Spinal Basis of Direction Control during Locomotion in Larval Zebrafish.

Navigation requires steering and propulsion, but how spinal circuits contribute to direction control during ongoing locomotion is not well understood. Here, we use drifting vertical gratings to evoke directed "fictive" swimming in intact but immobilized larval zebrafish while performing electrophysiological recordings from spinal neurons. We find that directed swimming involves unilateral changes in the duration of motor output and increased recruitment of motor neurons, without impacting the timing of spiking across or along the body. Voltage-clamp recordings from motor neurons reveal increases in phasic excitation and inhibition on the side of the turn. Current-clamp recordings from premotor interneurons that provide phasic excitation or inhibition reveal two types of recruitment patterns. A direction-agnostic pattern with balanced recruitment on the turning and nonturning sides is primarily observed in excitatory V2a neurons with ipsilateral descending axons, while a direction-sensitive pattern with preferential recruitment on the turning side is dominated by V2a neurons with ipsilateral bifurcating axons. Inhibitory V1 neurons are also divided into direction-sensitive and direction-agnostic subsets, although there is no detectable morphologic distinction. Our findings support the modular control of steering and propulsion by spinal premotor circuits, where recruitment of distinct subsets of excitatory and inhibitory interneurons provide adjustments in direction while on the move.SIGNIFICANCE STATEMENT Spinal circuits play an essential role in coordinating movements during locomotion. However, it is unclear how they participate in adjustments in direction that do not interfere with coordination. Here we have developed a system using larval zebrafish that allows us to directly record electrical signals from spinal neurons during "fictive" swimming guided by visual cues. We find there are subsets of spinal interneurons for coordination and others that drive unilateral asymmetries in motor neuron recruitment for direction control. Our findings suggest a modular organization of spinal premotor circuits that enables uninterrupted adjustments in direction during ongoing locomotion.

Determination of electric and thermoelectric properties of molecular junctions by AFM in peak force tapping mode.

Molecular thin films, such as self-assembled monolayers (SAMs), offer the possibility of translating the optimised thermophysical and electrical properties of high-Seebeck-coefficient single molecules to scalable device architectures. However, for many scanning probe-based approaches attempting to characterise such SAMs, there remains a significant challenge in recovering single-molecule equivalent values from large-area films due to the intrinsic uncertainty of the probe-sample contact area coupled with film damage caused by contact forces. Here we report a new reproducible non-destructive method for probing the electrical and thermoelectric (TE) properties of small assemblies (10-103) of thiol-terminated molecules arranged within a SAM on a gold surface, and demonstrate the successful and reproducible measurements of the equivalent single-molecule electrical conductivity and Seebeck values. We have used a modified thermal-electric force microscopy approach, which integrates the conductive-probe atomic force microscope, a sample positioned on a temperature-controlled heater, and a probe-sample peak-force feedback that interactively limits the normal force across the molecular junctions. The experimental results are interpreted by density functional theory calculations allowing quantification the electrical quantum transport properties of both single molecules and small clusters of molecules. Significantly, this approach effectively eliminates lateral forces between probe and sample, minimising disruption to the SAM while enabling simultaneous mapping of the SAMs nanomechanical properties, as well as electrical and/or TE response, thereby allowing correlation of the film properties.

Reimagining drug manufacturing paradigm in today's pharmacy landscape.

The coronavirus disease 2019 pandemic has escalated the ongoing problem of critical medication shortages, which has serious implications for the health of our patients. Currently, active pharmaceutical ingredients (APIs) are synthesized in large-scale batch operations and shipped to drug product manufacturers, where they are produced on a large scale at centralized facilities. In the centralized drug manufacturing process, the formulation components, operations, and packaging are structured to favor long-term storage and shipment of resultant medicines to the point of care, making this process vulnerable to supply chain disruptions. We propose a rethinking of the drug manufacturing paradigm with an upgraded pharmaceutical compounding-based manufacturing paradigm. This paradigm will be based on integration of continuous manufacturing of APIs and manufacturing of medicines at the point of care with application of machine learning, artificial intelligence, and 3-dimensional printing. This paradigm will support implementation of precision medicine and customization according to patients' needs. The new model of drug manufacturing will be less dependent on the supply chain while ensuring availability of medicines in a cost-effective manner.

