An Update on Gene Therapy for Inherited Retinal Dystrophy: Experience in Leber Congenital Amaurosis Clinical Trials

Inherited retinal dystrophies (IRDs) are a group of rare eye diseases caused by gene mutations that result in the degradation of cone and rod photoreceptors or the retinal pigment epithelium. Retinal degradation progress is often irreversible, with clinical manifestations including color or night blindness, peripheral visual defects and subsequent vision loss. Thus, gene therapies that restore functional retinal proteins by either replenishing unmutated genes or truncating mutated genes are needed. Coincidentally, the eye’s accessibility and immune-privileged status along with major advances in gene identification and gene delivery systems heralded gene therapies for IRDs. Among these clinical trials, voretigene neparvovec-rzyl (Luxturna), an adeno-associated virus vector-based gene therapy drug, was approved by the FDA for treating patients with confirmed biallelic RPE65 mutation-associated Leber Congenital Amaurosis (LCA) in 2017. This review includes current IRD gene therapy clinical trials and further summarizes preclinical studies and therapeutic strategies for LCA, including adeno-associated virus-based gene augmentation therapy, 11-cis-retinal replacement, RNA-based antisense oligonucleotide therapy and CRISPR-Cas9 gene-editing therapy. Understanding the gene therapy development for LCA may accelerate and predict the potential hurdles of future therapeutics translation. It may also serve as the template for the research and development of treatment for other IRDs.     

Fractal characteristic of three Chinese coals

Experimental and theoretical investigation about coal/char structure is presented. Surface structures of parent coal and char with different burn-off ratios were analyzed. We introduced the fractal theory into Scanning Electron Microscopy image analysis and utilize the particle surface fractal dimension (D_ps) to quantitatively describe the surface character of coal/char particles. D_ps of three Chinese coals reach their maximum in the 35-45 wt% char burn-off interval and then decrease with increasing carbon burn-off ratio. The inner-pore information of coal/char particles was determined by N₂ isotherm adsorption/desorption. Using fractal BET model, internal surface fractal dimension (D_s) of coal/char particles was calculated. The D_s change trend of three Chinese coals is similar to their S_BET development. It means the D_s can quantitatively describe the inner pore structure character of coal/char particles.     

Biomolecular and Genetic Prognostic Factors That Can Facilitate Fertility-Sparing Treatment (FST) Decision Making in Early Stage Endometrial Cancer (ES-EC): A Systematic Review

Endometrial cancer occurs in up to 29% of women before 40 years of age. Seventy percent of these patients are nulliparous at the time. Decision making regarding fertility preservation in early stage endometrial cancer (ES-EC) is, therefore, a big challenge since the decision between the risk of cancer progression and a chance to parenthood needs to be made. Sixty-two percent of women with complete remission of ES-EC after fertility-sparing treatment (FST) report to have a pregnancy wish which, if not for FST, they would not be able to fulfil. The aim of this review was to identify and summarise the currently established biomolecular and genetic prognostic factors that can facilitate decision making for FST in ES-EC. A comprehensive search strategy was carried out across four databases; Cochrane, Embase, MEDLINE, and PubMed; they were searched between March 1946 and 22nd December 2022. Thirty-four studies were included in this study which was conducted in line with the PRISMA criteria checklist. The final 34 articles encompassed 9165 patients. The studies were assessed using the Critical Appraisal Skills Program (CASP). PTEN and POLE alterations we found to be good prognostic factors of ES-EC, favouring FST. MSI, CTNNB1, and K-RAS alterations were found to be fair prognostic factors of ES-EC, favouring FST but carrying a risk of recurrence. PIK3CA, HER2, ARID1A, P53, L1CAM, and FGFR2 were found to be poor prognostic factors of ES-EC and therefore do not favour FST. Clinical trials with bigger cohorts are needed to further validate the fair genetic prognostic factors. Using the aforementioned good and poor genetic prognostic factors, we can make more confident decisions on FST in ES-EC.     

Diffusional behavior of amino acids in solid-phase reaction field as studied by H pulsed-field-gradient spin-echo NMR method

The diffusion coefficients (D) of tert-butyloxycarbonyl-l-phenylalanine (Boc-Phe) in Merrifield network polystyrene gels, used as a solid-phase reaction field have been determined as a function of the amino acid concentration over the temperature range from 30 to 50 °C by means of the ¹H pulsed-field-gradient spin-echo NMR method. From these experimental results, it was found that the D value of Boc-Phe in DMF-d₇ solution, in DVB 1 and 2% cross-linked network polystyrene gels depends on the amino acid concentration. The D value of Boc-Phe·Cs(tert-butyloxycarbonyl-l-phenylalanine cesium) salt in the solid-phase reaction field under chemical reaction was determined at 50 °C. Further, it was found that the D value depends on the NMR observation time, that is the applied two field-gradient pulse interval. Details of its analysis were discussed.     

