➊ Peter Pan Syndrome Analysis

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Peter Pan Syndrome Analysis

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S o-called navicular or caudal heel syndrome is one cause of lameness that can appear in horses of any breed or discipline. It can be limited to one limb; however, it most commonly affects both front hooves, causing bilateral lameness. While veterinarians have documented it in the rear feet, these cases are very rare. Tracy Turner. Veterinarians believe navicular is caused by mechanical stress and strain due to the constant pressure between the navicular bone and DDFT, which leads to the degeneration of those and other structures that make up the podotrochlear apparatus.

Poor foot conformation, such as a long toe and low heel, increases this stress and might potentiate development of the condition. The result is lameness, which can become chronic. Early on these horses might present with a shortened stride right out of their stalls or while warming up, says Dr. Duncan Peters. Because of the relationship between hoof angles and the podotrochlear apparatus structures, hoof care, as well as conformation, can also contribute to the condition. Strain and sports-related injury from highly physical disciplines requiring hard turns, fast stops, lateral movement, and jumping, can also compound the problem, Peters says. In recent years the prognosis for podotrochlosis has improved significantly due to the increased use of magnetic resonance imaging MRI and better communication between veterinarians and farriers to determine what is best for the horse, says Dr.

Craig S. New therapies have also emerged that could be invaluable for managing these horses and returning them to work. Tracy A. Turner operates Turner Equine Sports Medicine and Surgery , in Stillwater, Minnesota, which focuses on lameness, sports medicine, and surgery. Lesser has an interest in podiatry, lameness, and imaging. He also enjoys teaching, volunteering, doing research, and continuing to further his education.

Magnetic resonance imaging has become the gold standard for determining which structures are involved and the extent of damage, Lesser says. However, researchers have found in sound horses some of the same radiographic changes lame horses have. In some cases a bursogram is also helpful. In this procedure, says Turner, the veterinarian injects a dye into the navicular bursa the cushioning fluid-filled sac between the navicular bone and the deep digital flexor tendon that runs from the back of the knee down around the navicular bone to illuminate it, the cartilage, and the bursa edge of the deep digital flexor tendon on radiographs. Turner says a bursogram can be especially helpful when working up mounts used for lower-impact sports, such as trail riding.

These horses can be difficult to diagnose because they perform long, slow mileage and might only be used sporadically, he says. Peters adds that, in some cases, clinicians can also use ultrasound to image the soft tissue structures of the lower limb, such as the deep digital flexor tendon or the impar ligament, which could be involved with the lameness.

A Hands-On Lameness Exam. For that reason, all three veterinarians start every evaluation with a thorough hands-on lameness exam that includes a visual analysis of the hoof and its abnormalities, along with palpation and flexion tests. During a lameness exam, Peters likes to evaluate the horse on the longe line or under saddle to see how it moves. He also prefers watching the horse on both hard asphalt and soft arena sand surfaces. For example, the horse might have an exaggerated head bob and longer flight time on the affected leg due to the added concussion from trotting on hard ground.

Distal lower limb flexion tests are also part of this exam. With a bilateral forelimb lameness, he injects local anesthetic over the palmar digital nerves in the low pastern area of one limb, called a palmar digital nerve block. Turner also uses a wedge test , in which he places a 1-inch-thick block under the frog, the toe, and the inner and outer hoof walls for about 60 seconds, then trots the horse off to evaluate lameness. Because podotrochlosis can encompass a wide range of painful changes in the hoof, the lameness exam combined with diagnostic imaging are critical to determining the source of pain and developing a treatment plan for the horse.

P odotrochlosis often requires a multipronged treatment approach that might include rest, drug treatment, shock wave therapy, and hoof care that improves hoof angles, among others. His goal is to create a treatment and management plan that works for the individual horse. For acute pain, a veterinarian might prescribe a non-steroidal anti-inflammatory drug NSAID such as bute or firocoxib to help make the horse more comfortable and break the conditions initial pain cycle, Peters says. Bisphosphonates are another drug treatment option for specific navicular syndrome cases. These medications essentially reduce bone remodeling and the pain associated with it and were originally developed to treat osteoporosis in humans.

The U. Food and Drug Administration FDA has approved two bisphosphonate drugs specifically for treating navicular syndrome. Cells called osteoclasts play a key role in breaking down old bone while another type of cell—osteoblasts—creates new bone. This natural process ensures bones remain strong and healthy and allows them to adapt to changes in exercise level or musculoskeletal system stress. Bisphosphonates bind to osteoclasts to block excess bone resorption. Because bisphosphonates target bone rather than soft tissue, proper diagnosis to pinpoint the cause of podotrochlosis i. Lesser notes that bisphosphonates can cause colic and negatively affect the kidneys. Another approach gaining popularity in the early s is extracorporeal shock wave therapy.

