Engineering Synthetic Antibiotics in Non-Traditional Pathways to Counter Antibiotic Resistance

Antibiotic resistance is a growing threat to global healthcare that requires immediate action to avoid the post-antibiotic era. The inherent ability of bacteria to obtain resistance and the lack of new antibiotics has led to the current antibiotic crisis. Current antibiotics are typically found through soil compound screens and only target proteins within three cellular pathways: cellular replication, cell wall biosynthesis, and protein biosynthesis. In the last decade, strains have been isolated which have resistance to nearly all available antibiotics highlighting the urgent need for intervention. In this work we investigated the rational design of non-naturally derived antibiotics which target bacterial processes outside of the three traditional antibiotic target pathways. Antisense therapeutics are nucleic acid oligomers that bind sequence-specifically via Watson-Crick base pairing with native nucleic acids and inhibit translation of the targeted gene. For this work, we use non-natural nucleic acid analog oligomers, peptide nucleic acids (PNA), for their demonstrated intracellular stability and high binding affinity for native nucleic acids. In our initial study, we show PNA oligomers targeted to TEM-1 β-lactamase re-sensitized drug-resistant Escherichia coli to a β-lactam antibiotic. We further adapted E. coli to low levels of PNA and β-lactam antibiotic and observed high variability in expression of stress response genes possibly suggesting a bet-hedging type adaptive resistance. In our next study, we designed PNA to target essential bacterial genes in non-traditional antibiotic target pathways. We designed the PNA against the genome sequences of non-pathogenic, drug sensitive E. coli, Klebsiella pneumoniae, and Salmonella enterica and subsequently tested their antibacterial action in multidrug-resistant (MDR) clinical isolates of the same three species. We found that 54% of predicted targets were effective at inhibiting the MDR pathogens demonstrating the ability to design sequence-specific yet still broad-pathogen antisense therapeutics. We further demonstrated that combinations of these essential gene antisense PNA and small molecule antibiotics function synergistically to enhance bacterial inhibition despite the clinical strains high antibiotic resistance. We next focused our efforts on designing an antimicrobial agent for perturbing bacterial redox homeostasis. Reactive oxygen species (ROS) have been studied for their effect on antibiotic efficacy and the emergence of drug resistance. In this work, we studied one ROS in particular, superoxide, for its role as an oxidative stress catalyst and its demonstrated disruption of metal homeostasis in bacteria. To controllably produce superoxide, we investigated the design of quantum dot nanoparticles. When quantum dots are excited over their nominal bandgap, excited electrons and holes are available for redox half reactions in the biological environment. In this work, we demonstrated the tuning of quantum dots for superoxide production from molecular oxygen and further showed the tuned nanoparticles inhibition of clinical isolates. Further, we established that E. coli could be eradicated from co-culture with mammalian cells; leaving the mammalian cells intact. Given the role that ROS have been shown to have in bactericidal antibiotic efficacy, we hypothesized that our superoxide-producing nanoparticles would function synergistically with small molecule antibiotics. Indeed, our designed superoxide generating nanoparticles potentiated the activity of antibiotics in clinical MDR isolates in spite of their antibiotic resistance. In this study, superoxide potentiated the activity of bactericidal antibiotics as well as bacteriostatic antibiotics, which had not been shown previously. To better understand the effect of superoxide generation on bacteria, we performed analysis of E. coli’s transcriptome during treatment. We removed mat

Engineering Synthetic Antibiotics in Non-Traditional Pathways to Counter Antibiotic Resistance

