Public health responses to HIV epidemics have long relied on epidemiological modelling analyses to help prospectively project and retrospectively estimate the impact, cost-effectiveness, affordability, and investment returns of interventions, and to help plan the design of evaluations. But translating model output into policy decisions and implementation on the ground is challenged by the differences in background and expectations of modellers and decision-makers. As part of the PLoS Medicine Collection “Investigating the Impact of Treatment on New HIV Infections”—which focuses on the contribution of modelling to current issues in HIV prevention—we present here principles of “best practice” for the construction, reporting, and interpretation of HIV epidemiological models for public health decision-making on all aspects of HIV. Aimed at both those who conduct modelling research and those who use modelling results, we hope that the principles described here will become a shared resource that facilitates constructive discussions about the policy implications that emerge from HIV epidemiology modelling results, and that promotes joint understanding between modellers and decision-makers about when modelling is useful as a tool in quantifying HIV epidemiological outcomes and improving prevention programming.

Some individuals with intellectual disabilities do not respond to praise as a reinforcer, which may limit their ability to learn. We evaluated 2 procedures (stimulus pairing and response–stimulus pairing), both of which involved pairing previously neutral praise statements with preferred edible items, to determine their usefulness in establishing praise as a reinforcer. Results of Study 1 indicated that stimulus pairing was not effective in conditioning praise as a reinforcer for 3 of 4 subjects; results were inconclusive for the 4th subject. Results of Study 2 indicated that response–stimulus pairing was effective in conditioning praise as a reinforcer for 4 of 8 subjects. After conditioning, praise also increased the occurrence of additional target responses for these 4 subjects.

We evaluated an inexpensive, efficient, and noninvasive technique for measuring tissue damage produced by self-injurious behavior (SIB). The technique involved computerized measurement of wound surface area (WSA) based on digital photographs. In Study 1, we compared photographic measurement to a more commonly used procedure, transparency measurement, in estimating WSA of 20 wound models. Results showed that both methods were reliable and that there was a high degree of correspondence between the 2 sets of measures. In Study 2, we compared photographic WSA measures to direct-observation measures in documenting changes over time in the SIB exhibited by a woman with Prader-Willi syndrome. Results showed that increases and decreases in observed SIB during baseline and treatment conditions corresponded with changes in WSA measures, indicating that the computer-assisted photographic technique may be useful as a corroborative measure or as a primary measure when direct observation of SIB is not feasible.

Objective

Determine the acceptability and epidemiological impact of increases in HIV testing in gay men in New South Wales (NSW), Australia– particularly pertinent when considering treatment as prevention and the need to reduce undiagnosed infections.

Methods

We conducted an online survey and focus groups to assess whether increases in HIV testing would be acceptable to gay men in NSW. In parallel, we assessed the potential impact of increases in testing coverage and/or frequency using an individual-based model of HIV transmission.

Results

If sexual practices and the rate of initiating HIV treatment are unchanged then increasing HIV testing reduces infections. Increasing testing frequency has the largest impact, with a 13.8% reduction in HIV infections over 10 years if the ∼55–75% of men who test at least once per year increased their testing frequency to four times per year. If testing levels decrease from current levels then we expect an increase in HIV infections with a sharply rising trend over time. Increasing HIV testing would be acceptable if testing was more convenient. However, only ∼25% of men surveyed were ‘very likely’ to increase their level of HIV testing. Men delayed or avoided testing due to the slowness in obtaining results and if they believed they had not put themselves at risk.

Conclusions

An increase in HIV testing alone is unlikely to reduce HIV incidence substantially in NSW gay men– however, the relatively high testing levels need to continue to prevent an increase in HIV infections. In jurisdictions with lower levels of HIV testing, increases in testing coverage and frequency are likely to have a larger impact. Successful treatment as prevention interventions will require increases in testing rates; such increases would be acceptable to gay men in NSW but only if more convenient testing and rapid communication of results were available.

Background

There is an urgent need to improve the evidence base for provision of second-line antiretroviral therapy (ART) following first-line virological failure. This is particularly the case in Sub-Saharan Africa where 70% of all people living with HIV/AIDS (PHA) reside. The aim of this study was to simulate the potential risks and benefits of treatment simplification in second-line therapy compared to the current standard of care (SOC) in a lower-middle income and an upper-middle income country in Sub-Saharan Africa.

