Brain Sample Preparation After Performing in Utero Electroporation to Proliferating and Differentiated Cells in the Developing Mouse Neocortex.

In utero electroporation (IUE) enables labeling and manipulating specific types of cells by introducing DNA plasmids with desired promoters. After the surgery, mouse brains are fixed at any stage and analyzed after staining using specific antibodies. Here, we describe the flow of the IUE experiment from the preparation to microscopic observations.

Nonsense-mediated mRNA decay of mRNAs encoding a signal peptide occurs primarily after mRNA targeting to the endoplasmic reticulum.

Translation of messenger ribonucleic acids (mRNAs) encoding integral membrane proteins or secreted proteins occurs on the surface of the endoplasmic reticulum (ER). When a nascent signal peptide is synthesized from the mRNAs, the ribosome-nascent chain complex (RNC) is recognized by the signal recognition particle (SRP) and then transported to the surface of the ER. The appropriate targeting of the RNC-SRP complex to the ER is monitored by a quality control pathway, a nuclear cap-binding complex (CBC)-ensured translational repression of RNC-SRP (CENTRE). In this study, using ribosome profiling of CBC-associated and eukaryotic translation initiation factor 4E-associated mRNAs, we reveal that, at the transcriptomic level, CENTRE is in charge of the translational repression of the CBC-RNC-SRP until the complex is specifically transported to the ER. We also find that CENTRE inhibits the nonsense-mediated mRNA decay (NMD) of mRNAs within the CBC-RNC-SRP. The NMD occurs only after the CBC-RNC-SRP is targeted to the ER and after eukaryotic translation initiation factor 4E replaces CBC. Our data indicate dual surveillance for properly targeting mRNAs encoding integral membrane or secretory proteins to the ER. CENTRE blocks gene expression at the translation level before the CBC-RNC-SRP delivery to the ER, and NMD monitors mRNA quality after its delivery to the ER.

Physical Properties of Paste Synthesized from Wet- and Dry-Processed Silver Powders.

This study compares the characteristics and low-temperature curing properties of pastes prepared from silver (Ag) powders synthesized by either wet powder (WP) or dry powder (DP) processing. The WP synthesis of electrode particles has the advantage of controlling the average particle size and particle size distribution but the disadvantage of producing low-purity, crystalline particles because they are synthesized through chemical reduction at less than 100 °C. Conversely, the DP synthesis of electrode particles has the advantage of producing pure, highly crystalline particles (due to synthesis at high temperatures) but the disadvantage of a high processing cost. WP and DP were used to manufacture pastes for low-temperature curing, and the physical properties of the pastes and the electrode characteristics after low-temperature curing were compared between powder types. Shear stress as a function of the shear rate shows that the WP paste is a plastic fluid, whereas the DP paste is a pseudoplastic fluid, closer to a Newtonian fluid. Screen printing the Ag pastes and curing for 30 min at 130 °C produces a nonconductive WP paste, whereas it produces a DP paste with a conductivity of 61 mΩ/sq, indicating that the highly crystalline DP paste is advantageous for low-temperature curing.

Review of epilepsy care in the Democratic Republic of the Congo.

Epilepsy imposes a substantial burden on the Democratic Republic of Congo (DRC). These challenges encompass the lack of comprehensive disease surveillance, an unresolved understanding of its pathophysiology, economic barriers limiting access to essential care, the absence of epilepsy surgical capabilities, and deeply ingrained societal stigmas. Notably, the national prevalence of epilepsy remains undetermined, with research primarily concentrating on infectious factors like Onchocerca volvulus, leaving other potential causes underexplored. Most patients lack insurance, incurring out-of-pocket expenses that often lead them to opt for traditional medicine rather than clinical care. Social stigma, perpetuated by common misconceptions, intensifies the social isolation experienced by individuals living with epilepsy. Additionally, surgical interventions are unavailable, and the accessibility of anti-seizure medications and healthcare infrastructure remains inadequate. Effectively tackling these interrelated challenges requires a multifaceted approach, including conducting research into region-specific factors contributing to epilepsy, increasing healthcare funding, subsidizing the costs of treatment, deploying mobile tools for extensive screening, launching awareness campaigns to dispel myths and reduce stigma, and promoting collaborations between traditional healers and medical practitioners to enhance local understanding and epilepsy management. Despite the difficulties, significant progress can be achieved through sustained and compassionate efforts to understand and eliminate the barriers faced by epilepsy patients in the region. This review outlines essential steps for alleviating the epilepsy burden in the DRC. PLAIN LANGUAGE SUMMARY: There are not enough resources to treat epilepsy in the DRC. PWEs struggle with stigma and the lack of money. Many of them still use traditional medicine for treatment and hold wrong beliefs about epilepsy. That is why there is a need for more resources to make the lives of PWEs better in the DRC.

