RgIA4 Prevention of Acute Oxaliplatin-Induced Cold Allodynia Requires α9-Containing Nicotinic Acetylcholine Receptors and CD3+ T-Cells.

Chemotherapy-induced neuropathic pain is a debilitating and dose-limiting side effect. Oxaliplatin is a third-generation platinum and antineoplastic compound that is commonly used to treat colorectal cancer and commonly yields neuropathic side effects. Available drugs such as duloxetine provide only modest benefits against oxaliplatin-induced neuropathy. A particularly disruptive symptom of oxaliplatin is painful cold sensitivity, known as cold allodynia. Previous studies of the Conus regius peptide, RgIA, and its analogs have demonstrated relief from oxaliplatin-induced cold allodynia, yielding improvement that persists even after treatment cessation. Moreover, underlying inflammatory and neuronal protection were shown at the cellular level in chronic constriction nerve injury models, consistent with disease-modifying effects. Despite these promising preclinical outcomes, the underlying molecular mechanism of action of RgIA4 remains an area of active investigation. This study aimed to determine the necessity of the α9 nAChR subunit and potential T-cell mechanisms in RgIA4 efficacy against acute oxaliplatin-induced cold allodynia. A single dose of oxaliplatin (10 mg/kg) was utilized followed by four daily doses of RgIA4. Subcutaneous administration of RgIA4 (40 µg/kg) prevented cold allodynia in wildtype mice but not in mice lacking the α9 nAChR-encoding gene, chrna9. RgIA4 also failed to reverse allodynia in mice depleted of CD3+ T-cells. In wildtype mice treated with oxaliplatin, quantitated circulating T-cells remained unaffected by RgIA4. Together, these results show that RgIA4 requires both chrna9 and CD3+ T-cells to exert its protective effects against acute cold-allodynia produced by oxaliplatin.

Highly efficient and simple SSPER and rrPCR approaches for the accurate site-directed mutagenesis of large and small plasmids.

Advances are needed in the site-directed mutagenesis of large plasmids for protein structure-function studies, as current methods are often inefficient, complicated and time-consuming. Here two new methods are reported that overcome these difficulties, namely the single primer extension reaction (SSPER) strategy that reaches 100% efficiency and the reduce recycle PCR (rrPCR) method that is advantageous in generating single and pairwise combinations of mutations. Both methods are distinguished from current technologies by the addition of a step that easily removes the oligonucleotide primer(s) after the first reaction, thus allowing for the addition of a second reaction in chronological sequence to generate and isolate the appropriate DNA product with the site-directed mutation(s). High efficiency of the methods is demonstrated by generating single and paired combinations of the 11 site-directed mutations targeted on 5 different plasmid DNA templates ranging from 10 to 12 kb and 57-60% GC-content at a rate of 50-100%. Overall, the methods are demonstrated to be (i) highly accurate, allowing for screening of plasmids by DNA sequencing, (ii) streamlined to generate the mutations within a single day, (iii) cost-effective in requiring only two primers and two enzymes (DpnI and a proofreading DNA polymerase), (iv) straightforward in primer design, (v) applicable for both large and small plasmids, and (vi) easily implemented by entry level researchers.

Selective Penicillamine Substitution Enables Development of a Potent Analgesic Peptide that Acts through a Non-Opioid-Based Mechanism.

Venom-derived compounds are of broad interest in neuropharmacology and drug development. α-Conotoxins are small disulfide-containing peptides from Conus snails that target nicotinic acetylcholine receptors (nAChRs) and are in clinical development for non-opioid-based treatment of intractable pain. Although refined by evolution for interaction with target prey receptors, enhancements of pharmacological properties are needed for use in mammalian systems. Therefore, we synthesized analogues of α-conotoxin RgIA using a combination of selective penicillamine substitutions together with natural and non-natural amino acid replacements. This approach resulted in a peptide with 9000-fold increased potency on the human α9α10 nAChR and improved resistance to disulfide shuffling compared to the native peptide. The lead analogue, RgIA-5474, potently blocked α9α10 nAChRs, but not opioid- or other pain-related targets. In addition, RgIA-5474 effectively reversed chemotherapy-induced neuropathic pain.

Critical residue properties for potency and selectivity of α-Conotoxin RgIA towards α9α10 nicotinic acetylcholine receptors.