Carbazole-Based Tetrapodal Anchor Groups for Gold Surfaces: Synthesis and Conductance Properties.

As the field of molecular-scale electronics matures and the prospect of devices incorporating molecular wires becomes more feasible, it is necessary to progress from the simple anchor groups used in fundamental conductance studies to more elaborate anchors designed with device stability in mind. This study presents a series of oligo(phenylene-ethynylene) wires with one tetrapodal anchor and a phenyl or pyridyl head group. The new anchors are designed to bind strongly to gold surfaces without disrupting the conductance pathway of the wires. Conductive probe atomic force microscopy (cAFM) was used to determine the conductance of self-assembled monolayers (SAMs) of the wires in Au-SAM-Pt and Au-SAM-graphene junctions, from which the conductance per molecule was derived. For tolane-type wires, mean conductances per molecule of up to 10-4.37  G0 (Pt) and 10-3.78  G0 (graphene) were measured, despite limited electronic coupling to the Au electrode, demonstrating the potential of this approach. Computational studies of the surface binding geometry and transport properties rationalise and support the experimental results.

Influence of Manufacturing Process Variables on the Properties of Ophthalmic Ointments of Tobramycin.

PURPOSE: The main purpose of this study was to evaluate qualitative (Q1) and quantitative (Q2) equivalent oleaginous ophthalmic ointments of tobramycin (TOB) with different physicochemical properties and identify critical process/quality attributes using various in vitro methods of characterization. METHODS: Various sources of petrolatum and TOB, and two mixing methods were employed to generate Q1/Q2 equivalent ointments. Characterization studies included content uniformity, microscopy, modulated temperature differential scanning calorimetry (MTDSC), gas chromatography-mass spectrometry (GC/MS), thermogravimetric analysis (TGA) and rheology. RESULTS: The particle size distribution of TOB influenced the content uniformity of ointments. Differences in the MTDSC endothermic and exothermic peaks of TOB suggested the presence of different polymorphic forms. GC/MS revealed variations in the composition and distribution of linear and branched hydrocarbons of petrolatums. Differences were also observed in the TGA derivative weight loss peaks demonstrating differences in the composition of petrolatum that may be the source of the observed variations in the rheological parameters of the ointments. CONCLUSIONS: Source and composition of the petrolatum played a more critical role in determining the rheological properties compared to the method of preparation. Results demonstrated the impact of the source of TOB, excipients and manufacturing processes on the quality attributes of TOB ophthalmic ointments.

Solid-State Characterization of Three Polymorphs of an Orally Available Analog of Diethylenetriaminepentaacetic Acid.

The present work investigated the physical and thermal characteristics of three polymorphic forms (namely, PF1, PF2, and PF3) of a diethyl ester analog of diethylenetriaminepentaacetic acid (C2E2) produced under varying conditions. The identity of each form of C2E2 was confirmed by 1H-NMR, 13C-NMR, and mass spectroscopy. The different polymorphic forms exhibited solubilities ranging from 40 to 150 mg/mL. Powder X-ray diffraction (PXRD) and electron microscopy confirmed that all three forms were crystalline, two of which being scaly, and the third being well-formed. Infrared and Raman spectroscopy revealed differences in the C = O bonding region while differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA) showed widely different melting points with only one thermal event for each compound. The comparison of the melting points and heats of fusion show that the PF1 is monotropically related to both PF2 and PF3, while PF2 and PF3 are enantropically related. Our finding indicates that PF3 is the thermodynamically stable polymorph and will be used for in vitro and in vivo experiments.

A 3D Organically Synthesized Porous Carbon Material for Lithium-Ion Batteries.

We report the first organically synthesized sp-sp3 hybridized porous carbon, OSPC-1. This new carbon shows electron conductivity, high porosity, the highest uptake of lithium ions of any carbon material to-date, and the ability to inhibit dangerous lithium dendrite formation. The new carbon exhibits exceptional potential as anode material for lithium-ion batteries (LIBs) with high capacity, excellent rate capability, long cycle life, and potential for improved safety performance.

In-Situ Formation of Holmium Oxide in Pores of Mesoporous Carbon Nanoparticles as Substrates for Neutron-Activatable Radiotherapeutics.

