Early Developmental Processes Research Articles

Self-organization from organs to embryoids by activin in early amphibian development.

Embryonic development is a complex self-organizing process orchestrated by a series of regulatory events at the molecular and cellular levels, resulting in the formation of a fully functional organism. This review focuses on activin protein as a mesoderm-inducing factor and the self-organizing properties it confers. Activin has been detected in both unfertilized eggs and embryos, suggesting its involvement in early developmental processes. To explore its effects, animal cap cells-pluripotent cells from the animal pole of amphibian blastula-stage embryos-were treated with varying concentrations of activin. The results showed that activin induced mesodermal tissues, including blood, muscle, and notochord, in a dose-dependent manner. Co-treatment with activin and retinoic acid further promoted the development of kidney and pancreatic tissues, while activin alone stimulated the formation of beating cardiac tissue. In subsequent experiments, high concentrations of activin conferred an organizer-like activity on animal cap cells. The pretreatment duration affected outcomes: longer exposure induced anterior structures, such as eyes, while shorter exposure resulted in posterior structures, like tails. These findings reflect moderate self-assembly, where cells become increasingly organized. In another experiment, activin was used to create an artificial gradient. Explants cultured on this gradient developed into embryoids with well-defined anteroposterior, dorsoventral, and left-right axes, exemplifying higher-order self-organization. These results demonstrate that controlled activin gradients can drive the formation of nearly complete tadpole-like larvae, effectively recapitulating the processes of early embryogenesis. This system offers valuable insights into the mechanisms underlying axis formation and organogenesis, providing a promising platform for future research in developmental biology.

Coupling high-throughput and modeling approaches to streamline early-stage process development for biologics.

Platforms have long been implemented for downstream process development of monoclonal antibodies (mAbs) to streamline development and reduce timelines. These platforms are also increasingly being used for other complex biologics modalities. While development has traditionally been conducted at the lab bench scale in a sequential manner, automated miniaturized and parallelized approaches like RoboColumns and resin plates have also been implemented for chromatographic screening. Additionally, mechanistic modeling for chromatographic separations has also seen increased use for development applications. In this manuscript, we propose a workflow with elements of both high-throughput screening and modeling that provides a streamlined roadmap for early process development. The workflow utilizes automated resin plate screens to both narrow screening conditions and calibrate binding isotherm parameters. Mechanistic models are then used to characterize a robust range of conditions suitable for an early manufacturing process. Miniaturized RoboColumns then confirm the process space, thus completing the development without the use of any traditional lab-scale columns. Case studies demonstrate the utility of this workflow for both cation-exchange (CEX) and multimodal cation-exchange (MMCEX) processes. Process parameter sensitivities across process ranges for the models are compared with typical design-of-experiment (DOE) statistical models. The models are able to predict the mAb product as well as aggregate impurities. This workflow provides a practical method to enable increased process understanding while also reducing timeline and material requirements for development.

Investigating the metabolic load of monoclonal antibody production conveyed to an inducible CHO cell line using a transfer-rate online monitoring system.

Shake flasks are a foundational tool in early process development by allowing high throughput exploration of the design space. However, lack of online data at this scale can hamper rapid decision making. Oxygen transfer rate (OTR) monitoring has been readily applied as an online process characterization tool at the benchtop bioreactor scale. Recent advances in modern sensing technology have allowed OTR monitoring to be available at the shake flask level. It is now possible to multiplex time-of-action (e.g., Induction, temperature shift, pH shift, feeding initiation, point of harvest) characterization studies by relying on careful analysis of OTR profile kinetics. As a result, there is potential to save time and capital expenditures while exploring process intensification studies though accurate and physiologically relevant online data. In this article, we detail the application of OTR monitoring to characterize the impact that recombinant protein production has on an inducible CHO cell line expressing Palivizumab. We then test out time-of-action studies to intensify protein production outcomes. We observe that recombinant protein expression causes a metabolic load that diminishes potential biomass growth. As a result, when compared to a control standard process, delaying induction and temperature shift has the potential to improve viable cell densities (VCD) by 2-fold thus increasing recombinant protein yield by over 30 %. The study also demonstrates that OTR can serve as a useful tool to detect cessation of exponential growth. Consequently, time-of-action points that are characteristic of inducible systems can be formulated accurately and reliably to maximize production performance.

