Development of a porous layer-by-layer microsphere with branched aliphatic hydrocarbon porogens.

Porous polymer microspheres are employed in biotherapeutics, tissue engineering, and regenerative medicine. Porosity dictates cargo carriage and release that are aligned with the polymer physicochemical properties. These include material tuning, biodegradation, and cargo encapsulation. How uniformity of pore size affects therapeutic delivery remains an area of active investigation. Herein, we characterize six branched aliphatic hydrocarbon-based porogen(s) produced to create pores in single and multilayered microspheres. The porogens are composed of biocompatible polycaprolactone, poly(lactic-co-glycolic acid), and polylactic acid polymers within porous multilayered microspheres. These serve as controlled effective drug and vaccine delivery platforms.

The dental curing light: A potential health risk.

Powerful blue-light emitting dental curing lights are used in dental offices to photocure resins in the mouth. In addition, many dental personnel use magnification loupes. This study measured the effect of magnification loupes on the "blue light hazard" when the light from a dental curing light was reflected off a human tooth. Loupes with 3.5x magnification (Design for Vision, Carl Zeiss, and Quality Aspirator) and 2.5x magnification (Design for Vision and Quality Aspirator) were placed at the entrance of an integrating sphere connected to a spectrometer (USB 4000, Ocean Optics). A model with human teeth was placed 40 cm away and in line with this sphere. The light guide tip of a broad-spectrum Sapphire Plus (Den-Mat) curing light was positioned at a 45° angle from the facial surface of the central incisor. The spectral radiant power reflected from the teeth was recorded five times with the loupes over the entrance into the sphere. The maximum permissible cumulative exposure times in an 8-hr day were calculated using guidelines set by the ACGIH. It was concluded that at a 40 cm distance, the maximum permissible cumulative daily exposure time to light reflected from the tooth was approximately 11 min without loupes. The weighted blue irradiance values were significantly different for each brand of loupe (Fisher's PLSD p < 0.05) and were up to eight times greater at the pupil than when loupes were not used. However, since the linear dimensions of the resulting images would be 2.5 to 3.5x larger on the retina, the image area was increased by the square of the magnification and the effective blue light hazard was reduced compared to without the loupes. Thus, although using magnification loupes increased the irradiance received at the pupil, the maximum cumulative daily exposure time to reflected light was increased up to 28 min. Further studies are required to determine the ocular hazards of a focused stare when using magnification loupes and the effects of other curing lights used in the dental office.

Endosomal trafficking of nanoformulated antiretroviral therapy facilitates drug particle carriage and HIV clearance.

UNLABELLED: Limitations of antiretroviral therapy (ART) include poor patient adherence, drug toxicities, viral resistance, and failure to penetrate viral reservoirs. Recent developments in nanoformulated ART (nanoART) could overcome such limitations. To this end, we now report a novel effect of nanoART that facilitates drug depots within intracellular compartments at or adjacent to the sites of the viral replication cycle. Poloxamer 407-coated nanocrystals containing the protease inhibitor atazanavir (ATV) were prepared by high-pressure homogenization. These drug particles readily accumulated in human monocyte-derived macrophages (MDM). NanoATV concentrations were ∼1,000 times higher in cells than those that could be achieved by the native drug. ATV particles in late and recycling endosome compartments were seen following pulldown by immunoaffinity chromatography with Rab-specific antibodies conjugated to magnetic beads. Confocal microscopy provided cross validation by immunofluorescent staining of the compartments. Mathematical modeling validated drug-endosomal interactions. Measures of reverse transcriptase activity and HIV-1 p24 levels in culture media and cells showed that such endosomal drug concentrations enhanced antiviral responses up to 1,000-fold. We conclude that late and recycling endosomes can serve as depots for nanoATV. The colocalization of nanoATV at endosomal sites of viral assembly and its slow release sped antiretroviral activities. Long-acting nanoART can serve as a drug carrier in both cells and subcellular compartments and, as such, can facilitate viral clearance. IMPORTANCE: The need for long-acting ART is significant and highlighted by limitations in drug access, toxicity, adherence, and reservoir penetrance. We propose that targeting nanoformulated drugs to infected tissues, cells, and subcellular sites of viral replication may improve clinical outcomes. Endosomes are sites for human immunodeficiency virus assembly, and increasing ART concentrations in such sites enhances viral clearance. The current work uncovers a new mechanism by which nanoART can enhance viral clearance over native drug formulations.