To be effective the treatment must focus specifically on the painful or injured area. Lesser says shock wave therapy is useful when MRI shows signs of injury to structures such as the impar ligament, which attaches the navicular bone to the coffin bone and the suspensory ligaament. While new medical treatments offer promising outcomes, Lesser and Turner agree that trimming and shoeing are the first steps in managing horses with podotrochlosis.

Radiographs can guide trimming and shoeing decisions. They help the veterinarian and farrier determine exactly how much foot they can manipulate, Lesser says. One of the first shoes he reaches for is a wedged shoe with a welded heel plate. The heel plate helps prevent shock from transmitting directly through the frog and up to the navicular region. There is not a hard-and-fast formula farriers can refer to, such as simply applying a bar shoe or raising the heels. Shoe selection for horses with podotrochlosis also depends on discipline and disease severity, he adds. After trimming and shoeing, if additional pain management is necessary, Turner says veterinarians can inject two synovial cavities inside the hoof capsule with anti-inflammatory drugs.

He says he injects the coffin joint if the distal limb flexion was the most positive manipulative test or the navicular bursa if the frog wedge was positive. Other veterinarians prefer to have MRI dictate injections. Once the horse starts feeling good, Turner emphasizes the importance of putting him back into work. There is no one-size-fits-all treatment, and it requires continual follow-up and adjustments as needed. She called Turner, who ran through the set of diagnostic procedures he performs on any horse he suspects of having navicular-related problems: observation of the hoof, lower limb flexion tests, wedge tests, and nerve blocking. On this horse the flexion was the most positive indicator of a navicular issue. Moreover, the discrimination between perfectly complementary targets and two bases mismatch targets demonstrates that the assay is highly specific.

Therefore, this assay could be useful for detecting SARS-CoV-2, even in the presence of co-infection with other viruses that manifest similar respiratory symptoms. RNA and cDNA samples prepared from clinical samples were used as the template in RCA, using the one-step hybridization method and electrochemical detection. All samples used for the evaluation were prepared from nasopharyngeal swabs. The current signals from electrochemical measurements of the N gene blue bar graph and S gene orange bar graph are shown compared with the Cq result from qRT-PCR blue dots for N gene and orange dots for S gene. RCA uses strand displacing phi29 DNA polymerase to continuously amplify the circular nucleic acid template In comparison with PCR-based assays, RCA can be performed under isothermal conditions with minimal reagents and avoids the generation of false-positive results.

RCA assay is also less complicated compared with other isothermal amplification methods, such as a transcription-based system, strand displacement approach, invasive-cleavage reaction, or loop-mediated technology. RCA can be performed with a minimal pre-optimization step, hence it can be readily employed by non-skilled users Target amplification by RCA followed by electrochemical biosensor detection requires three steps of target recognition and hybridization to its complementary sequence. Next, the RCA amplicons are bound by the reporter and capture probes, followed by electrochemical detection of the redox-active dye.

This strategy ensures high specificity is achieved with the assay. Moreover, utilization of magnetic capture and separation of targets from non-targets reduces the chances of carry-over contamination and pipetting error. In areas where COVID virus circulation has been established, nucleic acid testing for a single discriminatory target is considered sufficient Therefore, in this study, we performed multiplex RCA for the simultaneous amplification of two genes, which are the N and S genes encoding the nucleocapsid and spike proteins, respectively.

Therefore, it is recommended that at least two targets are included in a SARS-CoV-2 diagnostic test to reduce the possibility of false-negatives due to mutations in the target genes. On the other hand, the ability of the assay to discriminate two bases mismatch could be useful for viral mutation studies. Therefore, wild-type and mutant strains could in principle be distinguished using probes tagged with different redox dyes as demonstrated in this study. Another advantage of our assay is the limit of detection LoD , which showed that it is highly sensitive and comparable to commercially available test kits and existing biosensors Table 1.

The application of the optimized assay for the diagnosis of COVID was evaluated with clinical samples. The ability of a test to distinguish between SARS-CoV-2 and other respiratory viruses is important because the clinical symptoms of COVID and influenza are very similar, however, the approach in management and control of the disease is different.

A multiplex test that can detect SARS-CoV-2, influenza, and other respiratory viruses simultaneously would save time and provide more accurate information to clinicians and public health officials At present, we have only tested multiplex RCA with two genes, however, it is possible to include additional gene targets for influenza and other respiratory viruses with additional optimization. In death cases, the viral load was 1. This shows that our assay fulfills the requirement for sensitivity and could potentially be used to diagnose COVID in the early stages of the illness when the viral load is still low. It is known that many factors can affect test performance and cause false-negative results.

Several studies have shown that the viral load varies from specimen types, collection methods, and time of collection 5 , Therefore, a robust diagnostic test with high sensitivity could reduce the chances of false negatives caused by low recovery of the virus from real samples. The detection of RCA amplicons using a USB or battery-powered, portable Palmsens4 potentiostat makes it easy to conduct the test in the field or in a community setting.

