Resistance to the Cyclotide Cycloviolacin O2 in Salmonella enterica Caused by Different Mutations That Often Confer Cross-Resistance or Collateral Sensitivity to Other Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are essential components of innate immunity in all living organisms, and these potent broad-spectrum antimicrobials have inspired several antibacterial development programs in the past 2 decades. In this study, the development of resistance to the Gram-negative bacterium-specific peptide cycloviolacin O2 (cyO2), a member of the cyclotide family of plant miniproteins, was characterized in Salmonella enterica serovar Typhimurium LT2. Mutants isolated from serial passaging experiments in increasing concentrations of cyO2 were characterized by whole-genome sequencing. The identified mutations were genetically reconstituted in a wild-type background. The additive effect of mutations was studied in double mutants. Fitness costs, levels of resistance, and cross-resistance to another cyclotide, other peptide and nonpeptide antibiotics, and AMPs were determined. A variety of resistance mutations were identified. Some of these reduced fitness and others had no effect on fitness in vitro, in the absence of cyO2. In mouse competition experiments, four of the cyO2-resistant mutants showed a significant fitness advantage, whereas the effects of the mutations in the others appeared to be neutral. The level of resistance was increased by combining several individual resistance mutations. Several cases of cross-resistance and collateral sensitivity between cyclotides, other AMPs, and antibiotics were identified. These results show that resistance to cyclotides can evolve via several different types of mutations with only minor fitness costs and that these mutations often affect resistance to other AMPs.

Immunolocalization of cyclotides in plant cells, tissues and organ supports their role in host defense.

MAIN CONCLUSION: The distribution of cyclotides was visualized in plant cells, tissues and organs using immunohistochemistry. Finding of cyclotides in tissues potentially vulnerable to pathogen attacks supports their role as defense molecules. The cyclotide family of plant peptides is characterized by the cyclic cystine knot motif and its diverse biological activities. Given their insecticidal and antimicrobial properties, the role of cyclotides in planta is probably associated with host defense. Our current understanding of the cellular compartmentalization of cyclotides in the vacuole is based on indirect studies on transgenic model plants that do not express cyclotides naturally. Matrix-assisted laser desorption ionization (MALDI) imaging has also been used to study the distribution of cyclotides, but the technique's resolution was insufficient to determine their tissue or cell distribution. To avoid the limitations of these approaches, immunohistochemical visualization methods were used. Antibodies were raised in rabbits using cycloviolacin O2 (cyO2), and their specificity was determined by Western and dot blot experiments. Slides for immunohistochemical analysis were prepared from leaf, petiole and root fragments of Viola odorata and Viola uliginosa, and specimens were visualized using indirect epifluorescence microscopy. The antibodies against cyclotides were specific against selected bracelet cyclotides with high similarity (cyO2, cyO3, cyO8, cyO13) and suitable for immunohistochemistry. The tissue distribution of the cyclotides visualized in this way is consistent with their proposed role in host defense-relatively large quantities were observed in the leaf and petiole epidermis in both Viola species. Cyclotides were also found in vascular tissue in all the assessed plant organs. The vacuole storage of cyclotides was directly shown.

Pharmacoinformatics approaches in the discovery of drug-like antimicrobials of plant origin.

Medicinal plants have served as an important source for addressing the ailments of humans and animals alike. The emergence of advanced technologies in the field of drug discovery and development has helped in isolating various bioactive phytochemicals and developing them as drugs. Owing to their significant pharmacological benefits and minimum adverse effects, they not only serve as good candidates for therapeutics themselves but also help in the identification and development of related drug like molecules against various metabolic and infectious diseases. The ever-increasing diversity, severity and incidence of infectious diseases has resulted in an exaggerated mortality and morbidity levels. Geno-proteomic mutations in microbes, irrational prescribing of antibiotics, antimicrobial resistance and human population explosion, all call for continuous efforts to discover and develop alternated therapeutic options against the microbes. This review article describes the pharmacoinformatics tools and methods which are currently used in the discovery of bioactive phytochemicals, thus making the process more efficient and effective. The pharmacological aspects of the drug discovery and development process have also been reviewed with reference to the in silico activities. Communicated by Ramaswamy H. Sarma.

Nanoscopic-Assisted Anterior Cruciate Ligament-Posterior Cruciate Ligament Reconstruction.

Knee arthroscopy has allowed us to continue performing surgeries that are minimally invasive and allow patients to have a quick recovery. Multiligamentous knee reconstruction with regards to the anterior cruciate ligament and posterior cruciate ligament can be done in a minimally invasive matter. Visualization is an issue during this surgery, especially looking in the posterior compartment of the knee. The NanoScope (Arthrex, Naples, FL) continues to provide increased possibilities for orthopaedic surgeons. Our technique provides a less-invasive way to observe the posterior compartment to assist the drilling of the tibial tunnel during the posterior cruciate ligament reconstruction. This technique provides distinct advantages over other treatments.

Preoperative supine traction radiographs often result in higher Lenke classifications than supine bending radiographs in adolescent idiopathic scoliosis.

STUDY DESIGN: Retrospective chart and radiographic review. OBJECTIVE: The purpose of this study is to determine if both traction and side-bending radiographs yield the same Lenke classification. Supine side-bending radiographs are used to evaluate curve flexibility and assign Lenke classification in Adolescent Idiopathic Scoliosis (AIS). Supine traction radiographs are another tool used by treating surgeons to gauge flexibility and appropriate levels for spinal fusion in AIS. METHODS: Retrospective chart and radiographic review were performed on AIS patients that underwent a posterior spinal fusion from 2008 to 2017. Cobb angles and Lenke classifications were determined on all upright posterioanterior (PA) spine radiographs, supine traction radiographs, and four supine bending radiographs. Statistical analysis using independent t tests and chi-square tests as appropriate were compared between patients with or without discordant Lenke classifications with p value set at < 0.05 for statistical significance. RESULTS: 184 patients met inclusion criteria, 36 males and 148 females. The average Cobb angle for the proximal thoracic (PT) curve was 27.2°, main thoracic (MT) curve was 60.5°, and thoracolumbar/lumbar (TL/L) curve was 48.0°. Significantly less curve correction was found with supine traction radiographs compared with bending radiographs: PT (23.1° vs 18.9°, p < 0.001), MT (38.9° vs 37.9°, p = 0.015), and TL/L (25.9° vs. 18.0°, p < 0.001). Lenke Classification was found concordant in 151/184 (82.1%). Traction views in the discordant Lenke classification group demonstrated less curve correction than those in the concordant group: PT (27.4° vs. 22.1°, p = 0.011), MT (45.3° vs. 37.5°, p < 0.001), and TL/L (29.3° vs 25.1°, p = 0.019). CONCLUSION: Supine traction and supine bending radiographs provided a concordant Lenke classification 82.1% of the time. However, supine traction radiographs demonstrate less curve correction, a higher Lenke classification, and underestimated the TL/L curve correction to a greater degree. A single supine traction film is not an adequate substitute to side-bending radiographs when determining Lenke classification in patients with Adolescent Idiopathic Scoliosis. LEVEL OF EVIDENCE: III.