Novel KDM6A (UTX) mutations and a clinical and molecular review of the X-linked Kabuki syndrome (KS2).

We describe seven patients with KDM6A (located on Xp11.3 and encodes UTX) mutations, a rare cause of Kabuki syndrome (KS2, MIM 300867) and report, for the first time, germ-line missense and splice-site mutations in the gene. We demonstrate that less than 5% cases of Kabuki syndrome are due to KDM6A mutations. Our work shows that similar to the commoner Type 1 Kabuki syndrome (KS1, MIM 147920) caused by KMT2D (previously called MLL2) mutations, KS2 patients are characterized by hypotonia and feeding difficulties during infancy and poor postnatal growth and short stature. Unlike KS1, developmental delay and learning disability are generally moderate-severe in boys but mild-moderate in girls with KS2. Some girls may have a normal developmental profile. Speech and cognition tend to be more severely affected than motor development. Increased susceptibility to infections, join laxity, heart, dental and ophthalmological anomalies are common. Hypoglycaemia is more common in KS2 than in KS1. Facial dysmorphism with KDM6A mutations is variable and diagnosis on facial gestalt alone may be difficult in some patients. Hypertrichosis, long halluces and large central incisors may be useful clues to an underlying KDM6A mutation in some patients.

Pharmacokinetics, biodistribution, and toxicity of folic acid-coated antiretroviral nanoformulations.

The drug delivery platform for folic acid (FA)-coated nanoformulated ritonavir (RTV)-boosted atazanavir (FA-nanoATV/r) using poloxamer 407 was developed to enhance cell and tissue targeting for a range of antiretroviral drugs. Such formulations would serve to extend the drug half-life while improving the pharmacokinetic profile and biodistribution to reservoirs of human immunodeficiency virus (HIV) infection. To this end, we now report enhanced pharmacokinetics and drug biodistribution with limited local and systemic toxicities of this novel nanoformulation. The use of FA as a targeting ligand for nanoATV/r resulted in plasma and tissue drug concentrations up to 200-fold higher compared to equimolar doses of native drug. In addition, ATV and RTV concentrations in plasma from mice on a folate-deficient diet were up to 23-fold higher for mice administered FA-nanoATV/r than for mice on a normal diet. Compared to earlier nanoATV/r formulations, FA-nanoATV/r resulted in enhanced and sustained plasma and tissue ATV concentrations. In a drug interaction study, ATV plasma and tissue concentrations were up to 5-fold higher in mice treated with FA-nanoATV/r than in mice treated with FA-nanoATV alone. As observed in mice, enhanced and sustained plasma concentrations of ATV were observed in monkeys. NanoATV/r was associated with transient local inflammation at the site of injection. There were no systemic adverse reactions associated with up to 10 weeks of chronic exposure of mice or monkeys to FA-nanoATV/r.

Development of an extended action fostemsavir lipid nanoparticle.

An extended action fostemsavir (FTR) lipid nanoparticle (LNP) formulation prevents human immunodeficiency virus type one (HIV-1) infection. This FTR formulation establishes a drug depot in monocyte-derived macrophages that extend the drug's plasma residence time. The LNP's physicochemical properties improve FTR's antiretroviral activities, which are linked to the drug's ability to withstand fluid flow forces and levels of drug cellular internalization. Each is, in measure, dependent on PEGylated lipid composition and flow rate ratios affecting the size, polydispersity, shape, zeta potential, stability, biodistribution, and antiretroviral efficacy. The FTR LNP physicochemical properties enable the drug-particle's extended actions.

Fixed prosthodontics provisional materials: making the right selection.

Clinicians have many choices when selecting an appropriate material for interim restorations for both single crowns and multi-units. Interim restorations serve as a diagnostic as well as biologic and biomechanical component of fixed prosthodontics treatment; in the anterior, they are also important in evaluating the esthetics for the definitive restoration. Factors to be considered when choosing provisional materials are physical properties, handling characteristics, patient response to the appearance of the interim restoration, durability of the restoration, and the cost of the material. Practitioners should, therefore, base their choice on the clinical needs for each situation.

