Choosing T-cell sources determines CAR-T cell activity in neuroblastoma.

Introduction: The clinical success of chimeric antigen receptor-modified T cells (CAR-T cells) for hematological malignancies has not been reproduced for solid tumors, partly due to the lack of cancer-type specific antigens. In this work, we used a novel combinatorial approach consisting of a versatile anti-FITC CAR-T effector cells plus an FITC-conjugated neuroblastoma (NB)-targeting linker, an FITC-conjugated monoclonal antibody (Dinutuximab) that recognizes GD2. Methods: We compared cord blood (CB), and CD45RA-enriched peripheral blood leukapheresis product (45RA) as allogeneic sources of T cells, using peripheral blood (PB) as a control to choose the best condition for anti-FITC CAR-T production. Cells were manufactured under two cytokine conditions (IL-2 versus IL-7+IL-15+IL-21) with or without CD3/CD28 stimulation. Immune phenotype, vector copy number, and genomic integrity of the final products were determined for cell characterization and quality control assessment. Functionality and antitumor capacity of CB/45RA-derived anti-FITC CAR-T cells were analyzed in co-culture with different anti-GD2-FITC labeled NB cell lines. Results: The IL-7+IL-15+IL-21 cocktail, in addition to co-stimulation signals, resulted in a favorable cell proliferation rate and maintained less differentiated immune phenotypes in both CB and 45RA T cells. Therefore, it was used for CAR-T cell manufacturing and further characterization. CB and CD45RA-derived anti-FITC CAR-T cells cultured with IL-7+IL-15+IL-21 retained a predominantly naïve phenotype compared with controls. In the presence of the NB-FITC targeting, CD4+ CB-derived anti-FITC CAR-T cells showed the highest values of co-stimulatory receptors OX40 and 4-1BB, and CD8+ CAR-T cells exhibited high levels of PD-1 and 4-1BB and low levels of TIM3 and OX40, compared with CAR-T cells form the other sources studied. CB-derived anti-FITC CAR-T cells released the highest amounts of cytokines (IFN-γ and TNF-α) into co-culture supernatants. The viability of NB target cells decreased to 30% when co-cultured with CB-derived CAR-T cells during 48h. Conclusion: CB and 45RA-derived T cells may be used as allogeneic sources of T cells to produce CAR-T cells. Moreover, ex vivo culture with IL-7+IL-15+IL-21 could favor CAR-T products with a longer persistence in the host. Our strategy may complement the current use of Dinutuximab in treating NB through its combination with a targeted CAR-T cell approach.

Prognostic value of translationally controlled tumor protein in colon cancer.

The translationally controlled tumor protein (TCTP) is a highly conserved protein involved in a variety of normal cell functions and disease processes. Preclinical studies revealed that TCTP has anti-apoptotic properties, promotes cell growth and division and is involved in cancer progression by promoting invasion and metastasis. The present study explored the potential value of TCTP as a prognostic marker in colon cancer. A retrospective analysis of 74 patients with colon cancer was performed. Using immunohistochemistry, TCTP levels in the primary tumor were assessed semi-quantitatively by the calculation of cytoplasmic and nuclear H-score. Cytoplasmic TCTP levels in the primary tumor had no statistically significant association with disease-free survival (DFS), progression-free survival (PFS) and overall survival (OS) in the present patient population. Patients whose primary tumors had a negative nuclear TCTP expression had significantly improved clinical outcomes. The PFS for the negative nuclear TCTP expression group was 7.7 months [95% confidence interval (CI), 5.8-9.5] compared with 5.5 months (95% CI, 3.2-7.8) in the group with positive nuclear expression (P=0.023, Mantel-Cox log-rank). Patients with a negative nuclear expression of TCTP had a significantly higher median OS (22.2 months; 95% CI, 16.1-28.3) compared with those with positive TCTP nuclear expression (median 13.2 months; 95% CI, 10.1-16.3; P=0.008, Mantel-Cox log-rank). In a multivariate Cox regression model, a positive nuclear TCTP H-score was an independent risk factor for worse PFS and OS. The 1-year OS rate in the group with negative nuclear TCTP expression was 86.3% compared with 56.5% in patients with positive nuclear TCTP expression (P=0.008). The present study suggested that semiquantitative H-score measurement of TCTP levels in the nuclei of tumor cells from the primary tumor is a potential prognostic marker for clinical outcomes in patients with colon cancer.

