis, regulation of cellular growth, apoptosis, as well as in hypoxia‑angiogenesis were altered in tumor‑derived MF cultures. Increased mRNA levels were obtained and activity of matrix metalloproteases in tumor‑derived MFs and these cells also exhibited a higher migratory capacity compared with the normal MFs. In summary, the results of the present study indicate that tumor‑associated MFs display an altered phenotype compared with healthy tissue derived counterparts. The results imply that epigenetic rather than genetic alterations are associated with the development of the distinct expressional and functional features, which define this MF phenotype in the tumor microenvironment.


Internet Addiction: A Review of the First Twenty Years.
Easy access to communication and information technologies has increased our dependence on technology for various aspects of our lives. Nevertheless, this remarkable growth of Internet Usage has been inextricably paired with a rise of excessive and dysfunctional Internet use. Conceptualized around 1996, a few years after the inception of the World Wide Web, Internet addiction has developed into a global issue influencing varying segments of the population at different levels. Despite heated debates on its addictive nature, consensus is emerging regarding the existence of this problematic behavior. In this paper we provide a comprehensive overview of the literature on Internet addiction in last 20 years. Purpose of this paper is to present crucial findings on Internet addiction to health profession. Besides numerous benefits of Internet use, the virtual environment brings various risks in every age group. The Internet is very significant in the everyday activities of children and youth and professional interventions with this age group should be specific considering their developmental characteristics. Exposure to online risks can have long-lasting and intense negative effects. Effective programs in prevention and treatment should include a multi-sectoral and interdisciplinary approach. Detail review of the symptomatology, diagnosis model an possibilities of treatment can be multiple beneficial to the health professionals and other helping professions due to actual needs for interventions in the field of the internet addiction treatment. Internet addiction is slowly becoming a societal concern as it particularly affects adolescents and children, who are more exposed and consequently more vulnerable. Findings presented in the paper can benefit in practice of treatment internet addiction and also as framework for further researches in the field.


Where's My Med? Improving Patient-Specific Medication Storage for Emergency Department Boarders.
There have been an increased number of patients boarding in the Emergency Department (ED) who often experience delays in receiving non-urgent medications not typically stocked in the ED automated dispensing cabinets (ADC). To describe a quality improvement project designed to decrease the amount of time it takes nursing staff to obtain patient-specific medications for patients boarding in the ED at a community hospital. Prior to this quality improvement project, standard practice was for pharmacy staff to send all medications to the ED based on inpatient cart fill lists, resulting in a 24-hour supply being sent at one time. Medications that were not immediately due were often misplaced by ED staff as there was no standard location to store them. A separate location for each exam room in the ED was created in one central ADC to store patient-specific medications. Pharmacy staff were instructed to load medications directly into the ADC for expected long-term behavioral health boarders. Nursing staff were instructed to place future doses in the newly created locations and were educated on how to locate medications utilizing the electronic MAR. ED nursing staff timed how long it took them to obtain medications for 40 boarding patients before and after implementation of the quality improvement initiatives. The post-improvement intervention decreased the amount of time it took to obtain medications for forty ED boarding patients by 66%, from an average of 37.7 minutes at baseline to 12.9 minutes post-interventions. This was a statistically significant improvement via an unpaired t-test (p < 0.0001 with a 95% CI from 15.3-34.47). This was rechecked after one year and the improvement was sustained. A multi-disciplinary team-based quality improvement initiative was successful in reducing the time it took to obtain medications for ED boarding patients.


Resistin: functional roles and therapeutic considerations for cardiovascular disease.
Resistin, originally described as an adipocyte-specific hormone, has been suggested to be an important link between obesity, insulin resistance and diabetes. Although its expression was initially defined in adipocytes, significant levels of resistin expression in humans are mainly found in mononuclear leukocytes, macrophages, spleen and bone marrow cells. Increasing evidence indicates that resistin plays important regulatory roles apart from its role in insulin resistance and diabetes in a variety of biological processes: atherosclerosis and cardiovascular disease (CVD), non-alcoholic fatty liver disease, autoimmune disease, malignancy, asthma, inflammatory bowel disease and chronic kidney disease. As CVD accounts for a significant amount of morbidity and mortality in patients with diabetes and without diabetes, it is important to understand the role that adipokines such as resistin play in the cardiovascular system. Evidence suggests that resistin is involved in pathological processes leading to CVD including inflammation, endothelial dysfunction, thrombosis, angiogenesis and smooth muscle cell dysfunction. The modes of action and signalling pathways whereby resistin interacts with its target cells are beginning to be understood. In this review, the current knowledge about the functions and pathophysiological implications of resistin in CVD development is summarized; clinical translations, therapeutic considerations and future directions in the field of resistin research are discussed. This article is part of a themed section on Fat and Vascular Responsiveness. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-3.