OCT4 Expression and Vasculogenic Mimicry Formation Positively Correlate with Poor Prognosis in Human Breast Cancer

To evaluate the prognostic value of OCT4 expression and vasculogenic mimicry (VM) in human breast cancer, we examined OCT4 expression and VM formation using immunohistochemistry and CD31/PAS (periodic acid-schiff) double staining on 90 breast cancer specimens. All patients were followed up for five–149 months following surgery. Survival curves were generated using Kaplan-Meier method. Multivariate analysis was performed using Cox regression model to assess the prognostic values. Results showed positive correlation between OCT4 expression and VM formation (p < 0.05). Both OCT4 expression and VM were also positively correlated with lymph node metastasis, higher histological grade, and Nottingham prognostic index (p < 0.05). Patients with OCT4 expression or VM formation exhibited poorer overall survival (OS) and disease-free survival (DFS) than OCT4-negative or VM-negative patients (p < 0.05). OCT4-positive/VM-positive patients also had the worst OS and DFS (p < 0.05). In multivariate survival analysis, VM, Nottingham prognostic index (NPI), and Her2 were independent prognostic factors related to OS and OCT4-positive/VM-positive patients, whereas NPI and Her2 were independent predictors of DFS. These results suggest that a combined OCT4 expression/VM could improve the prognostic judgment for breast cancer patients.     

Numerical simulation of flow focusing pattern and morphological changes in two-phase flow inside nozzle

The flow focusing nozzle is a new type of nozzle that performs effective atomization of the discrete phase by means of high-speed motion of the continuous phase.The flow pattern and its morphological changes have a significant effect on the atomization, but the influence of different parameters on the morphological change of the flow pattern remains unclear.The flow focusing pattern and morphological changes in the two-phase flow inside the nozzle were simulated numerically, based on the volume of fluid method.The results demonstrate that the ratio of the nozzle-to-capillary distance and capillary diameter, the gas–liquid velocity ratio, and capillary diameter have significant effects on the flow pattern.When the ratio of the nozzle-to-capillary distance H and capillary diameter D increases, or the capillary diameter D increases, the flow pattern tends to transform into a laminar form; however, when the gas–liquid velocity ratio V increases, the flow pattern tends to transform into a turbulence form.Furthermore, we define the cone-shaped expansion rate, cone-shaped focusing rate,and cone angle in order to study the morphological changes in the cone shape inside the nozzle.The results indicate that the morphological change of the cone shape and flow pattern transformation is interrelated.When the cone shape tends to be unstable, the flow pattern changes towards flow blurring, whereas, a stable cone indicates that the flow tends to exhibit a droplet pattern.     

Temperature and concentration dependence of diffusion coefficient in dilute solutions

Temperature and molecular weight dependence of k_D in D(C) = D(O) [1 + Ck_D], where D(C) is the diffusion coefficient for the density fluctuations in a dilute polymer solution, is investigated by first expressing D(C) as a function of the static structure factor S(q,C) within the framework of the Kirkwood-Riseman theory. The continuous transition of k_D from negative values under theta conditions to positive values in good solvents is calculated using various models for the intermolecular interaction potential and the results are presented graphically as function of a reduced variables $\text{S}\text{?}\text{R}{}_{\mathrm{H}}$ that combines both molecular weight and temperature effects. It is shown that the negative value of k_D at the theta temperature can be explained at least partially, in terms of an increase in the chain dimensions of two overlapping molecules. The concentration dependence of the self-diffusion coefficient is also discussed.     

Influence of heating and laser irradiation on the Raman D band in single-wall carbon nanotubes

Fine structure of the Raman D band in single-wall carbon nanotubes (SWNTs) was investigated by heating and laser irradiation. It is shown that the D band is composed of three components at ～ 1313, 1340, and 1355 cm^? 1, denoted by D₁, D₂, and D₃, respectively. The D₁ and D₂ intensities significantly increase with laser irradiation in air and vacuum, respectively. The D₃ intensity drastically increases with heating in air. From these results, it is suggested that the fine structure of the D band is attributed to different kinds of defects introduced in SWNTs.     

