In Vitro Regional Deposition of Nasal Sprays in an Idealized Nasal Inlet: Comparison with In Vivo Gamma Scintigraphy.

PURPOSE: To compare in vitro regional nasal deposition measurements using an idealized nasal airway geometry, the Alberta Idealized Nasal Inlet (AINI), with in vivo regional deposition for nasal drug products. MATERIALS AND METHODS: One aqueous solution formulation (NasalCrom), one aqueous suspension formulation (Nasonex) and one nasal pressurized metered dose spray device (QNASL) were selected. Two spray orientation angles, 60° and 45° from the horizontal, were selected. A steady inhalation flow rate of 7.5 L/min was selected to simulate slow inhalation through a single nostril. After actuation, the AINI was disassembled. The mass of drug deposited in each region and a downstream filter, representing penetration of drug to the lungs, was determined using ultraviolet-visible (UV-Vis) spectrophotometry. RESULTS: No filter (lung) deposition was detected for NasalCrom or Nasonex. Filter deposition ranged from 6 to 11% for QNASL. For NasalCrom, 45% to 69% of the dose deposited in the AINI was deposited in the vestibule and 31% to 55% was deposited in the turbinates; for Nasonex, 66% to 74% (vestibule) and 26% to 34% (turbinates); for QNASL, 90% to 100% (vestibule) and 0% to 10% (turbinates). No statistically significant difference was observed between regional deposition in vivo and in vitro for any of the formulations, except that nasopharyngeal deposition with Nasonex differed by less than 1.56% from in vivo, which while statistically significant, is unlikely to be clinically significant. CONCLUSIONS: The AINI was able to mimic regional in vivo deposition for nasal drug products, permitting differentiation between devices based on regional deposition.

Intranasal delivery of a synthetic Entamoeba histolytica vaccine containing adjuvant (LecA + GLA-3 M-052 liposomes): In vitro characterization.

Amebiasis, a disease caused by the parasite Entamoeba histolytica, is estimated to cause millions of infections and at least 55,000 deaths globally each year. With no vaccine currently available, there is an urgent need for an accessible means of stimulating protective mucosal immunity. The objective of this study was to characterize the nasal spray of a novel amebiasis vaccine candidate from a syringe-based liquid atomization device, the Teleflex MAD Nasal™, in both adult and infant nasal airways. Human ergonomic testing was completed to determine realistic actuation parameters. Spray pattern, plume geometry, and droplet size distribution were measured to evaluate reproducibility of free plume characteristics. The Alberta Idealized Nasal Inlet (AINI) and three realistic infant nasal airways were used to determine the in vitro deposition profile in adult and infant airways, respectively. Collectively, in vitro results demonstrated the feasibility of delivering the vaccine candidate to target sites within the nasal airways. Penetration through the nasal airways that could lead to deposition in the lungs was below the limit of quantification for both adult and infant geometries, indicating a low likelihood of adverse events due to lung exposure. These results support continued investigation of intranasal delivery of the synthetic Entamoeba histolytica vaccine.

Evolution of ß-lactamases and enzyme promiscuity.

ß-Lactamases represent one of the most prevalent resistance mechanisms against ß-lactam antibiotics. Beyond their clinical importance, they have also become key models in enzymology and evolutionary biochemistry. A global understanding of their evolution and sequence and functional diversity can therefore aid a wide set of different disciplines. Interestingly, ß-lactamases have evolved multiple times from distinct evolutionary origins, with ancestries that reach back billions of years. It is therefore no surprise that these enzymes exhibit diverse structural features and enzymatic mechanisms. In this review, we provide a bird's eye view on the evolution of ß-lactamases within the two enzyme superfamilies-i.e. the penicillin-binding protein-like and metallo-ß-lactamase superfamily-through phylogenetics. We further discuss potential evolutionary origins of each ß-lactamase class by highlighting signs of evolutionary connections in protein functions between ß-lactamases and other enzymes, especially cases of enzyme promiscuity.

Inhaled Nitric Oxide: In Vitro Analysis of Continuous Flow Noninvasive Delivery via Nasal Cannula.