Pitfalls in Early Bioprocess Development Using Shake Flask Cultivations.

For about 100years, the shake flask has been established for biotechnological cultivations as one of the most important cultivation systems in early process development. Its appeal lies in its simple handling and highly versatile application for a wide range of cell types-from bacteria to mammalian cells. In recent decades, extensive research has been conducted on the shake flask, to not perform processes blindly but to gain a deeper understanding of the various process parameters, phenomena, and their impact on the process. Although the characterization of the shake flask is now well-established in literature, many publications show that this knowledge is often inadequately applied. Therefore, this review provides an overview of the current state of knowledge on various topics related to the shake flask. We first present the key process parameters and their influence on different physical phenomena, such as power input, the largely unknown in-phase/out-of-phase phenomenon, as well as temperature and mass transfer. Then, the most common online monitoring systems that have been established for shake flasks are discussed. Finally, various pitfalls that often arise from inadequate knowledge of handling shake flask cultivations are discussed and guidance on how to avoid them is provided.

Mild Gestational Hypothyroidism in Mice Has Transient Developmental Effects and Long-Term Consequences on Neuroendocrine Systems.

Background: Thyroid hormones (TH) play a key role in fetal brain development. While severe thyroid dysfunction, has been shown to cause neurodevelopmental and reproductive disorders, the rising levels of TH-disruptors in the environment in the past few decades have increased the need to assess effects of subclinical (mild) TH insufficiency during gestation. Since embryos do not produce their own TH before mid-gestation, early development processes rely on maternal production. Notably, the reproductive network governed by gonadotropin-releasing hormone (GnRH) neurons develops during this critical period. Methods: The risk of mild maternal hypothyroidism on the development of GnRH neurons and long-term effect on neuroendocrine function in the offspring was investigated using a mouse model of gestational hypothyroidism induced by methimazole (MMI) treatment. Results: MMI treatment during gestation led to reduced litter size, consistent with increased miscarriages due to hypothyroidism. E12/13 embryos, collected from MMI-treated dams, had a decreased number of GnRH neurons, but the migration of the remaining GnRH neurons was normal. Cell proliferation was reduced in the vomeronasal organ (VNO), correlating with the reduced number of GnRH neurons detected in this region. Using a GnRH cell line confirmed attenuated proliferation in the absence of T3. Pups born from hypothyroid mothers had normal postweaning growth and estrus cycles, yet adult offspring had significantly more cells expressing estrogen receptor alpha in the arcuate nucleus. Notably, by adulthood, GnRH cell number and distribution was comparable with nontreated controls indicating that compensatory mechanisms occurred after E13. Conclusion: Overall, our work shows that mild TH disruption during gestation transiently affects proliferation of the pool of GnRH neurons within the VNO and has a long-term impact on neuroendocrine systems.

Study on the physiological mechanism and transcriptional regulatory network of early fruit development in Gleditsia sinensis Lam. (Fabaceae)

BackgroundGleditsia sinensis Lam. (Fabaceae) is a medicinal legume characterized by its spines and pods, which are rich in saponins, polysaccharides, and various specialized metabolites with potential medicinal and industrial applications. The low fruit set rate in artificially cultivated economic forests significantly impedes its development and utilization. A comprehensive understanding of the cellular events, physiological and biochemical processes, and molecular regulatory mechanisms underlying fruit initiation and early fruit development is essential for enhancing yield. However, such information for G. sinensis remains largely unexplored.ResultsIn this study, we identified that the early fruit development process in G. sinensis can be categorized into three distinct stages: pollination, the critical period of fertilization, and the initial fruit development followed by subsequent growth. The dynamic changes in non-structural carbohydrates and endogenous plant hormones within the ovary were found to play a significant role during fruit set and the early stages of fruit development. Additionally, the high activity of gibberellin, cytokinin, and sucrose-metabolizing enzymes in the ovary was conducive to early fruit development. Furthermore, we generated high-resolution spatiotemporal gene expression profiles in the ovary from the stage of efflorescence to early fruit development. Comparative transcriptomics and weighted gene co-expression network analysis revealed specific genes and gene modules predominant at distinct developmental stages, thereby highlighting unique genetic programming. Overall, we identified the potential regulatory network governing fruit initiation and subsequent development, as well as the sets of candidate genes involved, based on the aforementioned results.ConclusionsThe results offer a valuable reference and resource for the application of exogenous substances, such as hormones and sugars, during critical fruit development periods, and for the development of molecular tools aimed at improving yield.