Indeed, electrochemical based detection using techniques such as DPV, is highly sensitive, quantitative, cost-effective, and compatible with multiplexing. The test can be easily setup in places with limited resources such as in developing countries or in community centers where an outbreak has occurred. We are currently optimizing the RNA extraction process and exploring the use of a smartphone-based biosensor device that will further simplify the test procedure and reduce the sample-to-result turnaround time.

The high amplification capability of RCA and sensitivity of the electrochemical detection method enabled the detection of the viral N and S genes in synthetic linear targets as well as clinical samples. This approach may have a significant impact in places where rapid detection is required to minimize emerging SARS-CoV-2 outbreaks. All the reagent and buffer solutions were prepared with deionized DI water The oligonucleotide sequences are listed in Table 2. The mismatched targets for N and S genes contain two mismatched bases shown underlined in Table 2. First, 1. The washing step was repeated 4 times.

The redox-active dye was incorporated into the SiNPs using a modified method by Cheeveewattanagul et al. First, 0. The washing step was repeated four times. Subsequently, 0. The pellet was washed with 0. The washing step was repeated three times. The solution containing 0. A step-by-step protocol describing the assay can be found at Protocol Exchange For the ligation reaction, 0. The ligation product, called Padlock DNA, served as the template for amplification. The mixture containing the RCA amplicons was resolved by agarose gel electrophoresis. The resolved gel was visualized under UV light. Finally, the pellet was resuspended with 0. This solution was pipetted onto the SPCE. Another hybridization strategy, called one-step hybridization, was also tested to shorten the assay time.

This solution was used for DPV measurements. The assay specificity was evaluated with 1. Respiratory samples remaining from diagnostic tests conducted by the Institute for Urban Disease Control and Prevention, Department of Disease Control, Ministry of Public Health Thailand, were used to evaluate the performance of the assay. A total of anonymized respiratory clinical samples were used to evaluate the performance of the assay.

The samples include SARS-CoVpositive samples and samples that tested positive for other respiratory viruses such as the influenza virus and a respiratory syncytial virus. The institutional review board did not require written informed consent because the study samples were anonymous. Statistic significances were calculated by Microsoft Excel version Five technical replicates were performed to improve the statistics. Further information on research design is available in the Nature Research Reporting Summary linked to this article. The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files or from the corresponding author upon reasonable request.

Source data are provided with this paper. Clinical care of severe acute respiratory infections—Tool kit. Wiersinga, W. Pathophysiology, transmission, diagnosis, and treatment of coronavirus disease COVID : a review. JAMA , — Article Google Scholar. Sethuraman, N. Tang, Y. Feng, W. Santiago I. ChemBioChem 21 , — Kim, Y. Huang, W. Yan, C. Rapid and visual detection of novel coronavirus SARS-CoV-2 by a reverse transcription loop-mediated isothermal amplification assay. Baek, Y. Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV Microbes Infect. Broughton, J. Jiao, J. Xing, W.

A high-throughput, multi-index isothermal amplification platform for rapid detection of 19 types of common respiratory viruses including SARS-CoV Engineering Demidov, V. Rolling-circle amplification in DNA diagnostics: the power of simplicity. Expert Rev. Ouyang, X. Rolling circle amplification-based DNA origami nanostructrures for intracellular delivery of immunostimulatory drugs. Small 9 , — Shen, M. Recent advances and perspectives of nucleic acid detection for coronavirus. Wang, B. Rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification. Tian, B. Cheeveewattanagul, N. Loading of silicon nanoparticle labels with redox mediators for detection of multiple DNA targets within a single voltammetric sweep. Clark, K.

Ion-tagged oligonucleotides coupled with a magnetic liquid support for the sequence-specific capture of DNA. Henley, W. Spatially isolated reactions in a complex array: using magnetic beads to purify and quantify nucleic acids with digital and quantitative real-time PCR in thousands of parallel microwells. Lab Chip 20 , — Pearlman, S. Low-resource nucleic acid extraction method enabled by high-gradient magnetic separation.

ACS Appl. Interfaces 12 , — Palit, D. Adsorption of the dyes methylene blue and acridine orange and their mixtures from aqueous solutions on cholesterol surface. Colloid J. Molecular isothermal techniques for combating infectious diseases: towards low-cost point-of-care diagnostics. Asiello, P. Miniaturized isothermal nucleic acid amplification, a review. Lab Chip 11 , — T4 DNA Ligase. World Health Organization Zhou, P. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature , — Lu, X. Shirato, K. Development of genetic diagnostic methods for detection for novel coronavirus nCoV in Japan.

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The Peter Pan Syndrome Analysis involves sandwich Peter Pan Syndrome Analysis of RCA amplicons with probes that are functionalized with redox-active labels, which are subsequently detected by Peter Pan Syndrome Analysis pulse Peter Pan Syndrome Analysis DPV. The aim of this study was to explore gchq case summary role and mechanism of As Submit manuscript.