Radionuclide therapy with nano-sized carriers is a very promising approach to treat various types of cancer. The preparation of radioactive nanocarriers can be achieved with minimum handling using a neutron-activation approach. However, the nanocarrier material must possess certain characteristics such as low density, heat-resistance, high metal adsorption, easy surface modification and low toxicity in order to be useful. Mesoporous Carbon Nanoparticles (MCNs) in which holmium oxide is formed in their pores by a wet-impregnation process are investigated as a suitable material for this application. Holmium (165Ho) has a natural abundance of 100% and possesses a large cross-section for capturing thermal neutrons. After irradiation of Ho-containing MCNs in a neutron flux, 166Ho, which emits therapeutic high energy beta particles as well as diagnostic low energy gamma photons that can be imaged externally, is produced. The wet impregnation process (16 w/w% Ho loading) is shown to completely prevent the leaching of radioactive holmium from the MCNs without compromising their structural integrity. In vitro studies showed that the MCNs containing non-radioactive holmium do not exhibit toxicity and the same formulation with radioactive holmium (166Ho) demonstrated a tumoricidal effect. Post-irradiation PEGylation of the MCN surfaces endows dispersibility and biocompatibility.

Biotransformation Capacity of Carboxylesterase in Skin and Keratinocytes for the Penta-Ethyl Ester Prodrug of DTPA.

The penta-ethyl ester prodrug of the chelating agent diethylene triamine pentaacetic acid (DTPA), referred to as C2E5, effectively accelerated clearance of americium after transdermal delivery. Carboxylesterases (CESs) play important roles in facilitating C2E5 hydrolysis. However, whether CESs in human skin hydrolyze C2E5 remains unknown. We evaluated the gene and protein expression of CESs in distinctive human epidermal cell lines: HEKa, HEKn, HaCaT, and A431. The substrates p-nitrophenyl acetate (pNPA) and 4-nitrophenyl valerate (4-NPV) were used to access esterase and CES activity. C2E5 hydrolysis was measured by radiometric high-performance liquid chromatography after incubation of [(14)C]C2E5 with supernatant fractions after centrifugation at 9000g (S9) prepared from skin cell lines. CES-specific inhibitors were used to access metabolism in human skin S9 fractions with analysis by liquid chromatography-tandem mass spectrometry. We identified the human carboxylesterase 1 and 2 (CES1 and CES2) bands in a Western blot. The gene expression of these enzymes was supported by a real-time polymerase chain reaction (qPCR). pNPA and 4-NPV assays demonstrated esterase and CES activity in all the cell lines that were comparable to human skin S9 fractions. The prodrug C2E5 was hydrolyzed by skin S9 fractions, resulting in a primary metabolite, C2E4. In human skin S9 fractions, inhibition of C2E5 hydrolysis was greatest with a pan-CES inhibitor (benzil). CES1 inhibition (troglitazone) was greater than CES2 (loperamide), suggesting a primary metabolic role for CES1. These results indicate that human keratinocyte cell lines are useful for the evaluation of human cutaneous metabolism and absorption of ester-based prodrugs. However, keratinocytes from skin provide a small contribution to the overall metabolism of C2E5.

Enhanced toxicity of cisplatin with chemosensitizer phenethyl isothiocyanate toward non-small cell lung cancer cells when delivered in liposomal nanoparticles.

Naturally occurring phenethyl isothiocyanate (PEITC) was previously shown to sensitize human non-small cell lung cancer (NSCLC) cells to the platinum anticancer drug cisplatin (CDDP). Here, CDDP and PEITC were encapsulated in approximately 130 nm liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and L-α-phosphatidylglycerol (EPG). The liposomal formulation enhanced the toxicity of this doublet (1:2 molar ratio of CDDP/PEITC) toward NCI-H596 NSCLC cells; the percent survival of cells was 30.2±6.2% after treatment with the nanoparticle formulation, compared to 50.9±3.5% when administered together free. Thus, such a treatment modality could prove useful in the clinic for the treatment of NSCLC.

Solid dispersions of the penta-ethyl ester prodrug of diethylenetriaminepentaacetic acid (DTPA): formulation design and optimization studies.