Health effects of ambient particulate matter--biological mechanisms and inflammatory responses to in vitro and in vivo particle exposures.
In this article, we review and analyze different modes of exposure to ultrafine particles in order to assess particle-induced inflammatory responses and the underlying mechanisms in vitro and in vivo. Based on results from monocytic cells cultured under submerged conditions, we discuss (1) the impact of particle properties such as surface area and oxidative potential on lipid metabolism as a highly sensitive regulatory pathway and (2) the interference of diesel exhaust particles with toll-like receptor-mediated inflammatory responses. Furthermore, new developments of air-liquid interface exposure used as an alternative approach to simulate cell particle interactions are presented. In addition to the in vitro approaches, animal exposure studies are described that apply selected mouse models to elucidate potential allergic and inflammatory pulmonary responses and mast-cell-related mechanisms after particle exposure. Long-term inhalation of ultrafine particles might lead to irreversible changes in lung structure and function. Clinical studies addressing the characteristics of inflammatory airway cells are a promising approach to understand underlying pathophysiological mechanisms in chronic obstructive pulmonary disease. Finally, a potential outcome of human particle exposure is chronic cough in children. Here, discrimination between asthmatic and nonasthmatic cough by means of immunological parameters appears to be an important step toward improving diagnosis and therapy.


Linear response of mutans streptococci to increasing frequency of xylitol chewing gum use: a randomized controlled trial [ISRCTN43479664].
Xylitol is a naturally occurring sugar substitute that has been shown to reduce the level of mutans streptococci in plaque and saliva and to reduce tooth decay. It has been suggested that the degree of reduction is dependent on both the amount and the frequency of xylitol consumption. For xylitol to be successfully and cost-effectively used in public health prevention strategies dosing and frequency guidelines should be established. This study determined the reduction in mutans streptococci levels in plaque and unstimulated saliva to increasing frequency of xylitol gum use at a fixed total daily dose of 10.32 g over five weeks. Participants (n = 132) were randomized to either active groups (10.32 g xylitol/day) or a placebo control (9.828 g sorbitol and 0.7 g maltitol/day). All groups chewed 12 pieces of gum per day. The control group chewed 4 times/day and active groups chewed xylitol gum at a frequency of 2 times/day, 3 times/day, or 4 times/day. The 12 gum pieces were evenly divided into the frequency assigned to each group. Plaque and unstimulated saliva samples were taken at baseline and five-weeks and were cultured on modified Mitis Salivarius agar for mutans streptococci enumeration. There were no significant differences in mutans streptococci level among the groups at baseline. At five-weeks, mutans streptococci levels in plaque and unstimulated saliva showed a linear reduction with increasing frequency of xylitol chewing gum use at the constant daily dose. Although the difference observed for the group that chewed xylitol 2 times/day was consistent with the linear model, the difference was not significant. There was a linear reduction in mutans streptococci levels in plaque and saliva with increasing frequency of xylitol gum use at a constant daily dose. Reduction at a consumption frequency of 2 times per day was small and consistent with the linear-response line but was not statistically significant.


Nitric oxide is required for lung alveolarization revealed by deficiency of argininosuccinate lyase.
Inhaled nitric oxide (NO) therapy has been reported to improve lung growth in premature newborns. However, the underlying mechanisms by which NO regulates lung development remain largely unclear. NO is enzymatically produced by three isoforms of nitric oxide synthase (NOS) enzymes. NOS knockout mice are useful tools to investigate NO function in the lung. Each single NOS knockout mouse does not show obvious lung alveolar phenotype, likely due to compensatory mechanisms. While mice lacking all three NOS isoforms display impaired lung alveolarization, implicating NO plays a pivotal role in lung alveolarization. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing L-arginine, the sole precursor for NOS-dependent NO synthesis. ASL is also required for channeling extracellular L-arginine into a NO-synthetic complex. Thus, ASL deficiency (ASLD) is a non-redundant model for cell-autonomous, NOS-dependent NO deficiency. Here, we assessed lung alveolarization in ASL-deficient mice. Hypomorphic deletion of Asl (AslNeo/Neo) results in decreased lung alveolarization, accompanied with reduced level of S-nitrosylation in the lung. Genetic ablation of one copy of Caveolin-1, which is a negative regulator of NO production, restores total S-nitrosylation as well as lung alveolarization in AslNeo/Neo mice. Importantly, NO supplementation could partially rescue lung alveolarization in AslNeo/Neo mice. Furthermore, endothelial-specific knockout mice (VE-Cadherin Cre; Aslflox/flox) exhibit impaired lung alveolarization at 12 weeks old, supporting an essential role of endothelial-derived NO in the enhancement of lung alveolarization. Thus, we propose that ASLD is a model to study NO-mediated lung alveolarization.