Arginase deficiency in Bulgaria: first cases and potential endemic region for the disorder.

Arginase deficiency is an autosomal recessive urea cycle disorder caused by pathogenic variants in the ARG1 gene. The clinical features of the disease include spasticity, tremour, ataxia, hypotonia, microcephaly and seizures. Growth delay can also be observed in the affected individuals. Here we describe the results from molecular-genetic analysis of two patients with arginase deficiency. In the first case, we reported a novel homozygous missense variant c.775G>A p.(Gly259Ser) in a patient with Bulgarian ethnic origin. In the second case, a novel homozygous splice site variant c.329+1G>A was detected in a patient from a consanguineous family of Roma ethnic origin. A hundred samples of newborns of Roma origin were screened for variant c.329+1G>A and one individual was found to be a heterozygous carrier of variant c.329+1G> A. The results from this study indicated the necessity for screening of the Roma population with respect to the disease arginase deficiency in Bulgaria.

Technological Pathways to Produce Compressed and Highly Pure Hydrogen from Solar Power.

Hydrogen (H2 ) produced from renewables will have a growing impact on the global energy dynamics towards sustainable and carbon-neutral standards. The share of green H2 is still too low to meet the net-zero target, while the demand for high-quality hydrogen continues to rise. These factors amplify the need for economically viable H2 generation technologies. The present article aims at evaluating the existing technologies for high-quality H2 production based on solar energy. Technologies such as water electrolysis, photoelectrochemical and solar thermochemical water splitting, liquid metal reactors and plasma conversion utilize solar power directly or indirectly (as carbon-neutral electrons) and are reviewed from the perspective of their current development level, technical limitations and future potential.

The effect of separation of blocks on the crystallization kinetics and phase composition of poly(butylene adipate) in multi-block thermoplastic polyurethanes.

The influence of the hard segment nature on the crystallization kinetics of multi-block thermoplastic polyurethanes containing poly(butylene adipate) (PBA) as a soft segment was investigated. Using a combination of FTIR spectroscopy, time-domain 1H nuclear magnetic resonance (TD-NMR), differential scanning calorimetry (DSC), fast-scanning calorimetry (FSC) and wide-angle X-ray diffraction (WAXS), it was shown that aliphatic, cycloaliphatic and aromatic diisocyanates affect the phase separation efficiency of soft and hard segments. The best phase separation efficiency was observed for a sample containing aliphatic diisocyanate due to the development of a hydrogen bond network. The thermal history, phase separation and the degree of ordering of the polyurethane determine the polymorphic behavior of melt-crystallized PBA. The formation of a partially-ordered mesophase of linear aliphatic polyurethane leads to an increase in the crystallization rate of PBA at room temperature and the formation of thermodynamically stable α-crystals. The presence of bulk cycloaliphatic and aromatic diol-urethane fragments prevents the phase separation of PBA, which crystallizes after slow cooling in a mixture of α- and ß-crystalline forms. The new nanocalorimetry technique allows the identification of a direct correlation between the phase separation and crystallization kinetics of the melt-crystallized PBA in a wide range of cooling rates - from 2 to 30 000 K s-1. Particularly, ultra-fast cooling suppresses the nucleation of the ß-phase of PBA resulting in slow crystallization of only α-modification at room temperature. The role of the polyurethane mesophase in the crystallization of the soft segment was discussed for the first time.

A Novel Technique for Single-stage Reconstruction of Nipple-Areola Complex following Central Lumpectomy.