Deciphering the Role of Shank3 in Dendritic Morphology and Synaptic Function Across Postnatal Developmental Stages in the Shank3B KO Mouse.

Autism Spectrum Disorder (ASD) is marked by early-onset neurodevelopmental anomalies, yet the temporal dynamics of genetic contributions to these processes remain insufficiently understood. This study aimed to elucidate the role of the Shank3 gene, known to be associated with monogenic causes of autism, in early developmental processes to inform the timing and mechanisms for potential interventions for ASD. Utilizing the Shank3B knockout (KO) mouse model, we examined Shank3 expression and its impact on neuronal maturation through Golgi staining for dendritic morphology and electrophysiological recordings to measure synaptic function in the anterior cingulate cortex (ACC) across different postnatal stages. Our longitudinal analysis revealed that, while Shank3B KO mice displayed normal neuronal morphology at one week postnatal, significant impairments in dendritic growth and synaptic activity emerged by two to three weeks. These findings highlight the critical developmental window during which Shank3 is essential for neuronal and synaptic maturation in the ACC.

Advanced pharmacological and pharmacokinetic evaluation of 1,3 dimethylpurine-2,6-dione derivative (GR-14) with prominent mood-modulating activity in rats.

Research on new candidates for antidepressant/anxiolytic drugs from the long-chain arylpiperazines (LCAPs) group containing a 1,3-dimethylpurine-2,6-dione as a terminal amide fragment fits into the modern exploration trend. This study aimed to examine, for the first time in male Wistar rats, pharmacodynamic (antidepressant- and anxiolytic-like) and pharmacokinetic properties of 7-(5-(4-(3-chlorophenyl)piperazin-1-yl)pentyl)-1,3-dimethyl-3,7-dihydro-1 H-purine-2,6-dione hydrochloride (GR-14). Antidepressant- and anxiolytic-like activities have been assessed in the forced swim test (FST) and Vogel conflict drinking test, respectively. The pharmacokinetic characteristics of GR-14, its distribution into rat tissues, and several in vitro ADME-Tox parameters (hepatocytotoxic, neurocytotoxic, metabolic stability) have been defined. GR-14 produces strong and dose-dependent antidepressant- and anxiolytic-like effects in both tests used. Pharmacokinetic findings demonstrate that GR-14 reveals linear pharmacokinetics tested after intravenous (iv) and was rapidly absorbed after oral (po) administration. It rapidly crosses the blood-brain barrier (BBB) which is vital for therapeutic effects in vivo in psychiatric diseases, depression, and anxiety. Moreover, it is slowly eliminated from the brain, maintaining concentrations higher than those in plasma at the last time point measured. Further studies have also shown that GR-14 is an average high-clearance drug in rat liver microsomes and exerts neither hepatocytotoxic nor neurocytotoxic effects in vitro. The tested derivative GR-14 presents prominent mood-modulating activity in rats and has promising pharmacokinetic parameters and a good safety profile. The beneficial pharmacology and pharmacokinetics of GR-14 in vivo are in high concordance with its profile in vitro, thus underlining very hopeful properties to support the early development process.

