Full Maturation Research Articles

Differences of microbial growth and biofilm formation among periprosthetic joint infection-causing species: an animal study.

The most frequently used surgical procedures for periprosthetic joint infections (PJIs) are debridement, antibiotics, and implant retention (DAIR), as well as single- or two-stage revision arthroplasty. The choice of surgery is made depending on the full maturation of the biofilm layer. The purpose of this study was to evaluate the biofilm formation and microbial growth using common PJI-causing agents and compare its development on the implant surface. The in vivo study was performed using 40 Sprague-Dawley rats divided into five groups (n = 8/group): Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Candida albicans, and control. Six standard titanium alloy discs were placed into the subcutaneous air pouches of the interscapular areas of the rats. After the inoculation of microorganisms, disc and soft tissue cultures were collected at 2-week intervals for 6weeks, and the microbial load and the microscopic appearance of the biofilm were compared. The disc samples from the S. aureus group had the highest infection load at all time points; however, in soft tissue samples, this was only observed at week 4 and 6. Electron microscopic images showed no distinctive differences in the biofilm structures between the groups. S. aureus microbial burden was significantly higher in implant cultures at week 2 compared to other PJI-causing agents examined. These results may explain the higher failure rate seen if the DAIR procedure was performed at < 3-4weeks after the PJI symptom onset and support the observation that DAIR may not be effective against PJIs caused by S. aureus.

Molecular basis for the stepwise and faithful maturation of the 20S proteasome.

The proteasome degrades most superfluous and damaged proteins, and its decline is associated with many diseases. As the proteolytic unit, the 20S proteasome is assembled from 28 subunits assisted by chaperones PAC1/2/3/4 and POMP; then, it undergoes the maturation process, in which the proteolytic sites are activated and the assembly chaperones are cleared. However, mechanisms governing the maturation remain elusive. Here, we captured endogenous maturation intermediates of human 20S proteasome, which are low abundance and highly dynamic, and determined their structures by cryo-electron microscopy. Through structure-based functional studies, we identified the key switches that remodel and activate the proteolytic sites. Our results also revealed that the POMP degradation is tightly controlled by a dual-checking mechanism, while the α5 subunit senses POMP degradation to induce PAC1/2 release, achieving the full maturation. These findings elucidate mechanisms directing and safeguarding the proteasome maturation and set basis for building proteasomes to counteract the decline of protein degradation in aging and disease.

Huyang Yangkun formula regulates the mitochondria pathway of ovarian granulosa cell apoptosis through FTO/m6A-P53 pathway.

Premature ovarian insufficiency (POI) presents a significant challenge to female reproductive health. The Huyang Yangkun Formula (HYF), a traditional Chinese medicinal formulation, has been utilized in clinical settings for the treatment of POI for over a decade. Nevertheless, the therapeutic application of HYF is considerably constrained by the lack of clarity regarding its underlying mechanism of action. The experimental procedures entailed administering VCD to female Sprague-Dawley rats at a dosage of 160mg/kg/day over a period of 15 days, succeeded by a 100-day treatment with HYF. Blood serum samples were collected and analyzed using ELISA to quantify the concentrations of Anti-Müllerian Hormone (AMH), Follicle-Stimulating Hormone (FSH), and Estradiol (E2). The levels of N6-methyladenosine (m6A) were assessed through Dot blot analysis and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Western blotting was employed to validate the differential expression of m6A-related catalytic enzymes and apoptosis-related regulators, including BCL-2, BCL-XL, and MCL-1, which may be implicated in the effects of HYF. Certain shRNA-COV434 cell line was constructed for the exploration of molecular mechanism, and then the potential targets were finally verified by MeRIP-qPCR. HYF has been identified as having a significant influence on the development of residual ovarian follicles in rats with POI, especially during the initial stages. It was observed that HYF facilitates the progression of escaping antral follicles to full maturation. Additionally, HYF exhibited the capacity to enhance the proliferation of COV434, a human ovarian granulosa cell line, while concurrently inhibiting apoptosis within these cells. Notably, HYF treatment resulted in the downregulation of apoptotic proteins, including BCL-XL, cleaved-caspase 9, cleaved-caspase 3, and Bcl-2. Concurrently, m6A modification is implicated in the regulation of HYF. Both in vitro and in vivo studies indicate that FTO may play a role in the anti-apoptotic mechanisms mediated by m6A in ovarian granulosa cells influenced by HYF. Moreover, employing qPCR and MeRIP-qPCR techniques, P53 has been identified as the target gene for m6A modification mediated by FTO. These findings suggest that HYF holds promise as a potential treatment for POI and provide a more comprehensive understanding of the mechanism by which HYF operates, specifically its ability to prevent the BCL-2 mitochondrial apoptosis pathway mediated by P53 in ovarian granulosa cells of POI rats by regulating FTO/m6A-Tp53.

