Plasma Biomarkers of Traumatic Brain Injury in Adolescents With Sport-Related Concussion.

Importance: Blood-based biomarkers may clarify underlying neuropathology and potentially assist in clinical management of adolescents with sport-related concussion (SRC). Objective: To investigate the association between SRC and plasma biomarkers in adolescents. Design, Setting, and Participants: Prospective cohort study in Canadian sport and clinic settings (Surveillance in High Schools and Community Sport to Reduce Concussions and Their Consequences study; September 2019 to November 2022). Participants were a convenience sample of 849 adolescent (ages 10-18 years) sport participants with blood samples. Data were analyzed from February to September 2023. Exposures: Blood collection and clinical testing preseason (uninjured) and post-SRC follow-ups (ie, ≤72 hours, 1 week, and biweekly until medical clearance to return to play [RTP]). Main Outcomes and Measures: Plasma glial fibrillary acidic protein (GFAP), ubiquitin c-terminal hydrolase-L1 (UCH-L1), neurofilament light (NfL), and total tau (t-tau) were assayed. Group-level comparisons of biomarker levels were conducted between uninjured and post-SRC intervals (postinjury day [PID] 0-3, 4-10, 11-28, and >28) considering age and sex as modifiers. Secondary analyses explored associations between biomarker concentrations and clinical outcomes (Sport Concussion Assessment Tool, Fifth Edition [SCAT5] symptom scores and time to RTP). Results: This study included 1023 plasma specimens from 695 uninjured participants (467 male participants [67.2%]; median [IQR] age, 15.90 [15.13-16.84] years) and 154 participants with concussion (78 male participants [51.0%]; median [IQR] age, 16.12 [15.31-17.11] years). Acute (PID 0-3) differences relative to uninjured levels were found for GFAP (female participants: 17.8% increase; ß = 0.164; 95% CI, 0.064 to 0.263; P = .001; male participants: 17.1% increase; ß = 0.157; 95% CI, 0.086 to 0.229; P < .001), UCH-L1 (female participants: 43.4% increase; ß = 0.361; 95% CI, 0.125 to 0.596; P = .003), NfL (male participants: 19.0% increase; ß = 0.174; 95% CI, 0.087 to 0.261; P < .001), and t-tau (female participants: -22.9%; ß = -0.260; 95% CI, -0.391 to -0.130; P < .001; male participants: -18.4%; ß = -0.203; 95% CI, -0.300 to -0.106; P < .001). Differences were observed for all biomarkers at PID 4 to 10, 11 to 28, and greater than 28 compared with uninjured groups. GFAP, NfL, and t-tau were associated with SCAT5 symptom scores across several PID intervals. Higher GFAP after 28 days post-SRC was associated with earlier clearance to RTP (hazard ratio, 4.78; 95% CI, 1.59 to 14.31; P = .01). Male participants exhibited lower GFAP (-9.7%), but higher UCH-L1 (21.3%) compared with female participants. Age was associated with lower GFAP (-5.4% per year) and t-tau (-5.3% per year). Conclusions and Relevance: In this cohort study of 849 adolescents, plasma biomarkers differed between uninjured participants and those with concussions, supporting their continued use to understand concussion neuropathology. Age and sex are critical considerations as these biomarkers progress toward clinical validation.

Beacon Success Rate versus Gateway Density in Sub-GHz Sensor Networks.

Multiple Gateways (GWs) provide network connectivity to Internet of Things (IoT) sensors in a Wide Area Network (WAN). The End Nodes (ENs) can connect to any GW by discovering and acquiring its periodic beacons. This provides GW diversity, improving coverage area. However, simultaneous periodic beacon transmissions among nearby GWs lead to interference and collisions. In this study, the impact of such intra-network interference is analyzed to determine the maximum number of GWs that can coexist. The paper presents a new collision model that considers the combined effects of the Medium Access Control (MAC) and Physical (PHY) layers. The model takes into account the partial overlap durations and relative power of all colliding events. It also illustrates the relationship between the collisions and the resulting packet loss rates. A performance evaluation is presented using a combination of analytical and simulation methods, with the former validating the simulation results. The system models are developed from experimental data obtained from field measurements. Numerical results are provided with Gaussian Frequency Shift Keying (GFSK) modulation. This paper provides guidance on selecting GFSK modulation parameters for low bit-rate and narrow-bandwidth IoT applications. The analysis and simulation results show that larger beacon intervals and frequency hopping help in reducing beacon loss rates, at the cost of larger beacon acquisition latency. On the flip side, the gateway discovery latency reduces with increasing GW density, thanks to an abundance of beacons.

