Unveiling the vulnerability of the human abducens nerve: insights from comparative cranial base anatomy in mammals and primates.

The topographic anatomy of the abducens nerve has been the subject of research for more than 150 years. Although its vulnerability was initially attributed to its length, this hypothesis has largely lost prominence. Instead, attention has shifted toward its intricate anatomical relations along the cranial base. Contrary to the extensive anatomical and neurosurgical literature on abducens nerve anatomy in humans, its complex anatomy in other species has received less emphasis. The main question addressed here is why the human abducens nerve is predisposed to injury. Specifically, we aim to perform a comparative analysis of the basicranial pathway of the abducens nerve in mammals and primates. Our hypothesis links its vulnerability to cranial base flexion, particularly around the sphenooccipital synchondrosis. We examined the abducens nerve pathway in various mammals, including primates, humans (N = 40; 60% males; 40% females), and human fetuses (N = 5; 60% males; 40% females). The findings are presented at both the macroscopic and histological levels. To associate our findings with basicranial flexion, we measured the cranial base angles in the species included in this study and compared them to data in the available literature. Our findings show that the primitive state of the abducens nerve pathway follows a nearly flat (unflexed) cranial base from the pontomedullary sulcus to the superior orbital fissure. Only the gulfar segment, where the nerve passes through Dorello's canal, demonstrates some degree of variation. We present evidence indicating that the derived state of the abducens pathway, which is most pronounced in humans from an early stage of development, is characterized by following the significantly more flexed basicranium. Overall, the present study elucidates the evolutionary basis for the vulnerability of the abducens nerve, especially within its gulfar and cavernous segments, which are situated at the main synchondroses between the anterior, middle, and posterior cranial fossae-a unique anatomical relation exclusive to the abducens nerve. The principal differences between the pathways of this nerve and those of other cranial nerves are discussed. The findings suggest that the highly flexed human cranial base plays a pivotal role in the intricate anatomical relations and resulting vulnerability of the abducens nerve.

Sequential Fabrication of a Three-Layer Retina-like Structure.

Tissue engineering is considered a promising approach to treating advanced degenerative maculopathies such as nonexudative age-related macular degeneration (AMD), the leading cause of blindness worldwide. The retina consists of several hierarchical tissue layers, each of which is supported by a layer underneath. Each of these layers has a different morphology and requires distinct conditions for proper assembly. In fact, a prerequisite step for the assembly of each of these layers is the organization of the layer underneath. Advanced retinal degeneration includes degeneration of the other retina layers, including the choroid, the retinal pigmented epithelium (RPE), and the photoreceptors. Here, we report a step-by-step fabrication process of a three-layer retina-like structure. The process included the 3D printing of a choroid-like structure in an extracellular matrix (ECM) hydrogel, followed by deposition of the RPE monolayer. After the formation of the blood vessel-RPE interface, the photoreceptor cells were deposited to interact with the RPE layer. At the end of the fabrication process, each layer was characterized for its morphology and expression of specific markers, and the integration of the three-layer retina was evaluated. We envision that such a retina-like structure may be able to attenuate the deterioration of a degenerated retina and improve engraftment and regeneration. This retinal implant may potentially be suitable for a spectrum of macular degenerative diseases for which there are currently no cures and may save millions from complete blindness.

Birth outcomes associated with a natural delivery approach in a perinatal center: A comparative retrospective study.

OBJECTIVES: At our center, natural home-like delivery settings have been established in or near conventional labor wards, for the care of pregnant women who prefer little or no medical intervention during labor and birth. We compared obstetrical and neonatal outcomes of women in active spontaneous labor, between those who chose to deliver in a natural-delivery setup and those who chose a conventional setting. METHODS: This retrospective study included low-risk women who delivered at term between March 1, 2020 and December 31, 2022, in a single tertiary university affiliated medical center. Birth outcomes were compared between 124 women who delivered by natural birth (the study group) and 244 who gave birth in a conventional setting (the control group). RESULTS: No cesarean deliveries were performed in the study group, compared to 18 (7.4%) of the control group, p = 0.004. Intrapartum fever, postpartum hemorrhage, and uterotonic administration were similar between the groups. For the study compared to the control group, breastfeeding was more common (71.3% vs. 12.3%, p < 0.001), analgesia administration within 48 h delivery was lower (4.1% vs. 10.7%, p = 0.033), and maternal and neonatal length of hospitalization were shorter. Of the women initially admitted to the natural-delivery room, 14 (11.5%) were transferred to a conventional-delivery room. CONCLUSIONS: Birth in a hospital natural-delivery setting was associated with increased likelihood of vaginal birth, increased immediate breastfeeding and breastfeeding at discharge, and lower postpartum pain.