Several oncoplastic techniques have been proposed for subareolar breast cancer, some of which may require contralateral operation for symmetry, or more than one operation for delayed reconstruction of the nipple-areola complex (NAC). We herein developed a simple and effective oncoplastic approach following central quadrantectomy, aiming to achieve the following: (1) preservation of breast shape and contour for patients who are not accepting of a notably smaller breast or bilateral operation; (2) single procedure with advantages of single anesthetic and the ability to reconstruct a breast that has not yet been affected by radiation; (3) no autologous grafts with disadvantage of an extra donor site; (4) creation of natural neo-NAC with only incision within the region of the areola complex; and (5) maintaining long-term nipple projection. In this technique, the medial and lateral peri-areolar flap was advanced and rotated to restore partial neo-NAC, and to fill the defect after central tumor and NAC resection. The whole neo-NAC margin was created using the "round block" technique. Additionally, the nipple reconstruction was performed at the mitten of the neo-areola area using the C-V nipple flaps with an internal strut of rolled dermal grafts harvested from excised skin during the round block procedures. Lastly, a purse-string suture is placed in the outer skin margin for control of areola diameter and project. Six patients with central tumors of the breast were treated in this easily achieved approach. Patient satisfaction was noted as high, and the cosmetic results were evaluated as good to excellent.

Processing Ceramic Proton Conductor Membranes for Use in Steam Electrolysis.

Steam electrolysis constitutes a prospective technology for industrial-scale hydrogen production. The use of ceramic proton-conducting electrolytes is a beneficial option for lowering the operating temperature. However, a significant challenge with this type of electrolyte has been upscaling robust planar type devices. The fabrication of such multi-layered devices, usually via a tape casting process, requires careful control of individual layers' shrinkages to prevent warping and cracks during sintering. The present work highlights the successful processing of 50 × 50 mm2 planar electrode-supported barium cerium yttrium zirconate BaZr0.44Ce0.36Y0.2O2.9 (BZCY(54)8/92) half cells via a sequential tape casting approach. The sintering parameters of the half-cells were analyzed and adjusted to obtain defect-free half-cells with diminished warping. Suitably dense and gas-tight electrolyte layers are obtained after co-sintering at 1350 °C for 5 h. We then assembled an electrolysis cell using Ba0.5La0.5CoO3-δ as the steam electrode, screen printed on the electrolyte layer, and fired at 800 °C. A typical Ba0.5La0.5CoO3-δ|BaZr0.44Ce0.36Y0.2O3-δ(15 µm)|NiO-SrZr0.5Ce0.4Y0.1O3-δ cell at 600 °C with 80% steam in the anode compartment reached reproducible terminal voltages of 1.4 V @ 500 mA·cm-2, achieving ~84% Faradaic efficiency. Besides electrochemical characterization, the morphology and microstructure of the layered half-cells were analyzed by a combination of high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and energy-dispersive X-ray spectroscopy. Our results also provide a feasible approach for realizing the low-cost fabrication of large-sized protonic ceramic conducting electrolysis cells (PCECs).

Direction observation of the grain boundary segregation in molybdenum substituted lanthanum tungstate membranes.

Molybdenum substituted lanthanum tungstate membranes (LWO-Mo) offer a good alternative for the separation of hydrogen from gas mixtures. During several essential steps of the membrane processing, an intensive employment of ZrO2 milling balls is usually inevitable. However, how these milling balls affect the final LWO-Mo membranes, is still largely unknown. Employing comprehensive transmission electron microscopy (TEM) techniques, the residual Zr was found to segregate to the grain boundaries (GBs) of the LWO-Mo, either as thin layers or as individual nanograins. At atomic scale, structural and chemical analyses on these GB features were carried out quantitatively. The segregated Zr took more than half of the W sites of the LWO-Mo, resulting in a strained LWO structure and locally concentrated oxygen vacancies. To minimize any Zr contamination, either a competent alternative for ZrO2 or a careful introduction of certain secondary phases (SPs) was proposed. Our results unravel the processing-induced GB behaviors in LWO-Mo, which pave the way towards further optimized processing for various types of functional membranes.