Cephalometric evaluation of deep overbite correction using anterior bite turbos.

OBJECTIVES: To evaluate the outcome of treating deep overbite (OB) using anterior bite elevators concurrently with a pre-adjusted edgewise appliance. MATERIALS AND METHODS: The Case Western Reserve University (CWRU) cephalometric analysis was used to isolate tipping movement of upper (TUI) and lower incisors (TLI), bodily tooth movement of upper (BUI), and lower incisors (BLI), as well as vertical skeletal changes in the anterior region of the maxilla (MXSK) and mandible (MNSK). Thirty treated subjects were examined at pretreatment (T1) and posttreatment (T2) and compared to an untreated control group matched on age, sex, and Angle malocclusion from the Bolton Brush Growth Study Collection (CWRU, Cleveland, Ohio). RESULTS: Overbite (OB) in the treated group was decreased significantly (P < .001) (-5.6 mm) compared to controls. Statistically significant (P < .001) changes were found for BUI (-0.7 mm), TUI (0.9 mm), TLI (-1.4 mm), BLI (-1.1 mm), and MNSK (-1.6 mm). Most of the overbite correction was in the lower arch and included tipping and intrusion of the lower incisors along with an increase in lower vertical facial height. CONCLUSIONS: Deep OB correction was achieved efficiently using anterior bite elevators with pre-adjusted edgewise appliance. Correction using bite turbos would be a treatment option for individuals presenting with decreased lower facial height and deep bite.

Activated Charcoal Dental Products: Evidence of Effectiveness Is Lacking.

With society trending toward the avoidance of artificial components, so-called "natural" products have been gaining space and people's attention in recent years. Activated charcoal-based dental products are a prominent example of this movement because of their promise of removal of extrinsic stains or whitening of teeth by a natural means. Such products have gained popularity among patients, and companies have explored this market, launching charcoal-based and activated charcoal-based dentifrices, mouthrinses, toothbrushes, and whitening products that can be easily found at nearby stores and on the internet.1.

Development of an extended half-life GM-CSF fusion protein for Parkinson's disease.

Transformation of CD4+ T cell effector to regulatory (Teff to Treg) cells have been shown to attenuate disease progression by restoring immunological balance during the onset and progression of neurodegenerative diseases. In our prior studies, we defined a safe and effective pathway to restore this balance by restoring Treg numbers and function through the daily administration of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). These studies were conducted as a proof-of-concept testing in Parkinson's disease (PD) preclinical models and early phase I clinical investigations. In both instances, they served to ameliorate disease associated signs and symptoms. However, despite the recorded efficacy, the cytokine's short half-life, low bioavailability, and injection site reactions proved to be limitations for any broader use. To overcome these limitations, mRNA lipid nanoparticles encoding an extended half-life albumin-GM-CSF fusion protein were developed for both mouse (Msa-GM-CSF) and rat (Rsa-GM-CSF). These formulations were tested for immunomodulatory and neuroprotective efficacy using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and human wild-type alpha-synuclein (αSyn) overexpression preclinical models of PD. A single dose of the extended half-life mouse and rat mRNA lipid nanoparticles generated measurable GM-CSF plasma cytokine levels up to four days. Increased Treg frequency and function were associated with a resting microglial phenotype, nigrostriatal neuroprotection, and restoration of brain tissue immune homeostasis. These findings were substantively beyond the recorded efficacy of daily recombinant wild-type GM-CSF with a recorded half-life of six hours. Mechanistic evaluation of neuropathological transcriptional profiles performed in the disease-affected nigral brain region demonstrated an upregulation of neuroprotective CREB and synaptogenesis signaling and neurovascular coupling pathways. These findings highlight the mRNA-encoded albumin GM-CSF fusion protein modification linked to improvements in therapeutic efficacy. The improvements achieved were associated with the medicine's increased bioavailability. Taken together, the data demonstrate that mRNA LNP encoding the extended half-life albumin-GM-CSF fusion protein can serve as a benchmark for PD immune-based therapeutics. This is especially notable for improving adherence of drug regimens in a disease-affected patient population with known tremors and gait abnormalities.