An interaction between eIF4A3 and eIF3g drives the internal initiation of translation.

An RNA structure or modified RNA sequences can provide a platform for ribosome loading and internal translation initiation. The functional significance of internal translation has recently been highlighted by the discovery that a subset of circular RNAs (circRNAs) is internally translated. However, the molecular mechanisms underlying the internal initiation of translation in circRNAs remain unclear. Here, we identify eIF3g (a subunit of eIF3 complex) as a binding partner of eIF4A3, a core component of the exon-junction complex (EJC) that is deposited onto spliced mRNAs and plays multiple roles in the regulation of gene expression. The direct interaction between eIF4A3-eIF3g serves as a molecular linker between the eIF4A3 and eIF3 complex, thereby facilitating internal ribosomal entry. Protein synthesis from in vitro-synthesized circRNA demonstrates eIF4A3-driven internal translation, which relies on the eIF4A3-eIF3g interaction. Furthermore, our transcriptome-wide analysis shows that efficient polysomal association of endogenous circRNAs requires eIF4A3. Notably, a subset of endogenous circRNAs can express a full-length intact protein, such as ß-catenin, in an eIF4A3-dependent manner. Collectively, our results expand the understanding of the protein-coding potential of the human transcriptome, including circRNAs.

Lamin B1 as a key modulator of the developing and aging brain.

Lamin B1 is an essential protein of the nuclear lamina that plays a crucial role in nuclear function and organization. It has been demonstrated that lamin B1 is essential for organogenesis and particularly brain development. The important role of lamin B1 in physiological brain development and aging has only recently been at the epicenter of attention and is yet to be fully elucidated. Regarding the development of brain, glial cells that have long been considered as supporting cells to neurons have overturned this representation and current findings have displayed their active roles in neurogenesis and cerebral development. Although lamin B1 has increased levels during the differentiation of the brain cells, during aging these levels drop leading to senescent phenotypes and inciting neurodegenerative disorders such as Alzheimer's and Parkinson's disease. On the other hand, overexpression of lamin B1 leads to the adult-onset neurodegenerative disease known as Autosomal Dominant Leukodystrophy. This review aims at highlighting the importance of balancing lamin B1 levels in glial cells and neurons from brain development to aging.

CUSUM learning curves: what they can and can't tell us.

INTRODUCTION: There has been increased interest in assessing the surgeon learning curve for new skill acquisition. While there is no consensus around the best methodology, one of the most frequently used learning curve assessments in the surgical literature is the cumulative sum curve (CUSUM) of operative time. To demonstrate the limitations of this methodology, we assessed the CUSUM of console time across cohorts of surgeons with differing case acquisition rates while varying the total number of cases used to calculate the CUSUM. METHODS: We compared the CUSUM curves of the average console times of surgeons who completed their first 20 robotic-assisted (RAS) cases in 13, 26, 39, and 52 weeks, respectively, for their first 50 and 100 cases, respectively. This analysis was performed for prostatectomy (1094 surgeons), malignant hysterectomy (737 surgeons), and inguinal hernia (1486 surgeons). RESULTS: In all procedures, the CUSUM curve of the cohort of surgeons who completed their first 20 procedures in 13 weeks demonstrated a lower slope than cohorts of surgeons with slower case acquisition rates. The case number at which the peak of the CUSUM curve occurs uniformly increases when the total number of cases used in generation of the CUSUM chart changes from 50 to 100 cases. CONCLUSION: The CUSUM analyses of these three procedures suggests that surgeons with fast initial case acquisition rates have less variability in their operative times over the course of their learning curve. The peak of the CUSUM curve, which is often used in surgical learning curve literature to denote "proficiency" is predictably influenced by the total number of procedures evaluated, suggesting that defining the peak as the point at which a surgeon has overcome the learning curve is subject to routine bias. The CUSUM peak, by itself, is an insufficient measure of "conquering the learning curve."