Modified ECM-Based Bioink for 3D Printing of Multi-Scale Vascular Networks.

The survival and function of tissues depend on appropriate vascularization. Blood vessels of the tissues supply oxygen, and nutrients and remove waste and byproducts. Incorporating blood vessels into engineered tissues is essential for overcoming diffusion limitations, improving tissue function, and thus facilitating the fabrication of thick tissues. Here, we present a modified ECM bioink, with enhanced mechanical properties and endothelial cell-specific adhesion motifs, to serve as a building material for 3D printing of a multiscale blood vessel network. The bioink is composed of natural ECM and alginate conjugated with a laminin adhesion molecule motif (YIGSR). The hybrid hydrogel was characterized for its mechanical properties, biochemical content, and ability to interact with endothelial cells. The pristine and modified hydrogels were mixed with induced pluripotent stem cells derived endothelial cells (iPSCs-ECs) and used to print large blood vessels with capillary beds in between.

Complete human day 14 post-implantation embryo models from naive ES cells.

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs)3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions)4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.

Optic pathway glioma and endocrine disorders in patients with and without NF1.

BACKGROUND: Optic pathway gliomas (OPGs) are classified by anatomic location and the association with neurofibromatosis type 1 (NF1). Children with OPGs face sequelae related to tumor location and treatment modalities. We assessed the prevalence of endocrine dysfunction in children with OPGs and compared outcomes between those with and without NF1. METHODS: We performed a retrospective medical record review of medical history, and clinical and laboratory data, of children diagnosed with OPGs (n = 59, 61% with NF1) during 1990-2020, followed at a tertiary endocrine clinic. Growth and puberty parameters and occurrence of endocrine dysfunction were evaluated. RESULTS: Isolated optic nerve involvement was higher among patients with than without NF1. Patients without NF1 were younger at OPG diagnosis and more often treated with debulking surgery or chemotherapy. At the last endocrine evaluation, patients without NF1 had comparable height SDS, higher BMI SDS, and a higher rate of endocrine complications (78.3% vs. 41.7%, p = 0.006). Younger age at diagnosis, older age at last evaluation, and certain OPG locations were associated with increased endocrine disorder incidence. CONCLUSIONS: Endocrine dysfunction was more common in patients without NF1; this may be related to younger age at presentation, tumor locations, a greater progressive rate, and more aggressive treatments. IMPACT: The literature is sparse regarding sporadic OPGs, and the mean duration of follow-up is shorter than at our study. Our data show a higher rate of endocrine dysfunction in patients with OPGs than previously described. We also found a higher prevalence of endocrine dysfunctions among patients without compared to those with NF-1. A better understanding of the true prevalence of endocrine disabilities that may evolve along time can help in guiding physicians in the surveillance needed in patients with OPG.

Pan-genome and multi-parental framework for high-resolution trait dissection in melon (Cucumis melo).

Linking genotype with phenotype is a fundamental goal in biology and requires robust data for both. Recent advances in plant-genome sequencing have expedited comparisons among multiple-related individuals. The abundance of structural genomic within-species variation that has been discovered indicates that a single reference genome cannot represent the complete sequence diversity of a species, leading to the expansion of the pan-genome concept. For high-resolution forward genetics, this unprecedented access to genomic variation should be paralleled and integrated with phenotypic characterization of genetic diversity. We developed a multi-parental framework for trait dissection in melon (Cucumis melo), leveraging a novel pan-genome constructed for this highly variable cucurbit crop. A core subset of 25 diverse founders (MelonCore25), consisting of 24 accessions from the two widely cultivated subspecies of C. melo, encompassing 12 horticultural groups, and 1 feral accession was sequenced using a combination of short- and long-read technologies, and their genomes were assembled de novo. The construction of this melon pan-genome exposed substantial variation in genome size and structure, including detection of ~300 000 structural variants and ~9 million SNPs. A half-diallel derived set of 300 F2 populations, representing all possible MelonCore25 parental combinations, was constructed as a framework for trait dissection through integration with the pan-genome. We demonstrate the potential of this unified framework for genetic analysis of various melon traits, including rind color intensity and pattern, fruit sugar content, and resistance to fungal diseases. We anticipate that utilization of this integrated resource will enhance genetic dissection of important traits and accelerate melon breeding.