Antibiotic resistance is a growing threat to global healthcare that requires immediate action to avoid the post-antibiotic era. The inherent ability of bacteria to obtain resistance and the lack of new antibiotics has led to the current antibiotic crisis. Current antibiotics are typically found through soil compound screens and only target proteins within three cellular pathways: cellular replication, cell wall biosynthesis, and protein biosynthesis. In the last decade, strains have been isolated which have resistance to nearly all available antibiotics highlighting the urgent need for intervention. In this work we investigated the rational design of non-naturally derived antibiotics which target bacterial processes outside of the three traditional antibiotic target pathways. Antisense therapeutics are nucleic acid oligomers that bind sequence-specifically via Watson-Crick base pairing with native nucleic acids and inhibit translation of the targeted gene. For this work, we use non-natural nucleic acid analog oligomers, peptide nucleic acids (PNA), for their demonstrated intracellular stability and high binding affinity for native nucleic acids. In our initial study, we show PNA oligomers targeted to TEM-1 β-lactamase re-sensitized drug-resistant Escherichia coli to a β-lactam antibiotic. We further adapted E. coli to low levels of PNA and β-lactam antibiotic and observed high variability in expression of stress response genes possibly suggesting a bet-hedging type adaptive resistance. In our next study, we designed PNA to target essential bacterial genes in non-traditional antibiotic target pathways. We designed the PNA against the genome sequences of non-pathogenic, drug sensitive E. coli, Klebsiella pneumoniae, and Salmonella enterica and subsequently tested their antibacterial action in multidrug-resistant (MDR) clinical isolates of the same three species. We found that 54% of predicted targets were effective at inhibiting the MDR pathogens demonstrating the ability to design sequence-specific yet still broad-pathogen antisense therapeutics. We further demonstrated that combinations of these essential gene antisense PNA and small molecule antibiotics function synergistically to enhance bacterial inhibition despite the clinical strains high antibiotic resistance. We next focused our efforts on designing an antimicrobial agent for perturbing bacterial redox homeostasis. Reactive oxygen species (ROS) have been studied for their effect on antibiotic efficacy and the emergence of drug resistance. In this work, we studied one ROS in particular, superoxide, for its role as an oxidative stress catalyst and its demonstrated disruption of metal homeostasis in bacteria. To controllably produce superoxide, we investigated the design of quantum dot nanoparticles. When quantum dots are excited over their nominal bandgap, excited electrons and holes are available for redox half reactions in the biological environment. In this work, we demonstrated the tuning of quantum dots for superoxide production from molecular oxygen and further showed the tuned nanoparticles inhibition of clinical isolates. Further, we established that E. coli could be eradicated from co-culture with mammalian cells; leaving the mammalian cells intact. Given the role that ROS have been shown to have in bactericidal antibiotic efficacy, we hypothesized that our superoxide-producing nanoparticles would function synergistically with small molecule antibiotics. Indeed, our designed superoxide generating nanoparticles potentiated the activity of antibiotics in clinical MDR isolates in spite of their antibiotic resistance. In this study, superoxide potentiated the activity of bactericidal antibiotics as well as bacteriostatic antibiotics, which had not been shown previously. To better understand the effect of superoxide generation on bacteria, we performed analysis of E. coli’s transcripto

Tear Osmolarity and Dry Eye Symptoms in Women Using Oral Contraception and Contact Lenses

Purpose—To examine the relationship between oral contraceptive pill (OCP) use, contact lens wear, and dry eye signs and symptoms in healthy young females. Methods—Fifty-two women using OCPs and forty-five women not using any form of hormonal contraception were enrolled. Medical, menstrual, and contact lens histories were obtained and dry eye symptoms were assessed using the Ocular Surface Disease Index (OSDI) and Symptom Assessment iN Dry Eye (SANDE) questionnaires. Tear osmolarity testing was performed using the TearLab™ Osmolarity System. Results—Mean age of all subjects was 26.0 ± 3.7 years. There were no significant differences in any of the measurements between the follicular and luteal phases. While SANDE scores were significantly higher in subjects with OCP and recent contact lens use (p\u3c0.01), there were no significant differences in OSDI and tear osmolarity amongst the same subject groups. Subjects who reported both OCP and recent contact lens use had significantly higher OSDI and SANDE scores (p=0.015 and p\u3c0.001, respectively). Conclusions—There were no differences between the phases of the menstrual cycle. Tear osmolarity was not affected by OCP or contact lens use in young females. However, the combination of OCP use and contact lens wear may increase the severity of dry eye symptoms

Patterns of hospitalisation before and following initiation of haemodialysis: a 5 year single centre study