BACKGROUND: Inhaled nitric oxide (NO) is most frequently delivered to mechanically ventilated patients in critical care, but it can also be administered noninvasively. The delivered dose and efficiency of continuous flow NO supplied through a nasal cannula has yet to be established. This study aimed to determine the influence of nasal cannula type, supply flow, and breathing pattern on delivered NO using a realistic adult airway replica and lung simulator. METHODS: Simulated breathing patterns were selected to represent rest, sleep, and light exercise, and were varied to investigate the effects of tidal volume and breathing frequency independently. Supplied gas flows targeted tracheal concentrations at rest of 5 or 20 ppm NO and were supplied with 2 L/min O2. Three different cannulas were tested. Tracheal NO concentrations and NO mass flow past the trachea were evaluated. RESULTS: Cannula type had a minor influence on delivered dose. Tracheal NO concentrations differed significantly based on breathing pattern (P < 0.01); for a target NO concentration of 20 ppm at rest, average inhaled NO concentrations were 23.3 ± 0.5 ppm, 36.5 ± 1.4 ppm, and 17.2 ± 0.3 ppm for the rest, sleep, and light exercise breathing patterns, respectively. For the same test conditions, mass flow of NO past the trachea was less sensitive to breathing pattern: 20.3 ± 0.5 mg/h, 19.9 ± 0.8 mg/h, and 24.3 ± 0.4 mg/h for the rest, sleep, and light exercise breathing patterns, respectively. Mass flow and delivery efficiency increased when minute volume increased. CONCLUSIONS: These results indicate that inhaled NO concentration is strongly influenced by breathing pattern, whereas inhaled NO mass flow is not. NO mass flow may therefore be a useful dose metric for continuous flow delivery via nasal cannula.

Statistical analysis of mutational epistasis to reveal intramolecular interaction networks in proteins.

Epistasis occurs when the combined effect of two or more mutations differs from the sum of their individual effects, and reflects molecular interactions that affect the function and fitness of a protein. Epistasis is widely recognized as a key phenomenon that drives the dynamics of evolution. It can profoundly affect our ability to understand sequence-structure-function relationships, and thus has important implications for protein engineering and design. Characterizing higher-order epistasis, i.e., interactions between three or more mutations, can unveil hidden intramolecular interaction networks that underlie essential protein functions and their evolution. For this chapter, we developed an analytical pipeline that can standardize the study of intramolecular epistasis. We describe the generation and characterization of a combinatorial library, the statistical analysis of mutational epistasis, and finally, the depiction of epistatic networks on the 3D structure of a protein. We anticipate that this pipeline will benefit the increasing number of scientists that are interested in the functional characterization of mutational libraries to provide a deeper understanding of the molecular mechanisms of protein evolution.

Comprehensive exploration of the translocation, stability and substrate recognition requirements in VIM-2 lactamase.

Metallo-ß-lactamases (MBLs) degrade a broad spectrum of ß-lactam antibiotics, and are a major disseminating source for multidrug resistant bacteria. Despite many biochemical studies in diverse MBLs, molecular understanding of the roles of residues in the enzyme's stability and function, and especially substrate specificity, is lacking. Here, we employ deep mutational scanning (DMS) to generate comprehensive single amino acid variant data on a major clinical MBL, VIM-2, by measuring the effect of thousands of VIM-2 mutants on the degradation of three representative classes of ß-lactams (ampicillin, cefotaxime, and meropenem) and at two different temperatures (25°C and 37°C). We revealed residues responsible for expression and translocation, and mutations that increase resistance and/or alter substrate specificity. The distribution of specificity-altering mutations unveiled distinct molecular recognition of the three substrates. Moreover, these function-altering mutations are frequently observed among naturally occurring variants, suggesting that the enzymes have continuously evolved to become more potent resistance genes.

In vitro assessment of an idealized nose for nasal spray testing: Comparison with regional deposition in realistic nasal replicas.