Developmental dysfunction in a preclinical model of Kcnq2 developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEE) are rare but severe neurodevelopmental disorders characterised by early-onset seizures often combined with developmental delay, behavioural and cognitive deficits. Treatment for DEEs is currently limited to seizure control and provides no benefits to the patients' developmental and cognitive outcomes. Genetic variants are the most common cause of DEE with KCNQ2 being one of the most frequently identified disease-causing genes. KCNQ2 encodes a voltage-gated potassium channel KV7.2 widely expressed in the central nervous system and critically involved in the regulation of neuronal excitability. In this study, we aimed to characterise a KCNQ2 variant (K556E) found in a female patient with DEE using a heterologous expression system and a knock-in mouse model. Wild-type KCNQ2 or K556E variant were expressed in Chinese Hamster Ovary (CHO) cells (with or without KCNQ3) and their biophysical properties assessed using patch clamp recordings. We further engineered a new Kcnq2 DEE mouse model (K557E) based on the K556E variant and characterised it using behavioural, electrophysiological, and transcriptome analysis. A mild loss of function was observed only when the mutant channel was co-expressed with KCNQ3 in the heterologous system. The heterozygous knock-in mice showed a reduced survival rate and increased susceptibility to induced seizures. Electrophysiology recordings in brain slices revealed a hyperexcitable phenotype for cortical layer 2/3 pyramidal neurons with retigabine (KV7 channel opener) able to rescue both the increased sensitivity to chemically-induced seizures in vivo and neuronal excitability ex vivo. Whole-brain RNA sequencing revealed numerous differentially expressed genes and biological pathways pointing at dysregulation of early developmental processes. Our study reports on a novel Kcnq2 DEE mouse model recapitulating aspects of the disease phenotype with the electrophysiological and transcriptome analysis providing insights into KCNQ2 DEE mechanisms that can be leveraged for future therapy development.

Does the impact of open innovation depend on contextual factors? A case of the Korean biopharmaceutical industry.

Investments in the strategic development of the biopharmaceutical industry are increasing in both developed and developing countries. The biopharmaceutical industry is a technology-intensive industry where securing original technology and intellectual property rights is important. The role of open innovation is becoming more important due to the enormous research and development (R&D) funds and long development period in the early development process, and open innovation (OI) is becoming more important in the corporate world. Many empirical studies have been conducted on the impact on performance. However, the contextual factors that affect the relationship between OI activities and innovation performance have received relatively little attention, and studies from the perspective of developing countries catching up with developed countries are even rarer. Accordingly, this study examined the moderating effects (government R&D support, absorptive capacity, and alliance management capacity) that affect open innovation and innovation performance in the biopharmaceutical industry using data from Korea, one of the most representative latecomer countries in the biopharmaceutical industry. The basic information, OI activities, and patent achievements of Korean biopharmaceutical firms were collected and organized into a database. Samples with missing or incorrect information were excluded, and 527 firms were analyzed. Negative binomial regression analysis was performed considering the characteristics of patent performance, which is the dependent variable, and a time lag of one to two years was assumed considering the time required to generate results. OI in the form of technological cooperation, rather than technology purchasing, has a positive effect on patent performance. Meanwhile, the greater the absorptive capacity and government R&D support, the greater the positive impact of technological cooperation on patent performance. Conversely, the greater the alliance management capacity, the greater the positive impact of technological cooperation. These results indicate that the impact of OI activities on technological innovation performance may vary depending on context.

Early Process Development of an LPAR1 Antagonist, GS-2278

Early Process Development of an LPAR1 Antagonist, GS-2278

Preparation of C-S-H seeds from waste glass powder and its effect on early performance of cement paste

This study investigates the hydrothermal synthesis of calcium silicate hydrate (C-S-H) seeds using nano-size glass powder (NGP) derived from siliceous raw materials and CaO as the calcareous precursor. The influence of varying NGP concentrations on the particle size, microstructure, and phase composition of the C-S-H seeds was systematically analyzed. The synthesized C-S-H seeds were subsequently employed as additives to evaluate their effects on the early hydration process and compressive strength development of cement paste. The results indicate that increasing the NGP concentration from 0.2 mol/L to 0.8 mol/L leads to an exponential increase in the median particle size of the C-S-H seeds from 147 nm to 1328 nm and a corresponding morphological transformation from flocculent to granular structures. Additionally, the purity of the C-S-H sample decreased from 74.8% to 54.5% as the NGP concentration increased. The addition of C-S-H seeds can increase the 12h compressive strength of cement pastes by 15.5%–53.5%. This improvement is due to the accelerated early hydration kinetics induced by the C-S-H seeds, which act as nucleation sites, thereby increasing the nucleation and growth rates by 31.4% and 53.2%, respectively.

Relationship of PSC to embryos: Extending and refining capture of PSC lines from mammalian embryos.

