Delivered complementation in planta (DCIP) enables measurement of peptide-mediated protein delivery efficiency in plants.

Using a fluorescence complementation assay, Delivered Complementation in Planta (DCIP), we demonstrate cell-penetrating peptide-mediated cytosolic delivery of peptides and recombinant proteins in Nicotiana benthamiana. We show that DCIP enables quantitative measurement of protein delivery efficiency and enables functional screening of cell-penetrating peptides for in-planta protein delivery. Finally, we demonstrate that DCIP detects cell-penetrating peptide-mediated delivery of recombinantly expressed proteins such as mCherry and Lifeact into intact leaves. We also demonstrate delivery of a recombinant plant transcription factor, WUSCHEL (AtWUS), into N. benthamiana. RT-qPCR analysis of AtWUS delivery in Arabidopsis seedlings also suggests delivered WUS can recapitulate transcriptional changes induced by overexpression of AtWUS. Taken together, our findings demonstrate that DCIP offers a new and powerful tool for interrogating cytosolic delivery of proteins in plants and highlights future avenues for engineering plant physiology.

Supervised learning model predicts protein adsorption to carbon nanotubes.

Engineered nanoparticles are advantageous for biotechnology applications including biomolecular sensing and delivery. However, testing compatibility and function of nanotechnologies in biological systems requires a heuristic approach, where unpredictable protein corona formation prevents their effective implementation. We develop a random forest classifier trained with mass spectrometry data to identify proteins that adsorb to nanoparticles based solely on the protein sequence (78% accuracy, 70% precision). We model proteins that populate the corona of a single-walled carbon nanotube (SWCNT)­based nanosensor and study the relationship between the protein's amino acid­based properties and binding capacity. Protein features associated with increased likelihood of SWCNT binding include high content of solvent-exposed glycines and nonsecondary structure­associated amino acids. To evaluate its predictive power, we apply the classifier to identify proteins with high binding affinity to SWCNTs, with experimental validation. The developed classifier provides a step toward undertaking the otherwise intractable problem of predicting protein-nanoparticle interactions.

Nanoparticle cellular internalization is not required for RNA delivery to mature plant leaves.

Rapidly growing interest in the nanoparticle-mediated delivery of DNA and RNA to plants requires a better understanding of how nanoparticles and their cargoes translocate in plant tissues and into plant cells. However, little is known about how the size and shape of nanoparticles influence transport in plants and the delivery efficiency of their cargoes, limiting the development of nanotechnology in plant systems. In this study we employed non-biolistically delivered DNA-modified gold nanoparticles (AuNPs) of various sizes (5-20 nm) and shapes (spheres and rods) to systematically investigate their transport following infiltration into Nicotiana benthamiana leaves. Generally, smaller AuNPs demonstrated more rapid, higher and longer-lasting levels of association with plant cell walls compared with larger AuNPs. We observed internalization of rod-shaped but not spherical AuNPs into plant cells, yet, surprisingly, 10 nm spherical AuNPs functionalized with small-interfering RNA (siRNA) were the most efficient at siRNA delivery and inducing gene silencing in mature plant leaves. These results indicate the importance of nanoparticle size in efficient biomolecule delivery and, counterintuitively, demonstrate that efficient cargo delivery is possible and potentially optimal in the absence of nanoparticle cellular internalization. Overall, our results highlight nanoparticle features of importance for transport within plant tissues, providing a mechanistic overview of how nanoparticles can be designed to achieve efficacious biocargo delivery for future developments in plant nanobiotechnology.

Polymer-Conjugated Carbon Nanotubes for Biomolecule Loading.

Nanomaterials have emerged as an invaluable tool for the delivery of biomolecules such as DNA and RNA, with various applications in genetic engineering and post-transcriptional genetic manipulation. Alongside this development, there has been an increasing use of polymer-based techniques, such as polyethylenimine (PEI), to electrostatically load polynucleotide cargoes onto nanomaterial carriers. However, there remains a need to assess nanomaterial properties, conjugation conditions, and biocompatibility of these nanomaterial-polymer constructs, particularly for use in plant systems. In this work, we develop mechanisms to optimize DNA loading on single-walled carbon nanotubes (SWNTs) with a library of polymer-SWNT constructs and assess DNA loading ability, polydispersity, and both chemical and colloidal stability. Counterintuitively, we demonstrate that polymer hydrolysis from nanomaterial surfaces can occur depending on polymer properties and attachment chemistries, and we describe mitigation strategies against construct degradation. Given the growing interest in delivery applications in plant systems, we also assess the stress response of plants to polymer-based nanomaterials and provide recommendations for future design of nanomaterial-based polynucleotide delivery strategies.