An idealized nasal replica that mimics average regional deposition of nasal spray pump droplets in human nasal airways would potentially be useful in expediting the development of nasal spray products. The aim of this study was to validate an idealized nose, previously developed using in silico simulations, by comparing with regional deposition in realistic, sectioned nasal replicas obtained from in vitro deposition experiments. The realistic nasal airway replicas of five subjects obtained from computerized tomography were manufactured in plastic using rapid prototyping. The idealized nose was made using the same build procedure. A commercial nasal spray pump (NasalCrom, 5.2 mg cromolyn sodium per spray) was then actuated repeatably into each replica under a steady inspiratory flow of 7.5 L/min at two different orientations (45° and 60° from the horizontal). Sectioned replicas were disassembled, and the mass fraction of drug deposited on the surface of each anatomical region was determined. It was found that regional deposition of spray droplets in the idealized replica agreed well with average regional deposition in the realistic replicas. Regional deposition also agreed with previously published in vivo regional deposition using the same spray pump.

Assessing the effectiveness of artificial intelligence methods for melanoma: A retrospective review.

BACKGROUND: Artificial intelligence methods for the classification of melanoma have been studied extensively. However, few studies compare these methods under the same standards. OBJECTIVE: To seek the best artificial intelligence method for diagnosis of melanoma. METHODS: The contrast test used 2200 dermoscopic images. Image segmentations, feature extractions, and classifications were performed in sequence for evaluation of traditional machine learning algorithms. The recent popular convolutional neural network frameworks were used for transfer learning training classification. RESULTS: The region growing algorithm has the best segmentation performance, with an intersection over union of 70.06% and a false-positive rate of 17.67%. Classification performance was better with logistic regression, with a sensitivity of 76.36% and a specificity of 87.04%. The Inception V3 model (Google, Mountain View, CA) worked best in deep learning algorithms: the accuracy was 93.74%, the sensitivity was 94.36%, and the specificity was 85.64%. LIMITATIONS: There was no division in the severity of melanoma samples used in this experiment. The data set was relatively small for deep learning. CONCLUSION: The performance of traditional machine learning is satisfactory for the small data set of melanoma dermoscopic images, and the potential for deep learning in the future big data era is enormous.

In Vitro-In Silico Comparison of Pulsed Oxygen Delivery From Portable Oxygen Concentrators Versus Continuous Flow Oxygen Delivery.

BACKGROUND: Portable oxygen concentrators (POCs) deliver oxygen in intermittent pulses. The challenge of establishing equivalence between continuous flow oxygen and nominal pulse flow settings on different POCs is well known. In vitro bench measurements and in silico mathematical modeling were used to compare the performance of 4 POCs versus continuous flow oxygen by predicting the FIO2 at the trachea and entering the acini. METHODS: Each of the 4 POCs was connected to a 3-dimensional printed replica of a human adult nasal airway via nasal cannula. A test lung simulated 3 breathing patterns representative of a patient with COPD at rest, during exercise, and while asleep. POCs were tested for each breathing pattern at all integer pulse flow settings. Volume-averaged FIO2 was calculated by analyzing oxygen concentrations and inhalation flow over time. In vitro oxygen waveforms were then combined with a single-path mathematical model of the lungs to assess oxygen transport through the conducting airways. In vitro experiments and mathematical modeling were repeated for continuous flow oxygen. RESULTS: Continuous flow oxygen consistently delivered more (>2% absolute) oxygen in terms of volume-averaged FIO2 for all nominally equivalent pulse flow settings of >2. Differences were also observed when comparing performances between different POCs, particularly at high device settings (5 and 6). Simulations showed that efficiency of delivery to the acinar region of the lungs was higher in pulse flow than in continuous flow oxygen but that continuous flow oxygen generally delivered a higher absolute volume of oxygen. Differences in absolute oxygen delivery per breath between continuous flow oxygen and pulse flow were smaller for acinar delivery than for tracheal delivery. CONCLUSIONS: Significant differences in POC performance based on volume-averaged FIO2 were found between pulse flow and continuous flow oxygen, and among pulse flow modes in different POCs. Although pulse flow was a more efficient mode of delivery than continuous flow oxygen, continuous flow oxygen delivered a greater absolute volume of oxygen per breath.

Pulmonary Delivery of Therapeutic and Diagnostic Gases.

