Characterization and biodistribution of under-employed gene therapy vector AAV7.

IMPORTANCE: The use of adeno-associated viruses (AAVs) as gene delivery vectors has vast potential for the treatment of many severe human diseases. Over one hundred naturally existing AAV capsid variants have been described and classified into phylogenetic clades based on their sequences. AAV8, AAV9, AAVrh.10, and other intensively studied capsids have been propelled into pre-clinical and clinical use, and more recently, marketed products; however, less-studied capsids may also have desirable properties (e.g., potency differences, tissue tropism, reduced immunogenicity, etc.) that have yet to be thoroughly described. These data will help build a broader structure-function knowledge base in the field, present capsid engineering opportunities, and enable the use of novel capsids with unique properties.

The presence of CpGs in AAV gene therapy vectors induces a plasmacytoid dendritic cell-like population very early after administration.

AAV-mediated gene transfer is a promising platform still plagued by potential host-derived, antagonistic immune responses to therapeutic components. CpG-mediated TLR9 stimulation activates innate immune cells and leads to cognate T cell activation and suppression of transgene expression. Here, we demonstrate that CpG depletion increased expression of an antibody transgene product by 2-3-fold as early as 24 h post-vector administration in mice. No significant differences were noted in anti-transgene product/ anti-AAV capsid antibody production or cytotoxic gene induction. Instead, CpG depletion significantly reduced the presence of a pDC-like myeloid cell population, which was able to directly bind the antibody transgene product via Fc-FcγR interactions. Thus, we extend the mechanisms of TLR9-mediated antagonism of transgene expression in AAV gene therapy to include the actions of a previously unreported pDC-like cell population.

Pulmonary influenza A virus infection leads to suppression of the innate immune response to dermal injury.

The innate immune system is responsible for many important functions in the body including responding to infection, clearing cancerous cells, healing wounds, and removing foreign substances. Although many of these functions happen simultaneously in life, most laboratory studies of the innate immune response focus on one activity. How the innate immune system responds to concurrent insults in different parts of the body is not well understood. This study explores the impact of a lung infection on the cutaneous wound healing process. We used two complimentary models of injury: the excisional tail wound and subcutaneous implantation of polyvinyl alcohol (PVA) sponges. These models allow for assessment of the rate of closure and measurement of cellular and cytokine responses during acute wound healing, respectively. When mice with these healing wounds were infected with influenza A virus (IAV) in the lung there was a delay in wound healing. The viral lung infection suppressed the innate immune response in a healing wound, including cellular infiltrate, chemokines, growth factors, and cytokines. However, there was not a global immune suppression as there was an increase in inflammation systemically in mice with both infection and healing wounds compared to mice with only wounds or IAV infection. In addition, the lung immune response was largely unaffected indicating that responding to a lung infection is prioritized over a healing wound. This study introduces the concept of immune triage, in that when faced with multiple insults the immune system prioritizes responses. This paradigm likely applies to many situations that involve the innate immune system, and understanding how the innate immune system handles multiple insults is essential to understanding how it can efficiently clear pathogens while responding to other inflammatory events.

Acute and subchronic toxicity of metal complex azo acid dye and anionic surfactant oil on fish Oreochromis niloticus.

The acute toxicity study of metal complex dark green azo acid dye, anionic surfactant oil and their mixture determined the 96 hr LC50, and fish behaviours. Subchronic toxicity determined haematology parameters and concentrations of copper and chromium in blood. The 96 hr LC50 was determined by probit analysis and subchronic toxicity was conducted in 90 days. No mortalities were observed in control and anionic surfactant oil treatments. The 96 hr LC50 value of mixture was 26.7 mg I(-1) (95% CL = 20.7 - 46.8) and that of metal complex dark green azo acid dye was not met as the percentage of dead was below 50% of tested organisms. In a treatment of anionic surfactant oil and that of mixture observed behaviours were respiration response, uncoordinated movement, loss of equilibrium, erratic posture and loss of responsiveness. Subchronic toxicity indicated fluctuations in number of erythrocytes, leukocytes and thrombocytes in all chemical treatments. Erythrocyte morphology such as anisocytosis, erythrocytes hypertrophy, karyolysis, cytoplasm vacuolation, ghost cell were observed in fish blood in all chemical treatments. An inverse relation was observed between total copper and chromium concentration in blood. However, the toxicity effect was chemical dose dependent and length of exposure.