Do infant rats cry?
In the current revival of interest in the emotional and mental lives of animals, many investigators have focused attention on mammalian infants that emit distress vocalizations when separated from the home environment. Perhaps the most intensively studied distress vocalization is the ultrasonic vocalization of infant rats. Since its discovery, this vocalization has been interpreted both as a communicatory signal for the elicitation of maternal retrieval and as the manifestation of emotional distress. In contrast, the authors examined the cardiovascular causes and consequences of the vocalization, and on the basis of this work, they hypothesized that the vocalization is the acoustic by-product of the abdominal compression reaction (ACR), a maneuver that results in increased venous return to the heart. Therefore, the vocalization may be analogous to a sneeze, serving a physiological function while incidentally producing sound.


Analysis of pomegranate juice components in rat corpora cavernosal relaxation.
This study evaluated the action of pomegranate juice (PJ) and its five principal phenolic constituents on rat corpus cavernosum smooth muscle (CCSM). Isometric tension studies were performed after precontraction with phenylephrine in CCSM from rats. Relaxant responses to PJ and its constituents ellagic acid (EA), chlorogenic acid, caffeic acid, cumaric acid and rutin were investigated. PJ and EA caused CCSM relaxations (94.1 ± 3.7 and 51.3 ± 9.9%), while others induced limited relaxant responses. EA response was not inhibited by L-N(G)-nitroarginine methyl ester (100 μM) and 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (1 μM). Tetraethylammonium (100 μM) and apamin (10 μM) and nifedipine (10 μM) inhibited EA-induced relaxations at 10(-3) M by 84%, 82% and 78%, respectively. Glibenclamide (10 μM) inhibited EA response (97%, 100 μM). PJ-induced relaxation was not altered by several inhibitors. EA was estimated to be responsible for 13.3% of relaxation caused by PJ. Our study demonstrated that PJ and EA-induced marked relaxations in CCSM. The opening of Ca(2+)-activated K+ channels and the inhibition of Ca(2+)-channels regulate the relaxation by EA, but not PJ. EA has a minor contribution to the marked relaxation obtained by PJ, suggesting the presence of other PJ constituents, which induce nitric oxide-independent corporal relaxation. Further studies are needed to examine the potential of PJ in combination with a PDE5 inhibitor in ED.


Growing Bacteriophage M13 in Liquid Culture.
Stocks of bacteriophage M13 are usually grown in liquid culture. The infected bacteria do not lyse but, instead, grow at a slower than normal rate to form a dilute suspension. The inoculum of bacteriophage is almost always a freshly picked plaque or a suspension of bacteriophage particles obtained from a single plaque, as described here. Infected cells contain up to 200 copies of double-stranded, replicative-form DNA and extrude several hundred bacteriophage particles per generation. Thus, a 1-mL culture of infected cells can produce enough double-stranded viral DNA (1-2 mg) for restriction mapping and recovery of cloned DNA inserts and sufficient single-stranded DNA (∼5-10 mg) for site-directed mutagenesis, DNA sequencing, or synthesis of radiolabeled probes. The titer of bacteriophages in the supernatant from infected cells is so high (∼1012 pfu/mL) that a small aliquot serves as a permanent stock of the starting plaque.


The prognostic significance of beta(2)-microglobulin in patients with Hodgkin's lymphoma.
Serum beta(2)-microglobulin (s beta(2)m) is an established prognostic factor for multiple myeloma and non-Hodgkin's lymphoma, but only limited data suggest an adverse prognostic significance for Hodgkin's lymphoma (HL). This study was undertaken to examine the impact of s beta(2)m on the prognosis of patients with HL. s beta(2)m was measured by a radioimmunoassay (upper normal limit 2.4 mg/L), in pretreatment serum samples of 232 patients with HL, who were then treated with ABVD or equivalent regimens with or without radiotherapy. Multivariate survival analysis was based on Cox's proportional hazards model. Main patients' characteristics: median age 30.5 years (14-78); 58% males; 68% nodular sclerosis, 20% mixed cellularity and 12% lymphocyte predominance; 34% B-symptoms; 24% Ann Arbor stage I, 49% II, 18% III and 9% IV. Elevated s beta(2)m levels were detected in 65/232 patients (28%) and correlated with older age (p<0.001), mixed cellularity (p=0.03), B-symptoms (p=0.002), advanced stage (p=0.02), > or = 5 involved sites (p=0.02), inguinal/iliac involvement (p=0.009), lymphocytopenia (p=0.002) and elevated lactate dehydrogenase (p=0.01). The 7-year failure free survival (FFS) was 75% vs. 72% for patients with normal vs. elevated s beta(2)m (p=0.15). The corresponding 7-year overall survival (OS) rates were 86% vs. 52% (p=0.003). In multivariate analysis, elevated s beta(2)m was not predictive of FFS, but was independently associated with inferior OS (p=0.01), along with the number of involved sites (p<0.001). s beta(2)m is not a potent prognostic factor for FFS in optimally treated patients with HL. However s beta(2)m may be predictive of OS, probably due to its effect on the timing of treatment failure.