Transcriptomic Analysis During Olive Fruit Development and Expression Profiling of Fatty Acid Desaturase Genes.

The olive fruit is a drupe whose development and ripening takes several months from flowering to full maturation. During this period, several biochemical and physiological changes occur that affect the skin color, texture, composition, and size of the mesocarp. The final result is a fruit rich in fatty acids, phenolic compounds, tocopherols, pigments, sterols, terpenoids, and other compounds of nutritional interest. In this work, a transcriptomic analysis was performed using flowers (T0) and mesocarp tissue at seven different stages during olive fruit development and ripening (T1-T7) of the 'Picual' cultivar. A total of 1755 genes overexpressed at any time with respect to the flowering stage were further analyzed. These genes were grouped into eight clusters based on their expression profile. The gene enrichment analysis revealed the most relevant biological process of every cluster. Highlighting the important role of hormones at very early stages of fruit development (T1, Cluster 1), whereas genes involved in fatty acid biosynthesis were relevant throughout the fruit developmental process. Hence, genes coding for different fatty acid desaturase (SAD, FAD2, FAD3, FAD4, FAD5, FAD6, and FAD7) enzymes received special attention. In particular, 26 genes coding for different fatty acid desaturase enzymes were identified in the 'Picual' genome, contributing to the improvement of the genome annotation. The expression pattern of these genes during fruit development corroborated their role in determining fatty acid composition.

Exploring the late maturation of an intrinsic episodic memory network: A resting-state fMRI study

Previous research suggests that episodic memory relies on functional neural networks,which are present even in the absence of an explicit task. The regions that integrate.these networks and the developmental changes in intrinsic functional connectivity.remain elusive. In the present study, we outlined an intrinsic episodic memory network.(iEMN) based on a systematic selection of functional connectivity studies, and.inspected network differences in resting-state fMRI between adolescents (13–17 years.old) and adults (23–27 years old) from the publicly available NKI-Rockland Sample.Through a review of brain regions commonly associated with episodic memory.networks, we identified a potential iEMN composed by 14 bilateral ROIs, distributed.across temporal, frontal and parietal lobes. Within this network, we found an increase.in resting-state connectivity from adolescents to adults between the right temporal pole.and two regions in the right lateral prefrontal cortex. We argue that the coordination of.these brain regions, connecting areas of semantic processing and areas of controlled.retrieval, arises as an important feature towards the full maturation of the episodic.memory system. The findings add to evidence suggesting that adolescence is a key.period in memory development and highlights the role of intrinsic functional.connectivity in such development.

Have orthodontists changed their retention and follow-up protocols due to contemporary orthodontics? An online survey.