Methods

We developed a microsimulation model to compare outcomes associated with reducing treatment discontinuations between current SOC for second-line therapy in South Africa and Nigeria and an alternative regimen: ritonavir-boosted lopinavir (LPV/r) combined with raltegravir (RAL). We used published studies and collaborating sites to estimate efficacy, adverse effect and cost. Model outcomes were reported as incremental cost effectiveness ratios (ICERs) in 2011 USD per quality adjusted life year ($/QALY) gained.

Results

Reducing treatment discontinuations with LPV/r+RAL resulted in an additional 0.4 discounted QALYs and increased the undiscounted life expectancy by 0.8 years per person compared to the current SOC. The average incremental cost was $6,525 per treated patient in Nigeria and $4,409 per treated patient in South Africa. The cost-effectiveness ratios were $16,302/QALY gained and $11,085/QALY gained for Nigeria and South Africa, respectively. Our results were sensitive to the probability of ART discontinuation and the unit cost for RAL.

Conclusions

The combination of raltegravir and ritonavir-boosted lopinavir was projected to be cost-effective in South Africa. However, at its current price, it is unlikely to be cost-effective in Nigeria.

The navel orangeworm, Amyelois transitella is a major agricultural pest causing large losses in a variety of tree crops. Control of this insect pest may be achieved by interfering with olfactory pathways to block detection of female-produced sex pheromones and consequently, disrupt mating. The first component of this pathway is the pheromone-binding protein AtraPBP1, which recognizes the pheromone and presents it to the odorant receptor housed in a sensory neuron of the male antennae. Release of the ligand depends on a pH-induced conformational change associated with the acidity of the membrane surface. To characterize this conformational change and to understand how pheromones bind, we have determined the high resolution crystal structures of AtraPBP1 in complex with two main constituents of the sex pheromone, i.e., (11Z,13Z)-hexadecadienal and (11Z,13Z)-hexadecadienol. Comparison with the structure of the unliganded form demonstrates a large ∼90° movement of the C-terminal helix which is observed in other pheromone- or odorant-binding proteins accompanied by an unpredicted 37° displacement of the N-terminal helix. Molecular dynamic trajectories suggest that the conformational change of the α1 helix facilitates the movement of the C-terminal helix.

Juvenile hormone (JH) is a key insect developmental hormone that is found at low nanomolar levels in larval insects. The methyl ester of JH is hydrolyzed in many insects by an esterase that shows high specificity for JH. We have previously determined a crystal structure of the JH esterase (JHE) of the tobacco hornworm Manduca sexta (MsJHE) [Wogulis, M., Wheelock, C. E., Kamita, S. G., Hinton, A. C., Whetstone, P. A., Hammock, B. D., and Wilson, D. K. (2006) Biochemistry 45, 4045-4057]. Our molecular modeling indicates that JH fits very tightly within the substrate binding pocket of MsJHE. This tight fit places two non-catalytic amino acid residues, Phe-259 and Thr-314, within the appropriate distance and geometry to potentially interact with the α,β-unsaturated ester and epoxide, respectively, of JH. These residues are highly conserved in numerous biologically active JHEs. Kinetic analyses of mutants of Phe-259 or Thr-314 indicate that these residues contribute to the low KM that MsJHE shows for JH. This low KM, however, comes at the cost of reduced substrate turnover. Neither nucleophilic attack of the resonance stabilized ester by the catalytic serine nor the availability of a water molecule for attack of the acyl-enzyme intermediate appear to be a rate-determining step in the hydrolysis of JH by MsJHE. We hypothesize that the release of the JH acid metabolite from the substrate binding pocket limits the catalytic cycle. Our findings also demonstrate that chemical bond strength does not necessarily correlate with how reactive the bond will be to metabolism.

Purpose

To demonstrate dynamic imaging of a diffusible perfusion tracer, hyperpolarized [13C]urea, for regional measurement of blood flow in preclinical cancer models.