ABSTRACT Background The utilisation of healthcare resources by prevalent haemodialysis patients has been robustly evaluated with regard to the provision of outpatient haemodialysis; however, the impact of hospitalisation among such patients is poorly defined. Minimal information is available in the UK to estimate the health and economic burden associated with the inpatient management of prevalent haemodialysis patients. The aim of this study was to assess the pattern of hospitalisation among a cohort of haemodialysis patients, before and following their initiation of haemodialysis. In addition the study sought to assess the impact of their admissions on bed occupancy in a large tertiary referral hospital in a single region in the UK. Methods All admission episodes were reviewed and those receiving dialysis with the Belfast City Hospital Programme were identified over a 5 year period from January 2001 to December 2005. This tertiary referral centre provides dialysis services for a population of approximately 700 000 and additional specialist renal services for the remainder of Northern Ireland. The frequency and duration of hospitalisation, and contribution to bed day occupancy of haemodialysis patients, was determined and compared to other common conditions which are known to be associated with high bed occupancy. In addition, the pattern and timing of admissions in dialysis patients in relation to their dialysis initiation date was assessed. Results Over the 5 year study period, 798 haemodialysis patients were admitted a total of 2882 times. These accounted for 2.5% of all admissions episodes; the median number of admissions for these patients was 3 (2–5) which compared with 1 (1–2) for non-dialysis patients. The majority of first hospitalisations (54%) were within 100 days before or after commencement of maintenance dialysis therapy. In all clinical specialties the median length of stay for haemodialysis patients was significantly longer than for patients not on haemodialysis (p=0.004). In multivariate analysis with adjustment for age, gender, and other clinically relevant diagnostic codes, maintenance haemodialysis patients stayed on average 3.75 times longer than other patient groups (ratio of geometric means 3.75, IQR 3.46–4.06). Conclusions Maintenance haemodialysis therapy is an important risk factor for prolonged hospitalisation regardless of the primary reason for admission. Such patients require admission more frequently than the general hospital population, particularly within 100 days before and after initiation of their first dialysis treatment. </jats:sec

Conversion Total Knee Arthroplasty Needs Its Own Diagnosis Related Group Facility Reimbursement Code

Introduction: Conversion from a prior surgery to a total knee arthroplasty (TKA) is a more technically difficult procedure than primary TKA and is associated with worse short-term outcomes and increased complication and readmission rates, despite being undifferentiated under the current bundled payment model. The aim of this study was to determine differences in facility costs between primary TKA and conversion TKA, which we hypothesize are significant, to ensure providers are not penalized for treatment and high-risk patients have the same access to care. Methods: We retrospectively reviewed a consecutive series of patients undergoing primary TKA at two hospitals within Rothman Orthopaedic Institute from 2015-2017, comparing itemized facility costs between primary and conversion TKA patients. Secondary endpoints included length of stay, discharge disposition, and additional implants used. A multivariate regression analysis was performed to identify independent risk factors for increased facility costs, the need for additional implants, length of stay, and discharge disposition. Results: Of 2447 primary TKA procedures, 678 (27.7%) underwent conversion to TKA, which was associated with greater implant costs ($3424.25 vs. 3272.29, P\u3c0.0001), preoperative personnel costs ($1269.89 vs. $1217.72, p\u3c0.0001), and total costs ($6859.16 vs. $6703.55, p=0.0015). Presence of prior surgical hardware was a risk factor for increased implant costs ($501.1 increase, p=0.0024) and total cost ($501.4 increase, p=0.0024). Discussion: Conversion TKA is associated with significantly greater facility costs than primary TKA, thus confirming our hypothesis, and should be adjusted for in alternative payment models to ensure these patients do not encounter difficulties in accessing quality orthopaedic care

The Use of Cementless Components Does Not Significantly Increase Procedural Costs in Total Knee Arthroplasty

Introduction: Modern cementless total knee arthroplasty (TKA) designs have shown promising early clinical success; however, concerns exist regarding the higher cost of the cementless implants. The purpose of this study is to evaluate the total facility cost of cementless vs traditional cemented TKA along with the effect of cementless fixation on short-term outcomes. Methods: We reviewed a consecutive series of patients between 2015 and 2017 who underwent either cementless or cemented primary TKA. Itemized facility costs were calculated for every procedure using a time-driven activity-based costing algorithm. Controlling for demographic variables and medical comorbidities, we performed a multivariate analysis to identify independent risk factors for facility costs following TKA. Short-term outcome metrics including complications, readmissions, and patient-reported outcomes were compared between groups. Results: Among the 2426 primary TKA patients in this study, 119 (4.91%) were performed using cementless implants. When compared to cemented TKA, cementless TKA patients had higher implant costs, but lower supply costs and lower operating room personnel costs. When controlling for confounding variables, cementless fixation did not have a significant effect on total facility cost or outcomes. Discussion: The use of cementless TKA implants did not significantly increase total procedural costs when compared to traditional cemented TKA components at our institution. Our data suggest that the increased cost of a cementless implant is recouped through savings in cost of cement and supplies, as well as shorter operative times. The authors encourage investigators at other institutions to use the authors\u27 methodology to evaluate (preferably in a prospective manner) whether the findings from this study can be corroborated