Effects of electron-donating ability of binding sites on coordination number: the interactions of a cyclic Schiff base with copper ions.

The stepwise addition of Cu2+ ions to the nonplanar cyclic Schiff base 5,9,14,18-tetramethyl-1,4,10,13-tetraazacyclooctadeca-5,8,14,17-tetraene-7,16-dione (H4daaden, C18H28N4O2), yields a one-end-open dinuclear copper chelate. The pyridine adduct of the dinuclear copper chelate, namely, [µ-6,11-dimethyl-7,10-diazahexadeca-5,11-diene-2,4,13,15-tetraolato(4-)](pyridine)dicopper(II), [Cu2(C16H20N2O4)(C5H5N)], was characterized by single-crystal X-ray crystallography. The two CuII atoms of the copper chelate display different coordination modes, i.e. inner-N2O2 and outer-O2O2. The Cu atom which is bonded in the outer-O2O2 mode is axially bonded to a pyridine molecule, which suggests that the electron-donating ability of the O2O2 site to the Cu atom is poor. As a result, the O2O2-bonded Cu atom has a coordination number of five, showing square-bipyramidal geometry around the Cu atom. The N2O2-coordinated site provides sufficient electron density to the other Cu atom to be stabilized with a coordination number of four, showing square-planar geometry around the Cu atom. The electron-donating ability of the ligand coordination sites plays a key role in determining the coordination number of the Cu atoms of the dicopper chelate.

Acute angle-closure in juvenile X-linked retinoschisis.

Juvenile X-linked retinoschisis (JXR), the most common inherited retinal disorder in young males, presents with a wide range of phenotypic variations. Acute angle closure in children with JXR has been reported in the literature only once before. We present a case of acute-angle closure, temporally associated with pharmacologic dilation, in a 12-year-old boy with JXR.

Developing Functional Relationships between Soil Moisture Content and Corn Early-Season Physiology, Growth, and Development.

Drought is a severe threat to agriculture production that affects all growth stages of plants, including corn (Zea mays L.). Any factor affecting early seedling growth and development will significantly impact yield. Despite the recurrence of low rainfall during the growing seasons, corn responses to different early-season soil moisture content levels have not been investigated. In this study, we investigated how corn morpho-physiological and biomass traits responded to varied soil moisture content during the early vegetative stage. Two corn hybrids were grown in a pot-culture facility under five different soil moisture treatments (0.15, 0.12, 0.09, 0.06, and 0.03 m3 m-3 volumetric water content, VWC) to assess the growth and developmental responses to varied soil moisture content during early-season growth (V2 to V7) stage. Sub-optimal soil moisture content limited plant growth and development by reducing physiological and phenotypic expression. Stomatal conductance and transpiration were decreased by an average of 65% and 59% across stress treatments relative to optimum conditions. On average, soil moisture deficit reduced the total leaf area by 71% and 72% compared to the control in 'A6659VT2RIB' and 'P1316YHR', respectively. Shoot and root dry weights were reduced by 74% and 43% under 0.03 m3 m-3 VWC. An increase in the root-to-shoot ratio was noticed under low VWC conditions compared to the control. Based on the stress tolerance index, the physiology and leaf growth parameters were more sensitive to soil moisture deficit. Our results highlight the impact of sub-optimal soil moisture on physiology and morphological traits during early-season growth. 'P1316YHR' demonstrated better physiological performance under stress conditions, while 'A6659VT2RIB' produced relatively better root growth. The findings suggest that biomass partitioning between shoot and root components is dynamic and depends on stress intensity. The current findings can help to prioritize traits associated with the early-season drought tolerance in corn. The functional relationships developed between soil moisture content and growth and developmental responses can be integrated into corn crop modeling to allow better irrigation management decisions.

Measurement of the Induced Magnetic Polarisation of Rotated-Domain Graphene Grown on Co Film with Polarised Neutron Reflectivity.

In this paper, we determine the magnetic moment induced in graphene when grown on a cobalt film using polarised neutron reflectivity (PNR). A magnetic signal in the graphene was detected by X-ray magnetic circular dichroism (XMCD) spectra at the C K-edge. From the XMCD sum rules an estimated magnetic moment of 0.3 µB/C atom, while a more accurate estimation of 0.49 µB/C atom was obtained by carrying out a PNR measurement at 300 K. The results indicate that the higher magnetic moment in Co is counterbalanced by the larger lattice mismatch between the Co-C (1.6%) and the slightly longer bond length, inducing a magnetic moment in graphene that is similar to that reported in Ni/graphene heterostructures.