With advancements in orthodontic technology, treatment durations have shortened, often concluding at earlier ages. This shift prompts scrutiny of contemporary retention and post-treatment protocols. The study aimed to assess current professional preferences, compare them against patient age and treatment duration, and investigate the potential impacts of reduced treatment times on professional protocols, particularly when treatment concludes before pubertal growth. A questionnaire comprising 12 multiple-choice questions focused on active treatment and retention phases was developed using an online survey platform. It was distributed to licensed orthodontists engaged in patient treatment. Bivariate analysis was conducted using ANOVA and the Kruskal-Wallis test, with pairwise comparisons facilitated by the Dwass-Steel-Critchlow-Fligner method. Of 743 respondents, representing a 32% response rate, approximately 70% reported initiating treatment with fixed appliances in pre-pubertal patients. The most prevalent treatment combination involved commencing treatment during early permanent dentition and lasting between 12 to 24 months, resulting in treatment completion before full growth maturation. No discernible individualization was observed in retention protocols or post-retention follow-ups. Traditional retainer prescription post-orthodontic therapy was unanimous among respondents. Notably, experienced orthodontists tended to prefer regular patient visits for follow-up, while less experienced counterparts discharged patients after 12 months. Contemporary orthodontic treatments are characterized by shorter durations, yet orthodontists have not adopted retention and post-treatment follow-up practices accordingly. There is a pressing need for evidence-based guidelines to develop protocols tailored to the shorter treatment durations and the increasing prevalence of younger patients completing treatment.

Chemically Defined Organoid Culture System for Cholangiocyte Differentiation.

Cholangiocyte organoids provide a powerful platform for applications ranging from in vitro modeling to tissue engineering for regenerative medicine. However, their expansion and differentiation are typically conducted in animal-derived hydrogels, which impede the full maturation of organoids into functional cholangiocytes. In addition, these hydrogels are poorly defined and complex, limiting the clinical applicability of organoids. In this study, a novel medium composition combined with synthetic polyisocyanopeptide (PIC) hydrogels to enhance the maturation of intrahepatic cholangiocyte organoids (ICOs) into functional cholangiocytes is utilized. ICOs cultured in the presence of sodium butyrate and valproic acid, a histone deacetylase inhibitor, and a Notch signaling activator, respectively, in PIC hydrogel exhibit a more mature phenotype, as evidenced by increased expression of key cholangiocyte markers, crucial for biliary function. Notably, mature cholangiocyte organoids in PIC hydrogel display apical-out polarity, in contrast to the traditional basal-out polarization of ICOs cultured in Matrigel. Moreover, these mature cholangiocyte organoids effectively model the biliary pro-fibrotic response induced by transforming growth factor beta. Taken together, an animal-free, chemically defined culture system that promotes the ICOs into mature cholangiocytes with apical-out polarity, facilitating regenerative medicine applications and in vitro studies that require access to the apical membrane, is developed.

Combined effect of timing, position and management on Halyomorpha halys feeding injury assessment on apple cultivars

AbstractHalyomorpha halys is an invasive stink bug, native to east Asia, responsible for extensive economic loss in numerous crops. Since several factors can influence the severity of the recorded injury level, we performed field and semi‐field experiments in commercial apple orchards to monitor their interaction. To establish how position (both in the orchard and on the canopy), management and cultivar interact in determining pre‐harvest injury levels, more than 100,000 apples, distributed over 106 orchard blocks, were examined in the field. Apples located higher in the canopy, on edge rows and in orchards with organic management had a higher number of external injuries and such factors were more relevant when occurring in combination, and on mid and late‐season cultivars. Exclusion cages were used to assess if the injury severity changed with the apple growth stage and with the life stage of H. halys, exposing Red delicious apples to adults and nymphs for 48 hours, from May to July. Early‐season injuries did not evolve in distinct depressions nor in wide areas with necrotic tissue. Later in season, the injury severity was higher and similar for adults and older nymphs (fourth and fifth instars), with the development of depressions externally and necrotic tissue internally. Thus, even a low number of individuals could cause severe damage, over a short exposure period, when it happened near full maturation. The combined results offer new insights to support the development of informed and sustainable control strategies.