Accelerated Multiphosphorylated Peptide Synthesis.

Preparing phosphorylated peptides with multiple adjacent phosphorylations is synthetically difficult, leads to ß-elimination, results in low yields, and is extremely slow. We combined synthetic chemical methodologies with computational studies and engineering approaches to develop a strategy that takes advantage of fast stirring, high temperature, and a very low concentration of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to produce multiphosphorylated peptides at an extremely rapid time and high purity.

Occurrence of Macrophomina phaseolina in Israel: Challenges for Disease Management and Crop Germplasm Enhancement.

Macrophomina phaseolina is a soil-borne fungal pathogen infecting many important crop plants. The fungus, which can survive on crop debris for a long period of time, causes charcoal rot disease by secreting a diverse array of cell-wall degrading enzymes and toxins. M. phaseolina thrives during periods of high temperatures and arid conditions, as typically occur in Israel and other countries with a Mediterranean climate. Crop losses due to charcoal rot can be expected to increase and spread to other countries in a warming global climate. Management of this pathogen is challenging, requiring an array of approaches for the various crop hosts. Approaches that have had some success in Israel include grafting of melons and watermelons on resistant squash rootstocks and soil application of fungicide to reduce disease incidence in melons, fumigation and alterations in planting date and mulching of strawberries, and alteration in irrigation regime of cotton. Elsewhere, these approaches, as well as soil amendments and addition of organisms that are antagonistic to M. phaseolina, have had success in some crop situations. Management through host resistance would be the most sustainable approach, but it requires identifying a resistant germplasm for each crop and introgressing the resistance into the leading cultivars. Resistance to charcoal rot is under complex genetic control in most crops, posing a great challenge for its introgression into elite germplasm. Moreover, fast, reliable methods of screening for resistance would have to be developed for each crop. The toothpick-inoculation method used by us holds great promise for selecting resistant germplasm for melons and possibly for sesame, but other methodologies have to be devised for each individual crop.

Interactions between Magnaporthiopsis maydis and Macrophomina phaseolina, the Causes of Wilt Diseases in Maize and Cotton.

Fungal pathogens are a significant threat to crops worldwide. The soil fungus, Magnaporthiopsis maydis, severely affects sensitive maize hybrids by causing the rapid wilting of plants at the maturity stage. Similarly, the soil fungus, Macrophomina phaseolina, develops in a variety of host plants, which leads to rot and plant mortality. The presence of both pathogens together in diseased cotton plants in Israel suggests possible interactions between them. Here, these relationships were tested in a series of experiments accompanied by real-time PCR tracking in maize and cotton. Despite the fact that neither of the pathogens was superior in a growth plate confrontation assay, their co-inoculum had a significant influence under field conditions. In maize sprouts and fully matured plants, infection by both pathogens (compared to inoculation with each of them alone) led to lesser amounts of M. maydis DNA but to increased amounts of M. phaseolina DNA levels. These results were obtained under a restricted water regime, while optimal water irrigation led to less pronounced differences. In water-stressed cotton sprouts, infection with both pathogens led to an increase in DNA amounts of each of the pathogens. Whereas the M. maydis DNA levels in the double infection remain high at the end of the season, a reduction in the amount of M. phaseolina DNA was observed. The double infection caused an increase in growth parameters in maize and cotton and decreased levels of dehydration in maize plants accompanied by an increase in yield production. Dehydration symptoms were minor in cotton under an optimal water supply. However, under a restricted water regime, the double infection abolished the harmful effect of M. phaseolina on the plants' development and yield. These findings are the first report of interactions between these two pathogens in maize and cotton, and they encourage expanding the study to additional plant hosts and examining the potential involvement of other pathogens.

An Assay for Quantifying Protein-RNA Binding in Bacteria.

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

Synthetic 5' UTRs Can Either Up- or Downregulate Expression upon RNA-Binding Protein Binding.