The 21st Congress for the International Society for Aerosols in Medicine included, for the first time, a session on Pulmonary Delivery of Therapeutic and Diagnostic Gases. The rationale for such a session within ISAM is that the pulmonary delivery of gaseous drugs in many cases targets the same therapeutic areas as aerosol drug delivery, and is in many scientific and technical aspects similar to aerosol drug delivery. This article serves as a report on the recent ISAM congress session providing a synopsis of each of the presentations. The topics covered are the conception, testing, and development of the use of nitric oxide to treat pulmonary hypertension; the use of realistic adult nasal replicas to evaluate the performance of pulsed oxygen delivery devices; an overview of several diagnostic gas modalities; and the use of inhaled oxygen as a proton magnetic resonance imaging (MRI) contrast agent for imaging temporal changes in the distribution of specific ventilation during recovery from bronchoconstriction. Themes common to these diverse applications of inhaled gases in medicine are discussed, along with future perspectives on development of therapeutic and diagnostic gases.

Comparison of pulsed versus continuous oxygen delivery using realistic adult nasal airway replicas.

BACKGROUND: Portable oxygen concentrators (POCs) typically include pulse flow (PF) modes to conserve oxygen. The primary aims of this study were to develop a predictive in vitro model for inhaled oxygen delivery using a set of realistic airway replicas, and to compare PF for a commercial POC with steady flow (SF) from a compressed oxygen cylinder. METHODS: Experiments were carried out using a stationary compressed oxygen cylinder, a POC, and 15 adult nasal airway replicas based on airway geometries derived from medical images. Oxygen delivery via nasal cannula was tested at PF settings of 2.0 and 6.0, and SF rates of 2.0 and 6.0 L/min. A test lung simulated three breathing patterns representative of a chronic obstructive pulmonary disease patient at rest, during exercise, and while asleep. Volume-averaged fraction of inhaled oxygen (FiO2) was calculated by analyzing oxygen concentrations sampled at the exit of each replica and inhalation flow rates over time. POC pulse volumes were also measured using a commercial O2 conserver test system to attempt to predict FiO2 for PF. RESULTS: Relative volume-averaged FiO2 using PF ranged from 68% to 94% of SF values, increasing with breathing frequency and tidal volume. Three of 15 replicas failed to trigger the POC when used with the sleep breathing pattern at the 2.0 setting, and four of 15 replicas failed to trigger at the 6.0 setting. FiO2 values estimated from POC pulse characteristics followed similar trends but were lower than those derived from airway replica experiments. CONCLUSION: For the POC tested, PF delivered similar, though consistently lower, volume-averaged FiO2 than SF rates equivalent to nominal PF settings. Assessment of PF oxygen delivery using POC pulse characteristics alone may be insufficient; testing using airway replicas is useful in identifying possible cases of failure and may provide a better assessment of FiO2.

Treatment of infraorbital dark circles using 694-nm fractional Q-switched ruby laser.

The objective of this study was to evaluate the efficacy and safety of using a 694-nm fractional Q-switched ruby laser to treat infraorbital dark circles. Thirty women with infraorbital dark circles (predominant color: dark/brown) participated in this open-labeled study. The participants received eight sessions of 694-nm fractional Q-switched ruby laser treatment using a fluence of 3.0-3.5 J/cm2, at an interval of 7 days. The melanin deposition in the lesional skin was observed in vivo using reflectance confocal microscopy (RCM). The morphological changes were evaluated using a global evaluation, an overall self-assessment, and a Mexameter. Twenty-eight of the 30 patients showed global improvements that they rated as excellent or good. Twenty-six patients rated their overall satisfaction as excellent or good. The melanin index indicated a substantial decrease from 240.44 (baseline) to 194.56 (P < 0.05). The RCM results showed a dramatic decrease in melanin deposition in the upper dermis. The adverse effects were minimal. The characteristic finding of dark/brown infraorbital dark circles is caused by increased melanin deposition in the upper dermis. The treatment of these infraorbital dark circles using a 694-nm fractional QSR laser is safe and effective.

Split-face comparison of ultrapulse-mode and superpulse-mode fractionated carbon dioxide lasers on photoaged skin.