Intravitreal Triamcinolone Acetonide Presents a Diagnostic Challenge on Noncontrast Computed Tomography.

Triamcinolone acetonide (TA) is a widely used corticosteroid for various ophthalmological indications. We report a case of a 27-year-old female presented with upper eyelid edema and punctate corneal erosions and haze of the left eye, 1 week after pars plana vitrectomy with silicone oil (SO) tamponade and intravitreal TA for diabetic tractional retinal detachment. The condition persisted despite topical and systemic therapy. Computed tomography (CT) scan of the orbits was obtained to exclude postoperative SO migration. The scan showed a hyperintense lesion in the vitreous cavity of the left eye with no SO migration. The radiographic appearance of the lesion mimicked a foreign body; however, history and recent operative note excluded this possibility. A CT scan of various TA preparations revealed that the lesion's density is similar to those of TA. Improvement of corneal haze confirmed that the lesion was consistent with intravitreal TA. The patient developed eyelid edema of the right eye and later was diagnosed with nephrotic syndrome after further investigation. In conclusion, it is important to be familiar with the radiographic appearance of TA on CT to avoid incorrect diagnosis.

Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin.

Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

Activity of TDT 067 (terbinafine in Transfersome) against agents of onychomycosis, as determined by minimum inhibitory and fungicidal concentrations.

TDT 067 is a novel carrier-based dosage form (liquid spray) of 15 mg/ml of terbinafine in Transfersome that has been developed to deliver terbinafine to the nail bed to treat onychomycosis. In this study, we report the in vitro activities of TDT 067 against dermatophytes, compared with those of the Transfersome vehicle, naked terbinafine, and commercially available terbinafine (1%) spray. The MICs of TDT 067 and comparators against 25 clinical strains each of Trichophyton rubrum, T. mentagrophytes, and Epidermophyton floccosum were determined according to the CLSI M38-A2 susceptibility method (2008). Minimum fungicidal concentrations (MFCs) were determined by subculturing visibly clear wells from the MIC microtiter plates. TDT 067 demonstrated potent activity against the dermatophyte strains tested, with an MIC range of 0.00003 to 0.015 µg/ml. Overall, TDT 067 MIC(50) values (defined as the lowest concentrations to inhibit 50% of the strains tested) were 8-fold and 60-fold lower than those of naked terbinafine and terbinafine spray, respectively. The Transfersome vehicle showed minimal inhibitory activity. TDT 067 demonstrated lower MFC values for T. rubrum and E. floccosum than naked terbinafine and terbinafine spray. TDT 067 has more potent antifungal activity against dermatophytes that cause nail infection than conventional terbinafine preparations. The Transfersome vehicle appears to potentiate the antifungal activity of terbinafine. Clinical investigation of TDT 067 for the topical treatment of onychomycosis is warranted.

Paramagpy: software for fitting magnetic susceptibility tensors using paramagnetic effects measured in NMR spectra.

Paramagnetic metal ions with fast-relaxing electrons generate pseudocontact shifts (PCSs), residual dipolar couplings (RDCs), paramagnetic relaxation enhancements (PREs) and cross-correlated relaxation (CCR) in the nuclear magnetic resonance (NMR) spectra of the molecules they bind to. These effects offer long-range structural information in molecules equipped with binding sites for such metal ions. Here we present the new open-source software Paramagpy, which has been written in Python 3 with a graphic user interface. Paramagpy combines the functionalities of different currently available programs to support the fitting of magnetic susceptibility tensors using PCS, RDC, PRE and CCR data and molecular coordinates in Protein Data Bank (PDB) format, including a convenient graphical user interface. Paramagpy uses efficient fitting algorithms to avoid local minima and supports corrections to back-calculated PCS and PRE data arising from cross-correlation effects with chemical shift tensors. The source code is available from 10.5281/zenodo.3594568 .

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Birth of a Live Cria After Transfer of a Vitrified-Warmed Alpaca (Vicugna pacos) Preimplantation Embryo.