The penta-ethyl ester prodrug of diethylenetriaminepentaacetic acid (DTPA), which exists as an oily liquid, was incorporated into a solid dispersion for oral administration by the solvent evaporation method using blends of polyvinylpyrrolidone (PVP), Eudragit® RL PO and α-tocopherol. D-optimal mixture design was used to optimize the formulation. Formulations that had a high concentration of both Eudragit® RL PO and α-tocopherol exhibited low water absorption and enhanced stability of the DTPA prodrug. Physicochemical properties of the optimal formulation were evaluated using Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). In vitro release of the prodrug was evaluated using the USP Type II apparatus dissolution method. DSC studies indicated that the matrix had an amorphous structure, while FTIR spectrometry showed that DTPA penta-ethyl ester and excipients did not react with each other during formation of the solid dispersion. Dissolution testing showed that the optimized solid dispersion exhibited a prolonged release profile, which could potentially result in a sustained delivery of DTPA penta-ethyl to enhance bioavailability. In conclusion, DTPA penta-ethyl ester was successfully incorporated into a solid matrix with high drug loading and improved stability compared to prodrug alone.

Barriers to adoption of electric vehicles in Texas.

Sustainable mobility options such as electric vehicles (EVs) have the potential to improve the quality of life for Americans as well as those in other countries, as they can enhance the quality of the air we breathe, while reducing greenhouse gas emissions, fossil fuel consumption, and the adverse impacts of global warming. Despite their many benefits, however, the demand for EVs remains low. Therefore, this study aims to identify the barriers that affect the widespread EV adoption in the United States. Seventeen barriers were identified from the literature, and a questionnaire survey was designed and distributed to potential consumers of EVs. The survey yielded 733 responses, and various statistical tests like cluster analysis and chi-squared tests were performed. The results revealed that the high purchase price of the vehicle, high battery replacement cost, and the lack of public infrastructures for charging them were the primary concerns. The results also revealed that middle-aged men with high education and income are more enthusiastic about adopting EVs. The results presented in this study indicate a range of developments that different stakeholders could implement. To surmount the economic barriers to EV adoption, policymakers should strengthen incentives countrywide, and automakers should introduce more affordable EVs to the market. To overcome the challenges associated with charging, it is necessary to make investments in rapid charging infrastructure along the primary toll routes.

Photodynamic inactivation of chlorin e6-loaded CTAB-liposomes against Candida albicans.

BACKGROUND AND OBJECTIVES: Antimicrobial photodynamic inactivation (PDI) is a promising therapeutic modality for the treatment of local infections. To increase the efficacy of PDI, chlorine e6 (Ce6) was encapsulated in cationic CTAB-liposomes composed of various ratios of dimyristoyl-sn-glycero-phosphatidylcholine (DMPC) and the cationic surfactant, cetyltrimethyl ammonium bromide (CTAB). The PDI efficacy of the liposomal-Ce6 was assessed in vitro against susceptible and drug-resistant clinical isolates of Candida albicans (C. albicans) as well as in infected burn wounds. STUDY DESIGN/MATERIALS AND METHODS: Ce6 was encapsulated in CTAB-liposomes by the film hydration method. Particle size distribution and zeta potential of the cationic liposomes were measured using a Zetasizer Nano-ZS. UV-visible spectra were used to measure lipid/Ce6 (L/C) ratio and drug entrapment efficiency while differential scanning calorimetry (DSC) was used to study the thermotropic behavior of DMPC liposomes upon CTAB addition. In vivo PDI efficacy was carried out in an infected burn wound using a rat model. RESULTS: The increase in zeta potential and a shift in the phase transition temperature (Tm ) upon CTAB addition confirmed its entrapment within the lipid bilayers of the liposome. Meanwhile, the CTAB addition did not affect the Ce6 entrapment efficiency and physical attributes of the liposomes. In vitro studies showed that the PDI effect of the Ce6-loaded CTAB-liposomes was dependent on the lipid to Ce6 molar ratio (L/C), particle size and the concentration of CTAB in the liposomes. The lower L/C ratio and smaller liposomes exerted significantly higher PDI effects. In addition, an increase in the CTAB to lipid ratio led to a significant increase in the PDI effect of Ce6 against susceptible and drug-resistant clinical isolates of C. albicans after light illumination. CONCLUSIONS: Our results indicate that a low L/C ratio, high positive charge, and small particle size of CTAB-liposomes significantly enhances their PDI efficacy against C. albicans.