Testing the Sample Preparation Method and Oxygen Isotopic Analysis of Tooth Enamel for the Reconstruction of the Birth Seasonality of Ovicaprines (the Case of Teeth from Istykskaya Cave, Eastern Pamir)

Cyclic variations of δ18O along the growth line of M2 and M3 molars provide information about the seasonality of enamel formation and thereby about reproductive seasonality in animals, taking into account the time of tooth eruption and full enamel maturation. Determination of birth seasonality of small ruminants is relevant to the reconstruction of pastoralist strategies. Two peaks of reproductive activity per year are one of the most reliable indicators of human control of the small ruminants. As part of this work, for the first time in Russia, a method of sample preparation and analysis of oxygen isotope ratios in the tooth enamel of small ruminants is proposed. Traditionally, breeding seasonality was evaluated by the isotopic analysis of carbonates, the content of which in dental enamel is only 4 %. According to a new approach, oxygen from a sample is converted to gaseous CO by the interaction of enamel with carbon in a helium flow under 1300 °С, enabling one to measure δ18O in all components of the dental enamel, including phosphates, which make up 90 % of enamel mass and are resistant to diagenesis. In this study, four sample preparation protocols depending on the degree of preservation of teeth and their age were tested: (A) cleaning and sampling of enamel, (B) treatment by H2O2, (C) treatment by NaOCl and CH3COOH, and (D) extraction of Ag3PO4. Results show that for assessing seasonality breeding, it suffices to evaluate the lowest and the highest δ18O along the tooth growth line. If the preservation of sample is good, minimal chemical treatment is enough to observe these extremes.

The toxigenic potential of phytopathogenic fungi isolated from pineapple Ananas comosus (L.) Merr. var MD-2 associated with postharvest rot disease

MD-2 pineapple fruits are susceptible to phytopathogenic fungi producing mycotoxins, which can cause up to 40% of postharvest losses. This study aimed to isolate and identify fungi associated with postharvest diseases on pineapple fruits, and mycotoxins produced. Thirty-seven fungal strains were isolated from the pineapple fruit at the full maturation stage. About 51.37% of the fungal strains identified were belonging to Aspergillus. Those fungi that caused diseases in pineapple fruits were Aspergillus sclerotioniger, A. tubingensis, Penicillium citrinum, Fusarium equiseti, Trichoderma asperelliodes and T. asperellum. Aspergillus niger produced the highest ochratoxin A (OTA), scoring 85.51 µg/kg. On the other hand, P. citrinum scored 94.06 µg/kg, the highest quantity of total aflatoxins. This is the first report demonstrating mycotoxigenic fungi’s potential to cause infections in postharvest pineapple fruits in Mexico.

A rare case of intra-ovarian oocyte maturation.

The intra-ovarian presence of ootids, i.e. female gametes that have completed meiosis, is considered exceptional in the animal kingdom. The present study explores the first such case to be reported in a sea cucumber (Echinodermata: Holothuroidea). In the overwhelming majority of animals, including holothuroids, oocytes (i.e. immature female gametes) that are developing in the ovary undergo a primary arrest at the prophase stage of meiosis, which may last from days to decades. In free-spawning taxa, this arrest is normally lifted only during or shortly before transit in the gonoduct, when gamete release (spawning) is imminent. However, oocytes of the holothuroid Chiridota laevis were discovered to have resumed the second meiotic division including the completion of germinal vesicle breakdown and polar-body expulsion inside the ovary, effectively reaching the ootid stage concomitantly with ovulation (i.e. escape from follicle cells) prior to spawning. The potential drivers and significance of this exceptionally rare case of full intra-ovarian oogenic maturation are discussed.

Canopy Architecture and Sun Exposure Influence Berry Cluster-Water Relations in the Grapevine Variety Muscat of Alexandria.