A familial case of severe infantile nephronophthisis explained by oligogenic inheritance.

Renal cysts are common malformation during the prenatal and postnatal period and frequent cause of chronic kidney or ESRD. More than 70 genes have been shown to play role in their pathology. Part of them are responsible for the structure and function of the cilia, which assigns a large proportion of the renal cystic diseases in the ciliopathies. Another group of genes responsible for cystic kidneys encodes transcription factors with crucial role during organogenesis. We describe here a systematic approach for identifying the genetic cause(s) of an unusually severe form of renal cystic disease in a family with multiple affected siblings. High throughput mutations screening of the parents and one of the children was applied for identifying the genetic causes of the disease. The affected child was found to have inherited 3 deleterious mutations in two nephronophthisis genes, NPHP3 and NPHP4. The possibility for epistatic interaction of the NPHP mutations as well as the modifying effect of other inherited genetic variants is discussed.

Hydrogen separation through tailored dual phase membranes with nominal composition BaCe0.8Eu0.2O3-δ:Ce0.8Y0.2O2-δ at intermediate temperatures.

Hydrogen permeation membranes are a key element in improving the energy conversion efficiency and decreasing the greenhouse gas emissions from energy generation. The scientific community faces the challenge of identifying and optimizing stable and effective ceramic materials for H2 separation membranes at elevated temperature (400-800 °C) for industrial separations and intensified catalytic reactors. As such, composite materials with nominal composition BaCe0.8Eu0.2O3-δ:Ce0.8Y0.2O2-δ revealed unprecedented H2 permeation levels of 0.4 to 0.61 mL·min-1·cm-2 at 700 °C measured on 500 µm-thick-specimen. A detailed structural and phase study revealed single phase perovskite and fluorite starting materials synthesized via the conventional ceramic route. Strong tendency of Eu to migrate from the perovskite to the fluorite phase was observed at sintering temperature, leading to significant Eu depletion of the proton conducing BaCe0.8Eu0.2O3-δ phase. Composite microstructure was examined prior and after a variety of functional tests, including electrical conductivity, H2-permeation and stability in CO2 containing atmospheres at elevated temperatures, revealing stable material without morphological and structural changes, with segregation-free interfaces and no further diffusive effects between the constituting phases. In this context, dual phase material based on BaCe0.8Eu0.2O3-δ:Ce0.8Y0.2O2-δ represents a very promising candidate for H2 separating membrane in energy- and environmentally-related applications.

Creatine Deficiency Syndrome could be Missed Easily: A Case Report of Guanidinoacetate Methyltransferase Deficiency Presented with Neurodevelopmental Delay, Seizures, and Behavioral Changes, but Normal Structural MRI.

A case with GAMT deficiency (homozygous c.64dupG mutation) presented with neurodevelopmental delay, rare seizures, behavioral disturbances, and mild hypotonia, posing diagnostic challenges. Metabolic investigations showed low creatinine in plasma and urine (guanidinoacetate couldn't be investigated) and slightly elevated lactate. MRI was normal. Correct diagnosis was possible only after MR spectroscopy was performed at age 5½ years. A homozygous c.64dupG mutation of the GAMT gene was identified in the proband. In conclusion, every case with neurodevelopmental delay or arrest, especially when accompanied by seizures, behavioral impairment, muscle hypotonia or extrapyramidal symptoms should undergo MRI with MR spectroscopy. Normal structural MRI doesn't exclude a creatine deficiency syndrome. Biochemical investigations of guanidinoacetate, creatine, and creatinine in body fluid should be done to diagnose cerebral creatine deficiency syndromes and to specify the deficient enzyme. Thus, a treatable disease will not be missed.

Founder p.Arg 446* mutation in the PDHX gene explains over half of cases with congenital lactic acidosis in Roma children.