BACKGROUND: Both ultrapulse-mode and superpulse-mode fractional CO2 lasers (UPCO2 and SPCO2) could be successfully used in treating photoaged skin. OBJECTIVE: This evidence-based study was intended to compare the therapeutic and adverse effects of UPCO2 and SPCO2 in treating photoaged skin in Chinese subjects. METHODS: Eighteen Chinese subjects with Fitzpatrick skin type IV were enrolled in a randomized, split-face trial. Subjects received SPCO2 on one half of the face and UPCO2 on the other half. Before and after photos, skin color, epidermal water content, sebum level, periorbital wrinkles, skin roughness, and self-esteem questionnaires were used. RESULTS: Global evaluation and subjects' self-esteem assessments showed a similar trend at 1-month and 3-month follow-up visits on both sides. The UPCO2 laser has a shorter downtime of 6.25±2.71 days compared with 6.41±2.67 days for SPCO2, but has a higher incidence of edema, spot bleeding, prolonged redness and postinflammatory hyperpigmentation. More subjects prefer SPCO2 treatment because of similar efficacy and fewer adverse effects. CONCLUSION: The effectiveness of the SPCO2 laser in treating photoaged skin is very similar to the UPCO2 laser, with less erythema, but more crusting and longer downtime.

Treatment of psoriasis with interleukin-12/23 monoclonal antibody: a systematic review.

OBJECTIVE: To systematically review the efficacy and safety of interleukin-12/23 monoclonal antibody (IL-12/23 mAb) on psoriasis. METHODS: Relevant randomized controlled trials (RCTs) were identified by systematic literature searches in MEDLINE, OVID, EMBASE, Cochrane Library, and the metaRegister of Controlled Trials. The efficacy outcomes and adverse effects of included RCTs were critically assessed. RESULTS: A total of 3365 participants in 5 multicenter RCTs were included. The RRs of most efficacy outcomes showed significant differences between i) IL-12/23 mAb and placebo at week 12/16; ii) IL-12/23 mAb and etanercept at week 12; iii) IL-12/23 mAb in high dose and IL-12/23 mAb in low dose at week 24/28. Increasing treatment times did not obviously provide additional benefit to efficacy improvement. The adverse events of IL-12/23 mAb were similar to those of controls. Antibodies to IL-12/23 mAb were mostly undetected or shown at low titer. Treatment with IL-12/23 mAb did not influence related biochemical markers. CONCLUSIONS: IL-12/23 mAb was effective in the treatment of psoriasis on skin lesions, health-related quality of life and psoriatic arthritis in the short-term. The increase in treatment time points was not associated with additional efficacy and dose-dependence was observed with the ongoing treatment up to week 24/28. The adverse effects were minimal and tolerable.

Split-face study of topical 23.8% L-ascorbic acid serum in treating photo-aged skin.

BACKGROUND: L-ascorbic acid has been widely used to treat photo-aged skin. However, its aqueous formula is prone to oxidation. Therefore, a new formula that contains 23.8% L-ascorbic acid and a chemical penetration enhancer was developed. OBJECTIVE: Observe the efficacy and safety of topical 23.8% L-ascorbic acid serum on photo-aged skin. METHODS: Twenty Chinese women with photo-aged skin were enrolled in this split-face study. They were treated with topical L-ascorbic acid serum with iontophoresis on one side of the face once a day for 2 weeks; the other side of the face was spared treatment through participants' self-control. Changes in photo-aged skin were evaluated using a global evaluation, an overall self-assessment, a spectrophotometer, the phase-shift rapid in vivo measurement of skin (PRIMOS) 3D, and a corneometer. RESULTS: Sixteen of 20 patients (80%) experienced a score decrease of 2 or 3 grades, according to the dermatologist. Fifteen patients (75%) rated their overall satisfaction as excellent or good. Dyspigmentation, surface roughness, and fine lines on the treated side improved significantly. CONCLUSION: Topical 23.8% L-ascorbic acid serum is effective for the treatment of photo-aged skin and does not cause any obvious side effects.

A retrospective study of q-switched alexandrite laser in treating nevus of ota.

