Reconstitution of a biofilm adhesin system from a sulfate-reducing bacterium in Pseudomonas fluorescens.

Biofilms of sulfate-reducing bacterium (SRB) like Desulfovibrio vulgaris Hildenborough (DvH) can facilitate metal corrosion in various industrial and environmental settings leading to substantial economic losses. Although the mechanisms of biofilm formation by DvH are not yet well understood, recent studies indicate the large adhesin, DvhA, is a key determinant of biofilm formation. The dvhA gene neighborhood resembles the biofilm-regulating Lap system of Pseudomonas fluorescens but is curiously missing the c-di-GMP-binding regulator LapD. Instead, DvH encodes an evolutionarily unrelated c-di-GMP-binding protein (DVU1020) that we hypothesized is functionally analogous to LapD. To study this unusual Lap system and overcome experimental limitations with the slow-growing anaerobe DvH, we reconstituted its predicted SRB Lap system in a P. fluorescens strain lacking its native Lap regulatory components (ΔlapGΔlapD). Our data support the model that DvhA is a cell surface-associated LapA-like adhesin with a N-terminal "retention module" and that DvhA is released from the cell surface upon cleavage by the LapG-like protease DvhG. Further, we demonstrate DVU1020 (named here DvhD) represents a distinct class of c-di-GMP-binding, biofilm-regulating proteins that regulates DvhG activity in response to intracellular levels of this second messenger. This study provides insight into the key players responsible for biofilm formation by DvH, thereby expanding our understanding of Lap-like systems.

Microneedle Mediated Iontophoretic Delivery of Tofacitinib Citrate.

PURPOSE: To investigate in vitro transdermal delivery of tofacitinib citrate across human skin using microporation by microneedles and iontophoresis alone and in combination. METHODS: In vitro permeation studies were conducted using vertical Franz diffusion cells. Microneedles composed of polyvinyl alcohol and carboxymethyl cellulose were fabricated and successfully characterized using scanning electron microscopy. The microchannels created were further characterized using histology, dye binding study, scanning electron microscopy, and confocal microscopy studies. The effect of microporation on delivery of tofacitinib citrate was evaluated alone and in combination with iontophoresis. In addition, the effect of current density on iontophoretic delivery was also investigated. RESULTS: Total delivery of tofacitinib citrate via passive permeation was found out to be 11.04 ± 1 µg/sq.cm. Microporation with microneedles resulted in significant enhancement where a 28-fold increase in delivery of tofacitinib citrate was observed with a total delivery of 314.7±33.32 µg/sq.cm. The characterization studies confirmed the formation of microchannels in the skin where successful disruption of stratum corneum was observed after applying microneedles. Anodal iontophoresis at 0.1 and 0.5 mA/sq.cm showed a total delivery of 18.56 µg/sq.cm and 62.07 µg/sq.cm, respectively. A combination of microneedle and iontophoresis at 0.5 mA/sq.cm showed the highest total delivery of 566.59 µg/sq.cm demonstrating a synergistic effect. A sharp increase in transdermal flux was observed for a combination of microneedles and iontophoresis. CONCLUSION: This study demonstrates the use of microneedles and iontophoresis to deliver a therapeutic dose of tofacitinib citrate via transdermal route.

Enhanced Transdermal Delivery of N-Acetylcysteine and 4-Phenylbutyric Acid for Potential Use as Antidotes to Lewisite.