The α9α10 nicotinic acetylcholine receptor (nAChR) has been characterized as an effective anti-pain target that functions through a non-opioid mechanism. However, as a pentameric ion channel comprised of two different subunits, the specific targeting of α9α10 nAChRs has proven challenging. Previously the 13-amino-acid peptide, RgIA, was shown to block α9α10 nAChRs with high potency and specificity. This peptide, characterized from the venom of the carnivorous marine snail, Conus regius, produced analgesia in several rodent models of chronic pain. Despite promising pre-clinical data in behavioral assays, the number of specific α9α10 nAChR antagonists remains small and the physiological mechanisms of analgesia remain cryptic. In this study, we implement amino-acid substitutions to definitively characterize the chemical properties of RgIA that contribute to its activity against α9α10 nAChRs. Using this mutational approach, we determined the vital role of biochemical side-chain properties and amino acids in the second loop that are amenable to substitutions to further engineer next-generation analogs for the blockade of α9α10 nAChRs.

The Long-Term Efficacy of Radiofrequency Ablation With and Without Steroid Injection.

Background: Radiofrequency ablation (RFA) has been proven to be an effective option for treating chronic low back pain. In addition to RFA as a treatment modality, the administration of concomitantly to minimize the effect of hyperalgesia is common practice. However, there is insufficient evidence about the long-term outcomes of their use. Methods: This was a retrospective study that examined 239 patients who received spine, knee joint, and sacroiliac joint RFA between June 2014 and June 2018. Pre- and post-procedure pain scores, percent improvements, and duration of relief were included in our review. Subjects: This study included 239 patients of which 191 patients received steroids with their RFA. Results: These 191 patients experienced an average improvement of 48.48% relief for an average of 137.52 days. Forty-eight patients did not receive steroids with RFA and had an average improvement of 46.36% for an average of 126.10 days. The statistical analysis revealed there was no significant difference between the two groups for percent improvement (p = 0.71) and duration of relief (p = 0.67). Conclusions: Patients who received steroids with RFA compared to RFA alone did not differ significantly in percent improvement in pain and duration of relief.

RgIA4 Accelerates Recovery from Paclitaxel-Induced Neuropathic Pain in Rats.

Chemotherapeutic drugs are widely utilized in the treatment of human cancers. Painful chemotherapy-induced neuropathy is a common, debilitating, and dose-limiting side effect for which there is currently no effective treatment. Previous studies have demonstrated the potential utility of peptides from the marine snail from the genus Conus for the treatment of neuropathic pain. α-Conotoxin RgIA and a potent analog, RgIA4, have previously been shown to prevent the development of neuropathy resulting from the administration of oxaliplatin, a platinum-based antineoplastic drug. Here, we have examined its efficacy against paclitaxel, a chemotherapeutic drug that works by a mechanism of action distinct from that of oxaliplatin. Paclitaxel was administered at 2 mg/kg (intraperitoneally (IP)) every other day for a total of 8 mg/kg. Sprague Dawley rats that were co-administered RgIA4 at 80 µg/kg (subcutaneously (SC)) once daily, five times per week, for three weeks showed significant recovery from mechanical allodynia by day 31. Notably, the therapeutic effects reached significance 12 days after the last administration of RgIA4, which is suggestive of a rescue mechanism. These findings support the effects of RgIA4 in multiple chemotherapeutic models and the investigation of α9α10 nicotinic acetylcholine receptors (nAChRs) as a non-opioid target in the treatment of chronic pain.

Does the Proportion of Same-Day and 24-Hour Appointments Impact Patient Satisfaction?

BACKGROUND: The relationship between open access and patient satisfaction is mixed. Our study is the first to assess the relationship between open access appointment scheduling and patient satisfaction in the Military Health System (MHS). It is also unique in that we examine both same-day and 24-hour access through a relationship with satisfaction. METHODS: We conducted a panel time-series analysis with general estimating equations on the Army population of outpatient facilities (N = 32), with 32 364 957 total observations. Our primary independent variables were the proportion of a facility's appointments within 24 hours and same day from July 2013 to May 2015. RESULTS: We identified that a higher proportion of same-day appointments is associated with increased patient satisfaction with the ability to see their provider when needed. We did not find the same result when examining access within 24 hours. CONCLUSIONS: Open access appointment scheduling appears to have a greater impact on patient satisfaction with timeliness of care if that appointment is made the same day the patient presents to the facility. Facilities should consider opening more of their schedule to accommodate same-day appointments. This can result in less costly primary care instead of emergency department usage.

Targeted delivery of antisense oligonucleotides by chemically self-assembled nanostructures.