Promoting siRNA delivery via enhanced cellular uptake using an arginine-decorated amphiphilic dendrimer.
RNA interference (RNAi) with small interfering RNA (siRNA) is expected to offer an attractive means to specifically and efficiently silence disease-associated genes for treating various diseases provided that safe and efficient delivery systems are available. In this study, we have established an arginine-decorated amphiphilic dendrimer composed of a hydrophobic alkyl chain and a hydrophilic PAMAM dendron bearing arginine terminals as nonviral vector for siRNA delivery. Indeed, this dendrimer proved to be very effective at delivering siRNAs in human prostate cancer PC-3 cells and in human hematopoietic CD34+ stem cells, leading to improved gene silencing compared to the corresponding nonarginine decorated dendrimer. Further investigation confirmed that this dendrimer was granted with the capacity to form stable nanoparticles with siRNA and significantly enhance cellular uptake of siRNA. In addition, this dendrimer revealed no discernible cytotoxicity. All these findings demonstrate that decoration of the dendrimer surface with arginine residues is indeed a useful strategy to improve the delivery ability of dendrimers.


Cognitive and cardiac outcomes 5 years after off-pump vs on-pump coronary artery bypass graft surgery.
Conventional coronary artery bypass graft surgery with use of cardiopulmonary bypass (on-pump CABG) is associated with excellent long-term cardiac outcomes but also with a high incidence of cognitive decline. The effect of avoiding cardiopulmonary bypass (off-pump CABG) on long-term cognitive and cardiac outcomes is unknown. To compare the effect of off-pump CABG and on-pump CABG surgery on long-term cognitive and cardiac outcomes. The Octopus Study, a multicenter randomized controlled trial conducted in the Netherlands, which enrolled 281 low-risk CABG patients between 1998 and 2000. Five years after their surgery, surviving patients were invited for a follow-up assessment. Patients were randomly assigned to receive either off-pump (n = 142) or on-pump (n = 139) CABG surgery. The primary measure was cognitive status 5 years after surgery, which was determined by a psychologist blinded to treatment allocation who administered 10 standardized validated neuropsychological tests. Secondary measures were occurrence of cardiovascular events (all-cause mortality, stroke, myocardial infarction, and coronary reintervention), anginal status, and quality of life. After 5 years, 130 patients were alive in each group. Cognitive outcomes could be determined in 123 and 117 patients in the off-pump and on-pump groups, respectively. When using a standard definition of cognitive decline (20% decline in performance in 20% of the neuropsychological test variables), 62 (50.4%) of 123 in the off-pump group and 59 (50.4%) of 117 in the on-pump group had cognitive decline (absolute difference, 0%; 95% confidence interval [CI], -12.7% to 12.6%; P>.99). When a more conservative definition of cognitive decline was used, 41 (33.3%) in the off-pump group and 41 (35.0%) in the on-pump group had cognitive decline (absolute difference, -1.7%; 95% CI, -13.7% to 10.3%; P = .79). Thirty off-pump patients (21.1%) and 25 on-pump patients (18.0%) experienced a cardiovascular event (absolute difference, 3.1%; 95% CI, -6.1% to 12.4%; P = .55). No differences were observed in anginal status or quality of life. In low-risk patients undergoing CABG surgery, avoiding the use of cardiopulmonary bypass had no effect on 5-year cognitive or cardiac outcomes. isrctn.org Identifier: ISRCTN69438133.


Chromosome 14 translocations in non-Burkitt lymphomas.
Chromosome studies were performed on malignant cells obtained from 27 patients with non-Burkitt lymphomas. A marker chromosome affecting the long arm of No. 14 (14q+) was the single most frequent abnormality and was noted in 17 of these patients. The frequency of the 14q+ marker varied with the type of lymphoma. For patients with malignant lymphoma, histiocytic, the frequency was 5 of 8; for mixed-cell type, 1 of 3; for poorly differentiated lymphocytic, 8 of 8; for well-differentiated lymphocytic, 0 of 3; for lymphoblastic, 0 of 1; for Hodgkin's disease, 2 of 3; and for mycosis fungoides, 1 of 1. The donor chromosome involved in the 14q translocation was identified in 12 cases; certain chromosomes appeared to be affected more frequently than others. Although the break point was band 14q32 in most cases, the exact location of the receptor site on 14q was not always consistent. The distal part of 14q24 was also involved as a receptor site in at least one translocation. These findings suggest that, in some types of lymphoid malignancy, cells with a 14q translocation have a proliferative advantage over cells with other chromosome rearrangments. The presence of the 14q translocation may be important in the future for the distinction among morphologically different, but functionally comparable, subgroups of lymphoid malignancies.