Climate-change-related increases in the frequency and intensity of heatwaves affect viticulture, leading to losses in yield and grape quality. We assessed whether canopy-architecture manipulation mitigates the effects of summer stress in a Mediterranean vineyard. The Vitis vinifera L variety Muscat of Alexandria plants were monitored during 2019-2020. Two canopy shoot-positioning treatments were applied: vertical shoot positioning (VSP) and modulated shoot positioning (MSP). In MSP, the west-side upper foliage was released to promote partial shoot leaning, shading the clusters. Clusters were sampled at pea size (PS), veraison (VER), and full maturation (FM). Measurements included rachis anatomy and hydraulic conductance (Kh) and aquaporins (AQP) and stress-related genes expression in cluster tissues. The results show significant seasonal and interannual differences in Kh and vascular anatomy. At VER, the Kh of the rachis and rachis+pedicel and the xylem diameter decreased but were unaffected by treatments. The phloem-xylem ratio was either increased (2019) or reduced (2020) in MSP compared to VSP. Most AQPs were down-regulated at FM in pedicels and up-regulated at VER in pulp. A potential maturation shift in MSP was observed and confirmed by the up-regulation of several stress-related genes in all tissues. The study pinpoints the role of canopy architecture in berry-water relations and stress response during ripening.

Langerhans Cells in Sentinel Lymph Nodes from Melanoma Patients.

Langerhans cells (LCs) are professional Dendritic Cells (DCs) involved in immunoregulatory functions. At the skin level, LCs are immature. In response to tissue injuries, they migrate to regional Lymph Nodes (LNs), reaching a full maturation state. Then, they become effective antigen-presenting cells (APCs) that induce anti-cancer responses. Notably, melanoma patients present several DC alterations in the Sentinel Lymph Node (SLN), where primary antitumoral immunity is generated. LCs are the most represented DCs subset in melanoma SLNs and are expected to play a key role in the anti-melanoma response. With this paper, we aim to review the current knowledge and future perspectives regarding LCs and melanoma. A systematic review was carried out according to the PRISMA statement using the PubMed (MEDLINE) library from January 2004 to January 2024, searching for original studies discussing LC in melanoma. The final synthesis included 15 articles. Several papers revealed significant LCs-melanoma interactions. Melanoma immune escape mechanisms include SLN LC alterations, favoring LN metastasis arrival/homing and melanoma proliferation. The SLN LCs of melanoma patients are defective but not irreversibly, and their function may be restored by appropriate stimuli. Thus, LCs represent a promising target for future immunotherapeutic strategies and cancer vaccines.

Development of Prefrontal Circuits and Cognitive Abilities.

The prefrontal cortex is considered as the site of multifaceted higher-order cognitive abilities. These abilities emerge late in life long after full sensorimotor maturation, in line with the protracted development of prefrontal circuits that has been identified on molecular, structural, and functional levels. Only recently, as a result of the impressive methodological progress of the last several decades, the mechanisms and clinical implications of prefrontal development have begun to be elucidated, yet major knowledge gaps still persist. Here, we provide an overview on how prefrontal circuits develop to enable multifaceted cognitive processing at adulthood. First, we review recent insights into the mechanisms of prefrontal circuit assembly, with a focus on the contribution of early electrical activity. Second, we highlight the major reorganization of prefrontal circuits during adolescence. Finally, we link the prefrontal plasticity during specific developmental time windows to mental health disorders and discuss potential approaches for therapeutic interventions.

Defining healthy and breast tumor-associated neutrophils at a whole-organism, single-cell resolution

Abstract Neutrophils accumulate systemically in tumor-bearing organisms, and have been shown to promote primary tumor progression and metastasis through suppression of the anti-tumor immune response. However, neutrophils have specialized phenotypes in different organs, and their full transcriptional heterogeneity and maturation trajectory has yet to be defined across healthy and cancer states. Here, we performed single-cell RNA sequencing on isolated neutrophils from bone marrow, spleen, lymph nodes, blood, mammary fat pad/tumor, and lungs from wild-type and breast tumor-bearing MMTV-PyMT mice. Our analysis reveals distinct transcriptional programs which distinguish systemic neutrophil function in normal homeostasis and cancer. In wild-type mice, we describe a neutrophil maturation trajectory that begins with granulopoiesis in the bone marrow, continues development in the spleen and blood, and reaches a fully mature state in the lymph nodes, mammary fat pad, and lungs. This is captured by five maturation stages, N0-N4, each with its own characteristic gene signature. In MMTV-PyMT mice, we observe a systemic upregulation of genes including Ifitm1, Wfdc17, Clec4e, Igfbp6, and Saac, which have been implicated in cancer-induced immunosuppression. Taken altogether, this work provides a multi-organ perspective into neutrophil development across healthy and cancer states and identifies markers specific to breast tumor-associated neutrophils which may be potential immunotherapeutic targets.