Nanoparticles for protein delivery in planta.

Delivery of proteins into walled plant cells remains a challenge with few tractable solutions. Recent advances in biomacromolecule delivery using nanotechnology may evince methods to be exploited for protein delivery. While protein delivery remains no small feat, even in mammalian systems, the ability for nanoparticles to penetrate the cell wall and be decorated with a plethora of functional moieties makes them ideal protein vehicles in plants. As advances in protein biotechnology accelerate, so does the need for commensurate delivery systems. However, the road to nanoparticle-mediated protein delivery is fraught with challenges in regard to cell wall penetration, intracellular delivery, endosomal escape, and nanoparticle chemistry and design. The dearth of literature surrounding protein delivery in walled plant cells hints at the challenge of this problem but also indicates vast opportunity for innovations in plant-tailored nanotechnology.

Conception rates after fluoroscopy-guided fallopian tubal cannulation: an alternative to in vitro fertilization for patients with tubal occlusion.

OBJECTIVE: Previous studies show good technical success rates for fallopian tube recanalization. Scarce literature exists regarding advance techniques currently used by interventional radiologists during fallopian tube recanalization procedures. This study investigates the level of intervention and tubal patency and its association with technical success and associated pregnancy outcomes. METHODS: We retrospectively evaluated fallopian tube recanalization procedures performed at a single center in a 24-year period. A total of 160 couples undergoing a basic infertility evaluation were included. Hysterosalpingography with high pressure contrast injection followed by selective contrast, guidewire catheterization at the tubal ostium, and/or microcatheter/microwire recanalization were performed. Comparisons of the tubal fertilization rate by relevant characteristics were tested for statistical significance with t tests for continuous data or with Pearson chi-square tests for categorical data. RESULTS: Technical success rate was 94% (319 of 341 tubes). High pressure contrast injection alone (184 of 341, 54%), selective catheterization (40%), and microcatheter/microwire (6%) interventions yielded technical success rates of 98%, 90%, and 73%, respectively. The overall rate of conception was 35% (17 of 48). CONCLUSION: Current techniques of fallopian tube recanalization offer a desirable and safe option with high technical success for patients seeking treatment for infertility due to proximal fallopian tube obstruction.

Anticoagulation: a practical guide for strabismus surgeons.

An increasing number of surgical strabismus patients are taking oral anticoagulant and antiplatelet agents, with more diverse mechanisms of action than those used in the past. The decision as to whether to continue these drugs throughout the perioperative period is difficult and must be based on the balance between hemorrhagic and thrombotic risk. To help guide strabismus surgeons with clinical management in these cases, we review potential hemorrhagic complications of strabismus surgery and examine the use of anticoagulant and antiplatelet drugs during the perioperative period. Surgical strategies that might help minimize intraoperative hemorrhage in patients on anticoagulant therapy are also discussed.

One-sided triangle: A case of double vision.

A 51-year-old woman presented with acute diplopia and was found to have ptosis and complete bilateral external and internal ophthalmoplegia. She had normal reflexes and gait. Serological testing showed elevated levels of GQ1b ganglioside autoantibodies, making the diagnosis of Miller Fisher syndrome. This case illustrates an atypical presentation of the Miller Fisher variant of Guillain-Barre syndrome, which should be considered in all patients presenting with bilateral ophthalmoplegia.

Methicillin-Resistant Staphylococcus aureus: The Effects Are More Than Skin Deep.

OBJECTIVES: To assess the psychosocial effects of a methicillin-resistant Staphylococcus aureus (MRSA) diagnosis on the households of children with MRSA skin and soft tissue infection (SSTI). STUDY DESIGN: We constructed and administered an interview to the primary caregiver within the home of a child with a history of MRSA SSTI. RESULTS: Seventy-six households were enrolled. Survey responses were analyzed and grouped into 4 themes: health behavior changes, disclosure, social interactions, and knowledge/awareness. The most common theme was disclosure; 91% of participants reported sharing their child's MRSA diagnosis with someone outside of the household. Forty-two percent of respondents reported a change in the manner in which household contacts interacted as a result of the index patient's MRSA diagnosis, including isolating the index patient from other children in the household. Many households reported adopting enhanced personal hygiene behaviors and environmental cleaning routines. Thirty-eight percent of participating households reported altering how they interact with people outside of their home, largely to avoid spreading MRSA to vulnerable individuals. In addition, many participants perceived that others regarded them with caution, especially at daycare, whereas other affected households were excluded from family gatherings. CONCLUSION: Primary caregivers of children with MRSA SSTI reported changing their health behaviors, altering their interactions with people outside of their home, and feeling isolated by others in response to their child's MRSA diagnosis. The findings of our study highlight a need for community interventions and education to prevent the negative psychosocial repercussions associated with MRSA.