[Chemical and nutritional assessment of a soybean and cereal mixture for adults].
Legume seeds and fibre rich plant foods usually improve aspects of human diabetes control as they are potential sources of "delayed release" carbohydrates. A regional bakery mixture of soybean and cereals, interesting for its palatability and high content in non starch polysaccharides was chemically and nutritionally evaluated. Comparisons were made with the usual commercial laboratory chow and with a cafeteria mixture. Each one of the three diets was offered ad libitum to adult rats of line IIMb/Fm beta (beta), affected by obesity, hypertriglyceridemia and glucose intolerance or diabetes. Treatments lasted three months and were performed on two groups of male rats: (a) From 100 days old growing significantly. (b) From 200 days old. Meals had similar carbohydrate and calorie contents but acid followed by enzymatica hydrolysis was required to free monosaccharides from the soybean mixture. Cafeteria mixture lacked in fibre, was rich in saturated fats and sodium, and it caused hyperphagia. Each group of rats showed similar food intakes in both ages although weight gain was significantly higher in the younger animals. In the latter, values of glycemic response showed no difference between diets. Cafeteria mixture caused significant hyperglycemia to the elder rats, while the soybean bakery mixture produced a remarkably lower glycemic response; in one case it was even lower than the one produced by the commercial chow. Differential response showed more clearly with age. The results of the feces analysis demonstrated an increased proportion of fecal water for the bakery mixture group, probably due to the amount of undigestible fibre, inducing beneficial effects on large bowel functionality.


Inflammatory cytokines induce fibrosis and ossification of human ligamentum flavum cells.
In vitro experiment using degenerated human ligamentum flavum (LF) and various inflammatory cytokines. To examine the effect of inflammatory cytokines on LF cells and to identify their roles in the pathogenesis of LF hypertrophy and ossification. Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by the degenerative processes (ie, increased collagen synthesis and chondroid metaplasia) of ligament fibroblasts. Degenerated intervertebral disk spontaneously produces inflammatory cytokines, which might affect the adjacent LF through local milieu of the spinal canal. The interlaminar portion of the LF was collected during surgical spinal procedures in 15 patients (age range, 49-78 y) with lumbar spinal stenosis. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with various inflammatory cytokines: interleukin (IL)-1α, IL-6, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and nitric oxide (NO). Cytotoxicity was analyzed by MTT assays. DNA synthesis was measured with H-thymidine incorporation, and mRNA expression of types I, III, V, and XI collagen and osteocalcin were performed by reverse transcription-polymerase chain reaction. Histochemical stains such as Von Kossa were also performed to detect bone nodule formation. There was no cytotoxicity in the LF cells treated with each cytokine. There were significant increases in DNA synthesis and upregulated mRNA expression of types I, V, XI collagen and osteocalcin in LF cultures treated with various cytokines. LF cultures treated with IL-6, TNF-α, PGE2, and NO showed positive Von Kossa staining, indicating bone nodule formation from LF cells. Inflammatory cytokines (IL-6, TNF-α, PGE2, and NO) seem to play a crucial role in hypertrophy and ossification of LF. Degenerated, herniated intervertebral disks, and facet arthrosis may influence LF through inflammatory cytokines and cause hypertrophy and ossification of LF.


Structure-activity relationships for enhancement of adenosine A1 receptor binding by 2-amino-3-benzoylthiophenes.
The structural requirements for stimulation of adenosine A1 agonist binding by 2-amino-3-benzoylthiophenes and related compounds were investigated. Slowing of the dissociation of [3H]N6cyclohexyladenosine binding was used as a specific measure of the allosteric effects of these compounds. The thiophene ring could be replaced with benzene but not with several nitrogen-containing heterocycles. The 2-amino group was required, and at least one hydrogen on the amino group appeared to be necessary for activity. The keto carbonyl was also essential. Alkyl substitution at the 4-position of the thiophene ring increased activity, whereas 5-position substitution appeared to have little effect. Activity was also increased by various substitutions on the phenyl ring, with 3-(trifluoromethyl) showing optimal activity. The phenyl ring could be replaced with cyclohexyl without major loss of activity. 1-Aminofluoren-9-one, a conformationally locked derivative, was active. Based in part in the latter observation, the active conformation is proposed to have an intramolecular hydrogen bond between the amino nitrogen and the carbonyl oxygen. Because the 2-amino-3-benzoylthiophenes showed competitive adenosine antagonism as well as allosteric enhancement, their affinities as competitive inhibitors of [3H]8-cyclopentyl-1,3-dipropylxanthine binding to A1 receptors were also assessed. Structure-activity relationships for competitive antagonism were distinct from those for allosteric enhancement, with ratios between the two activities varying by more than 1000-fold. Of the analogs tested, (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone (PD 81,723) had the most favorable ratio of enhancement to antagonism.