Chairside resin-based provisional restorative materials for fixed prosthodontics.

Provisional restorations are vital to fixed prosthodontics treatment, providing an important diagnostic function while in place. In addition to protecting the prepared teeth, provisionalization enables clinicians to refine biologic and biomechanical issues before the final restoration is fabricated. Adjustments can be made in the provisional restoration to achieve both the clinician's and patient's desired results. The fabrication of temporary restorations requires that clinicians be proficient with a variety of materials and techniques that can be used to make well-adapted and functional provisionals. There are many material choices available to temporize a single crown as well as multi-unit fixed partial dentures, and the selection of provisional materials should be made based on a case-by-case evaluation. This article provides a review of polymeric resin provisional materials.

CRISPR-Cas9 Mediated Exonic Disruption for HIV-1 Elimination.

BACKGROUND: A barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high HIV-1 mutation rate leads to viral diversity, immune evasion, and consequent antiretroviral drug resistance. While CRISPR-spCas9 can eliminate latent proviral DNA, its efficacy is limited by HIV strain diversity and precision target cell delivery. METHODS: A library of guide RNAs (gRNAs) designed to disrupt five HIV-1 exons (tat1-2/rev1-2/gp41) was constructed. The gRNAs were derived from a conseensus sequence of the transcriptional regulator tat from 4004 HIV-1 strains. Efficacy was affirmed by gRNA cell entry through transfection, electroporation, or by lentivirus or lipid nanoparticle (LNP) delivery. Treated cells were evaluated for viral excision by monitoring HIV-1 DNA, RNA, protein, and progeny virus levels. FINDINGS: Virus was reduced in all transmitted founder strains by 82 and 94% after CRISPR TatDE transfection or lentivirus treatments, respectively. No recorded off-target cleavages were detected. Electroporation of TatDE ribonucleoprotein and delivery of LNP TatDE gRNA and spCas9 mRNA to latently infected cells resulted in up to 100% viral excision. Protection against HIV-1-challenge or induction of virus during latent infection, in primary or transformed CD4+ T cells or monocytes was achieved. We propose that multi-exon gRNA TatDE disruption delivered by LNPs enables translation for animal and human testing. INTERPRETATION: These results provide "proof of concept' for CRISPR gRNA treatments for HIV-1 elimination. The absence of full-length viral DNA by LNP delivery paired with undetectable off-target affirms the importance of payload delivery for effective viral gene editing. FUNDING: The work was supported by the University of Nebraska Foundation, including donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, and individual donor support from the Frances and Louie Blumkin Foundation and from Harriet Singer. The research received support from National Institutes of Health grants T32 NS105594, 5R01MH121402, 1R01Al158160, R01 DA054535, PO1 DA028555, R01 NS126089, R01 NS36126, PO1 MH64570, P30 MH062261, and 2R01 NS034239.

Suppression of Ionic Doping by Molecular Dopants in Conjugated Polymers for Improving Specificity and Sensitivity in Biosensing Applications.

Ionic doping effects in conjugated polymers often cause nonspecific signaling and a low selectivity of bioelectronic sensing. Using remote-gate field-effect transistor characterization of molecular and ionic doping in poly(3-hexylthiophene) (P3HT) and acid-functionalized polythiophene, poly[3-(3-carboxypropyl) thiophene-2,5-diyl] (PT-COOH), we discovered that proton doping effects on the interfacial potential occurring in P3HT could be suppressed by sequentially doping P3HT by 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (F4TCNQ). To be specific, intrinsic pH sensitivity shown by pure P3HT (18 mV/pH in a range from pH 3 to 9) was fully dissipated for doped P3HT:F4TCNQ. However, F4TCNQ sequential doping instead increases pH sensitivity of acid-functionalized polythiophene, PT-COOH (40 mV/pH), compared to that of a pure PT-COOH (30 mV/pH). Interactions between polythiophene backbone and side chains, which constrain the activity of COOH, are weakened by stronger F4TCNQ doping leaving behind responsive COOH groups exposed to aqueous solutions. This is supported by the reduced pH sensitivity of PT-COOH sequentially doped by a weaker dopant, tetracyanoethylene (TCNE) (21 mV/pH). Thus, doping is shown to stabilize a nonpolar conjugated polymer to pH-induced fluctuations on one hand, and to activate a COOH side chain to pH-induced response on the other.