The first composite increment: Dependency on placement technique for interaction behavior with maturing dentin-adhesive bond at pulpal floors in deep occlusal cavities – A comprehensive review

Abstract Introduction: When the traditional incremental technique is used for direct placement of composite resin in a deep occlusal cavity, each increment (2 mm or thicker) is individually cured where it undergoes hardening and shrinkage. As increments are bonded to all cavity walls, a constrained shrinkage develops in tooth, composite, and/or interfaces. In the first composite increment, this constrained shrinkage generates tensile stresses which are distributed in a nonuniform pattern within that increment, resulting in premature stressing of the dentin bond of the hybrid layer at the pulpal floor before it reaches full maturation. This premature stressing leads to initiation of interfacial debonding and propagation to form a microgap. This behavior is associated with persistent postoperative sensitivity and tooth pain, over the time, which subsequently results in restoration failure. Aims: This paper provides a comprehensive review on the first composite increment and its dependency on placement technique for the interaction behavior with the maturing dentin-adhesive bond at pulpal floors in deep occlusal cavities. Materials and Methods: The dental database was searched, and 59 articles were collected and included in this review, spanning the years from 1984 to 2023. Results and Discussion: Three biomimetic direct restorative techniques were reported in the literature for incrementally restoring large occlusal cavities. These techniques are the decoupling with time, the decoupling with fiber, and the decoupling with split-increment. They all aim at minimizing the generated shrinkage stresses in the first composite increment for protecting the developing dentin bond of the hybrid layer until it reaches full maturation and thus preventing the initiation and propagation of interfacial defects at pulpal floors of deep occlusal cavities. Finally, the restoration is completed by placing and curing successive increments of 1.5 mm each to fill the cavity; their number depends on the cavity depth. These increments can each be cured immediately following placement. Conclusions: In deep occlusal cavities, the interaction behavior between the first composite increment and the maturing dentin-adhesive bond at the pulpal floor depends on the technique used for the increment placement. This behavior is either favorable with the direct biomimetic techniques or unfavorable with the traditional incremental technique. With the direct biomimetic techniques, no premature shrinkage stressing of the dentin bond is induced at the pulpal floor. This prevents the initiation of interfacial debonding and propagation to form microgaps and results in the absence of postoperative sensitivity and persistent tooth pain.

Macrophages play a nutritive role in post-metamorphic maturation in Drosophila.

In the body of multicellular organisms, macrophages play an indispensable role in maintaining tissue homeostasis by removing old, apoptotic and damaged cells. In addition, macrophages allow significant remodeling of body plans during embryonic morphogenesis, regeneration and metamorphosis. Although the huge amount of organic matter that must be removed during these processes represents a potential source of nutrients, their further use by the organism has not yet been addressed. Here, we document that, during metamorphosis, Drosophila larval adipose tissue is infiltrated by macrophages, which remove dying adipocytes by efferocytosis and engulf leaking RNA-protein granules and lipids. Consequently, the infiltrating macrophages transiently adopt the adipocyte-like metabolic profile to convert remnants of dying adipocytes to lipoproteins and storage peptides that nutritionally support post-metamorphic development. This process is fundamental for the full maturation of ovaries and the achievement of early fecundity of individuals. Whether macrophages play an analogous role in other situations of apoptotic cell removal remains to be elucidated.

Intracellular Accumulation and Secretion of YKL-40 (CHI3L1) in the Course of DMSO-Induced HL-60 Cell Differentiation.