Markers of stress and inflammation as potential mediators of the relationship between exercise and depressive symptoms: findings from the TRAILS study.
The hypothalamic-pituitary-adrenal axis, autonomic nervous system, and immune system have been proposed to underlie the antidepressant effect of exercise. Using a population sample of 715 adolescents, we examined whether pathways from exercise to affective and somatic symptoms of depression were mediated by these putative mechanisms. Exercise (hours/week) and depressive symptoms were assessed at age 13.5 (± 0.5) and 16.1 (± 0.6). Cortisol and heart rate responses to a standardized social stress test and C-reactive protein levels were measured at age 16. Exercise was prospectively and inversely related to affective (B = -0.16, 95% CI = -0.30 to -0.03) but not somatic symptoms (B = -0.04, 95% CI = -0.21 to 0.13). Heart rate during social stress partially mediated this relationship (B = -0.03, 95% CI = -0.07 to -0.01). No other mediating effects were found. Hence, the autonomic stress system may play a role in the relationship between exercise and depressive symptoms.


Changing Perceptions of Harm of E-Cigarettes Among U.S. Adults, 2012-2015.
Although the impact of long-term use of electronic cigarettes (e-cigarettes) on health is still unknown, current scientific evidence indicates that e-cigarettes are less harmful than combustible cigarettes. The study examined whether perceived relative harm of e-cigarettes and perceived addictiveness have changed during 2012-2015 among U.S. adults. Data were from Tobacco Products and Risk Perceptions surveys of probability samples representative of U.S. adults in 2012, 2014, and 2015. Changes over time in perceived harmfulness of e-cigarettes were examined using pairwise comparisons of proportions and multinomial logistic regression analysis. Analyses were conducted in January 2016. Whereas 11.5% and 1.3% of adults perceived e-cigarettes to have about the same level of harm and to be more harmful than cigarettes, respectively, in 2012, 35.7% and 4.1% did so in 2015. The proportion of adults who thought e-cigarettes were addictive more than doubled during 2012-2015 (32.0% in 2012 vs 67.6% in 2015). Compared with 2012, the odds of perceiving e-cigarettes to be equally or more harmful (than to be less harmful) doubled (95% CI=1.64, 2.41) in 2014, and tripled (95% CI=2.60, 3.81) in 2015. There is an increase in the proportion of U.S. adults who misperceive the harm of e-cigarettes and consider them to be as harmful as combustible cigarettes. The study highlights the need to design public health messages that accurately interpret the scientific data on the potential harm of e-cigarettes and clearly differentiate the absolute from the relative harm of e-cigarettes.


S-adenosylmethionine exerts a protective effect against thioacetamide-induced injury in primary cultures of rat hepatocytes.
S-adenosylmethionine (SAMe) has been shown to protect hepatocytes from toxic injury, both experimentally-induced in animals and in isolated hepatocytes. The mechanisms by which SAMe protects hepatocytes from injury can result from the pathways of SAMe metabolism. Unfortunately, data documenting the protective effect of SAMe against mitochondrial damage from toxic injury are not widely available. Thioacetamide is frequently used as a model hepatotoxin, which causes in vivo centrilobular necrosis. Even though thioacetamide-induced liver necrosis in rats was alleviated by SAMe, the mechanisms of this protective effect remain to be verified. The aim of our study was to determine the protective mechanisms of SAMe on thioacetamide-induced hepatocyte injury by using primary hepatocyte cultures. The release of lactate dehydrogenase (LDH) from cells incubated with thioacetamide for 24 hours, was lowered by simultaneous treatment with SAMe, in a dose-dependent manner. The inhibitory effect of SAMe on thioacetamide-induced lipid peroxidation paralleled the effect on cytotoxicity. A decrease in the mitochondrial membrane potential, as determined by Rhodamine 123 accumulation, was also prevented. The attenuation by SAMe of thioacetamide-induced glutathione depletion was determined after subsequent incubation periods of 48 and 72 hours. SAMe protects both cytoplasmic and mitochondrial membranes. This effect was more pronounced during the development of thioacetamide-induced hepatocyte injury that was mediated by lipid peroxidation. Continuation of the SAMe treatment then led to a reduction in glutathione depletion, as a potential consequence of an increase in glutathione production, for which SAMe is a precursor.