Materials and Methods

A pulse sequence using balanced steady state free precession (bSSFP) was developed, with progressively increasing flip angles for efficient sampling of the hyperpolarized magnetization. This allowed temporal and volumetric imaging of the [13C]urea signal. Regional signal dynamics were quantified for kidneys and liver, and estimates of relative blood flows were derived from the data. Detailed perfusion simulations were performed to validate the methodology.

Results

Significant differences were observed in the signal patterns between normal and cancerous murine hepatic tissues. In particular, a 19% reduction in mean blood flow was observed in tumors, with 26% elevation in the tumor rim. The blood flow maps were also compared with metabolic imaging results with hyperpolarized [1-13C]pyruvate.

Conclusion

Regional assessment of perfusion is possible by imaging of hyperpolarized [13C]urea, which is significant for the imaging of cancer.

Background

To investigate the associations of aural symptoms, headache and depression with the presence of temporomandibular disorder (TMD) symptoms in a young adult population in Japan.

Methods

A personal interview survey was conducted on first-year university students (n = 1,930) regarding symptoms of TMD, aural problems, headache, shoulder pain and depression. Logistic regression was applied to assess the associations of these problems with the presence of TMD symptoms after controlling for age and gender.

Results

Among the 1,930 students, 543 students exhibited TMD symptoms and were classified into 7 groups: clicking only (Group I, n = 319), pain in the TMJ only (Group II, n = 21), difficulty in mouth opening only (Group III, n = 18), clicking and pain (Group IV, n = 29), clicking and difficulty in mouth opening (Group V, n = 48), difficulty in mouth opening and pain (Group VI, n = 11), and combination of three symptoms (Group VII, n = 97). The control group (n = 1,387) were subjects without any TMD symptoms. After adjusting for age and gender, a strong association was observed between TMD symptoms (Group II and IV) and tinnitus (OR = 12.1 and 13.2, respectively). TMD symptoms (Group I, II and III) were also associated with vertigo and headache. Otalgia and depression were significantly associated with the presence of clicking only.

Conclusions

TMD symptoms were significantly correlated to aural symptoms and headache. A functional evaluation of the stomatognathic system should be considered in subjects with unexplained aural symptoms and headache.

The kidney has an intrinsic ability to repair itself when injured. Epithelial cells of distal tubules may participate in regeneration. Stem cell marker, TRA-1-60 is linked to pluripotency in human embryonic stem cells and is lost upon differentiation. TRA-1-60 expression was mapped and quantified in serial sections of human foetal, adult and diseased kidneys. In 8- to 10-week human foetal kidney, the epitope was abundantly expressed on ureteric bud and structures derived therefrom including collecting duct epithelium. In adult kidney inner medulla/papilla, comparisons with reactivity to epithelial membrane antigen, aquaporin-2 and Tamm–Horsfall protein, confirmed extensive expression of TRA-1-60 in cells lining collecting ducts and thin limb of the loop of Henle, which may be significant since the papillae were proposed to harbour slow cycling cells involved in kidney homeostasis and repair. In the outer medulla and cortex there was rare, sporadic expression in tubular cells of the collecting ducts and nephron, with positive cells confined to the thin limb and thick ascending limb and distal convoluted tubules. Remarkably, in cortex displaying tubulo-interstitial injury, there was a dramatic increase in number of TRA-1-60 expressing individual cells and in small groups of cells in distal tubules. Dual staining showed that TRA-1-60 positive cells co-expressed Pax-2 and Ki-67, markers of tubular regeneration. Given the localization in foetal kidney and the distribution patterns in adults, it is tempting to speculate that TRA-1-60 may identify a population of cells contributing to repair of distal tubules in adult kidney.

Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs), adult chondrocytes and STRO-1+ marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2) and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development, informing and opening new possibilities in development of strategies for bone repair/tissue engineering.

Five human RecQ helicases are involved in genome maintenance. RECQL5, one of the important members of this helicase family, is involved in DNA single-strand break repair and base excision DNA repair.