Novel TTLL5 Variants Associated with Cone-Rod Dystrophy and Early-Onset Severe Retinal Dystrophy

Variants of the TTLL5 gene, which encodes tubulin tyrosine ligase-like family member five, are a rare cause of cone dystrophy (COD) or cone-rod dystrophy (CORD). To date, only a few TTLL5 patients have been clinically and genetically described. In this study, we report five patients harbouring biallelic variants of TTLL5. Four adult patients presented either COD or CORD with onset in the late teenage years. The youngest patient had a phenotype of early onset severe retinal dystrophy (EOSRD). Genetic analysis was performed by targeted next generation sequencing of gene panels and assessment of copy number variants (CNV). We identified eight variants, of which six were novel, including two large multiexon deletions in patients with COD or CORD, while the EOSRD patient harboured the novel homozygous p.(Trp640*) variant and three distinct USH2A variants, which might explain the observed rod involvement. Our study highlights the role of TTLL5 in COD/CORD and the importance of large deletions. These findings suggest that COD or CORD patients lacking variants in known genes may harbour CNVs to be discovered in TTLL5, previously undetected by classical sequencing methods. In addition, variable phenotypes in TTLL5-associated patients might be due to the presence of additional gene defects.     

Structure and steady-state spray performance of an effervescent diesel injector

We describe, for the first time, the structure and steady-state performance of an effervescent Diesel injector (EDI) having a needle and sac and being entirely contained in a conventional Diesel injector body. Experimental results help address key questions, including the single influences of needle lift, exit orifice diameter, aerator pore size, atomizing gas–liquid ratio, and injection pressure on injector performance (mean drop size, drop size distribution, and cone half-angle), as well as any coupling that results when these quantity are varied in tandem. D₃₂ data demonstrate that there is a small, but experimentally significant, influence of needle lift on performance when it increases from 0.025 to 0.076 mm, but no effect with a further increase to 0.318 mm. There is no significant effect of either atomizing gas-to-liquid ratio (GLR), exit orifice diameter, or aerator pore size, but injection pressure does play a key role. No coupling between the effects of needle lift and any other parameter on D₃₂ was observed. Drop size distribution data show that the distribution width increases at smaller needle lifts and with an increase in injection pressure—there is a small effect of exit orifice diameter at the largest GLRs. Neither GLR nor aerator pore size has any effect, nor is there any coupling between the effects of needle lift and any other parameter. Finally, spray cone angle data show that increasing needle lift from 0.025 to 0.318 mm reduces cone half-angle from 11 to 7°. Other parameters have no effect, nor is there any coupling between needle lift changes and variations in other parameters. The effects of needle lift, when they occur, are ascribed to corresponding changes in atomizer exit velocity.     

Conditional Loss of the Exocyst Component Exoc5 in Retinal Pigment Epithelium (RPE) Results in RPE Dysfunction,Photoreceptor Cell Degeneration,and Decreased Visual Function

To characterize the mechanisms by which the highly conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on Exoc5 (also known as sec10), a central exocyst component. We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice. Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE. In this study, we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency. Exoc5^−/− zebrafish had smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments. At 3.5 days post-fertilization, loss of rod and cone opsins were observed in zebrafish exoc5 mutants. Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function and loss of visual photoreceptor pigments. Abnormal levels of RPE65 together with a reduced c-wave amplitude indicate a dysfunctional RPE. The retinal phenotype in Exoc5^−/− mice was present at 20 weeks, but was more pronounced at 27 weeks, indicating progressive disease phenotype. We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development. Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision. Together, these data suggest that exocyst-mediated signaling in the RPE is required for RPE structure and function, indirectly leading to photoreceptor degeneration.     

Hydrodynamic study of fine metallic powders in an original spouted bed contactor in view of chemical vapor deposition treatments

An original gas-solid contactor was developed so as to treat by chemical vapor deposition, fine (mean diameter 23 μm) and dense (bulk density 7700 kg/m³) NiCoCrAlYTa powders with large size distribution. In order to avoid the presence of a distributor in the reactive zone, a spouted bed configuration was selected, consisting in a glass cylindrical column associated through a 60° cone to an inlet tube, connected at its bottom to a grid so as to support the powders at rest. A hydrodynamic study was conducted at ambient temperature and pressure, combining pressure drop measurements and visual observations as a function of gas velocity and of the ratio H/D of the height of the bed at rest over the bed diameter. Using conventional alumina particles belonging to Geldart's group B, it was shown that this equipment is able to ensure conventional spouted bed behavior, especially for H/D ratio equal to 1. From numerous experiments conducted with the fine metallic powders of interest, it was shown that (i) conventional pressure drop curves for spouted beds are obtained for H/D ratios between 1 and 1.8, (ii) due to the large grain size distribution of particles, minimum spouted bed velocities occur in a range rather than at precise values. Visual observations reveal the presence of the spout and fountain at the minimum spouted bed velocity and for H/D equal to 1.     

