First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill.

Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the phytoene desaturase gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.     

Removal of an Azo Dye from Wastewater through the Use of Two Technologies: Magnetic Cyclodextrin Polymers and Pulsed Light

Water pollution by dyes is a huge environmental problem; there is a necessity to produce new decolorization methods that are effective, cost-attractive, and acceptable in industrial use. Magnetic cyclodextrin polymers offer the advantage of easy separation from the dye solution. In this work, the β-CD-EPI-magnetic (β-cyclodextrin-epichlorohydrin) polymer was synthesized, characterized, and tested for removal of the azo dye Direct Red 83:1 from water, and the fraction of non-adsorbed dye was degraded by an advanced oxidation process. The polymer was characterized in terms of the particle size distribution and surface morphology (FE-SEM), elemental analysis (EA), differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), infrared spectrophotometry (IR), and X-ray powder diffraction (XRD). The reported results hint that 0.5 g and pH 5.0 were the best conditions to carry out both kinetic and isotherm models. A 30 min contact time was needed to reach equilibrium with a qmax of 32.0 mg/g. The results indicated that the pseudo-second-order and intraparticle diffusion models were involved in the assembly of Direct Red 83:1 onto the magnetic adsorbent. Regarding the isotherms discussed, the Freundlich model correctly reproduced the experimental data so that adsorption was confirmed to take place onto heterogeneous surfaces. The calculation of the thermodynamic parameters further demonstrates the spontaneous character of the adsorption phenomena (ΔG° = −27,556.9 J/mol) and endothermic phenomena (ΔH° = 8757.1 J/mol) at 25 °C. Furthermore, a good reusability of the polymer was evidenced after six cycles of regeneration, with a negligible decline in the adsorption extent (10%) regarding its initial capacity. Finally, the residual dye in solution after treatment with magnetic adsorbents was degraded by using an advanced oxidation process (AOP) with pulsed light and hydrogen peroxide (343 mg/L); >90% of the dye was degraded after receiving a fluence of 118 J/cm²; the discoloration followed a pseudo first-order kinetics where the degradation rate was 0.0196 cm²/J. The newly synthesized β-CD-EPI-magnetic polymer exhibited good adsorption properties and separability from water which, when complemented with a pulsed light-AOP, may offer a good alternative to remove dyes such as Direct Red 83:1 from water. It allows for the reuse of both the polymer and the dye in the dyeing process.     

Anatomical Laser Microdissection of the Ileum Reveals mtDNA Depletion Recovery in A Mitochondrial Neuro-Gastrointestinal Encephalomyopathy (MNGIE) Patient Receiving Liver Transplant

mitochondrial neuro-gastrointestinal encephalomyopathy (MNGIE) is a rare genetic disorder characterized by thymidine phosphorylase (TP) enzyme defect. The absence of TP activity induces the imbalance of mitochondrial nucleotide pool, leading to impaired mitochondrial DNA (mtDNA) replication and depletion. Since mtDNA is required to ensure oxidative phosphorylation, metabolically active tissues may not achieve sufficient energy production. The only effective life-saving approach in MNGIE has been the permanent replacement of TP via allogeneic hematopoietic stem cell or liver transplantation. However, the follow-up of transplanted patients showed that gut tissue changes do not revert and fatal complications, such as massive gastrointestinal bleeding, can occur. The purpose of this study was to clarify whether the reintroduction of TP after transplant can recover mtDNA copy number in a normal range. Using laser capture microdissection and droplet-digital-PCR, we assessed the mtDNA copy number in each layer of full-thickness ileal samples of a naive MNGIE cohort vs. controls and in a patient pre- and post-TP replacement. The treatment led to a significant recovery of gut tissue mtDNA amount, thus showing its efficacy. Our results indicate that a timely TP replacement is needed to maximize therapeutic success before irreversible degenerative tissue changes occur in MNGIE.     