BACKGROUND: The Q-switched alexandrite laser (QSAL) has been clinically proven to be effective in treating nevus of Ota, but a large-scale retrospective study with long-term follow-up has never been performed. OBJECTIVE: To evaluate the efficacy of the QSAL in treating nevus of Ota, the laser's long-term side effects, complications associated with the treatments, and the recurrence rate. MATERIALS AND METHODS: Eight hundred six patients (651 female, 155 male) with a diagnosis of nevus of Ota who had received a series of QSAL (wavelength 755 nm) treatments were recruited. The typical settings were fluences of 3.8 to 4.8 J/cm(2) and a spot size of 3 mm. Follow-ups were conducted via questionnaire with 590 patients who had completed the treatment at least 3 years earlier. RESULTS: Overall, 93.9% of patients achieved complete clearance after an average of 5.2 sessions. All recurrences (0.8%) appeared beyond the previously treated sites. No long-term adverse effects, including hypopigmentation, hyperpigmentation, textural changes, and malignant transformation, were observed. CONCLUSION: The QSAL is a safe and effective modality for treating nevus of Ota. Recurrence was rare and appeared beyond the previously treated sites. The authors have indicated no significant interest with commercial supporters.

BRAF exon 15 T1799A mutation is common in melanocytic nevi, but less prevalent in cutaneous malignant melanoma, in Chinese Han.

Frequent somatic mutations of BRAF (v-raf murine sarcoma viral oncogene homolog B) exon T1799A, which are implicated in the initial events of promutagenic cellular proliferation, are detected in both malignant melanomas (MM) and melanocytic nevi (MN). Most of the data regarding BRAF exon T1799A mutation have been from Caucasian cohorts, and a comprehensive screening of a homogeneous population is lacking. A total of 379 cases of MN and 195 cases of MM were collected from Chinese Han living in three geographical regions in China, i.e., northeast, southwest, and northwest China. BRAF exon T1799A mutation was detected by PCR and sequencing from microdissected tumors. In all, 59.8% cases of MN harbored BRAF exon T1799A mutation. Samples from regions with high UV exposure had higher detection rates than regions with lower UV exposure (73.5, 67.0, and 38.9%, respectively; χ(2) = 31.674, P = 1.59E-7). There were no differences in mutation rates between congenital and acquired MN; however, acquired MN with advanced age of onset had a higher mutation rate than those with younger age of onset (χ(2) = 13.23, P = 0.02). In all, 15.0% cases of MM harbored the BRAF mutation. The mutation rate in MM was not affected by region, histological type, gender, pattern of UV exposure, and age. The study suggests that the mutation is not necessarily associated with malignant transformation.

A Chinese experience of fractional ultrapulsed CO2laser for skin rejuvenation.

BACKGROUND AND OBJECTIVES: The ablative fractional CO2 laser has been successfully used in treating photoaged skin in the Caucasian population. However, its application in Asian skin has not been widely reported. The purpose of this study is to observe the efficacy and safety of the protocol 'ActiveFX' for photodamaged facial skin in Chinese patients. METHODS: A non-sequential fractional ultrapulsed CO2 laser with specific settings is used in addition to a new computer pattern generator (CPG). Twenty patients received a single-session, single-pass, ablative fractional treatment on a split face. The patients were evaluated at baseline, and 1 and 3 months (M1, M3) after the treatment using a quartile grading scale. They were also asked to assess their overall satisfaction using a 4-point scale. The improvement of the coarse wrinkles was also quantitatively analyzed with a 3D in vivo imaging system. RESULTS: At M1 and M3, the blinded investigator rated global improvement as 2.8 ± 0.95 and 3.00 ± 0.73, which was consistent with the patients' assessment (2.55 ± 0.83 at M1 and 3.15 ± 0.88 at M3). The roughness analysis (Rz) demonstrated a significant decrease in periorbital wrinkles (p < 0.0001). Minimal and reversible adverse side effects and rapid healing were noted. CONCLUSIONS: Non-sequential fractional ultrapulsed CO2 laser resurfacing (ActiveFX) is considered an excellent treatment modality for photodamaged Chinese facial skin.