The Natural History, Pathobiology, and Clinical Manifestations of SARS-CoV-2 Infections.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (COVID-19). SARS-CoV-2, is a positive-sense single-stranded RNA virus with epithelial cell and respiratory system proclivity. Like its predecessor, SARS-CoV, COVID-19 can lead to life-threatening disease. Due to wide geographic impact affecting an extremely high proportion of the world population it was defined by the World Health Organization as a global public health pandemic. The infection is known to readily spread from person-to-person. This occurs through liquid droplets by cough, sneeze, hand-to-mouth-to-eye contact and through contaminated hard surfaces. Close human proximity accelerates SARS-CoV-2 spread. COVID-19 is a systemic disease that can move beyond the lungs by blood-based dissemination to affect multiple organs. These organs include the kidney, liver, muscles, nervous system, and spleen. The primary cause of SARS-CoV-2 mortality is acute respiratory distress syndrome initiated by epithelial infection and alveolar macrophage activation in the lungs. The early cell-based portal for viral entry is through the angiotensin-converting enzyme 2 receptor. Viral origins are zoonotic with genomic linkages to the bat coronaviruses but without an identifiable intermediate animal reservoir. There are currently few therapeutic options, and while many are being tested, although none are effective in curtailing the death rates. There is no available vaccine yet. Intense global efforts have targeted research into a better understanding of the epidemiology, molecular biology, pharmacology, and pathobiology of SARS-CoV-2. These fields of study will provide the insights directed to curtailing this disease outbreak with intense international impact. Graphical Abstract.

Initial experience and outcome of a new hemodialysis access device for catheter-dependent patients.

OBJECTIVE: The effects of a new long-term subcutaneous vascular access device were studied in access-challenged patients who were poor candidates for fistulas or grafts due to venous obstruction. Bacteremia rates, patency, and function of the Hemodialysis Reliable Outflow (HeRO) Vascular Access Device (Hemosphere Inc, Minneapolis, Minn) were evaluated. METHODS: The HeRO device consists of a 6-mm expanded polytetrafluoroethylene graft attached to a 5-mm nitinol-reinforced silicone outflow component designed to bypass venous stenoses and enter the internal jugular vein directly, providing continuous arterial blood flow into the right atrium. The HeRO device was studied in a multicenter clinical trial to test the hypothesis that access-challenged patients would experience a statistically significant reduction in bacteremia rates compared with a tunneled dialysis catheter (TDC) literature control of 2.3/1000 days. HeRO-related bacteremia rates, adequacy of dialysis, patency, and adverse events were analyzed. RESULTS: The HeRO device was implanted in 36 access-challenged patients who were followed for a mean 8.6 months (9931 HeRO days). The HeRO-related bacteremia rate was 0.70/1000 days. All HeRO-related bacteremias occurred during the bridging period when a TDC was still implanted before HeRO graft incorporation. HeRO adequacy of dialysis (mean Kt/V) was 1.7. HeRO primary patency was 38.9%, and secondary patency was 72.2%. CONCLUSIONS: In access-challenged patients, a statistically significant reduction in HeRO-related bacteremia was noted compared with TDC literature. The device had similar function and patency compared with conventional arteriovenous graft literature.

In vitro interactions between splenocytes and dansylamide dye-embedded nanoparticles detected by flow cytometry.

Engineered nanoparticles (NPs) possess a range of biological activity. In vitro methods for assessing toxicity and efficacy would be enhanced by simultaneous quantitative information on the behavior of NPs in culture systems and signals of cell response. We have developed a method for visualizing NPs within cells using standard flow-cytometric techniques and uniquely designed spherical siloxane NPs with an embedded (covalently bound) dansylamide dye. This method allowed NP visualization without obscuring detection of relevant biomarkers of cell subtype, activation state, and other events relevant to assessing bioactivity. We determined that NPs penetrated cells and induced a range of biological signals consistent with activation and costimulation. These results indicate that NPs may affect cell function at concentrations below those inducing cytotoxicity or apoptosis and demonstrate a novel method to image both localization of NPs and cell-level effects. FROM THE CLINICAL EDITOR: A method for visualizing NPs within cells using standard flow-cytometric techniques is reported in this paper. The novel method allowed NP visualization without obscuring detection of relevant biomarkers of cell subtype, activation state, and other events relevant to assessing bioactivity. NPs also induced a range of biological signals consistent with activation and costimulation.