Advance care and resuscitation plans in a tertiary hospital: a multimodal approach.

OBJECTIVES: Advance care planning (ACP) is the process of documenting a person's preference for medical treatment in the event of future deterioration. This audit aimed to improve discussion and documentation of ACP in patients who die during a hospital admission. METHODS: We performed a clinical audit in 2021 of inpatients at a tertiary hospital in Sydney, Australia to evaluate the benefit of multimodal interventions to improve ACP compared with previous audits from 2016 and 2011. RESULTS: In 2021, 97% of audited patients had a documented ACP prior to death compared with 80% in the 2016 audit. The completion of NFR documentation on admission in 2016 was 33%, while in 2021 65% of ACPs were completed within 24 hours of admission.In 2021, 94% of patients had a paper resuscitation form filled; however, identification stickers, which are associated with risk of error, were used in 64%; and 25% of forms were only partially completed. Palliative care was consulted for 44% of patients prior to death; 33% on the day of or prior to death. CONCLUSIONS: Improvement in prevalence and timing of ACP prior to death is seen in the postintervention audit. A repeat audit in 5 years will be conducted, with interventions focused on improving documentation of ACP.

Outcomes of early hospital readmission after kidney transplantation: Perspectives from a Canadian transplant centre.

BACKGROUND: Early hospital readmissions (EHRs) after kidney transplantation range in incidence from 18%-47% and are important and substantial healthcare quality indicators. EHR can adversely impact clinical outcomes such as graft function and patient mortality as well as healthcare costs. EHRs have been extensively studied in American healthcare systems, but these associations have not been explored within a Canadian setting. Due to significant differences in the delivery of healthcare and patient outcomes, results from American studies cannot be readily applicable to Canadian populations. A better understanding of EHR can facilitate improved discharge planning and long-term outpatient management post kidney transplant. AIM: To explore the burden of EHR on kidney transplant recipients (KTRs) and the Canadian healthcare system in a large transplant centre. METHODS: This single centre cohort study included 1564 KTRs recruited from January 1, 2009 to December 31, 2017, with a 1-year follow-up. We defined EHR as hospitalizations within 30 d or 90 d of transplant discharge, excluding elective procedures. Multivariable Cox and linear regression models were used to examine EHR, late hospital readmissions (defined as hospitalizations within 31-365 d for 30-d EHR and within 91-365 d for 90-d EHR), and outcomes including graft function and patient mortality. RESULTS: In this study, 307 (22.4%) and 394 (29.6%) KTRs had 30-d and 90-d EHRs, respectively. Factors such as having previous cases of rejection, being transplanted in more recent years, having a longer duration of dialysis pretransplant, and having an expanded criteria donor were associated with EHR post-transplant. The cumulative probability of death censored graft failure, as well as total graft failure, was higher among the 90-d EHR group as compared to patients with no EHR. While multivariable models found no significant association between EHR and patient mortality, patients with EHR were at an increased risk of late hospital readmissions, poorer kidney function throughout the 1st year post-transplant, and higher hospital-based care costs within the 1st year of follow-up. CONCLUSION: EHRs are associated with suboptimal outcomes after kidney transplant and increased financial burden on the healthcare system. The results warrant the need for effective strategies to reduce post-transplant EHR.

A lipoglycopeptide antibiotic for Gram-positive biofilm-related infections.

Drug-resistant Gram-positive bacterial infections are still a substantial burden on the public health system, with two bacteria (Staphylococcus aureus and Streptococcus pneumoniae) accounting for over 1.5 million drug-resistant infections in the United States alone in 2017. In 2019, 250,000 deaths were attributed to these pathogens globally. We have developed a preclinical glycopeptide antibiotic, MCC5145, that has excellent potency (MIC90 ≤ 0.06 µg/ml) against hundreds of isolates of methicillin-resistant S. aureus (MRSA) and other Gram-positive bacteria, with a greater than 1000-fold margin over mammalian cell cytotoxicity values. The antibiotic has therapeutic in vivo efficacy when dosed subcutaneously in multiple murine models of established bacterial infections, including thigh infection with MRSA and blood septicemia with S. pneumoniae, as well as when dosed orally in an antibiotic-induced Clostridioides difficile infection model. MCC5145 exhibited reduced nephrotoxicity at microbiologically active doses in mice compared to vancomycin. MCC5145 also showed improved activity against biofilms compared to vancomycin, both in vitro and in vivo, and a low propensity to select for drug resistance. Characterization of drug action using a transposon library bioinformatic platform showed a mechanistic distinction from other glycopeptide antibiotics.

