Large collections of purified proteins have become essential to systems biology programs for generating protein ligands and for validating protein interactions. These large protein collections will likewise benefit structural studies, and quests for protein-based therapeutics. Whether many protein targets need to be produced, such as a microbial proteome, or a few poorly expressing protein targets need to be expressed as soluble fragments (divide and conquer approach), efficient high-throughput processing can be a bottleneck (1). This chapter describes a method for efficient high-throughput purification of hexahistidine-tagged proteins that are expressed in Eschericia coli (E. coli) using immobilized metal affinity chromatography (IMAC) (2) in a 96-well format. This approach is particularly suitable for proteomic applications that require modest amounts of highly purified proteins to be generated very efficiently. This approach is also very useful for identifying protein targets that are most amenable to scaled-up production for use in structural studies. The typical yield of proteins purified using this system is 50 150 micrograms, which is generally greater than that of many in vitro expression systems and much less costly. The method as described has been optimized for purifying approximately 150 micrograms of hexahistidine-tagged protein, but the method is flexible so that the amount of affinity matrix and culture volumes can be adjusted for optimal binding capacity and consequently highest purity. Although the method detailed here uses IMAC to purify hexahistidine-tagged proteins, this basic platform can be used with many other tags and affinity resins.

After over 70 years of research on the association between stressful life events and health, it is generally accepted that we have a good understanding of the role of stressors in disease risk. In this review, we highlight that knowledge but also emphasize misunderstandings and weaknesses in this literature with the hope of triggering further theoretical and empirical development. We organize this review in a somewhat provocative manner, with each section focusing on an important issue in the literature where we feel that there has been some misunderstanding of the evidence and its implications. Issues that we address include the definition of a stressful event, characteristics of diseases that are impacted by events, differences in the effects of chronic and acute events, the cumulative effects of events, differences in events across the life course, differences in events for men and women, resilience to events, and methodological challenges in the literature.

Background

Surgical site infection (SSI) rates are publicly reported as quality metrics and increasingly used to determine financial reimbursement.

Objective

To evaluate the volume-outcome relationship as well as the year-to-year stability of performance rankings following coronary artery bypass graft (CABG) surgery and hip arthroplasty.

Research design

We performed a retrospective cohort study of Medicare beneficiaries who underwent CABG surgery or hip arthroplasty at US hospitals from 2005 to 2011, with outcomes analyzed through March 2012. Nationally validated claims-based surveillance methods were used to assess for SSI within 90 days of surgery. The relationship between procedure volume and SSI rate was assessed using logistic regression and generalized additive modeling. Year-to-year stability of SSI rates was evaluated using logistic regression to assess hospitals' movement in and out of performance rankings linked to financial penalties.

Results

Case-mix adjusted SSI risk based on claims was highest in hospitals performing <50 CABG/year and <200 hip arthroplasty/year compared with hospitals performing ≥200 procedures/year. At that same time, hospitals in the worst quartile in a given year based on claims had a low probability of remaining in that quartile the following year. This probability increased with volume, and when using 2 years' experience, but the highest probabilities were only 0.59 for CABG (95% confidence interval, 0.52-0.66) and 0.48 for hip arthroplasty (95% confidence interval, 0.42-0.55).

Conclusions

Aggregate SSI risk is highest in hospitals with low annual procedure volumes, yet these hospitals are currently excluded from quality reporting. Even for higher volume hospitals, year-to-year random variation makes past experience an unreliable estimator of current performance.

The value of an anticipated rewarding event is a crucial component of the decision to engage in its pursuit. But little is known of the networks responsible for encoding and retrieving this value. By using biosensors and pharmacological manipulations, we found that basolateral amygdala (BLA) glutamatergic activity tracks and mediates encoding and retrieval of the state-dependent incentive value of a palatable food reward. Projection-specific, bidirectional chemogenetic and optogenetic manipulations revealed that the orbitofrontal cortex (OFC) supports the BLA in these processes. Critically, the function of ventrolateral and medial OFC→BLA projections is doubly dissociable. Whereas lateral OFC→BLA projections are necessary and sufficient for encoding of the positive value of a reward, medial OFC→BLA projections are necessary and sufficient for retrieving this value from memory. These data reveal a new circuit for adaptive reward valuation and pursuit and provide insight into the dysfunction in these processes that characterizes myriad psychiatric diseases.

Background

Deep and organ/space surgical site infections (D/OS SSI) cause significant morbidity, mortality, and costs. Rates are publicly reported and increasingly used as quality metrics affecting hospital payment. Lack of standardized surveillance methods threaten the accuracy of reported data and decrease confidence in comparisons based upon these data.

Methods

We analyzed data from national validation studies that used Medicare claims to trigger chart review for SSI confirmation after coronary artery bypass graft surgery (CABG) and hip arthroplasty. We evaluated code performance (sensitivity and positive predictive value) to select diagnosis codes that best identified D/OS SSI. Codes were analyzed individually and in combination.

Results

Analysis included 143 patients with D/OS SSI after CABG and 175 patients with D/OS SSI after hip arthroplasty. For CABG, 9 International Classification of Diseases, 9th Revision (ICD-9) diagnosis codes identified 92% of D/OS SSI, with 1 D/OS SSI identified for every 4 cases with a diagnosis code. For hip arthroplasty, 6 ICD-9 diagnosis codes identified 99% of D/OS SSI, with 1 D/OS SSI identified for every 2 cases with a diagnosis code.