Lindemann-like size-independent glass-transition criterion for polymers

It is well known that the intrinsic melting mechanism is independent of crystal size according to Lindemann's melting criterion. In order to probe whether the glass transition mechanism is also size-independent, segment dynamics of free-standing polystyrene (PS) films is determined by considering the temperature- and thickness-dependent number of styrene segments N_α(T,D) in the cooperative rearranging region (CRR). Under the help of Adams-Gibbs glass transition theory and molecular dynamics simulation, N_α(T,D) function is established and it decreases as D decreases or T increases. However, N_α[T_g(D),D] at the glass transition temperature T_g(D) is size-independent, which is consistent with the simulation results obtained by Donth's method. Meanwhile, its relative temperature function N_α{[T − T_g(D)]/T_g(D)} is also size-independent. Therefore, N_α[T_g(D),D] function as a criterion for glass transition, which describes the physical nature of the glass transition, is similar to the vibrational amplitude in Lindemann's melting criterion.     

The influence of sorption hysteresis on diffusion coefficients represented with different moisture potentials

This paper continues the discussion in a previous paper [Saeidpour and Wadsö doi:10.1016/j.cemconres.2016.02.003] where a clear effect of sorption hysteresis was seen in moisture diffusion coefficients for cement-based materials. Coefficients with vapor content as potential (D_v) had different values depending on whether the sample was in absorption or in desorption. In this paper we recalculate these D_v coefficients into coefficients with other potentials and draw them as a function of different potentials. The aim is to see if the effect of hysteresis disappears with an optimal choice of representation. The best choice was to draw D_v is as a function of the degree of saturation (concentration, moisture content).     

Fractional coefficient of macromolecules in concentrated solution and in the vicinity of the critical solution temperature. Diffusion and sedimentation studies of polystyrene in toluene and trans-decalin

The concentration dependence of the frictional coefficient (f) in concentrated solutions of well-defined polystyrene fractions has been studied both in a good solvent (toluene) and in a solvent where θ conditions and condition in the vicinity of phase separation can be realized (trans-decalin). f and the self-diffusion coefficient (D⁺) have been calculated from a combination of measured translational diffusion coefficients (D) and osmotic pressure data for the systems; polystyrene $\text{(M = 390 000)}\text{trans-}\text{decalin}$ up to 90 kg/m³ at 20° (θ-conditions), 30° and 40°C; polystyrene $\text{(M = 110 000)}\text{toluene}$ up to 120 kg/m³ at 25°C. Sedimentation measurements are also reported. The main results are: (a) over a concentration interval 0–100 kg/m³, f shows more than a ten-fold and almost linear increase; (b) in trans-decalin when the temperature is raised from 20° (θ-conditions) to 40°C (good solvent conditions), keeping the concentration constant, f increases only slightly, the increase being somewhat more pronounced at higher concentrations; (c) the concentration dependence of the ratio $\text{D}\text{D}{}^{\mathrm{+}}$ is considerable under good solvent conditions but becomes gradually less pronounced when θ-conditions are approached; (d) diffusion/osmosis and velocity sedimentation give identical values of f over the entire concentration interval.     

Solvent self-diffusion in semidilute polystyrene solutions. Pulsed-gradient spin-echo measurements on a standard Fourier transform n.m.r. spectrometer

Measurements of solvent self-diffusion coefficients in the systems polystyrene/dichloromethane and polystyrene/cyclopentane recorded on a standard Fourier transform n.m.r. spectrometer are reported. The ratio $\text{D}{}_{\mathrm{s}}\text{D}{}_{\mathrm{s,0}}$ was found to decrease linearly with increasing volume fraction of polystyrene, over the interval studied (ø_PS<0.27), for both systems. For PS/cyclopentane an increase in temperature from 12.5°C (close to UCST) to 40°C was not found to alter the concentration dependence of the ratio $\text{D}{}_{\mathrm{s}}\text{D}{}_{\mathrm{s,0}}$ significantly.     

Gravity pipe flow of polymeric bulk solids in pneumatic conveying system

Various physical parameters of gravity pipes such as gravity pipe diameter, the inclination of the gravity pipe, the cone angle, temperature of the discharged material and the rate of air counter flow into the gravity pipe were studied. The results obtained from the pipe diameter and cone angle experiments showed that the mass flow rate was proportional to (D-1.4d)^2.5 and also to θ^-0.5. The results from the pipe inclination experiment showed the existence of an angle of inclination for maximum flow, which was also reported by Wieghardt [Uber einige versuche an stromungen in sand. Ingenieur Archived 20, 109-115]. The air flow experiment also showed that the mass flow rate was inversely proportional to the air counter-flow rate, and strongly influenced by the material properties. Results from the temperature experiment showed that the temperature of the material had slight effect on the mass flow rate for the temperature range that was used in the experiment. Flow visualization images showed formation of solid plugs in the pipe that played a part in influencing the behaviour and mass flow rate of solids in the system.