A comparison study of superovulation strategies for C57BL/6J and B6D2F1 mice in CRISPR-Cas9 mediated genome editing.

Reproductive techniques such as superovulation and in vitro fertilisation (IVF) have been widely used in generating genetically modified animals. The current gold standard for superovulation in mice is using coherent treatments of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). An alternative method using inhibin antiserum (IAS) instead of eCG has been recently reported. Here, we evaluate different superovulation strategies in C57BL/6J and B6D2F1 mice. Firstly, we found that using 5-week-old C57BL/6J and 4-week-old B6D2F1 donors could achieve better superovulation outcomes. Then, we compared eCG-hCG, IAS-hCG and eCG-IAS-hCG with different dosages in both mouse strains. Significantly increased numbers of oocytes were obtained by using IAS-hCG and eCG-IAS-hCG methods. However, low fertilisation rates (36.3-38.8%) were observed when natural mating was applied. We then confirmed that IVF could dramatically ameliorate the fertilisation rates up to 89.1%. Finally, we performed CRISPR-Cas9 mediated genome editing targeting Scn11a and Kcnh1 loci, and successfully obtained mutant pups using eCG-hCG and IAS-hCG induced zygotes, which were fertilised by either natural mating or IVF. Our results showed that IAS is a promising superovulation reagent, and the efficiency of genome editing is unlikely to be affected by using IAS-induced zygotes.

Mapping multicenter randomized controlled trials in anesthesiology: a scoping review.

BACKGROUND: Evidence suggests that there are substantial inconsistencies in the practice of anesthesia. There has not yet been a comprehensive summary of the anesthesia literature that can guide future knowledge translation interventions to move evidence into practice. As the first step toward identifying the most promising interventions for systematic implementation in anesthesia practice, this scoping review of multicentre RCTs aimed to explore and map the existing literature investigating perioperative anesthesia-related interventions and clinical patient outcomes. METHODS: Multicenter randomized controlled trials were eligible for inclusion if they involved a tested anesthesia-related intervention administered to adult surgical patients (≥ 16 years old), with a control group receiving either another anesthesia intervention or no intervention at all. The electronic databases Embase (via OVID), MEDLINE, and MEDLINE in Process (via OVID), and Cochrane Central Register of Control Trials (CENTRAL) were searched from inception to February 26, 2021. Studies were screened and data were extracted by pairs of independent reviewers in duplicate with disagreements resolved through consensus or a third reviewer. Data were summarized narratively. RESULTS: We included 638 multicentre randomized controlled trials (n patients = 615,907) that met the eligibility criteria. The most commonly identified anesthesia-related intervention theme across all studies was pharmacotherapy (n studies = 361 [56.6%]; n patients = 244,610 [39.7%]), followed by anesthetic technique (n studies = 80 [12.5%], n patients = 48,455 [7.9%]). Interventions were most often implemented intraoperatively (n studies = 233 [36.5%]; n patients = 175,974 [28.6%]). Studies typically involved multiple types of surgeries (n studies = 187 [29.2%]; n patients = 206 667 [33.5%]), followed by general surgery only (n studies = 115 [18.1%]; n patients = 201,028 [32.6%]) and orthopedic surgery only (n studies = 94 [14.7%]; n patients = 34,575 [5.6%]). Functional status was the most commonly investigated outcome (n studies = 272), followed by patient experience (n studies = 168), and mortality (n studies = 153). CONCLUSIONS: This scoping review provides a map of multicenter RCTs in anesthesia which can be used to optimize future research endeavors in the field. Specifically, we have identified key knowledge gaps in anesthesia that require further systematic assessment, as well as areas where additional research would likely not add value. These findings provide the foundation for streamlining knowledge translation in anesthesia in order to reduce practice variation and enhance patient outcomes.

Construction of a full-length antibody phage display vector.