Synthetic nucleic acids have shown great potential in the treatment of various diseases. Nevertheless, the selective delivery to a target tissue has proved challenging. The coupling of nucleic acids to targeting peptides, proteins, and antibodies has been explored as an approach for their selective tissue delivery. Nevertheless, the preparation of covalently coupled peptides and proteins that can also undergo intracellular release as well as deliver more than one copy of the nucleic acid has proved challenging. Recently, we have developed a novel method for the rapid noncovalent conjugation of nucleic acids to targeting single chain antibodies (scFv) using chemically self-assembled nanostructures (CSANs). CSANs have been prepared by the self-assembly of two dihydrofolate reductase molecules (DHFR(2)) and a targeting scFv in the presence of bis-methotrexate (bis-MTX). The valency of the nanorings can be tuned from one to eight subunits, depending on the length and composition of the linker between the dihydrofolate reductase molecules. To explore their potential for the therapeutic delivery of nucleic acids as well as the ability to expand the capabilities of CSANs by incorporating smaller cyclic targeting peptides, we prepared DHFR(2) proteins fused through a flexible peptide linker to cyclic-RGD, which targets αvß3 integrins, and a bis-MTX chemical dimerizer linked to an antisense oligonucleotide (bis-MTX-ASO) that has been shown to silence expression of eukaryotic translation initiation factor 4E (eIF4E). Monomeric and multimeric cRGD-CSANs were then prepared with bis-MTX-ASO and shown to undergo endocytosis in the breast cancer cell line, MDA-MB-231, which overexpresses αvß3. The bis-MTX-ASO was shown to undergo endosomal escape resulting in the knock down of eIF4E with at least the same efficiency as ASO delivered by oligofectamine. The modularity, flexibility, and common method of conjugation may prove to be a useful general approach for the targeted delivery of ASOs, as well as other nucleic acids to cells.

A new technique involving a spherical stainless steel device to optimize positioning of the umbilicus.

BACKGROUND: Creating an aesthetically pleasing umbilicus may be challenging due to various factors that involve the patient limitations and suboptimal techniques available to the surgeon. Although many techniques aim to locate the umbilicus after abdominoplasty, none are ideal. The authors use a new technique involving a stainless steel spherical device for definite location of the new neo-umbilicus site. METHODS: Abdominoplasty with full muscle plication and umbilicoplasty was performed to test the effectiveness of this new technique that involves a stainless steel marble called the Umbilicator. It has a diameter of 1.5 cm and three 2-mm holes drilled 120° apart in an equilateral triangle. The Umbilicator is secured to the inferior and superior dermis of the umbilical stalk to help identify the future location of the umbilicus on the abdominal skin. Once the marble is secured, the superior abdominal flap is redraped and trimmed, the suture is repaired, and the location of the umbilicus is determined by feeling for the smooth spherical surface bump with gentle downward pressure on the overlying abdominal skin located within the proximity of the umbilicus. RESULTS: The result of this technique produced a definitive means of identifying and delivering the umbilical stalk during abdominoplasty. This technique has been performed in 23 consecutive abdominoplasty procedures with no difficulties locating the umbilical stalk and no infections resulting from the procedure. CONCLUSIONS: Accurate identification of the umbilicus provides the ability to create an aesthetically pleasing neo-umbilicus, thus optimizing abdominoplasty results. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article.

Cortical thickness parameters for endoscopic browlift fixation.

BACKGROUND: Techniques for endoscopic browlift include bony fixation over the lateral frontal region and soft tissue fixation over the temporal region. Although bony fixation over the lateral frontal region is advocated universally, limited information exists about bicortical thickness in this area. OBJECTIVES: The authors provide bicortical thickness measurements between the frontal midline and the most inferior temporal region to assist surgeons in identifying appropriate fixation planes. METHODS: Bicortical thickness was measured in the hemicraniums of 13 female cadavers, along the coronal planes that travel through the anterior border of the mandibular condyles and at the junction of the posterior mandibular condyles and the external auditory meatuses. Measurements began at the midline and coursed laterally at 1-cm intervals. RESULTS: Average cranial thickness along the frontal region ranged from 8.9 ± 2.4 mm to 6.4 ± 2.8 mm over the anterior coronal line and 8.8 ± 2.2 mm to 5.6 ± 1.8 mm over the posterior line. Average thickness along the temporal region ranged from 5.6 ± 2.8 mm to 2.8 mm ± 1.4 mm over the anterior coronal line and 5.1 ± 1.8 mm to 3.4 ± 1.4 mm over the posterior line. Minimum thickness was 3.7 mm and 1.3 mm over the frontal and temporal regions, respectively. There was no significant difference between left and right hemicranial thickness. CONCLUSIONS: To avoid violation of the inner cortex during surgery, endoscopic browlift procedures should include measurement of cortical thickness at various fixation points. Bony fixation over the temporal region should be avoided. Minimal bicortical thickness was observed in the lateral frontal region.