Current Concepts and Best Evidence on Strategies to Prevent Dental Erosion.

Dental erosion is a multifactorial condition associated with chemical, biological, and behavioral factors whereby a non-bacterial chemical process leads to an irreversible loss of dental structure. Consequences of this erosive process include painful sensitivity, susceptibility to further erosion, mechanical wear, changes in occlusion, exposure of dental pulp, and poor esthetics. Substantial evidence has revealed new insights to diagnosing early stages of dental erosion and enabling novel preventive approaches to control its progression. In the context of outpatient medical/dental practice, clinicians often encounter patients with progressive dental erosion. This article summarizes published research in this area of dentistry to suggest guidelines that are clinically oriented but scientifically fundamental. It is aimed at helping clinicians effectively integrate this information into their professional evaluations of dental erosion with regard to diagnosis, risk factors, clinical signs, assessment, and clinical preventive strategies and treatment. Clinicians should address patient diet habits, educate patients on prevalence data, and inform them regarding potential acidic interactions, such as medically induced acidic conditions, that may ultimately lead to tooth destruction. Prevention of dental erosion, including the recognition of initial erosive lesions and the implementation of the early intervention, involves the clinical expertise of both the dentist and physician.

A Humid-Air-Operable, NO2-Responsive Polymer Transistor Series Circuit with Improved Signal-to-Drift Ratio Based on Polymer Semiconductor Oxidation.

A subparts per million-sensitive nitrogen dioxide (NO2) sensing circuit with improved humid air stability was realized incorporating UV-ozone treatment on a poly(bisdodecylquaterthiophene)/polystyrene blend film. The circuit consisted of a pair of organic field-effect transistors (OFETs) in series, one OFET with and one without this treatment. In contrast to most previous OFET sensors, the readout was obtained from the voltage Vout at a point between the OFETs. The circuit showed a low detection limit (200 ppb) toward NO2 and greatly reduced the voltage drift in the humid environment compared to the current drift of the circuit or the individual OFETs because of the balance of conductance drifts on either side of the readout point, which differs from the existing OFET-based sensors. By using Vout as the detection parameter, the sensitivity of the circuit approaches 25 and 400% for NO2 concentrations of 200 ppb and 20 ppm, respectively. Moreover, the Vout is substantial enough to be easily measured by a voltmeter, which could remove the need for complex equipment (semiconductor analyzer system) for the sensing test. We thus demonstrate a simplified approach to stabilized OFET circuits that could be used in printable, flexible, or wearable sensors.

Synthesis of a long acting nanoformulated emtricitabine ProTide.

While antiretroviral therapy (ART) has revolutionized treatment and prevention of human immunodeficiency virus type one (HIV-1) infection, regimen adherence, viral mutations, drug toxicities and access stigma and fatigue are treatment limitations. These have led to new opportunities for the development of long acting (LA) ART including implantable devices and chemical drug modifications. Herein, medicinal and formulation chemistry were used to develop LA prodrug nanoformulations of emtricitabine (FTC). A potent lipophilic FTC phosphoramidate prodrug (M2FTC) was synthesized then encapsulated into a poloxamer surfactant (NM2FTC). These modifications extended the biology, apparent drug half-life and antiretroviral activities of the formulations. NM2FTC demonstrated a >30-fold increase in macrophage and CD4+ T cell drug uptake with efficient conversion to triphosphates (FTC-TP). Intracellular FTC-TP protected macrophages against an HIV-1 challenge for 30 days. A single intramuscular injection of NM2FTC, at 45 mg/kg native drug equivalents, into Sprague Dawley rats resulted in sustained prodrug levels in blood, liver, spleen and lymph nodes and FTC-TP in lymph node and spleen cells at one month. In contrast, native FTC-TPs was present for one day. These results are an advance in the transformation of FTC into a LA agent.