The alpaca (Vicugna pacos) is an important species for the production of fiber and food. Genetic improvement programs for alpacas have been hindered, however, by the lack of field-practical techniques for artificial insemination and embryo transfer. In particular, successful techniques for the cryopreservation of alpaca preimplantation embryos have not been reported previously. The objective of this study was to develop a field-practical and efficacious technique for cryopreservation of alpaca preimplantation embryos using a modification of a vitrification protocol originally devised for horses and adapted for dromedary camels. Four naturally cycling non-superovulated Huacaya females serving as embryo donors were mated to males of proven fertility. Donors received 30 µg of gonadorelin at the time of breeding, and embryos were non-surgically recovered 7 days after mating. Recovered embryos (n = 4) were placed individually through a series of three vitrification solutions at 20°C (VS1: 1.4 M glycerol; VS2: 1.4 M glycerol + 3.6 M ethylene glycol; VS3: 3.4 M glycerol + 4.6 M ethylene glycol) before loading into an open-pulled straw (OPS) and plunging directly into liquid nitrogen for storage. At warming, each individual embryo was sequentially placed through warming solutions (WS1: 0.5 M galactose at 37°C; WS2: 0.25 M galactose at 20°C), and warmed embryos were incubated at 37°C in 5% CO2 in humidified air for 20-22 h in 1 ml Syngro® holding medium supplemented with 10% (v/v) alpaca serum to perform an initial in vitro assessment of post-warming viability. Embryos whose diameter increased during culture (n = 2) were transferred individually into synchronous recipients, whereas embryos that did not grow (n = 2) were transferred together into a single recipient to perform an in vivo assessment of post-warming viability. Initial pregnancy detection was performed ultrasonographically 29 days post-transfer when fetal heartbeat could be detected, and one of three recipients was pregnant (25% embryo survival rate). On November 13, 2019, the one pregnant recipient delivered what is believed to be the world's first cria produced from a vitrified-warmed alpaca embryo.

Experimental harvest regulations reveal that water availability during spring, not harvest, affects change in a waterfowl population.

Population change is regulated by vital rates that are influenced by environmental conditions, demographic stochasticity, and, increasingly, anthropogenic effects. Habitat destruction and climate change threaten the future of many wildlife populations, and there are additional concerns regarding the effects of harvest rates on demographic components of harvested organisms. Further, many population managers strictly manage harvest of wild organisms to mediate population trends of these populations. The goal of our study was to decouple harvest and environmental variability in a closely monitored population of wild ducks in North America, where we experimentally regulated harvest independently of environmental variation over a period of 4 years. We used 9 years of capture-mark-recapture data to estimate breeding population size during the spring for a population of wood ducks in Nevada. We then assessed the effect of one environmental variable and harvest pressure on annual changes in the breeding population size. Climatic conditions influencing water availability were strongly positively related to population growth rates of wood ducks in our study system. In contrast, harvest regulations and harvest rates did not affect population growth rates. We suggest efforts to conserve waterfowl should focus on the effects of habitat loss in breeding areas and climate change, which will likely affect precipitation regimes in the future. We demonstrate the utility of capture-mark-recapture methods to estimate abundance of species which are difficult to survey and test the impacts of anthropogenic harvest and climate on populations. Finally, our results continue to add to the importance of experimentation in applied conservation biology, where we believe that continued experiments on nonthreatened species will be critically important as researchers attempt to understand how to quantify and mitigate direct anthropogenic impacts in a changing world.

Quantitative comparison between single-photon emission computed tomography and positron emission tomography imaging of lung ventilation with 99mTc-technegas and 68Ga-gallgas in patients with chronic obstructive pulmonary disease: A pilot study.

The aim of this study was quantitative comparison between 68Ga-Gallgas positron emission tomography (PET) and 99mTc-Technegas single photon emission computed tomography (SPECT) for lung ventilation function assessment in patients with moderate-to-severe obstructive pulmonary disease and to identify image-derived texture features correlating to the physiologic parameters. Five patients with moderate-to-severe chronic obstructive pulmonary disease with PET and SPECT lung ventilation scans were selected for this study. Threshold-based segmentations were used to compare ventilated regions between both imaging techniques. Histograms of both scans were compared to reveal main differences in distributions of radiotracers. Volumes of segmentation as well as 50 textural features measured in the pulmonary region were correlated to the forced expiratory volume in 1 s (FEV1) as the relevant physiological variable. A better peripheral distribution of the radiotracer was observed in PET scans for three out of five patients. A segmentation threshold of 27% and 31% for normalized scans, for PET and SPECT respectively, was found optimal for volume correlation with FEV1. A high correlation (Pearson correlation coefficient >0.9) was found between 16 texture features measured from SPECT and 7 features measured from PET and FEV1. Quantitative measurements revealed different tracer distribution in both techniques. These results suggest that tracer distribution patterns may depend on the cause of the pulmonary obstruction. We found several texture features measured from SPECT to correlate to FEV1.