Superior Cerebellar Atrophy: An Imaging Clue to Diagnose ITPR1-Related Disorders

The inositol 1,4,5-triphosphate receptor type 1 (ITPR1) gene encodes an InsP₃-gated calcium channel that modulates intracellular Ca²⁺ release and is particularly expressed in cerebellar Purkinje cells. Pathogenic variants in the ITPR1 gene are associated with different types of autosomal dominant spinocerebellar ataxia: SCA15 (adult onset), SCA29 (early-onset), and Gillespie syndrome. Cerebellar atrophy/hypoplasia is invariably detected, but a recognizable neuroradiological pattern has not been identified yet. With the aim of describing ITPR1-related neuroimaging findings, the brain MRI of 14 patients with ITPR1 variants (11 SCA29, 1 SCA15, and 2 Gillespie) were reviewed by expert neuroradiologists. To further evaluate the role of superior vermian and hemispheric cerebellar atrophy as a clue for the diagnosis of ITPR1-related conditions, the ITPR1 gene was sequenced in 5 patients with similar MRI pattern, detecting pathogenic variants in 4 of them. Considering the whole cohort, a distinctive neuroradiological pattern consisting in superior vermian and hemispheric cerebellar atrophy was identified in 83% patients with causative ITPR1 variants, suggesting this MRI finding could represent a hallmark for ITPR1-related disorders.     

NMDA and P2X7 Receptors Require Pannexin 1 Activation to Initiate and Maintain Nociceptive Signaling in the Spinal Cord of Neuropathic Rats

Pannexin 1 (Panx1) is involved in the spinal central sensitization process in rats with neuropathic pain, but its interaction with well-known, pain-related, ligand-dependent receptors, such as NMDA receptors (NMDAR) and P2X7 purinoceptors (P2X7R), remains largely unexplored. Here, we studied whether NMDAR- and P2X7R-dependent nociceptive signaling in neuropathic rats require the activation of Panx1 channels to generate spinal central sensitization, as assessed by behavioral (mechanical hyperalgesia) and electrophysiological (C-reflex wind-up potentiation) indexes. Administration of either a selective NMDAR agonist i.t. (NMDA, 2 mM) or a P2X7R agonist (BzATP, 150 μM) significantly increased both the mechanical hyperalgesia and the C-reflex wind-up potentiation, effects that were rapidly reversed (minutes) by i.t. administration of a selective pannexin 1 antagonist (10panx peptide, 300 μM), with the scores even reaching values of rats without neuropathy. Accordingly, 300 μM 10panx completely prevented the effects of NMDA and BzATP administered 1 h later, on mechanical hyperalgesia and C-reflex wind-up potentiation. Confocal immunofluorescence imaging revealed coexpression of Panx1 with NeuN protein in intrinsic dorsal horn neurons of neuropathic rats. The results indicate that both NMDAR- and P2X7R-mediated increases in mechanical hyperalgesia and C-reflex wind-up potentiation require neuronal Panx1 channel activation to initiate and maintain nociceptive signaling in neuropathic rats.     

Nutritional Calcium Supply Dependent Calcium Balance,Bone Calcification and Calcium Isotope Ratios in Rats

Serum calcium isotopes (δ^44/42Ca) have been suggested as a non-invasive and sensitive Ca balance marker. Quantitative δ^44/42Ca changes associated with Ca flux across body compartment barriers relative to the dietary Ca and the correlation of δ^44/42Ca_Serum with bone histology are unknown. We analyzed Ca and δ^44/42Ca by mass-spectrometry in rats after two weeks of standard-Ca-diet (0.5%) and after four subsequent weeks of standard- and of low-Ca-diet (0.25%). In animals on a low-Ca-diet net Ca gain was 61 ± 3% and femur Ca content 68 ± 41% of standard-Ca-diet, bone mineralized area per section area was 68 ± 15% compared to standard-Ca-diet. δ^44/42Ca was similar in the diets, and decreased in feces and urine and increased in serum in animals on low-Ca-diet. δ^44/42Ca_Bone was higher in animals on low-Ca-diet, lower in the diaphysis than the metaphysis and epiphysis, and unaffected by gender. Independent of diet, δ^44/42Ca_Bone was similar in the femora and ribs. At the time of sacrifice, δ^44/42Ca_Serum inversely correlated with intestinal Ca uptake and histological bone mineralization markers, but not with Ca content and bone mineral density by µCT. In conclusion, δ^44/42Ca_Bone was bone site specific, but mechanical stress and gender independent. Low-Ca-diet induced marked changes in feces, serum and urine δ^44/42Ca in growing rats. δ^44/42Ca_Serum inversely correlated with markers of bone mineralization.     