Enhancement of the signal-to-noise ratio in fiber-optics based SERS detection by rough-cutting the end surface.

Fiber-optics based surface-enhanced Raman scattering (FO-SERS) has an unique advantage of being able to remotely detect analyte molecules because the fiber length can be adjusted as desired. However, the Raman signal of the fiber-optic material is so strong that it is an important challenge in utilization of optical fiber for remote SERS sensing. In this study, we found that the background noise signal was greatly reduced by ca. 32% compared to conventional fiber-optics with a flat surface cut. To confirm the feasibility of FO-SERS detection, silver nanoparticles labeled with 4-fluorobenzenethiol were attached onto the end surface of an optical fiber to form a SERS-signaling substrate. The SERS intensity from the fiber-optics with a roughened surface as SERS substrate was increased significantly with respect to signal-to-noise ratio (SNR) values compared to optical fibers with flat end surface. This result implies that the fiber-optics with roughened surface could be used as an efficient alternative for FO-SERS sensing platform.

Demographic and Clinical Factors Affecting Pediatric Survival in South Kivu, the Democratic Republic of the Congo.

Promoting children's health is challenging in underresourced regions, with worse outcomes in areas of sociopolitical instabilities. This encapsulates the difficulties faced by the Panzi General Referral Hospital (PGRH) in South Kivu, the Democratic Republic of the Congo. In this retrospective, cross-sectional study of 456 children ≤ 18 years who presented to the pediatric emergency department of PGRH between December 2018 and May 2019, we present demographic and clinical predictors that affect pediatric survival. We note that referrals from external clinics (odds ratio [OR], 0.37; 95% CI, 0.18-0.75), poor maternal education (OR, 0.21; 95% CI, 0.07-0.67), diagnoses of meningitis (OR, 0.37; 95% CI, 0.18-0.75) or malnutrition (OR, 0.21; 95% CI, 0.07-0.67) are risk factors hindering pediatric survival. Paternal unemployment or longer durations of hospital stay, on the other hand, are protective toward survival. These predictors confirm the importance of accessibility and availability of medical resources and knowledge as levers to establish an effective, robust network of pediatric care delivery capable of withstanding South Kivu's unresolved political tumult.

Utility of illness symptoms for predicting COVID-19 infections in children.

BACKGROUND: The Centers for Disease Control and Prevention and the American Academy of Pediatrics recommend that symptomatic children remain home and get tested to identify potential coronavirus disease 2019 (COVID-19) cases. As the pandemic moves into a new phase, approaches to differentiate symptoms of COVID-19 versus other childhood infections can inform exclusion policies and potentially prevent future unnecessary missed school days. METHODS: Retrospective analysis of standardized symptom and exposure screens in symptomatic children 0-18 years tested for SARS-CoV-2 at three outpatient sites April to November 2020. Likelihood ratios (LR), number needed to screen to identify one COVID-19 case, and estimated missed school days were calculated. RESULTS: Of children studied (N = 2,167), 88.9% tested negative. Self-reported exposure to COVID-19 was the only factor that statistically significantly increased the likelihood of a positive test for all ages (Positive LR, 5-18 year olds: 5.26, 95% confidence interval (CI): 4.37-6.33; 0-4 year olds: 5.87, 95% CI: 4.67-7.38). Across ages 0-18, nasal congestion/rhinorrhea, sore throat, abdominal pain, and nausea/vomiting/diarrhea were commonly reported, and were either not associated or had decreased association with testing positive for COVID-19. The number of school days missed to identify one case of COVID-19 ranged from 19 to 48 across those common symptoms. CONCLUSIONS: We present an approach for identifying symptoms that are non-specific to COVID-19, for which exclusion would likely lead to limited impact on school safety but contribute to school-days missed. As variants and symptoms evolve, students and schools could benefit from reconsideration of exclusion and testing policies for non-specific symptoms, while maintaining testing for those who were exposed.