Lewisite is a highly toxic chemical warfare agent that leads to cutaneous and systemic damage. N-acetylcysteine (NAC) and 4-phenylbutryic acid (4-PBA) are two novel antidotes developed to treat toxicity caused by lewisite and similar arsenicals. Our in vivo studies demonstrated safety and effectiveness of these agents against skin injury caused by surrogate lewisite (Phenylarsine oxide) proving their potential for the treatment of lewisite injury. We further focused on exploring various enhancement strategies for an enhanced delivery of these agents via skin. NAC did not permeate passively from propylene glycol (PG). Iontophoresis as a physical enhancement technique and chemical enhancers were investigated for transdermal delivery of NAC. Application of cathodal and anodal iontophoresis with the current density of 0.2 mA/cm2 for 4 h followed by passive diffusion till 24 h significantly enhanced the delivery of NAC with a total delivery of 65.16 ± 1.95 µg/cm2 and 87.23 ± 7.02 µg/cm2, respectively. Amongst chemical enhancers, screened oleic acid, oleyl alcohol, sodium lauryl ether sulfate, and dimethyl sulfoxide (DMSO) showed significantly enhanced delivery of NAC with DMSO showing highest delivery of 28,370.2 ± 2355.4 µg/cm2 in 24 h. Furthermore, 4-PBA permeated passively from PG with total delivery of 1745.8 ± 443.5 µg/cm2 in 24 h. Amongst the chemical enhancers screened for 4-PBA, oleic acid, oleyl alcohol, and isopropyl myristate showed significantly enhanced delivery with isopropyl myristate showing highest total delivery of 17,788.7 ± 790.2 µg/cm2. These studies demonstrate feasibility of delivering these antidotes via skin and will aid in selection of excipients for the development of topical/transdermal delivery systems of these agents.

Effects of Sodium Salts of Fatty Acids and Their Derivatives on Skin Permeation of Cromolyn Sodium.

Atopic dermatitis is a chronic inflammatory disorder with rising prevalence. The safety concerns over usually used steroids are driving the need for developing an effective atopic dermatitis treatment. The use of therapeutic agents such as cromolyn sodium (CS) is suggested. However, due to its physicochemical properties, CS permeation across the skin is a challenge. The aim of this study was to investigate the effect of sodium salts of fatty acids or their derivatives with varied carbon chain lengths as potential enhancers on the skin permeation of CS. These included sodium caprylate, salcaprozate sodium, sodium decanoate, sodium palmitate, and sodium oleate dissolved in propylene glycol along with CS (4% w/w). In vitro permeation of the formulations across the dermatomed porcine ear skin was investigated over 24 h using Franz Diffusion cells. The amount of CS permeation from propylene glycol was 5.54 ± 1.06 µg/cm2 after 24 h. Initial screening of enhancers (enhancer: drug::1:1) showed enhancement in permeation of CS using sodium oleate and sodium caprylate, which were then investigated in higher ratio of drug: enhancer (1:2). Among all the formulations tested, sodium oleate (enhancer: drug::1:2) was observed to significantly (p < 0.05) enhance the permeation of CS with the highest total delivery of 359.79 ± 78.92 µg/cm2 across skin in 24 h and higher drug retention in the skin layers (153.0 ± 24.93 µg/cm2) as well. Overall, sodium oleate was found to be the most effective enhancer followed by sodium caprylate for improving the topical delivery of CS.

Development and Evaluation of a Topical Foam Formulation for Decontamination of Warfare Agents.

Lewisite is a highly toxic and potent chemical warfare vesicating agent capable of causing pain, inflammation, and blistering. Therapeutic strategies that safely and effectively attenuate this damage are important. Early and thorough decontamination of these agents from skin is required to prevent their percutaneous absorption. In our studies, we used phenylarsine oxide (PAO), a surrogate for arsenicals, to simulate lewisite exposure. Various parameters such as determination of extraction solvents, skin extraction efficiency, donor volume, and donor concentration were optimized for decontamination of PAO. We aimed to develop a novel, easy to apply foam formulation that can decontaminate arsenicals. We screened various foaming agents, vehicles, and chemical enhancers for the development of foam. Lead formulation foam F30 was further characterized for foam density, foam expansion, foam liquid stability, foam volume stability, and foam gas fraction. The amount of PAO delivered into human skin in 30 min of exposure was 228.57 ± 28.44 µg/sq·cm. The amount of PAO remaining in human skin after decontamination with blank foam F30 was 50.09 ± 9.71, demonstrating an overall percentage decontamination efficiency of over 75%. Furthermore, the decontamination efficacy of F30 was also tested in the porcine skin model and results indicated an even higher decontamination efficacy. These studies demonstrated that the developed foam formulation can be used for effective decontamination of chemical warfare agents.

Strategic optimization of conditions for the solubilization of GST-tagged amphipathic helix-containing ciliary proteins overexpressed as inclusion bodies in E. coli.