Antibody phage display technology plays an important role in the development of monoclonal antibodies, humanization, and affinity evolution of antibodies. Thus far, antibody phage display mainly focuses on the display of antibody variable region or antigen-binding fragments. In this study, we constructed a new phage display system that can display full-length IgG antibodies on M13 phage. The phage display vector contains open reading frames (ORFs) encoding full-length the heavy and light chains of the antibody. NcoI/XhoI restriction enzyme sites were used to clone the variable region of the heavy chain into the heavy chain ORF, and SalI/NotI sites were used to clone the light chain variable region. SnaBI and SbfI restriction enzyme sites were designed between the cloning sites of heavy and light chains, respectively, to increase the cloning efficiency. The full-length antibodies of nivolumab against programmed death factor 1, trastuzumab against human epidermal growth factor 2, diL2K against the cluster of differentiation 3 epsilon, and adalimumab against tumor necrosis factor- alpha were displayed on phage with the vector. Phage-displayed antibodies showed their original antigen-binding activity. An amber codon shifted the vector to express IgG in non-suppressed Escherichia coli. The heavy and light chains of the E. coli-expressed antibodies could be detected through western blotting, and the antigen-binding activity was confirmed using an enzyme-linked immunosorbent assay. Biopanning was carried out with a model phage display antibody library, and the results showed that the novel phage system could be used for antibody library construction and highly efficient antibody screening. The reported system is the first full-length antibody phage display system.

What Are the Burden, Causes, and Costs of Early Hospital Readmissions After Kidney Transplantation?

INTRODUCTION: Kidney transplant recipients are at risk for complications resulting in early hospital readmission. This study sought to determine the incidences, risk factors, causes, and financial costs of early readmissions. DESIGN: This single-centre cohort study included 1461 kidney recipients from 1 Jul 2004 to 31 Dec 2012, with at least 1-year follow-up. Early readmission was defined as hospitalization within 30 or 90-days postdischarge from transplant admission. Associations between various parameters and 30 and 90-days posttransplant were determined using multivariable Cox proportional hazards models. The hospital-associated costs of were assessed. RESULTS: The rates of early readmission were 19.4% at 30 days and 26.8% at 90 days posttransplant. Mean cost per 30-day readmission was 11 606 CAD. Infectious complications were the most common reasons and resulted in the greatest cost burden. Factors associated with 30 and 90-days in multivariable models were recipient history of chronic lung disease (hazard ratio or HR 1.78 [95%CI: 1.14, 2.76] and HR 1.68 [1.14, 2.48], respectively), median time on dialysis (HR 1.07 [95% CI: 1.01, 1.13]and HR 1.06 [95% CI: 1.01, 1.11], respectively), being transplanted preemptively (HR 1.75 [95% CI: 1.07, 2.88] and HR 1.66 [95% CI: 1.07, 2.57], respectively), and having a transplant hospitalization lasting of and more than 11 days (HR 1.52 [95% CI: 1.01, 2.27] and HR 1.65 [95% CI: 1.16, 2.34], respectively). DISCUSSION: Early hospital readmission after transplantation was common and costly. Strategies to reduce the burden of early hospital readmissions are needed for all patients.

A Systematic Review of the Effect of N-Acetylcysteine on Serum Creatinine and Cystatin C Measurements.

INTRODUCTION: N-acetylcysteine (NAC) is an antioxidant that can regenerate glutathione and is primarily used for acetaminophen overdose. NAC has been tested and used for preventing iatrogenic acute kidney injury or slowing the progression of chronic kidney disease, with mixed results. There are conflicting reports that NAC may artificially lower measured serum creatinine without improving kidney function, potentially by assay interference. Given these mixed results, we conducted a systematic review of the literature to determine whether there is an effect of NAC on kidney function as measured with serum creatinine and cystatin C. METHODS: A literature search was conducted to identify all study types reporting a change in serum creatinine after NAC administration. The primary outcome was change in serum creatinine after NAC administration. The secondary outcome was a change in cystatin C after NAC administration. Subgroup analyses were conducted to assess effect of creatinine assay (Jaffe vs. non-Jaffe and intravenous vs. oral). RESULTS: Six studies with a total of 199 participants were eligible for the systematic review and meta-analysis. There was a small but significant decrease in serum creatinine after NAC administration overall (weighted mean difference [WMD], -2.80 µmol/L [95% confidence interval {CI} -5.6 to 0.0]; P = 0.05). This was greater with non-Jaffe methods (WMD, -3.24 µmol/L [95% CI -6.29 to -0.28]; P = 0.04) than Jaffe (WMD, -0.51 µmol/L [95% CI -7.56 to 6.53]; P = 0.89) and in particular with intravenous (WMD, -31.10 µmol/L [95% CI -58.37 to -3.83]; P = 0.03) compared with oral NAC (WMD, -2.5 µmol/L [95% CI -5.32 to 0.32]; P = 0.08). There was no change in cystatin C after NAC administration. DISCUSSION: NAC causes a decrease in serum creatinine but not in cystatin C, suggesting analytic interference rather than an effect on kidney function. Supporting this, the effect was greater with non-Jaffe methods of creatinine estimation. Future studies of NAC should use the Jaffe method of creatinine estimation when kidney outcomes are being reported. Even in clinical settings, the use of an enzymatic assay when high doses of intravenous NAC are being used may result in underdiagnosis or delayed diagnosis of acute kidney injury.