Maternal suboptimal selenium intake and low-level lead exposure affect offspring's microglial immune profile and its reactivity to a subsequent inflammatory hit.
Micronutrients such as selenium (Se) are essentials since prenatal life to support brain and cognitive development. Se deficiency, which affects up to 1 billion people worldwide, can interact with common adverse environmental challenges including (Pb), exacerbating their toxic effects. Exploiting our recently validated rat model of maternal Se restriction and developmental low Pb exposure, our aims were to investigate: (i) the early consequences of suboptimal Se intake and low-Pb exposure on neuroinflammation in neonates' whole brains; (ii) the potential priming effect of suboptimal Se and low-Pb exposure on offspring's glial reactivity to a further inflammatory hit. To these aims female rats were fed with suboptimal (0.04 mg/kg; Subopt) and optimal (0.15 mg/kg; Opt) Se dietary levels throughout pregnancy and lactation and exposed or not to environmentally relevant Pb dose in drinking water (12.5 µg/mL) since 4 weeks pre-mating. We found an overall higher basal expression of inflammatory markers in neonatal brains, as well as in purified microglia and organotypic hippocampal slice cultures, from the Subopt Se offspring. Subopt/Pb cultures were highly activated than Subopt cultures and showed a higher susceptibility to the inflammatory challenge lipopolysaccharide than cultures from the Opt groups. We demonstrate that even a mild Se deficiency and low-Pb exposure during brain development can influence the neuroinflammatory tone of microglia, exacerbate the toxic effects of Pb and prime microglial reactivity to subsequent inflammatory stimuli. These neuroinflammatory changes may be responsible, at least in part, for adverse neurodevelopmental outcomes.


Development of a skin colonization model in gnotobiotic piglets for the study of the microbial ecology of meticillin-resistant Staphylococcus aureus ST398.
Meticillin-resistant Staphylococcus aureus (MRSA) ST398 continues to spread amongst pigs and other domestic animals and man. This highlights the need for models to examine MRSA colonization and investigate control strategies. This study aimed to develop a gnotobiotic pig model and assess the potential of bacterial interference from selected coagulase-negative staphylococci (CNS) against MRSA ST398. Groups of 2-week-old piglets were atraumatically inoculated either with MRSA and/or CNS. Skin and mucosae were swabbed, and bacterial counts compared over a period of 21 days. Piglets developed healthily, and bacterial populations increased similarly for both MRSA and CNS until day 32. On day 37, MRSA counts in groups with CNS reduced significantly compared with MRSA alone (P = 0·03). The results showed that inoculation of piglet skin with MRSA resulted in spontaneous colonization and that MRSA ST398 has a low pathogenic potential in gnotobiotic piglets. Quantitative bacteriology indicated that initial MRSA colonization was unaffected by concurrent CNS colonization but that interference may occur over a longer period. Gnotobiotic piglets provide a reproducible model suitable for bacterial interference studies, which should be further explored as an alternative to antimicrobials in the control of MRSA.


ChAP-MS: a method for identification of proteins and histone posttranslational modifications at a single genomic locus.
The field of epigenomics has been transformed by chromatin immunoprecipitation approaches that provide for the localization of a defined protein or posttranslationally modified protein to specific chromosomal sites. While these approaches have helped us conceptualize epigenetic mechanisms, the field has been limited by the inability to define features such as the proteome and histone modifications at a specific genomic locus in an unbiased manner. We developed an unbiased approach whereby a unique native genomic locus was isolated, which was followed by high-resolution proteomic identification of specifically associated proteins and histone posttranslational modifications. This chromatin affinity purification with mass spectrometry (ChAP-MS) technique was used to specifically enrich a ~1,000 base pair section of GAL1 chromatin under transcriptionally active and repressive conditions, as well as to identify the specifically bound proteins and histone posttranslational modifications. ChAP-MS should yield insight into the regulatory mechanisms of transcription and help identify factors that epigenetically control chromatin function.