Pluripotent stem cell lines derived from preimplantation mouse embryos have opened opportunities for the study of early mammalian development and generation of genetically uncompromised material for differentiation into specific cell types. Murine embryonic stem cells are highly versatile and can be engineered and introduced into host embryos, transferred to recipient females, and gestated to investigate gene function at multiple levels as well as developmental mechanisms, including lineage segregation and cell competition. In this review, we summarize the biomedical motivation driving the incremental modification to culture regimes and analyses that have advanced stem cell research to its current state. Ongoing investigation into divergent mechanisms of early developmental processes adopted by other species, such as agriculturally beneficial mammals and birds, will continue to enrich knowledge and inform strategies for future in vitro models.

Distinct contributions of microbial and plant residues to SOC during ecosystem primary succession in a Tibetan glacier foreland

Soil organic carbon (SOC) rapidly accumulates during ecosystem primary succession in glacier foreland. This makes it an ideal model for studying soil carbon sequestration and stabilization, which are urgently needed to mitigate climate change. Here, we investigated SOC dynamics in the Kuoqionggangri glacier foreland on the Tibetan Plateau. The study area along a deglaciation chronosequence of 170-year comprising three ecosystem succession stages, including barren ground, herb steppe, and legume steppe. We quantified amino sugars, lignin phenols, and relative expression of genes associated with carbon degradation to assess the contributions of microbial and plant residues to SOC, and used FT-ICR mass spectroscopy to analyze the composition of dissolved organic matter. We found that herbal plant colonization increased SOC by enhancing ecosystem gross primary productivity, while subsequent legumes development decreased SOC, due to increased ecosystem respiration from labile organic carbon inputs. Plant residues were a greater contributor to SOC than microbial residues in the vegetated soils, but they were susceptible to microbial degradation compared to the more persistent and continuously accumulating microbial residues. Our findings revealed the organic carbon accumulation and stabilization process in early soil development, which provides mechanism insights into carbon sequestration during ecosystem restoration under climate change.

Early Brain Development and Public Health.

Early brain development research is important to public health professionals for understanding early development and strengthening systemic supports for young children's healthy brain growth. This overview describes basic processes of early brain development, including prenatal development and the "fetal programming" of brain and behavior, neural proliferation and the essential influence of experience in the creation of neural networks, the maturational timing of different brain systems and their behavioral consequences, the myelination of neural pathways and its influence on children's action and thinking, and the capacity of the brain to create new neural connections throughout life, which contributes to its continuing adaptability to new experiences. The implications of this research for public health and for strengthening support for early brain development are considered throughout this discussion.

Calculating the surface energies of crystals on a face-specific and whole particle basis: Case study of the α- and β-polymorphic forms of L-glutamic acid

Molecular-scale modelling for predicting surface energies on a face-specific and whole particle basis is applied to all the crystallographically-independent surfaces of L-glutamic acid forms. The predicted data is found to be in good general agreement with measured surface energies using inverse gas chromatography and Washburn capillary rise techniques with the former revealing higher values compared to the prediction, perhaps consistent with the polar (zwitterionic) nature of this material. This fusion of experimental and computational data provides a high-fidelity definition of the face-by-face breakdown of the energetic anisotropy of the crystals. There is increasing industrial interest in defining the potential impact of whole particle properties on the performance of formulated drug product and their manufacturability especially as the community accelerates the molecule to medicine journey. The overall molecular modelling approach highlights its application in designing ingredients for optimising face-specific particle surface energies for product formulatability particularly in early phase process development.

Co-exposure effect of different colour of LED lights and increasing temperature on zebrafish larvae (Danio rerio): Immunohistochemical, metabolomics, molecular and behaviour approaches