Human RECQL5 is a member of the RecQ helicase family, which maintains genome stability via participation in many DNA metabolic processes, including DNA repair. Human cells lacking RECQL5 display chromosomal instability. We find that cells depleted of RECQL5 are sensitive to oxidative stress, accumulate endogenous DNA damage, and increase the cellular poly(ADP-ribosyl)ate response. In contrast to the RECQ helicase family members WRN, BLM, and RECQL4, RECQL5 accumulates at laser-induced single-strand breaks in normal human cells. RECQL5 depletion affects the levels of PARP-1 and XRCC1, and our collective results suggest that RECQL5 modulates and/or directly participates in base excision repair of endogenous DNA damage, thereby promoting chromosome stability in normal human cells.

Rationale: As computed tomography (CT) screening for lung cancer becomes more widespread, volumetric analyses, including doubling times, of CT-screen detected lung nodules and lung cancers may provide useful information in the follow-up and management of CT-detected lung nodules and cancers.

Objectives: To analyze doubling times in CT screen detected lung cancers and compare prevalent and nonprevalent cancers and different cell types on non small cell lung cancer.

Methods: We performed volumetric and doubling time analysis on 63 non–small cell lung cancers detected as part of the Pittsburgh Lung Screening Study using a commercially available VITREA 2 workstation and VITREA VITAL nodule segmentation software.

Measurements and Main Results: Doubling times (DT) were divided into three groups: rapid (DT < 183 d), typical (DT 183–365 d), and slow (DT > 365 d). Adenocarcinoma/bronchioloalveolar carcinoma comprised 86.7% of the slow DT group compared with 20% of the rapid DT group. Conversely, squamous cell cancer comprised 60% of the rapid DT group compared with 3.3% of the slow DT group. Twenty-eight of 42 (67%) prevalent and 2 of 21 (10%) nonprevalent cancers were in the slow DT group (P < 0.0001; Fisher's exact test). Twenty-four of 32 (75%) prevalent and 1 of 11 (9%) nonprevalent adenocarcinomas were in the slow DT group (P < 0.0002; Fisher's exact test).

Conclusions: Volumetric analysis of CT-detected lung cancers is particularly useful in AC/BAC. Prevalent cancers have a significantly slower DT than nonprevalent cancers and a higher percentage of adenocarcinoma/bronchioloalveolar carcinoma. These results should affect the management of indeterminant lung nodules detected on screening CT scans.

Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from M. marinum (MMNAT). To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against M. tuberculosis with MIC values of 2.3–16.9 µM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action different from known anti-tubercular drugs.

ABSTRACT

Lignocellulosic biomass, the most abundant polymer on Earth, is typically composed of three major constituents: cellulose, hemicellulose, and lignin. The crystallinity of cellulose, hydrophobicity of lignin, and encapsulation of cellulose by the lignin-hemicellulose matrix are three major factors that contribute to the observed recalcitrance of lignocellulose. By means of designer cellulosome technology, we can overcome the recalcitrant properties of lignocellulosic substrates and thus increase the level of native enzymatic degradation. In this context, we have integrated six dockerin-bearing cellulases and xylanases from the highly cellulolytic bacterium, Thermobifida fusca, into a chimeric scaffoldin engineered to bear a cellulose-binding module and the appropriate matching cohesin modules. The resultant hexavalent designer cellulosome represents the most elaborate artificial enzyme composite yet constructed, and the fully functional complex achieved enhanced levels (up to 1.6-fold) of degradation of untreated wheat straw compared to those of the wild-type free enzymes. The action of these designer cellulosomes on wheat straw was 33 to 42% as efficient as the natural cellulosomes of Clostridium thermocellum. In contrast, the reduction of substrate complexity by chemical or biological pretreatment of the substrate removed the advantage of the designer cellulosomes, as the free enzymes displayed higher levels of activity, indicating that enzyme proximity between these selected enzymes was less significant on pretreated substrates. Pretreatment of the substrate caused an increase in activity for all the systems, and the native cellulosome completely converted the substrate into soluble saccharides.