Expression of affinity-tagged recombinant proteins for crystallography, protein-protein interaction, antibody generation, therapeutic applications, etc. mandates the generation of high-yield soluble proteins. Although recent developments suggest the use of yeast, insect, and mammalian cell lines as protein expression platforms, Escherichia coli is still the most popular, due mainly to its ease of growth, feasibility in genetic manipulation and economy. However, some proteins have a spontaneous tendency to form inclusion bodies (IBs) when over-expressed in bacterial expression systems such as E. coli, thus posing a challenge in purification and yield. At times, small peptides undergo degradation during protein production and hence using suitable tags could circumvent the problem. Although several independent techniques have been used to solubilize IBs, these cannot always be applied in a generic sense. Although tagging a GST moiety is known to enhance the solubility of fusion proteins in E. coli, resulting in yields of 10-50 mg/L of the culture, the inherent nature of the protein sequence at times could lead to the formation of IBs. We have been working on a Myc Binding Protein-1 orthologue, viz. Flagellar Associated Protein 174 (FAP174) from the axoneme of Chlamydomonas reinhardtii that binds to an A-Kinase Anchoring Protein 240 (AKAP240) which has been annotated as Flagellar Associated Protein 65 (FAP65). Using an in-silico approach, we have identified two amphipathic helices on FAP65 (CrFAP65AH1 and CrFAP65AH2) that are predicted to bind to FAP174. To test this prediction, we have cloned the GST-tagged peptides, and overexpressed them in E. coli that have resulted in insoluble IBs. The yields of these over-expressed recombinant proteins dropped considerably due to IB formation, indicating aggregation. An integrated approach has been used to solubilize four highly hydrophobic polypeptides, viz. two amphipathic helices and the respective proline variants of FAP65. For solubilizing these polypeptides, variables such as non-denaturing detergents (IGEPAL CA-630), changing the ionic strength of the cell lysis and solubilization buffer, addition of BugBuster®, diluting the cell lysate and sonication were introduced. Our statistically viable results yielded highly soluble and functional polypeptides, indiscreet secondary structures, and a yield of ~ 20 mg/L of the E. coli culture. Our combinatorial strategy using chemical and physical methods to solubilize IBs could prove useful for hydrophobic peptides and proteins with amphipathic helices.

Biomechanical and histomorphometric analysis of endosteal implants placed by using the osseodensification technique in animal models: A systematic review and meta-analysis.

STATEMENT OF PROBLEM: Osseodensification, a counterclockwise drilling technique for the placement of endosseous implants is a popular clinical technique. However, the effect of the osseodensification technique on primary implant stability, bone-implant contact, and bone area frequency occupancy is unclear. PURPOSE: The purpose of this systematic review and meta-analysis was to investigate the biomechanical and histomorphometric outcomes of endosteal implants placed by using the osseodensification technique in animal models. MATERIAL AND METHODS: An electronic search through Medline/PubMed, Lilacs, and Science Direct databases, and an additional manual search of the reference list of included articles was conducted by using specific keywords and Medical Subject Headings (MeSH) terms for articles in the English language and published up to April 31, 2020. Only animal studies comparing the biomechanical and histomorphometric outcomes of endosteal implants placed by using the osseodensification and conventional drilling protocol were included. The SYstematic Review Center for Laboratory animal Experimentation (SYRCLE) tool was used to determine the risk of bias assessment, and the quality of included studies was assessed by using Animal Research: Reporting in Vivo Experiments (ARRIVE) guidelines. RESULTS: Nine studies were included. The results of the meta-analysis showed that the pooled weighted mean difference of the insertion torque value for the primary implant stability of endosseous dental implants placed by using the osseodensification technique was 2.270 (95% confidence interval [CI]=1.147 to 3.393; P<.001), the weighted mean difference of the percentage of bone-implant contact at 3 weeks was 0.487 (95% CI=0.220 to 0.754; P=.114), the weighted mean difference of the percentage of bone-implant contact at 6 weeks was 0.565 (95% CI=0.219 to 0.911; P=.448), the weighted mean difference of the percentage of bone area frequency occupancy at 3 weeks was 0.679 (95% CI=0.265 to 1.093; P=.073), and the weighted mean difference of the percentage of bone area frequency occupancy at 6 weeks was 0.391 (95% CI=-0.204 to 0.986; P=.027). CONCLUSIONS: Limited data from animal studies suggest that the primary implant stability, bone-implant contact, and bone area frequency occupancy significantly improved for the endosteal implants placed by using the osseodensification technique compared with conventional drilling protocol. However, additional laboratory and clinical studies are recommended to provide stronger evidence.