Direct visualization of the transition status during neural differentiation by dual-fluorescent reporter human pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) can differentiate into neurons and glia via neural progenitor cells and are widely used for neurogenic studies. However, directly visualizing the transition status during the neural differentiation of live cells is difficult. Here, targeting NEUROG2 (NGN2) and TUBB3 as markers of neurogenic cells and neurons, respectively, we established TUBB3EGFP/NGN2TagRFP dual-reporter hiPSCs using CRISPR-Cas9 technology. We induced the differentiation of cortical neurons from dual-reporter hiPSCs, successfully visualizing cell-fate conversion in two-dimensional (2D) culture and 3D organoids. The reporter cells were used to monitor drug effects to enhance neural induction, responses to gene knockdown, transplantation to the embryonic mouse brain, and live imaging at single-cell resolution. Notably, the earliest REELIN-positive neurons showed a distinctive migration pattern, and their production was accelerated by HES1-function loss. Together, these results demonstrate the potential for dual-reporter hiPSCs in therapeutic neural regeneration strategies and studies on human cortical development.

Mycobacterium intracellulare induces a Th17 immune response via M1-like macrophage polarization in canine peripheral blood mononuclear cells.

Mycobacterium avium-intracellulare complex (MAC) is one of the most prevalent pathogenic nontuberculous mycobacteria that cause chronic pulmonary disease. The prevalence of MAC infection has been rising globally in a wide range of hosts, including companion animals. MAC infection has been reported in dogs; however, little is known about interaction between MAC and dogs, especially in immune response. In this study, we investigated the host immune response driven by M. intracellulare using the co-culture system of canine T helper cells and autologous monocyte-derived macrophages (MDMs). Transcriptomic analysis revealed that canine MDMs differentiated into M1-like macrophages after M. intracellulare infection and the macrophages secreted molecules that induced Th1/Th17 cell polarization. Furthermore, canine lymphocytes co-cultured with M. intracellulare-infected macrophages induced the adaptive Th17 responses after 5 days. Taken together, our results indicate that M. intracellulare elicits a Th17 response through macrophage activation in this system. Those findings might help the understanding of the canine immune response to MAC infection and diminishing the potential zoonotic risk in One Health aspect.

UPF1 promotes rapid degradation of m6A-containing RNAs.

N6-methyladenosine (m6A) is the most prevalent internal modification in eukaryotic mRNAs and affects RNA processing and metabolism. When YTHDF2, an m6A-recognizing protein, binds to m6A, it facilitates the destabilization of m6A-containing RNAs (m6A RNAs). Here, we demonstrate that upstream frameshift 1 (UPF1), a key factor for nonsense-mediated mRNA decay, interacts with YTHDF2, thereby triggering rapid degradation of m6A RNAs. The UPF1-mediated m6A RNA degradation depends on a specific interaction between UPF1 and N-terminal residues 101-168 of YTHDF2, UPF1 ATPase/helicase activities, and UPF1 interaction with proline-rich nuclear receptor coactivator 2 (PNRC2), a decapping-promoting factor preferentially involved in nonsense-mediated mRNA decay. Furthermore, transcriptome-wide analyses show that YTHDF2-bound mRNAs that are not substrates for HRSP12-RNase P/MRP-mediated endoribonucleolytic cleavage are destabilized with a higher dependency on UPF1. Collectively, our data indicate dynamic and multilayered regulation of the stability of m6A RNAs and highlight the multifaceted role of UPF1 in mRNA decay.

Regulation of Hepatic Gluconeogenesis by Nuclear Receptor Coactivator 6.