Tandem reactions of 1,3-diacid chlorides with 2-methylimidazoline and 2-methyl-1,4,5,6-tetrahydropyrimidine: one-pot synthesis of 1,8-naphthyridinetetraones.

The reactions of 2-methylimidazoline and 2-methyl-1,4,5,6-tetrahydropyrimidine with 1,3-diacid chlorides, in the presence of Et3N in refluxing MeCN give highly functionalized potentially bioactive 1,8-naphthyridinetetraones. 2-Methylimidazoline and 2-methyl-1,4,5,6-tetrahydropyrimidine can be viewed as tridentate nucleophiles which give four consecutive tandem nucleophilic attacks on electrophiles.

The early picosecond photophysics of Ru(II) polypyridyl complexes: a tale of two timescales.

The early picosecond time scale excited-state dynamics of the paradigm tris(2,2'-bipyridyl)Ruthenium(II) ([Ru(bpy)(3)](2+)) and related complexes have been examined by picosecond Kerr-gated time-resolved resonance Raman (ps-TR(3)) spectroscopy. The evolution of the signature Raman bands of the lowest thermally equilibrated excited (THEXI) state under two-color pump/probe conditions show that this state is not fully populated within several hundred femtoseconds as proposed previously but rather only within the first 20 ps following excitation. In addition to an emission observed within the instrument rise time (τ < 3 ps), the early picosecond dynamics are characterized by a rise in the intensity of the Raman marker bands of the THEXI-(3)MLCT state, a rise time which, within experimental uncertainty, is not influenced by either partial or complete ligand deuteriation or the presence of ligands other than bpy, as in the heteroleptic complexes [Ru(bpy)(2)(L1)](+) and [Ru(bpy)(2)(Hdcb)](+) (where H(2)dcb is 4,4'-dicarboxy-2,2'-bipyridine and L1 is 2,-(5'-phenyl-4'-[1,2,4]triazole-3'-yl)pyridine). Overall, although the results obtained in the present study are consistent with those obtained from examination of this paradigm complex on the femtosecond timescale, regarding initial formation of the vibrationally hot (3)MLCT state by ISC from the singlet Franck-Condon state, the observation that the THEXI-(3)MLCT state reaches thermal equilibration over a much longer time period than previously suggested warrants a re-examination of views concerning the rapidity with which thermal equilibration of transition metal complex excited states takes place.

Comparison of diffuse reflectance fourier transform mid-infrared and near-infrared spectroscopy with grating-based near-infrared for the determination of fatty acids in forages.

Diffuse reflectance Fourier transform mid infrared (FTMIR) and near-infrared spectroscopy (FTNIR) were compared to scanning monochromator-grating-based near-infrared spectroscopy (SMNIR), for their ability to quantify fatty acids (FA) in forages. A total of 182 samples from thirteen different forage cultivars and three different harvest times were analyzed. Three calibration analyses were conducted for lauric (C12:0), myristic (C14:0), palmitic (C16:0), stearic (C18:0), palmitoleic (C16:1), oleic (C18:1), linoleic (C18:2), and alpha-linolenic (C18:3) acids. When all samples were used in a one-out partial least squares (PLS) calibration, the average R (2) were FTNIR (0.95) > SMNIR (0.94) > FTMIR (0.91). Constituents C18:2 and C16:0 had among the highest R (2) regardless of the spectroscopic method used. The FTNIR did better for C12:0, C14:0, and C18:3. The SMNIR did better for C16:0, C16:1, C18:0, C18:1, and C18:2. A second set of calibrations developed with half of the samples as the calibration set and the rest as the validation set showed that all the methods produce acceptable calibrations, with calibration R (2) above 0.9 for most constituents. However, the SMNIR had a better average calibration relative error than the FTNIR, which was slightly better than the FTMIR. A third set of calibration equations developed using 100 random PLS runs with the 182 samples split randomly also shows that the three spectral methods are satisfactory for predicting FA. It is not clear whether any of the spectral methods is distinctly better than another. Calibration R (2) and validation R (2) were higher for most FA with the SMNIR than the FTMIR and FTNIR.