The construction of complex gene-regulatory networks requires both inhibitory and upregulatory modules. However, the vast majority of RNA-based regulatory "parts" are inhibitory. Using a synthetic biology approach combined with SHAPE-seq, we explored the regulatory effect of RNA-binding protein (RBP)-RNA interactions in bacterial 5' UTRs. By positioning a library of RNA hairpins upstream of a reporter gene and co-expressing them with the matching RBP, we observed a set of regulatory responses, including translational stimulation, translational repression, and cooperative behavior. Our combined approach revealed three distinct states in vivo: in the absence of RBPs, the RNA molecules can be found in either a molten state that is amenable to translation or a structured phase that inhibits translation. In the presence of RBPs, the RNA molecules are in a semi-structured phase with partial translational capacity. Our work provides new insight into RBP-based regulation and a blueprint for designing complete gene-regulatory circuits at the post-transcriptional level.

An in Vivo Binding Assay for RNA-Binding Proteins Based on Repression of a Reporter Gene.

We study translation repression in bacteria by engineering a regulatory circuit that functions as a binding assay for RNA binding proteins (RBP) in vivo. We do so by inducing expression of a fluorescent protein-RBP chimera, together with encoding its binding site at various positions within the ribosomal initiation region (+11-13 nt from the AUG) of a reporter module. We show that when bound by their cognate RBPs, the phage coat proteins for PP7 (PCP) and Qß (QCP), strong repression is observed for all hairpin positions within the initiation region. Yet, a sharp transition to no-effect is observed when positioned in the elongation region, at a single-nucleotide resolution. Employing in vivo Selective 2'-hydroxyl acylation analyzed by primer extension followed by sequencing (SHAPE-seq) for a representative construct, established that in the translationally active state the mRNA molecule is nonstructured, while in the repressed state a structured signature was detected. We then utilize this regulatory phenomena to quantify the binding affinity of the coat proteins of phages MS2, PP7, GA, and Qß to 14 cognate and noncognate binding sites in vivo. Using our circuit, we demonstrate qualitative differences between in vitro to in vivo binding characteristics for various variants when comparing to past studies. Furthermore, by introducing a simple mutation to the loop region for the Qß-wt site, MCP binding is abolished, creating the first high-affinity QCP site that is completely orthogonal to MCP. Consequently, we demonstrate that our hybrid transcriptional-post-transcriptional circuit can be utilized as a binding assay to quantify RNA-RBP interactions in vivo.

A Synthetic Oligo Library and Sequencing Approach Reveals an Insulation Mechanism Encoded within Bacterial σ54 Promoters.

We use an oligonucleotide library of >10,000 variants to identify an insulation mechanism encoded within a subset of σ54 promoters. Insulation manifests itself as reduced protein expression for a downstream gene that is expressed by transcriptional readthrough. It is strongly associated with the presence of short CT-rich motifs (3-5 bp), positioned within 25 bp upstream of the Shine-Dalgarno (SD) motif of the silenced gene. We provide evidence that insulation is triggered by binding of the ribosome binding site (RBS) to the upstream CT-rich motif. We also show that, in E. coli, insulator sequences are preferentially encoded within σ54 promoters, suggesting an important regulatory role for these sequences in natural contexts. Our findings imply that sequence-specific regulatory effects that are sparsely encoded by short motifs may not be easily detected by lower throughput studies. Such sequence-specific phenomena can be uncovered with a focused oligo library (OL) design that mitigates sequence-related variance, as exemplified herein.

Memory, Executive Skills, and Psychosocial Phenotype in Children with Pharmacoresponsive Epilepsy: Reactivity to Intervention.

BACKGROUND: Recent studies on pharmacoresponsive epilepsies demonstrate specific memory, executive functions (EF), and psychosocial deficits in this group. These deficits are often undertreated, and little is known about the neuropsychological factors that may support moderation of the deficits through intervention. The aim of this study was to explore the effects of a structured cognitive behavioral group intervention on both memory and emotional domains and to evaluate the factors influencing its efficacy. METHODS: The feasibility study implemented a newly designed intervention for children with pharmacoresponsive epilepsies (N = 33, aged 9-14 years, 51% girls), hypothesizing that memory and psychosocial symptoms in children with pharmacoresponsive epilepsies are sensitive to intervention using structured memory and psychosocial modules in a weekly group session setting. Comparable memory and psychosocial assessments were used to evaluate performance at baseline and post-intervention. Results were compared to age- and education-matched healthy controls (N = 27, aged 9-14 years). RESULTS: Pre-post-intervention comparisons show improvements in STM (p < 0.01, η2 = 0.358), optimism (p < 0.05, η2 = 0.245), and self-efficacy (p < 0.05, η2 = 0.164). Unique negative relations between memory deficits and psychosocial phenotype were seen in epilepsy patients and not in controls in response to the intervention. EF moderated this intervention effect (p < 0.05, η2 = 0.252), whereas psychosocial status and pharmacological profile did not. CONCLUSION: Cognitive behavioral therapy focusing on memory and psychosocial perceptions for children with pharmacoresponsive epilepsies seems promising, with greater improvement in memory and psychosocial functioning in children with more affected EF.