Impact of paternal presence and parental social-demographic characteristics on birth outcomes.

OBJECTIVES: Maternal race, marital status, and social environment impact risk of preterm delivery and size for gestational age. Although some paternal characteristics such as age are associated with pregnancy outcomes, the influence of the paternal presence, race/ethnicity and adverse life events is not well known. The objective of the study was to assess birth outcomes in mothers with a paternal presence compared to those without during the post-partum period. The secondary aim was to determine whether paternal race is associated with birth outcomes. METHODS: This was a cross-sectional study using parental surveys linked with birth certificate data from 2016 to 2018. Adverse birth composite outcomes (ABCO) including small for gestational age (SGA), prematurity or neonatal intensive care unit admission (NICU) were assessed. RESULTS: A total of 695 parents were analyzed (239 single mothers and 228 mother-father pairs). Compared to mothers with a father present, mothers without a father present exhibited increased odds of ABCO, prematurity and NICU. Non-Hispanic Black fathers had increased odds of ABCO and NICU compared to Non-Hispanic Whites (NHW). Hispanic fathers had increased odds of NICU compared to NHW. CONCLUSIONS: Paternal absence in the post-partum period and paternal race were both independently associated with ABCO and NICU. Assessment of paternal presence and paternal race in clinical practice may help identify opportunities for additional support necessary to optimize birth outcomes.

Autogenous bone ring for vertical bone augmentation procedure with simultaneous implant placement: A systematic review of histologic and histomorphometric outcomes in animal studies.

STATEMENT OF PROBLEM: The performance of the autogenous bone ring (ABR) technique used for the vertical bone augmentation procedure has not yet been determined. PURPOSE: The purpose of this systematic review was to investigate whether the ABR technique for the vertical bone augmentation procedure with simultaneous implant placement enhances the histologic and histomorphometric outcomes in surgically created bone defects in animal models. MATERIAL AND METHODS: An electronic search of 3 databases, Medline/PubMed, Science Direct, Lilacs, and a manual search of a reference list of relevant studies were performed. Only animal studies regarding vertical bone augmentation by using the ABR technique with simultaneous implant placement in surgically created bone defects were included in the review. The SYstematic Review Center for Laboratory Animal Experimentation tool was used to determine the risk of bias assessment, and the quality of included studies was assessed by using the Animal Research: Reporting in Vivo Experiments guidelines. RESULTS: Five studies were included in the qualitative analysis based on the inclusion and exclusion criteria. One study comparing the ABR technique with the bovine and biphasic ring reported a high percentage of bone area and bone implant contact of the ABR at 5 weeks of osseointegration. Similar histologic findings were reported in another study at 4 months of healing, but contrasting data were reported at 8 months. Another study reported an increase in the percentage of bone area and bone implant contact of the ABR technique from 3 months to 6 months of osseointegration. Two studies reported an increase in the percentage of bone area, and 1 study reported a decrease in the mean of bone implant contact, both with simultaneous and staged implant procedures. CONCLUSION: Based on the limited available studies, the use of the ABR technique for the vertical bone augmentation procedure with simultaneous implant placement presented optimal histologic and histomorphometric outcomes in surgically created bone defects in animal models. However, the results of the current review are not sufficiently robust to support the use of the ABR technique for vertical bone augmentation in humans.

Comparison of Plaque Removal and Wear between Charcoal Infused Bristle and Nylon Bristle Toothbrushes: A Randomized Clinical Crossover Study.