YKL-40 (CHI3L1) is a matrix glycoprotein stored in human neutrophil-specific granules and released upon activation. While it is implicated in inflammation, cancer progression, and cell differentiation, its exact physiological role remains unclear. This study investigated the intracellular expression and secretion of YKL-40 by untreated and DMSO-treated HL-60 cells in association with surface expression of CD11b and CD66b throughout the differentiation process (up to 120 h). Secreted YKL-40 protein and mRNA levels of YKL-40, CD66b, and CD11b were measured by ELISA and quantitative RT-PCR, respectively. The intracellular YKL-40 and surface CD11b and CD66b expression were assessed by flow cytometry. A significant increase in CD11b expression confirmed DMSO-induced differentiation of HL-60 cells. Upon DMSO stimulation, YKL-40 mRNA expression increased in a time-dependent manner, unlike CD66b. The lack of CD66b (a granulocyte maturation and activation marker) on the surface of HL-60 cells might suggest that DMSO treatment did not induce full maturation or activation. The intracellular YKL-40 protein expression was increasing up to 96 h of DMSO treatment and then declined. YKL-40 secretion into the culture medium was detectable only at later time points (96 and 120 h), which was correlated with a decreased proliferation of DMSO-treated HL-60 cells. These findings suggest sequential changes in YKL-40 production and secretion during DMSO-induced differentiation of HL-60 cells and might contribute to a better understanding of YKL-40's involvement in both physiological processes and disease development, including multiple sclerosis.

CRISPR/Cas9-Based Disease Modeling and Functional Correction of Interleukin 7 Receptor Alpha Severe Combined Immunodeficiency in T-Lymphocytes and Hematopoietic Stem Cells.

Interleukin 7 Receptor alpha Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the IL7RA gene. Defective IL7R expression in humans hampers T cell precursors' proliferation and differentiation during lymphopoiesis resulting in the absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukemia, suggesting the application of targeted gene editing to insert a correct copy of the IL7RA gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modeling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knockin strategy that targets IL7RA exon 1 and introduces through homology-directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signaling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.

Physiological quality of white oat seeds produced with Nitrogen, Iron and Zinc biofortification

The main function of the seed is the perpetuation of the species, which means it is able to disperse to new environments and give rise to new plants. The use of high quality seeds is one of the determining factors for the establishment of the crop and is directly related to maximum germination potential and vigor, resulting in uniformity of the plant stand. Therefore, the objective was to evaluate the physiological quality of white oat seeds produced under different nitrogen management and foliar biofortification with iron and zinc. In the second week of June 2021, white oat cultivar URS Corona was sown in plots of 5 lines, 5 m long each and spacing between lines of 0.20 m, totaling an experimental unit of 5 m². The treatments consisted of different doses of biofortification with Iron and Zinc applied foliarly to the plant when the inflorescence was exposed, at the beginning of grain filling. For biofortification, the source of iron used was the compound of FeSO4 7H2O and zinc, ZnSO4 5H2O was used, each nutrient was applied separately and the combination of iron + zinc at doses of 0, 1000, 2000 and 4000 g ha-1, and to aplication of nitrogen use ureia 45%. Applications were carried out with a knapsack sprayer, in a spray volume of 500 liters ha-1. The experiment was harvested 130 days after emergence, in the full maturation phase, at phenological stage 90, according to the ZADOKS scale. The tests carried out to evaluate the physiological quality of the seeds were: first germination count, germination, accelerated aging, root length, shoot length, total length, seedling dry mass and electrical conductivity. The data were subjected to analysis of variance (ANOVA) at a 5% probability of error. For the variables that showed a significant effect, an average test was carried out for qualitative sources of variation and polynomial regression for quantitative sources of variation with a 5% probability of error, carried out with the aid of the GENES software. From the evaluations, it was concluded that the use of foliar biofortification with iron and zinc alone or in combination, aplication with nitrogen influenced the physiological quality of white oat seeds.