Conclusions

This standardized and efficient approach for identifying D/OS SSI can be used by hospitals to improve case detection and public reporting. This method can also be used to identify potential D/OS SSI cases for review during hospital audits for data validation.

Objective

To compare the accuracy of surveillance of severe sepsis using electronic health record clinical data vs claims and to compare incidence and mortality trends using both methods.

Design

We created an electronic health record-based surveillance definition for severe sepsis using clinical indicators of infection (blood culture and antibiotic orders) and concurrent organ dysfunction (vasopressors, mechanical ventilation, and/or abnormal laboratory values). We reviewed 1,000 randomly selected medical charts to characterize the definition's accuracy and stability over time compared with a claims-based definition requiring infection and organ dysfunction codes. We compared incidence and mortality trends from 2003-2012 using both methods.

Setting

Two US academic hospitals.

Patients

Adult inpatients.

Results

The electronic health record-based clinical surveillance definition had stable and high sensitivity over time (77% in 2003-2009 vs 80% in 2012, P=.58) whereas the sensitivity of claims increased (52% in 2003-2009 vs 67% in 2012, P=.02). Positive predictive values for claims and clinical surveillance definitions were comparable (55% vs 53%, P=.65) and stable over time. From 2003 to 2012, severe sepsis incidence imputed from claims rose by 72% (95% CI, 57%-88%) and absolute mortality declined by 5.4% (95% CI, 4.6%-6.7%). In contrast, incidence using the clinical surveillance definition increased by 7.7% (95% CI, -1.1% to 17%) and mortality declined by 1.7% (95% CI, 1.1%-2.3%).

Conclusions

Sepsis surveillance using clinical data is more sensitive and more stable over time compared with claims and can be done electronically. This may enable more reliable estimates of sepsis burden and trends.

BACKGROUND: Optimal behavior and decision making result from a balance of control between two strategies, one cognitive/goal-directed and one habitual. These systems are known to rely on the anatomically distinct dorsomedial and dorsolateral striatum, respectively. However, the transcriptional regulatory mechanisms required to learn and transition between these strategies are unknown. Here we examined the role of one chromatin-based transcriptional regulator, histone modification via histone deacetylases (HDACs), in this process. METHODS: We combined procedures that diagnose behavioral strategy in rats with pharmacological and viral-mediated HDAC manipulations, chromatin immunoprecipitation, and messenger RNA quantification. RESULTS: The results indicate that dorsal striatal HDAC3 activity constrains habit formation. Systemic HDAC inhibition following instrumental (lever press → reward) conditioning increased histone acetylation throughout the dorsal striatum and accelerated habitual control of behavior. HDAC3 was removed from the promoters of key learning-related genes in the dorsal striatum as habits formed with overtraining and with posttraining HDAC inhibition. Decreasing HDAC3 function, either by selective pharmacological inhibition or by expression of dominant-negative mutated HDAC3, in either the dorsolateral striatum or the dorsomedial striatum accelerated habit formation, while HDAC3 overexpression in either region prevented habit. CONCLUSIONS: These results challenge the strict dissociation between dorsomedial striatum and dorsolateral striatum function in goal-directed versus habitual behavioral control and identify dorsostriatal HDAC3 as a critical molecular directive of the transition to habit. Because this transition is disrupted in many neurodegenerative and psychiatric diseases, these data suggest a potential molecular mechanism for the negative behavioral symptoms of these conditions and a target for therapeutic intervention.

Both smoking during pregnancy and secondhand smoke exposure are associated with reduced health outcomes. However, limited consistent evidence exists of risks of secondhand smoke exposure in pregnancy. Currently, inadequate smoking cessation services exist in Irish maternity hospitals. To identify the number of pregnant women smoking during pregnancy and to identify their exposure to secondhand smoke, we conducted a cross-sectional observational pilot study in one regional maternity hospital in Ireland in July/August 2018. Respondents were (1) women attending antenatal clinics and (2) postpartum women before discharge. Variables measured included smoking status of pregnant women and partner status, demographic variables, secondhand smoke exposure, and support for hospital smoke-free policy and development of smoking cessation services. The overall response rate was 42.2% in this study. The response rate was 56.5% (111/196) from postnatal wards and 37.3% (215/577) from antenatal clinics. Over 40% of respondents reported they had smoked during their lifetime. The majority of women (70%) reported quitting smoking before their pregnancy. Few women were active smokers. Almost 40% reported exposure to tobacco smoke in the previous week (38.5%); 16.9% reported living with a smoker, a critical factor in increased risk (Odds Ratio (OR) 3.89, 95% CI = 1.86-8.15, p < 0.001). Approximately 10% of postnatal mothers reported that their newborn would travel home with a smoker. Support for a no-smoking hospital policy was very high as was support for the development of cessation services. No documentation of secondhand smoke exposure for pregnant women or newborns is sought or recorded routinely in the hospital. A systems approach to develop smoking cessation programmes in maternity care should include screening and documenting of secondhand smoke exposure risks for women during pregnancy, and for their newborns at discharge, to improve health outcomes and protect human rights.