AIM: To compare plaque removal and wear between charcoal infused bristle toothbrushes (T1) and nylon bristle toothbrushes (T2) in a randomized clinical crossover study. MATERIALS AND METHODS: A cross-over study was conducted in 2 phases of 6 weeks duration each with an intervening 2-week washout. Twenty-five participants meeting inclusion criteria were randomly allocated into groups A (13) and B (12). In phase 1: group A was assigned T1 and group B was assigned T2. Toothbrushing was advised twice daily for 2 minutes by modified bass technique after meals. At baseline, 3 weeks and 6 weeks the wear index (WI), plaque index (PI) and gingival index (GI) were recorded. Following washout in phase 2 group A was assigned T2 and group B was assigned T1 and the same study protocol was followed. RESULTS: Intra-group comparison between baseline, 3 and 6 weeks by the paired t-test resulted in significant reduction in PI, GI and increase in WI (p <0.05) for T1 and T2. Inter-group comparison using the unpaired t-test resulted in WI for T1 being significantly higher (p <0.05) at 3 weeks and lower at 6 weeks (p <0.05) compared to T2. PI for T1 was significantly higher at 3 weeks (p <0.05) and lower at 6 weeks (p <0.05) compared to T2. No significant difference in GI scores between T1 and T2 at 3 and 6 weeks was observed (p >0.05). CONCLUSION: Charcoal infused bristles demonstrated less wear and more plaque removal compared to nylon bristles. CLINICAL SIGNIFICANCE: Charcoal infused bristles demonstrate less wear compared to nylon bristles.

A-kinase anchoring proteins are enriched in the central pair microtubules of motile cilia in Chlamydomonas reinhardtii.

Cilia are microtubule-based sensory organelles present in a number of eukaryotic cells. Mutations in the genes encoding ciliary proteins cause ciliopathies in humans. A-kinase anchoring proteins (AKAPs) tether ciliary signaling proteins such as protein kinase A (PKA). The dimerization and docking domain (D/D) on the RIIα subunit of PKA interacts with AKAPs. Here, we show that AKAP240 from the central-pair microtubules of Chlamydomonas reinhardtii cilia uses two C-terminal amphipathic helices to bind to its partner FAP174, an RIIα-like protein with a D/D domain at the N-terminus. Co-immunoprecipitation using anti-FAP174 antibody with an enriched central-pair microtubule fraction isolated seven interactors whose mass spectrometry analysis revealed proteins from the C2a (FAP65, FAP70, and FAP147) and C1b (CPC1, HSP70A, and FAP42) microtubule projections and FAP75, a protein whose sub-ciliary localization is unknown. Using RII D/D and FAP174 as baits, we identified two additional AKAPs (CPC1 and FAP297) in the central-pair microtubules.

Highly Efficient Carbon Dioxide Electroreduction via DNA-Directed Catalyst Immobilization.

Electrochemical reduction of carbon dioxide (CO2) is a promising route to up-convert this industrial byproduct. However, to perform this reaction with a small-molecule catalyst, the catalyst must be proximal to an electrode surface. Efforts to immobilize molecular catalysts on electrodes have been stymied by the need to optimize the immobilization chemistries on a case-by-case basis. Taking inspiration from nature, we applied DNA as a molecular-scale "Velcro" to investigate the tethering of three porphyrin-based catalysts to electrodes. This tethering strategy improved both the stability of the catalysts and their Faradaic efficiencies (FEs). DNA-catalyst conjugates were immobilized on screen-printed carbon and carbon paper electrodes via DNA hybridization with nearly 100% efficiency. Following immobilization, a higher catalyst stability at relevant potentials is observed. Additionally, lower overpotentials are required for the generation of carbon monoxide (CO). Finally, high FE for CO generation was observed with the DNA-immobilized catalysts as compared to the unmodified small-molecule systems, as high as 79.1% FE for CO at -0.95 V vs SHE using a DNA-tethered catalyst. This work demonstrates the potential of DNA "Velcro" as a powerful strategy for catalyst immobilization. Here, we demonstrated improved catalytic characteristics of molecular catalysts for CO2 valorization, but this strategy is anticipated to be generalizable to any reaction that proceeds in aqueous solutions.

Metal nanoparticles as inhibitors of enzymes and toxins of multidrug-resistant Staphylococcus aureus.