A systematic review on the efficacy and safety of low molecular weight heparin as an anticancer therapeutic in preclinical animal models.

OBJECTIVE: The therapeutic effects of low molecular weight heparins (LMWH) may extend past thrombosis prevention, with preclinical evidence demonstrating anti-metastatic properties. Clinical evidence on the topic, however, remains controversial. A systematic review of preclinical evidence may help elucidate reasons for this contradictory evidence. The objective of our systematic review is to assess the anti-metastatic properties of LMWHs in solid tumour animal models. METHODS: MEDLINE, Embase, Web of Science and PubMed were searched from inception to May 12th, 2020. All articles were screened independently and in duplicate. Studies that compared LMWH to a placebo or no treatment arm in solid tumour animal models were included. The primary outcome was the burden of metastasis. Secondary outcomes included primary tumour growth and mortality. The risk of bias was assessed in duplicate using a modified Cochrane Risk of Bias tool. RESULTS: Forty-two studies were included in the review. Administration of a LMWH was associated with a significant decrease in the burden of metastasis (SMD -2.18; 95% CI -2.66 to -1.70). Additionally, the administration of a LMWH was also associated with a significant reduction in primary tumour growth (SMD -1.95; 95% CI -2.56 to -1.34) and risk of death (RR 0.39; 95% CI 0.16-0.97). All included studies were deemed to be at an unclear risk of bias for at least one methodological criterion. CONCLUSIONS: Our results demonstrate that LMWH can effectively reduce metastatic burden and reduce tumour growth in preclinical animal models of solid tumour malignancies. Reasons for the contradiction with clinical evidence require further exploration.

An amphipathic peptide with antibiotic activity against multidrug-resistant Gram-negative bacteria.

Peptide antibiotics are an abundant and synthetically tractable source of molecular diversity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an amphipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina. The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC, a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3-4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the 'no observable adverse effect level' (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models.

Impact of post-haemorrhoidectomy pain relief checklists on pain outcomes: a randomized controlled trial.

BACKGROUND: Haemorrhoidectomy is associated with significant post-operative pain which is primarily managed pharmacologically. Whether a non-pharmacological adjunct such as a checklist can improve pain outcomes after an open haemorrhoidectomy has yet to be studied. The purpose of this study was to determine if a patient-completed checklist of prescribed post-haemorrhoidectomy pain medications would improve pain management after surgery. METHODS: We conducted a dual-centre randomized controlled trial of patients undergoing a Milligan-Morgan haemorrhoidectomy for symptomatic third or fourth degree haemorrhoids. Thirty-five patients were randomized into either a control group which received post-operative pain medication plus a visual analogue scale (VAS) form, or an intervention group which received a post-operative medication checklist in addition to the items the control group received. Both groups recorded their pain levels on the VAS forms at 10.00, 14.00 and 20.00 hours each day for 14 days post-operatively. RESULTS: Patients in the checklist group reported a significantly greater reduction in mean VAS pain score of 2.51 (95% confidence interval (CI) 1.34-3.68; P < 0.001) between day 1 post-op and day 14 post-op compared to 1.86 (95% CI 0.77-2.95; P = 0.001) for the control group. There was no significant difference between mean pain experienced by patients in either group over each of the 14 days individually or overall (P = 0.07). CONCLUSION: The pain medication checklist lead to a greater reduction in pain between day 1 and 14 after an open haemorrhoidectomy compared to standard care but did not significantly reduce mean pain across any individual days or overall.

Approach to treatment of cicatricial ectropion: a systematic review and meta-analysis comparing surgical and minimally invasive options.