Residential Proximity to Freeways is Associated with Uncontrolled Asthma in Inner-City Hispanic Children and Adolescents.

Background. Proximity to heavy traffic has been linked to increased asthma severity. However, it is unknown whether exposure to heavy traffic is associated with the ability to maintain asthma control. Objectives. This study examines whether exposure to heavy traffic is associated with the ability to maintain asthma control in inner-city children. Methods. 756 inner-city asthmatic Hispanic children were followed for one year in a pediatric asthma management program (Breathmobile). At each scheduled visit, asthma specialist tracked patients' asthma severity and managed their asthma based on the NAEPP guidelines. The patients' residential distance from the nearest freeway was calculated based on residential address at study entry. Distance to nearest freeway was used as a surrogate marker for high exposure from traffic-related air pollutants. Results. Patients who lived near a freeway were significantly more likely to have asthma that was not well controlled (P = .03). Patients with intermittent and mild baseline severity have a two-fold increased risk of having asthma that is uncontrolled if they lived <2 miles from a freeway (OR = 2.2, P = .04). Conclusion. In children with asthma, residential proximity to freeways is associated with uncontrolled asthma.

Long-term maintenance of pediatric asthma: focus on budesonide/formoterol inhalation aerosol.

Current national and international asthma guidelines recommend treatment of children with asthma towards achieving and maintaining asthma control. These guidelines provide more stringent recommendations to increase therapy for patients with uncontrolled asthma in order to reduce asthma-related morbidity and mortality. Newer combination agents such as budesonide and formoterol have been shown to be safe and effective in treatment of asthma in children. Use of long-term controller agents like this in combination with improved compliance and treatment of co-morbid conditions have been successful in this endeavor. This review discusses control of pediatric asthma with focus on the use of budesonide in combination with formoterol.

Loss of asthma control in inner city children with asthma after withdraw of asthma controller medication.

To determine what percentage of inner-city children with asthma would lose asthma control when taken off asthma controllers, a retrospective analysis was performed on inner-city asthmatic children who achieved asthma control in an asthma specific disease management program. Once disease control was achieved patients had stepwise reduction of asthma controllers based on the National Asthma Education and Prevention Program (NAEPP) Expert Review Panel (EPR) 2 guidelines. In patients who were taken off all controllers, probability of maintaining asthma control at the first visit after cessation of these medications was significantly lower compared to patients kept on inhaled corticosteroids. We conclude that cessation of asthma controllers in previously well controlled inner-city asthmatic children results in loss of asthma control in a significant number of these patients. Data support recommendations from national asthma guidelines to step down controller therapy, but clinical monitoring is important to reduce impairment due to loss of control.

Continuous glucose monitoring technology for personal use: an educational program that educates and supports the patient.

PURPOSE: The purpose of this article is to describe the development and implementation of an educational program for the initiation of real-time continuous glucose monitoring (CGM) technology for personal use, not 3-day CGMS diagnostic studies. The education program was designed to meet the needs of patients managing their diabetes with either diabetes medications or insulin pump therapy in an outpatient diabetes education center using a team-based approach. METHODS: Observational research, complemented by literature review, was used to develop an educational program model and teaching strategies. Diabetes educators, endocrinologists, CGM manufacturer clinical specialists, and patients with diabetes were also interviewed for their clinical observations and experience. RESULTS: The program follows a progressive educational model. First, patients learn in-depth about real-time CGM technology by attending a group presensor class that provides detailed information about CGM. This presensor class facilitates self-selection among patients concerning their readiness to use real-time CGM. If the patient decides to proceed with real-time CGM use, CGM initiation is scheduled, using a clinic-centered protocol for both start-up and follow-up. CONCLUSIONS: Successful use of real-time CGM involves more than just patient enthusiasm or interest in a new technology. Channeling patient interest into a structured educational setting that includes the benefits and limitations of real-time CGM helps to manage patient expectations.