Hyperamylasemia post living donor nephrectomy does not relate to pain

Introduction The aetiology of pain after laparoscopic donor nephrectomy remains unclear. Given the proximity of the left kidney to the tail of the pancreas, we aimed to assess whether mobilisation and retrieval of the left kidney might inflame the pancreas, leading to pain and hyperamylasaemia in the post-operative period. Patient and methods In the present study, 16 consecutive live kidney donors were analysed in the same three months period. Amylase levels were measured on days 1 and 2. For each 24-hour period postoperatively analgesia consumption was recorded, as well as pain scores at rest on a visual analogue scale (VAS). Results Three out of 16 donors presented hyperamylasemia. A multiple regression analysis found levobupivacaine dose, propofol dose, transversus abdominis plane block and day 1 amylase did not significantly predict pain scores. Interestingly, body mass index significantly correlated with increased pain scores (p = 0.041). Also, increasing CO2 insufflation pressure and use of local anaesthetic infusion catheters predicted a decreased deep pain score (p = 0.036 and p = 0.037). Conclusion There was no correlation of amylase levels and pain scores. Pancreatitis is a rare complication of nephrectomy and no overt cases were seen in the case of donor nephrectomy

Comprehensive Investigation of the Caveolin 2 Gene: Resequencing and Association for Kidney Transplant Outcomes

Caveolae are plasma membrane structures formed from a complex of the proteins caveolin-1 and caveolin-2. Caveolae interact with pro-inflammatory cytokines and are dysregulated in fibrotic disease. Although caveolae are present infrequently in healthy kidneys, they are abundant during kidney injury. An association has been identified between a CAV1 gene variant and long term kidney transplant survival. Chronic, gradual decline in transplant function is a persistent problem in kidney transplantation. The aetiology of this is diverse but fibrosis within the transplanted organ is the common end point. This study is the first to investigate the association of CAV2 gene variants with kidney transplant outcomes. Genomic DNA from donors and recipients of 575 kidney transplants performed in Belfast was investigated for common variation in CAV2 using a tag SNP approach. The CAV2 SNP rs13221869 was nominally significant for kidney transplant failure. Validation was sought in an independent group of kidney transplant donors and recipients from Dublin, Ireland using a second genotyping technology. Due to the unexpected absence of rs13221869 from this cohort, the CAV2 gene was resequenced. One novel SNP and a novel insertion/deletion in CAV2 were identified; rs13221869 is located in a repetitive region and was not a true variant in resequenced populations. CAV2 is a plausible candidate gene for association with kidney transplant outcomes given its proximity to CAV1 and its role in attenuating fibrosis. This study does not support an association between CAV2 variation and kidney transplant survival. Further analysis of CAV2 should be undertaken with an awareness of the sequence complexities and genetic variants highlighted by this study

Twenty Years of Capacity Building across the Cancer Prevention and Control Research Network

PURPOSE: to improve population health, community members need capacity (i.e., knowledge, skills, and tools) to select and implement evidence-based interventions (EBIs) to fit the needs of their local settings. Since 2002, the Centers for Disease Control and Prevention has funded the national Cancer Prevention and Control Research Network (CPCRN) to accelerate the implementation of cancer prevention and control EBIs in communities. The CPCRN has developed multiple strategies to build community members\u27 capacity to implement EBIs. This paper describes the history of CPCRN\u27s experience developing and lessons learned through the use of five capacity-building strategies: (1) mini-grant programs, (2) training, (3) online tools, (4) evidence academies, and (5) evaluation support for partners\u27 capacity-building initiatives. METHODS: We conducted a narrative review of peer-reviewed publications and grey literature reports on CPCRN capacity-building activities. Guided by the Interactive Systems Framework, we developed histories, case studies, and lessons learned for each strategy. Lessons were organized into themes. RESULTS: Three themes emerged: the importance of (1) community-engagement prior to and during implementation of capacity-building strategies, (2) establishing and sustaining partnerships, and (3) co-learning at the levels of centers, networks, and beyond. CONCLUSION: CPCRN activities have increased the ability of community organizations to compete for external funds to support implementation, increased the use of evidence in real-world settings, and promoted the broad-scale implementation of cancer control interventions across more than eight states. Lessons from this narrative review highlight the value of long-term thematic networks and provide useful guidance to other research networks and future capacity-building efforts