Cicatricial ectropion can involve the skin, subcutaneous tissue, muscle, and septum to result in chronic tearing and keratopathy. Surgery involving the orbital rim or eyelid is a common cause. Minimally invasive techniques may provide alternative options for correction, but the comparative benefit to surgery is unknown. To compare the efficacy of surgical and minimally invasive minimally invasive treatment options for cicatricial ectropion. A comprehensive literature search of Medline, EMBASE, and the Cochrane Library published from 1960 to August 2019 was performed for studies that described any treatment of cicatricial ectropion. 1391 studies were found, of which 31 had extractable data for 299 patients. Pooling of outcome data occurred for the primary and secondary outcomes. The complete and partial response rates to treatment (primary outcomes) as well as the recurrence rate and physician global assessment of cosmesis (secondary outcomes) were analyzed. Surgical correction resulted in complete correction in 79% of patients compared to 63% of hyaluronic acid treated patients. Hyaluronic acid injection had a better aesthetic outcome, but a higher recurrence rate overall. Hyaluronic acid filler with a high G' along with delayed dissolution trended toward a lower recurrence rate. Other minimally invasive treatments had little data. The literature found was limited to mostly single-center, observational studies. Hyaluronic acid may be a viable alternative for cicatricial ectropion in those patients who cannot undergo surgery. Further prospective studies are required to routinely recommend minimally invasive techniques.

Genome-Wide Off-Target Analysis in CRISPR-Cas9 Modified Mice and Their Offspring.

The emergence of the CRISPR-Cas9 system has triggered a technical revolution in mammalian genome editing. Compared to traditional gene-targeting strategies, CRISPR-Cas9 technology offers a more efficient and cost-effective approach for generating genetically modified animal models. However, off-target cleavage in CRISPR-mediated genome editing is a major concern in the analysis of phenotypes as well as the selection of therapeutic targets. Here, we analyzed whole-genome sequencing (WGS) data from two knock-out (KO) mouse strains generated by using the CRISPR-Cas9 system targeting the Mmd and Paqr8 loci. A total of nine individuals were sequenced including two parents, four F1 offspring and three uninjected control mice. Using GATK and bcftools software, we identified two off-target events in the founder mice. The two CRISPR-Cas9-induced off-target events were predictable using Cas-OFFinder and were not passed on to the offspring that we investigated. In addition, our results indicated that the number of CRISPR-Cas9-induced mutations was not statistically distinguishable from the background de novo mutations (DNMs).

A novel murine antibody and an open sandwich immunoassay for the detection of clenbuterol.

Clenbuterol (CLEN) is a sympathomimetic amine used as a decongestant and bronchodilator while treating breathing disorders. It is also used in food-producing animals as it improves the rate of red meat production. However, it is prohibited in many countries nowadays due to human health and safety concerns. Unfortunately, the illegal use of CLEN is still rampant. Thus, monitoring it in food and livestock is important. Here, we report a novel murine antibody and an open sandwich enzyme linked immunosorbent assay (OS-ELISA) to detect CLEN based on antigen-antibody reactions. The genes of antibody variable regions in mice immunized with CLEN conjugated with bovine serum albumin were cloned into a phagemid (pDong1/Fab) to construct a phage-display antibody library, from which a novel antibody, A12, was selected. Then, an OS-ELISA was developed to detect CLEN using separated variable regions of the A12 antibody. The limit of detection of the assay was found to be 8 ng/mL, which was useful for monitoring CLEN usage.

Structure-Function Studies of Polymyxin B Lipononapeptides.

The emerging threat of infections caused by highly drug-resistant bacteria has prompted a resurgence in the use of the lipodecapeptide antibiotics polymyxin B and colistin as last resort therapies. Given the emergence of resistance to these drugs, there has also been a renewed interest in the development of next generation polymyxins with improved therapeutic indices and spectra of action. We report structure-activity studies of 36 polymyxin lipononapeptides structurally characterised by an exocyclic FA-Thr²-Dab³ lipodipeptide motif instead of the native FA-Dab¹-Thr²-Dab³ tripeptide motif found in polymyxin B, removing one of the positively charged residues believed to contribute to nephrotoxicity. The compounds were prepared by solid phase synthesis using an on-resin cyclisation approach, varying the fatty acid and the residues at position 2 (P2), P3 and P4, then assessing antimicrobial potency against a panel of Gram-negative bacteria, including polymyxin-resistant strains. Pairwise comparison of N-acyl nonapeptide and decapeptide analogues possessing different fatty acids demonstrated that antimicrobial potency is strongly influenced by the N-terminal L-Dab-1 residue, contingent upon the fatty acid. This study highlights that antimicrobial potency may be retained upon truncation of the N-terminal L-Dab-1 residue of the native exocyclic lipotripeptide motif found in polymyxin B. The strategy may aid in the design of next generation polymyxins.