IMPORTANCE

Cellulosic biomass is a potential alternative resource which could satisfy future demands of transportation fuel. However, overcoming the natural lignocellulose recalcitrance remains challenging. Current research and development efforts have concentrated on the efficient cellulose-degrading strategies of cellulosome-producing anaerobic bacteria. Cellulosomes are multienzyme complexes capable of converting the plant cell wall polysaccharides into soluble sugar products en route to biofuels as an alternative to fossil fuels. Using a designer cellulosome approach, we have constructed the largest form of homogeneous artificial cellulosomes reported to date, which bear a total of six different cellulases and xylanases from the highly cellulolytic bacterium Thermobifida fusca. These designer cellulosomes were comparable in size to natural cellulosomes and displayed enhanced synergistic activities compared to their free wild-type enzyme counterparts. Future efforts should be invested to improve these processes to approach or surpass the efficiency of natural cellulosomes for cost-effective production of biofuels.

In Vietnam, premature mortality due to AIDS-related conditions is commonly associated with late initiation to antiretroviral therapy (ART). This study explores reasons for late ART initiation among people living with HIV (PLHIV) from the perspectives of health care providers and PLHIV. The study was undertaken in six clinics from five provinces in Vietnam. Baseline CD4 counts were collected from patient records and grouped into three categories: very late initiators (≤100 cells/mm3 CD4), late initiators (100–200 cells/mm3) and timely initiators (200–350 cells/mm3). Thirty in-depth interviews with patients who started ART and 15 focus group discussions with HIV service providers were conducted and thematic analysis of the content performed. Of 934 patients, 62% started ART very late and 11% initiated timely treatment. The proportion of patients for whom a CD4 count was obtained within six months of their HIV diagnosis ranged from 22% to 72%. The proportion of patients referred to ART clinics by voluntary testing and counselling centres ranged from 1% to 35%. Structural barriers to timely ART initiation were poor linkage between HIV testing and HIV care and treatment services, lack of patient confidentiality and a shortage of HIV/AIDS specialists. If Vietnam’s treatment practice is to align with WHO recommendations then the connection between voluntary counselling and testing service and ART clinics must be improved. Expansion and decentralization of HIV/AIDS services to allow implementation at the community level increased task sharing between doctors and nurses to overcome limited human resources, and improved patient confidentiality are likely to increase timely access to HIV treatment services for more patients.

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a central participant in the base excision repair pathway, exhibiting AP endonuclease activity that incises the DNA backbone 5′ to an abasic site. Besides its prominent role as a DNA repair enzyme, APE1 was separately identified as a protein called redox effector factor 1, which is able to enhance the DNA binding activity of several transcription factors through a thiol-exchange-based reduction–oxidation mechanism. In the present study, we found that human APE1 is S-glutathionylated under conditions of oxidative stress both in the presence of glutathione in vitro and in cells. S-glutathionylated APE1 displayed significantly reduced AP endonuclease activity on abasic-site-containing oligonucleotide substrates, a result stemming from impaired DNA binding capacity. The combination of site-directed mutagenesis, biochemical assays, and mass spectrometric analysis identified Cys99 in human APE1 as the critical residue for the S-glutathionylation that leads to reduced AP endonuclease activity. This modification is reversible by reducing agents, which restore APE1 incision function. Our studies describe a novel posttranslational modification of APE1 that regulates the DNA repair function of the protein.

Advanced diagnostic imaging and histopathological investigation were performed in an adult horse with chronic facial swelling due to a bone sequestrum and abscessation. In contrast to other cases, there were no draining tracts, difficulty eating, weight loss, head shaking, or fistula formation between the oral cavity and the maxillary sinus.

Background

Commercial sex workers within the population of men who have sex with men (MSM) in China, known as ‘money boys’ (MBs), are perceived to be at higher risk for HIV and other sexually-transmissible infections (STIs).

Methods

We conducted a systematic review and meta-analyses from peer-reviewed literature accessed in two English (PubMed and Embase) and three Chinese databases (CNKI, CQVIP, Wanfang data). A data synthesis exercise was carried out to determine the extent and patterns of behaviours and HIV/STI epidemics. Pooled estimates, with 95% confidence intervals, for each study variable were calculated.