Planar aromatic anchors control the electrical conductance of gold|molecule|graphene junctions.

The synthesis of a family of alkanethiol molecules with planar aromatic head groups, designed to anchor molecules effectively to graphene electrodes, is reported. Characterisation of self-assembled monolayers of these molecules on a gold surface via conductive atomic force microscopy shows that when an aromatic head group is present, the conductance G graphene obtained using a graphene coated probe is higher than the conductance G Pt obtained using a platinum (Pt) probe. For Pt probe and graphene probe junctions, the tunnelling decay constant of benzyl ether derivatives with an alkanethiol molecular backbone is determined as ß = 5.6 nm-1 and 3.5 nm-1, respectively. The conductance ratio G graphene/G Pt increases as the number of rings present in the aromatic head unit, n, increases. However, as the number of rings increases, the conductance path length increases because the planar head groups lie at an angle to the plane of the electrodes. This means that overall conductance decreases as n increases. Density functional theory-based charge transport calculations support these experimental findings. This study confirms that planar aromatic head groups can function as effective anchoring units for graphene electrodes in large area molecular junctions. However, the results also indicate that the size and geometry of these head groups must be considered in order to produce effective molecular designs.

Preparation of neutron-activatable holmium nanoparticles for the treatment of ovarian cancer metastases.

Nanoparticles containing stable holmium ((165) Ho) are prepared by nanotemplate engineering and subsequently irradiated in a neutron flux to yield (166) Ho, a beta-emitting radiotherapeutic isotope. After intraperitoneal injection to mice bearing SKOV-3 ovarian tumors, significant tumor accumulation of the (166) Ho-nanoparticles is observed by SPECT imaging indicating the potential of these neutron activatable nanoparticles for internal radiation therapy of ovarian cancer metastases.

High payload dual therapeutic-imaging nanocarriers for triggered tumor delivery.

The in vitro and in vivo characterization of an optimized formulation of nanoparticles (NPs) loaded with a high content of dexamethasone palmitate (DEX-P), a chemotherapeutic adjuvant that decreases interstitial fluid pressure in tumors, and (111) In, a signaling agent, is described. These NPs are uniform in size and composition. Single photon emission computed tomography imaging demonstrates significant tumor uptake of (111) In-labeled DEX-P NPs in tumor-bearing mice. As with many nanoparticle-based drug delivery systems, significant liver accumulation is observed. Assessment of liver histology and blood tests show no apparent hepatic or renal toxicity of the DEX-P NPs. Conversion of DEX-P to DEX occurs when DEX-P NPs are incubated with mouse plasma, human tumor homogenate and ascites from tumor bearing mice, but not with human plasma. This conversion is slower in plasma from Es1(e) ((-/-)) /SCID mice, a potential alternative animal model that better mimics humans; however, plasma from these mice are not completely devoid of esterase activity. The difference between blood and tumor esterase activity in humans facilitates the delivery of DEX-P NPs to tumors and the release of dexamethasone by an esterase trigger.

Pharmacokinetics, bioavailability, tissue distribution, excretion, and metabolite identification of methoxyflavones in Kaempferia parviflora extract in rats.

Kaempferia parviflora (KP) is an herbal plant in the family of Zingiberaceae. KP mainly contains methoxyflavones, especially 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF), and 3,5,7,3',4'-pentamethoxyflavone (PMF). The present study was designed to characterize the pharmacokinetics, including bioavailability, distribution, excretion, and identification of metabolites after administration of a KP ethanolic extract. Male rats were orally or intravenously administered a 250 mg/kg concentration of a KP extract, and blood samples were obtained at selected times to determine pharmacokinetic parameters of PMF, TMF, and DMF. For distribution and excretion studies, the organs, urine, and feces samples were collected at various times after oral administration of a larger (750 mg/kg) dose of KP extract. Methoxyflavones in the biological samples were quantified by high-performance liquid chromatography-UV, and the metabolites in urine and feces were further identified by using liquid chromatography-tandem mass spectrometry. After oral administration, concentrations of the three methoxyflavones quickly approached their maximal concentration, ranging from 0.55 to 0.88 µg/ml within 1 to 2 h after administration, and then were gradually excreted with half-lives of 3 to 6 h. The methoxyflavones showed low oral bioavailability of 1 to 4%. Three methoxyflavones were detected at their highest levels in liver followed by kidney. They were also found in lung, testes, and brain. After absorption, organ distribution, and metabolism, the components of KP were mainly eliminated through urine in the forms of demethylated, sulfated, and glucuronidated products and as demethylated metabolites in the feces. The parent compounds were found to have 0.79, 1.76, and 3.10% dose recovery in urine and 1.06, 1.77, and 0.96% dose recovery in feces for PMF, TMF, and DMF, respectively. These studies are the first to describe the pharmacokinetics of KP extract to provide the information on blood and tissue levels.