Investigation of 31 of Roma patients with congenital lactic acidosis (CLA) from Bulgaria identified homozygosity for the R446* mutation in the PDHX gene as the most common cause of the disorder in this ethnic group. It accounted for around 60% of patients in the study and over 25% of all CLA cases referred to the National Genetic Laboratory in Bulgaria. The detection of a homozygous patient from Hungary and carriers among population controls from Romania and Slovakia suggests a wide spread of the mutation in the European Roma population. The clinical phenotype of the twenty R446* homozygotes was relatively homogeneous, with lactic acidosis crisis in the first days or months of life as the most common initial presentation (15/20 patients) and delayed psychomotor development and/or seizures in infancy as the leading manifestations in a smaller group (5/20 patients). The subsequent clinical picture was dominated by impaired physical growth and a very consistent pattern of static cerebral palsy-like encephalopathy with spasticity and severe to profound mental retardation seen in over 80% of cases. Most patients had a positive family history. We propose testing for the R446* mutation in PDHX as a rapid first screening in Roma infants with metabolic acidosis. It will facilitate and accelerate diagnosis in a large proportion of cases, allow early rehabilitation to alleviate the chronic clinical course, and prevent further affected births in high-risk families.

Synthesis and characterization of nonsubstituted and substituted proton-conducting La(6-x)WO(12-y).

Mixed proton-electron conductors (MPEC) can be used as gas separation membranes to extract hydrogen from a gas stream, for example, in a power plant. From the different MPEC, the ceramic material lanthanum tungstate presents an important mixed protonic-electronic conductivity. Lanthanum tungstate La(6-x)WO(12-y) (with y = 1.5x + δ and x = 0.5-0.8) compounds were prepared with La/W ratios between 4.8 and 6.0 and sintered at temperatures between 1300 and 1500 °C in order to study the dependence of the single-phase formation region on the La/W ratio and temperature. Furthermore, compounds substituted in the La or W position were prepared. Ce, Nd, Tb, and Y were used for partial substitution at the La site, while Ir, Re, and Mo were applied for W substitution. All substituents were applied in different concentrations. The electrical conductivity of nonsubstituted La(6-x)WO(12-y) and for all substituted La(6-x)WO(12-y) compounds was measured in the temperature range of 400-900 °C in wet (2.5% H2O) and dry mixtures of 4% H2 in Ar. The greatest improvement in the electrical characteristics was found in the case of 20 mol % substitution with both Re and Mo. After treatment in 100% H2 at 800 °C, the compounds remained unchanged as confirmed with XRD, Raman, and SEM.

Enhanced H2 separation through mixed proton-electron conducting membranes based on La5.5 W0.8 M0.2 O11.25-δ.

La(5.5) WO11.25-δ is a proton-conducting oxide that shows high protonic conductivity, sufficient electronic conductivity, and stability in moist CO2 environments. However, the H2 flows achieved to date when using La(5.5) WO11.25-δ membranes are still below the threshold for practical application in industrial processes. With the aim of improving the H2 flow obtained with this material, La(5.5) WO11.25-δ was doped in the W position by using Re and Mo; the chosen stoichiometry was La(5.5) W0.8 M0.2 O11.25-δ . This work presents the electrochemical characterization of these two compounds under reducing conditions, the H2 separation properties, as well as the influence of the H2 concentration in the feed stream, degree of humidification, and operating temperature. Doping with both Re and Mo enabled the magnitude of H2 permeation to be enhanced, reaching unrivaled values of up to 0.095 mL min(-1) cm(-2) at 700 °C for a La(5.5) W0.8 Re0.2 O11.25-δ membrane (760 µm thick). The spent membranes were investigated by using XRD, SEM, and TEM on focused-ion beam lamellas. Furthermore, the stability in CO2 -rich and H2 S-containing atmospheres was evaluated, and the compounds were shown to be stable in the atmospheres studied.