Results

Thirty-two eligible articles (9 in English and 23 in Chinese) were identified. Our analysis indicated that Chinese MBs are generally young, currently employed, at low literacy levels and highly mobile. The prevalence of HIV, syphilis and co-infection among MBs were estimated to be 6.0% (4.2–8.5%), 12.4% (9.9–15.3%) and 2.2% (1.1–4.1%) over the period of 2004–2011. Level of condom use among MBs is generally higher than the broader MSM population (69.2% at last act, and 48.5% consistently over the past 6 months). One-third of the Chinese MBs identified themselves as bisexual and 8.7% (5.6–13.5%) are currently married to a female. Further, 40.9% (34.5–47.7%) of MBs participated in group sex in the past 12 months and 14.8% (10.6–20.3%) concurrently use illicit drugs.

Conclusions

HIV/STI epidemics have affected Chinese MBs but the evidence suggests that the extent of infections is not greater than among other MSM in China.

Background

The purpose of this study was to determine the effects of low-flow cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest followed by postbypass recovery on the phosphorylation state of transcription factor, cyclic adenosine 3′, 5′-monophosphate response element– binding protein (CREB), in the striatum of neonatal brain.

Methods

Neonatal piglets (1.4 to 2.5 kg) anesthetized with isoflurane and fentanyl were put on CPB. The animals were cooled to 18°C during a 20-minute period. The CPB circuit flow was then either reduced to 20 mL · kg−1 · min−1 for 90 minutes (low-flow CPB) or turned off for 90 minutes (deep hypothermic circulatory arrest), following with a gradual increase in the flow and rewarming during a 30-minute period and a 2-hour recovery. At the end of the recovery period, the animals were rapidly euthanized, and the striata were removed and frozen for immunochemical analysis by Western blot technique using antibodies against phosphorylated and total CREB. The results are presented as mean ± standard deviation (p < 0.05 was significant).

Results

Deep hypothermic circulatory arrest did not result in alteration in either the level of CREB or its degree of phosphorylation in the piglet striatum whereas after low-flow CPB, CREB phosphorylation was significantly increased (p < 0.005) and there was also an increase in CREB expression (p < 0.01).

Conclusions

This study indicates that at 2 hours of recovery, low-flow CPB but not deep hypothermic circulatory arrest causes an increase in CREB phosphorylation and expression. Future studies will determine the degree to which the increase in CREB phosphorylation correlates with cell survival and neuronal injury after CPB.

We are developing a robust, minimally invasive device for detecting progression toward hemorrhagic shock in trauma patients. To accomplish this, oxygen micro-sensors are being developed that contain a solution of oxygen sensitive phosphorescent probes within gas permeable tubing attached to optical fibers. These micro-sensors can be inserted into peripheral tissue to accurately measure tissue oxygenation. As the blood volume decreases (hemorrhage), physiological mechanisms progressively restrict blood flow to “non essential” peripheral tissues, redirecting that flow to the essential internal organs. It is hypothesized that the sensors will detect the shut down of peripheral blood flow well before the decreasing blood volume reaches the threshold where multi-organ failure begins. Proactive treatment with volume expanders or blood, guided by peripheral oxygen measurements, would significantly reduce multi-organ failure and other complications in trauma cases.

Background

Microorganisms employ a multiplicity of enzymes to efficiently degrade the composite structure of plant cell wall cellulosic polysaccharides. These remarkable enzyme systems include glycoside hydrolases (cellulases, hemicellulases), polysaccharide lyases, and the carbohydrate esterases. To accomplish this challenging task, several strategies are commonly observed either separately or in combination. These include free enzyme systems, multifunctional enzymes, and multi-enzyme self-assembled designer cellulosome complexes.

Results

In order to compare these different paradigms, we employed a synthetic biology approach to convert two different cellulases from the free enzymatic system of the well-studied bacterium, Thermobifida fusca, into bifunctional enzymes with different modular architectures. We then examined their performance compared to those of the combined parental free-enzyme and equivalent designer-cellulosome systems. The results showed that the cellulolytic activity displayed by the different architectures of the bifunctional enzymes was somewhat inferior to that of the wild-type free enzyme system.

Conclusions

The activity exhibited by the designer cellulosome system was equal or superior to that of the free system, presumably reflecting the combined proximity of the enzymes and high flexibility of the designer cellulosome components, thus enabling efficient enzymatic activity of the catalytic modules.