De novo KCNB1 mutations in infantile epilepsy inhibit repetitive neuronal firing.

The voltage-gated Kv2.1 potassium channel encoded by KCNB1 produces the major delayed rectifier potassium current in pyramidal neurons. Recently, de novo heterozygous missense KCNB1 mutations have been identified in three patients with epileptic encephalopathy and a patient with neurodevelopmental disorder. However, the frequency of KCNB1 mutations in infantile epileptic patients and their effects on neuronal activity are yet unknown. We searched whole exome sequencing data of a total of 437 patients with infantile epilepsy, and found novel de novo heterozygous missense KCNB1 mutations in two patients showing psychomotor developmental delay and severe infantile generalized seizures with high-amplitude spike-and-wave electroencephalogram discharges. The mutation located in the channel voltage sensor (p.R306C) disrupted sensitivity and cooperativity of the sensor, while the mutation in the channel pore domain (p.G401R) selectively abolished endogenous Kv2 currents in transfected pyramidal neurons, indicating a dominant-negative effect. Both mutants inhibited repetitive neuronal firing through preventing production of deep interspike voltages. Thus KCNB1 mutations can be a rare genetic cause of infantile epilepsy, and insufficient firing of pyramidal neurons would disturb both development and stability of neuronal circuits, leading to the disease phenotypes.

Auditory verbal memory and psychosocial symptoms are related in children with idiopathic epilepsy.

OBJECTIVE: Idiopathic epilepsies are considered to have relatively good prognoses and normal or near normal developmental outcomes. Nevertheless, accumulating studies demonstrate memory and psychosocial deficits in this population, and the prevalence, severity and relationships between these domains are still not well defined. We aimed to assess memory, psychosocial function, and the relationships between these two domains among children with idiopathic epilepsy syndromes using an extended neuropsychological battery and psychosocial questionnaires. METHODS: Cognitive abilities, neuropsychological performance, and socioemotional behavior of 33 early adolescent children, diagnosed with idiopathic epilepsy, ages 9-14years, were assessed and compared with 27 age- and education-matched healthy controls. RESULTS: Compared to controls, patients with stabilized idiopathic epilepsy exhibited higher risks for short-term memory deficits (auditory verbal and visual) (p<0.0001), working memory deficits (p<0.003), auditory verbal long-term memory deficits (p<0.0021), and more frequent psychosocial symptoms (p<0.0001). The severity of auditory verbal memory deficits was related to severity of psychosocial symptoms among the children with epilepsy but not in the healthy controls. SIGNIFICANCE: Results suggest that deficient auditory verbal memory may be compromising psychosocial functioning in children with idiopathic epilepsy, possibly underscoring that cognitive variables, such as auditory verbal memory, should be assessed and treated in this population to prevent secondary symptoms.

A novel mutation in the endosomal Na+/H+ exchanger NHE6 (SLC9A6) causes Christianson syndrome with electrical status epilepticus during slow-wave sleep (ESES).

Mutations in the solute carrier family 9, subfamily A member 6 (SLC9A6) gene, encoding the endosomal Na+/H+ exchanger 6 (NHE6) are associated with Christianson syndrome, a syndromic form of X-linked intellectual disability characterized by microcephaly, severe global developmental delay, autistic behavior, early onset seizures and ataxia. In a 7-year-old boy with characteristic clinical and neuroimaging features of Christianson syndrome and epileptic encephalopathy with continuous spikes and waves during sleep, we identified a novel splice site mutation (IVS10-1G>A) in SLC9A6. These findings expand the clinical spectrum of the syndrome and indicate NHE6 dysfunction as a new cause of electrical status epilepticus during slow-wave sleep (ESES).