Diversity of Staphylococcus aureus strains colonizing various niches of the human body.

OBJECTIVES: As individuals may be colonized with multiple strains of Staphylococcus aureus at different body sites, the objectives of this study were to determine whether S. aureus polyclonal colonization exists within one body niche and the optimal sampling sites and culture methodology to capture the diversity of S. aureus strains in community-dwelling individuals. METHODS: Swabs were collected from the nares, axillae, and inguinal folds of 3 children with community-associated S. aureus infections and 11 household contacts, all with known S. aureus colonization. S. aureus isolates were recovered from each body niche using 4 culture methods and evaluated for polyclonality using phenotypic and genotypic strain characterization methodologies. RESULTS: Within individuals, the mean (range) number of phenotypes and genotypes was 2.4 (1-4) and 3.1 (1-6), respectively. Six (43%) and 10 (71%) participants exhibited phenotypic and genotypic polyclonality within one body niche, respectively. Broth enrichment yielded the highest analytical sensitivity for S. aureus recovery, while direct plating to blood agar yielded the highest genotypic strain diversity. CONCLUSIONS: This study revealed S. aureus polyclonality within a single body niche. Culture methodology and sampling sites influenced the analytical sensitivity of S. aureus colonization detection and the robustness of phenotypic and genotypic strain recovery.

Eosinophil localization to the basement membrane zone is autoantibody- and complement-dependent in a human cryosection model of bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by antibodies (IgG and IgE) targeting cell-substrate adhesion proteins. A variety of BP models suggest that autoantibody-dependent neutrophil degranulation is essential for blister formation. However, lesional biopsies reveal a predominance of eosinophils and few neutrophils. Our goal was to evaluate the role of antibodies and complement in eosinophil localization, degranulation and split formation at the dermo-epidermal junction (DEJ) utilizing a human skin cryosection model of BP paired with a human eosinophilic cell line, 15HL-60. Expression of receptors for IgG (FcγRII), IgE (FcεRI) and complement (CR1 and CR3) was confirmed on 15HL-60 cells using flow cytometry. 15HL-60 expression of granule protein [eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO)] mRNA and their degranulation in vitro was confirmed using RT-PCR and ELISA, respectively. For cryosection experiments, BP or control sera or IgG and IgE antibodies purified from BP sera were utilized in combination with 15HL-60 cells ± fresh complement. Both BP serum and fresh complement were required for localization of 15-HL60 cells to the DEJ. Interestingly, eosinophil localization to the DEJ was dependent on IgG, but not IgE, and complement. However, no subepidermal split was observed. Additionally, the 15HL-60 cells did not degranulate under any experimental conditions and direct application of cell lysate to cryosections did not result in a split. Our observation that eosinophil localization to the DEJ is dependent on IgG mediated complement fixation provides additional insight into the sequence of events during the development of BP lesions.

Antimicrobial Susceptibility Profiles of Staphylococcus aureus Isolates Recovered from Humans, Environmental Surfaces, and Companion Animals in Households of Children with Community-Onset Methicillin-Resistant S. aureus Infections.

Our objective was to determine the antibiotic susceptibility profiles of Staphylococcus aureus isolates recovered from 110 households of children with community-onset methicillin-resistant S. aureus (MRSA) infections. Cultures were obtained from household members, household objects, and dogs and cats, yielding 1,633 S. aureus isolates. The S. aureus isolates were heterogeneous, although more than half were methicillin resistant. The highest proportion of MRSA was found in bathrooms. The majority of isolates were susceptible to antibiotics prescribed in outpatient settings.

Evaluation of Environmental Sampling Methods for Detection of Staphylococcus aureus on Fomites.

We evaluated a variety of methods to recover S. aureus from inanimate surfaces. Two contact agar plates and three swab sampling methods were tested on porous and non-porous surfaces and bar soap. The cost and ease of use of each method was also evaluated. S. aureus was recovered using all methods on both porous and non-porous surfaces. S. aureus could not be detected on three of four brands of soap.

Vitamin D sufficiency and Staphylococcus aureus infection in children.

Vitamin D promotes epithelial immunity by upregulating antimicrobial peptides, including LL-37, which have bactericidal activity against Staphylococcus aureus. We found that children with vitamin D deficiency or insufficiency [25-hydroxyvitamin D <30 ng/mL] were more likely to present with recurrent, rather than primary, S. aureus skin or soft tissue infection. Vitamin D sufficiency may be one of a myriad of host and environmental factors that can be directly impacted to reduce the frequency of S. aureus skin and soft tissue infection.