Staphylococcus aureus is an aerobic Gram-positive spherical bacterium known to cause a broad range of infections worldwide. It is a major cause of infective skin and soft infections and severe and life-threatening conditions, such as pneumonia, bloodstream infections, and endocarditis. The emergence of drug-resistant strains of S aureus, particularly methicillin-resistant S aureus (MRSA), has become a significant concern in the healthcare community. Antibiotic-resistant S aureus is commonly acquired in hospitals and long-term care facilities. It often affects patients with weakened immune systems, those undergoing invasive medical procedures, or those who have been hospitalized for extended periods. In the US, S aureus is known to cause potentially fatal illnesses, such as toxic shock syndrome (TSS) and acute-onset toxic shock syndrome (TSS), which are characterized by fever and hypotension. It develops resistance to antibiotics through several mechanisms, such as the production of enzymes that inactivate antibiotics, target site modification, efflux pumps, and plasmid-mediated resistance. Therefore, preventing the spread of drug-resistant S aureus is needed, and there is an urgent need to explore novel approaches in the development of anti-staphylococcal agents. This article reviews the principal infections caused by S aureus, major virulence factors, mechanisms of resistance development, and nanotechnology-based solutions for the control of drug-resistant S aureus.

Vasculature organotropism in drug delivery.

Over the past decades, there has been an exponential increase in the development of preclinical and clinical nanodelivery systems, and recently, an accelerating demand to deliver RNA and protein-based therapeutics. Organ-specific vasculature provides a promising intermediary for site-specific delivery of nanoparticles and extracellular vesicles to interstitial cells. Endothelial cells express organ-specific surface marker repertoires that can be used for targeted delivery. This article highlights organ-specific vasculature properties, nanodelivery strategies that exploit vasculature organotropism, and overlooked challenges and opportunities in targeting and simultaneously overcoming the endothelial barrier. Impediments in the clinical translation of vasculature organotropism in drug delivery are also discussed.

Transdermal delivery of naloxone hydrochloride using minimally invasive physical ablation techniques.

NAL's hydrophilicity and the inherent lipophilic properties of the stratum corneum hinders its capacity for immediate delivery through skin in opioid rescue cases. In this study, we had sought to investigate the feasibility of using minimally invasive physical ablative techniques including sonophoresis, laser, dermaplaning, microneedles, and microdermabrasion for systemically delivering NAL via the skin. These techniques reduced lag time to NAL delivery to about 3-12 min from 71.22 ± 9.62 min seen for passive delivery. Also, they all significantly enhanced the amount of NAL delivered in 1 h and over 24 h period of evaluation as compared to the passive group (p < 0.05). Sonophoresis and laser showed the greatest delivery in 1 h, followed by dermaplaning. The cumulative amount of drug delivered by these approaches in 1 h were 1277.95 ± 387.06, 83.33 ± 11.11, 30.66 ± 5.67 µg/cm2, respectively. Though the most remarkable, inconsistencies in in vitro permeation profile of NAL were observed with the 1 MHz ultrasound frequency used. With proper optimization of the conditions of use and design, the different approaches explored in this study can be potentially applied for the systemic delivery of naloxone in opioid overdose emergencies and opioid disaccustoming purposes.

Reconstitution of a Biofilm Adhesin System from a Sulfate-Reducing Bacterium in Pseudomonas fluorescens.

Biofilms of the sulfate reducing bacterium (SRB) Desulfovibrio vulgaris Hildenborough (DvH) can facilitate metal corrosion in various industrial and environmental settings leading to substantial economic losses; however, the mechanisms of biofilm formation by DvH are not yet well-understood. Evidence suggests that a large adhesin, DvhA, may be contributing to biofilm formation in DvH. The dvhA gene and its neighbors encode proteins that resemble the Lap system, which regulates biofilm formation by Pseudomonas fluorescens, including a LapG-like protease DvhG and effector protein DvhD, which has key differences from the previously described LapD. By expressing the Lap-like adhesion components of DvH in P. fluorescens, our data support the model that the N-terminal fragment of the large adhesin DvhA serves as an adhesin "retention module" and is the target of the DvhG/DvhD regulatory module, thereby controlling cell-surface location of the adhesin. By heterologously expressing the DvhG/DvhD-like proteins in a P. fluorescens background lacking native regulation (ΔlapGΔlapD) we also show that cell surface regulation of the adhesin is dependent upon the intracellular levels of c-di-GMP. This study provides insight into the key players responsible for biofilm formation by DvH, thereby expanding our understanding of Lap-like systems.