Informational Masking Effects of Similarity and Uncertainty on Early and Late Stages of Auditory Cortical Processing.
Understanding speech in a background of other people talking is a difficult listening situation for hearing-impaired individuals, and even for those with normal hearing. Speech-on-speech masking is known to contribute to increased perceptual difficulty over nonspeech background noise because of informational masking provided over and above the effects of energetic masking. While informational masking research has identified factors of similarity and uncertainty between target and masker that contribute to reduced behavioral performance in speech background noise, critical gaps in knowledge including the underlying neural-perceptual processes remain. By systematically manipulating aspects of acoustic similarity and uncertainty in the same auditory paradigm, the current study examined the time course and objectively quantified these informational masking effects at both early and late stages of auditory processing using auditory evoked potentials (AEPs). Thirty participants were included in a cross-sectional repeated measures design. Target-masker similarity was manipulated by varying the linguistic/phonetic similarity (i.e., language) of the talkers in the background. Specifically, four levels representing hypothesized increasing levels of informational masking were implemented: (1) no masker (quiet); (2) Mandarin; (3) Dutch; and (4) English. Stimulus uncertainty was manipulated by task complexity, specifically presentation of target-to-target interval (TTI) in the auditory evoked paradigm. Participants had to discriminate between English word stimuli (/bæt/ and /pæt/) presented in an oddball paradigm under each masker condition pressing buttons to either the target or standard stimulus. Responses were recorded simultaneously for P1-N1-P2 (standard waveform) and P3 (target waveform). This design allowed for simultaneous recording of multiple AEP peaks, as well as accuracy, reaction time, and d' behavioral discrimination to button press responses. Several trends in AEP components were consistent with effects of increasing linguistic/phonetic similarity and stimulus uncertainty. All babble maskers significantly affected outcomes compared to quiet. In addition, the native language English masker had the largest effect on outcomes in the AEP paradigm, including reduced P3 amplitude and area, as well as decreased accuracy and d' behavioral discrimination to target word responses. AEP outcomes for the Mandarin and Dutch maskers, however, were not significantly different across any measured component. Latency outcomes for both N1 and P3 also supported an effect of stimulus uncertainty, consistent with increased processing time related to greater task complexity. An unanticipated result was the absence of the interaction of linguistic/phonetic similarity and stimulus uncertainty. Observable effects of both similarity and uncertainty were evidenced at a level of the P3 more than the earlier N1 level of auditory cortical processing suggesting that higher-level active auditory processing may be more sensitive to informational masking deficits. The lack of significant interaction between similarity and uncertainty at either level of processing suggests that these informational masking factors operated independently. Speech babble maskers across languages altered AEP component measures, behavioral detection, and reaction time. Specifically, this occurred when the babble was in the native/same language as the target, while the effects of foreign language maskers did not differ. The objective results from this study provide a foundation for further investigation of how the linguistic content of target and masker and task difficulty contribute to difficulty understanding speech-in-noise.


Salmonella enterica Infection of Murine and Human Enteroid-Derived Monolayers Elicits Differential Activation of Epithelium-Intrinsic Inflammasomes.
Recent studies have determined that inflammasome signaling plays an important role in driving intestinal epithelial cell (IEC) responses to bacterial infections, such as Salmonella enterica serovar Typhimurium. There are two primary inflammasome pathways, canonical (involving caspase-1) and noncanonical (involving caspase-4 and -5 in humans and caspase-11 in mice). Prior studies identified the canonical inflammasome as the major pathway leading to interleukin-18 (IL-18) release and restriction of S Typhimurium replication in the mouse cecum. In contrast, the human C2Bbe1 colorectal carcinoma cell line expresses little caspase-1 but instead utilizes caspase-4 to respond to S Typhimurium infection. Intestinal enteroid culture has enabled long-term propagation of untransformed IECs from multiple species, including mouse and human. Capitalizing on this technology, we used a genetic approach to directly compare the relative importance of different inflammatory caspases in untransformed mouse and human IECs and transformed human IECs upon S Typhimurium infection in vitro We show that caspase-1 is important for restricting intracellular S Typhimurium replication and initiating IL-18 secretion in mouse IECs but is dispensable in human IECs. In contrast, restriction of intracellular S Typhimurium and production of IL-18 are dependent on caspase-4 in both transformed and untransformed human IECs. Notably, cytosolic replication in untransformed cells from both species was less pronounced than in transformed human cells, suggesting that transformation may impact additional pathways that restrict S Typhimurium replication. Taken together, these data highlight the differences between mouse and human IECs and the utility of studying transformed and untransformed cells in parallel.


Fidelity of Escherichia coli DNA polymerase IV. Preferential generation of small deletion mutations by dNTP-stabilized misalignment.
Escherichia coli DNA polymerase IV (pol IV), a member of the error-prone Y family, predominantly generates -1 frameshifts when copying DNA in vitro. T-->G transversions and T-->C transitions are the most frequent base substitutions observed. The in vitro data agree with mutational spectra obtained when pol IV is overexpressed in vivo. Single base deletion and base substitution rates measured in the lacZalpha gene in vitro are, on average, 2 x 10(-4) and 5 x 10(-5), respectively. The range of misincorporation and mismatch extension efficiencies determined kinetically are 10(-3) to 10(-5). The presence of beta sliding clamp and gamma-complex clamp loading proteins strongly enhance pol IV processivity but have no discernible influence on fidelity. By analyzing changes in fluorescence of a 2-aminopurine template base undergoing replication in real time, we show that a "dNTP-stabilized" misalignment mechanism is responsible for making -1 frameshift mutations on undamaged DNA. In this mechanism, a dNTP substrate is paired "correctly" opposite a downstream template base, on a "looped out" template strand instead of mispairing opposite a next available template base. By using the same mechanism, pol IV "skips" past an abasic template lesion to generate a -1 frameshift. A crystal structure depicting dNTP-stabilized misalignment was reported recently for Sulfolubus solfataricus Dpo4, a Y family homolog of Escherichia coli pol IV.