Evaluation and Characterization of Post-Stroke Lung Damage in a Murine Model of Cerebral Ischemia

After stroke and other brain injuries, there is a high incidence of respiratory complications such as pneumonia or acute lung injury. The molecular mechanisms that drive the brain-lung interaction post-stroke have not yet been elucidated. We performed transient middle cerebral artery occlusion (MCAO) and sham surgery on C57BL/6J mice and collected bronchoalveolar lavage fluid (BALF), serum, brain, and lung homogenate samples 24 h after surgery. A 92 proteins-panel developed by Olink Proteomics^® was used to analyze the content in BALF and lung homogenates. MCAO animals had higher protein concentration levels in BALF than sham-controls, but these levels did not correlate with the infarct volume. No alteration in alveolar-capillary barrier permeability was observed. A total of 12 and 14 proteins were differentially expressed between the groups (FDR < 0.1) in BALF and lung tissue homogenates, respectively. Of those, HGF, TGF-α, and CCL2 were identified as the most relevant to this study. Their protein expression patterns were verified by ELISA. This study confirmed that post-stroke lung damage was not associated with increased lung permeability or cerebral ischemia severity. Furthermore, the dysregulation of HGF, TGF-α, and CCL2 in BALF and lung tissue after ischemia could play an important role in the molecular mechanisms underlying stroke-induced lung damage.     

Applicability assessment of a real-time PCR assay for the specific detection of bovine,ovine and caprine material in feedstuffs

A TaqMan real-time polymerase chain reaction (PCR) method was developed for specific detection of bovine, ovine and caprine processed animal protein (PAP) in industrial feedstuffs. The method uses species-specific primers and probes targeting short mitochondrial D-loop sequences, and a positive amplification control based on 18S rRNA gene. The applicability of the real-time PCR protocol was assessed through analysis of 126 industrial feed samples that were manufactured to reproduce rendering processes of commercial feeds destined for farmed animals. The assay successfully classified samples as positive or negative according to the ruminant composition, enabling qualitative detection of banned material in feeds at levels as low as 0.1%. Although the method provides quantitative potential, results suggest that the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing treatments of the feeds, which affect the amount and quality of amplifiable DNA.     

Long‐Range Nanoparticle Surface‐Energy‐Transfer Ruler for Monitoring Photothermal Therapy Response

A recent gold nanotechnology‐driven approach opens up a new possibility for the destruction of cancer cells through photothermal therapy. Ultimately, photothermal therapy may enter into clinical therapy and, as a result, there is an urgent need for techniques to monitor the tumor response to therapy. Driven by this need, a nanoparticle surface‐energy‐transfer (NSET) approach to monitor the photothermal therapy process by measuring a simple fluorescence intensity change is reported. The fluorescence intensity change is due to the light‐controlled photothermal release of single‐stranded DNA/RNA via dehybridization during the therapy process. Time‐dependent results show that just by measuring the fluorescence intensity change, the photothermal therapy response during the therapy process can be monitored. The possible mechanism and operating principle of the NSET assay are discussed. Ultimately, this NSET assay could have enormous potential applications in rapid, on‐site monitoring of the photothermal therapy process, which is critical to providing effective treatment of cancer and multidrug‐resistant bacterial infections.