Carboxylesterase-triggered hydrolysis of nanoparticle PEGylating agents.

Despite the importance of PEGylation in achieving long nanoparticle circulation times, many nanoparticles are coated with PEGylating agents susceptible to enzymatic degradation. In this study, solid lipid nanoparticles (SLNs) prepared with ester-containing compounds were evaluated for their stability in the presence of carboxylesterase. SLN suspensions became turbid within 30 min of enzymatic exposure, indicating possible disassociation of a portion of the nanoparticles. The particle size of SLNs incubated with the enzyme was smaller than the size of controls, although their morphologies appeared similar in transmission electron microscopy images. Although SLNs offered some protection over micelles, PEG6000 monostearate was rapidly degraded within 15 min. Hydrolysis of polysorbate 60 was much slower, reaching only 36% in 2 h. These studies reveal the importance of confirming the stability of PEG surface coatings prior to undertaking in vivo experiments in small animal models, which can have considerably higher plasma esterase activity than humans.

Therapeutic complications in a patient with high-risk acute lymphoblastic leukemia and undiagnosed hereditary hemochromatosis.

Hereditary hemochromatosis (HH) is an autosomal-recessive disorder of iron metabolism that most commonly manifests in the fourth or fifth decade of life. Here, we describe a 14-year-old male who presented with high-risk acute lymphoblastic leukemia and previously undiagnosed HH. His treatment course was remarkable for significant therapeutic complications, including iron overload, hepatic failure, cardiac dysfunction, and death. Postmortem testing revealed homozygosity for the C282Y mutation, confirming the diagnosis of HH. Since HH mutations occur commonly in select populations, screening patients with leukemia for HH may better inform treatment decisions regarding chemotherapy, transfusions, and/or iron chelation therapy.

Optimization of the lyophilization process for long-term stability of solid-lipid nanoparticles.

OBJECTIVES: To optimize a lyophilization protocol for solid-lipid nanoparticles (SLNs) loaded with dexamethasone palmitate (Dex-P) and to compare the long-term stability of lyophilized SLNs and aqueous SLN suspensions at two storage conditions. MATERIALS AND METHODS: The effect of various parameters of the lyophilization process on SLN redispersibility was evaluated. A three month stability study was conducted to compare changes in the particle size and drug loading of lyophilized SLNs with SLNs stored as aqueous suspensions at either 4°C or 25°C/60% relative humidity (RH). RESULTS AND DISCUSSION: Of nine possible lyoprotectants tested, sucrose was shown to be the most efficient at achieving SLN redispersibility. Higher freezing temperatures, slower freezing rates, and longer secondary drying times were also shown to be beneficial. Loading of the SLNs with Dex-P led to slightly larger particle size and polydispersity index increases, but both parameters remained within an acceptable range. Drug loading and particle shape were maintained following lyophilization, and no large aggregates were detected. During the stability study, significant growth and drug loss were observed for aqueous SLN suspensions stored at 25°C/60% RH. In comparison, lyophilized SLNs stored at 4°C exhibited a consistent particle size and showed <20% drug loss. Other storage conditions led to intermediate results. CONCLUSIONS: A lyophilization protocol was developed that allowed SLNs to be reconstituted with minimal changes in their physicochemical properties. During a three month period, lyophilized SLNs stored at 4°C exhibited the greatest stability, showing no change in the particle size and a minimal reduction in drug retention.