Although there are studies in the literature on the effects of different coloured light-emitting diodes (LEDs) on different organisms, there is limited information on how these effects change with temperature increase. In this study, the effects of blue, green, red and white LED lights on the early development process of zebrafish (Danio rerio (Hamilton, 1822)) were comprehensively investigated. In addition, to simulate global warming, it was examined how a one-degree temperature increase affects this process. For this purpose, zebrafish embryos, which were placed at 4 hpf (hours post fertilization) in an incubator whose interior was divided into four areas, were kept at three different temperatures (28, 29 and 30 °C) for 120 h. The group kept in a dark environment was chosen as the control. The temperature of the control group was also increased at the same rate as the other groups. The results showed that at the end of the exposure period, temperature and light colour caused an increase in body malformations. Histopathological damage and immunopositive signals of HSP 70 and 8-OHdG biomarkers in larval brains, increase in free oxygen radicals, apoptotic cells and lipid accumulation throughout the body, increase in locomotor activity, decrease in heart rate and blood flow, and significant changes in more than thirty metabolite levels were detected. In addition, it has been determined that many metabolic pathways are affected, especially glutathione, vitamin B6 and pyrimidine metabolism. Moreover, it has been observed that a one-degree temperature increase worsens this negative effect. It was concluded that blue light was the closest light to the control group and was less harmful than other light colours. The study revealed that blue light produced results that were most similar to those seen in the control group.

Early differential impact of MeCP2 mutations on functional networks in Rett syndrome patient-derived human cerebral organoids.

Human cerebral organoids derived from induced pluripotent stem cells can recapture early developmental processes and reveal changes involving neurodevelopmental disorders. Mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene are associated with Rett syndrome, and disease severity varies depending on the location and type of mutation. Here, we focused on neuronal activity in Rett syndrome patient-derived organoids, analyzing two types of MeCP2 mutations - a missense mutation (R306C) and a truncating mutation (V247X) - using calcium imaging with three-photon microscopy. Compared to isogenic controls, we found abnormal neuronal activity in Rett organoids and altered network function based on graph theoretic analyses, with V247X mutations impacting functional responses and connectivity more severely than R306C mutations. These changes paralleled EEG data obtained from patients with comparable mutations. Labeling DLX promoter-driven inhibitory neurons demonstrated differences in activity and functional connectivity of inhibitory and excitatory neurons in the two types of mutation. Transcriptomic analyses revealed HDAC2-associated impairment in R306C organoids and decreased GABAA receptor expression in excitatory neurons in V247X organoids. These findings demonstrate mutation-specific mechanisms of vulnerability in Rett syndrome and suggest targeted strategies for their treatment.

The Construction of Stem Cell-Induced Hepatocyte Model and Its Application in Evaluation of Developmental Hepatotoxicity of Environmental Pollutants.

Stem cells, with their ability to self-renew and differentiate into various cell types, are a unique and valuable resource for medical research and toxicological studies. The liver is the most crucial metabolic organ in the human body and serves as the primary site for the accumulation of environmental pollutants. Enrichment with environmental pollutants can disrupt the early developmental processes of the liver and have a significant impact on liver function. The liver comprises a complex array of cell types, and different environmental pollutants have varying effects on these cells. Currently, there is a lack of well-established research models that can effectively demonstrate the mechanisms by which environmental pollutants affect human liver development. The emergence of liver cells and organoids derived from stem cells offers a promising tool for investigating the impact of environmental pollutants on human health. Therefore, this study systematically reviewed the developmental processes of different types of liver cells and provided an overview of studies on the developmental toxicity of various environmental pollutants using stem cell models.

Life cycle and development of the marine leech Branchellion lobata (Hirudinea: Piscicolidae), from round stingrays, Urobatis halleri, from southern California

During captivity, round stingrays, Urobatis halleri, became infected with the marine leech Branchellion lobata. When adult leeches were deprived of blood meal, they experienced a rapid decrease in body mass and did not survive beyond 25 days. If kept in aquaria with host rays, B. lobata fed frequently and soon produced cocoons, which were discovered adhered to sand grains. A single leech emerged from each cocoon (at ~ 21 days), and was either preserved for histology or molecular analysis, or monitored for development by introduction to new hosts in aquaria. Over a 74-day observation period, leeches grew from ~ 2 to 8 mm without becoming mature. Newly hatched leeches differed from adults in lacking branchiae and apparent pulsatile vesicles. The microbiome of the hatchlings was dominated by a specific, but undescribed, member of the gammaproteobacteria, also recovered previously from the adult leech microbiome. Raising B. lobata in captivity provided an opportunity to examine their reproductive strategy and early developmental process, adding to our limited knowledge of this common group of parasites.