Nuclear receptor coactivator 6 (NCOA6) is a transcriptional coactivator of nuclear receptors and other transcription factors. A general Ncoa6 knockout mouse was previously shown to be embryonic lethal, but we here generated liver-specific Ncoa6 knockout (Ncoa6 LKO) mice to investigate the metabolic function of NCOA6 in the liver. These Ncoa6 LKO mice exhibited similar blood glucose and insulin levels to wild type but showed improvements in glucose tolerance, insulin sensitivity, and pyruvate tolerance. The decrease in glucose production from pyruvate in these LKO mice was consistent with the abrogation of the fasting-stimulated induction of gluconeogenic genes, phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6pc). The forskolin-stimulated inductions of Pck1 and G6pc were also dramatically reduced in primary hepatocytes isolated from Ncoa6 LKO mice, whereas the expression levels of other gluconeogenic gene regulators, including cAMP response element binding protein (Creb), forkhead box protein O1 and peroxisome proliferator-activated receptor γ coactivator 1α, were unaltered in the LKO mouse livers. CREB phosphorylation via fasting or forskolin stimulation was normal in the livers and primary hepatocytes of the LKO mice. Notably, it was observed that CREB interacts with NCOA6. The transcriptional activity of CREB was found to be enhanced by NCOA6 in the context of Pck1 and G6pc promoters. NCOA6-dependent augmentation was abolished in cAMP response element (CRE) mutant promoters of the Pck1 and G6pc genes. Our present results suggest that NCOA6 regulates hepatic gluconeogenesis by modulating glucagon/cAMP-dependent gluconeogenic gene transcription through an interaction with CREB.

Chiral Plasmonic Nanowaves by Tilted Assembly of Unidirectionally Aligned Block Copolymers with Buckling-Induced Microwrinkles.

Chiral-structured nanoscale materials exhibit chiroptical properties with preferential absorptions of circularly polarized light. The distinctive optical responses of chiral materials have great potential for advanced optical and biomedical applications. However, the fabrication of three-dimensional structures with mirrored nanoscale geometry is still challenging. This study introduces chiral plasmonic nanopatterns in wavy shapes based on the unidirectional alignment of block copolymer thin films and their tilted transfer, combined with buckling processes. The cylindrical nanodomains of polystyrene-block-poly(2-vinylpyridine) thin films were unidirectionally aligned over a large area by the shear-rolling process. The aligned block copolymer thin films were transferred onto uniaxially prestrained polydimethylsiloxane films at certain angles relative to the stretching directions. The strain was then released to induce buckling. The aligned nanopatterns across the axis of the formed microwrinkles were selectively infiltrated with gold ions. After reduction by plasma treatment, chiral plasmonic nanowave patterns were fabricated with the presence of mirror-reflected circular dichroism spectra. This fabrication method does not require any lithography processing or innately chiral biomaterials, which can be advantageous over other conventional fabrication methods for artificial nanoscale chiral materials.

Shear-Rolling Process for Unidirectionally and Perpendicularly Oriented Sub-10-nm Block Copolymer Patterns on the 4 in Scale.

Shear alignment of the block copolymer (BCP) thin film is one of the promising directed self-assembly (DSA) methodologies for the unidirectional alignment of sub-10 nm microdomains of BCPs for next-generation nanolithography and nanowire-grid polarizers. However, because of the differences in the surface/interfacial energies at the top surface/bottom interface, the shear-induced ordering of BCP nanopatterns has been restricted to BCPs with spherical and cylindrical nanopatterns and cannot be realized for high-aspect-ratio perpendicular lamellar structures, which is essential for practical application to semiconductor pattern processes. It is still a difficult challenge to fabricate the unidirectional alignment in a short time over a large area. In this study, we propose an approach for combining the shear-rolling process with the filtered plasma treatment of BCP films for the fabrication of unidirectionally aligned and perpendicularly oriented lamellar nanostructures. This approach enables fabrication within 1 min on a 4 in scale. We treated filtered plasma on the BCP film for perpendicular orientation and executed the hot-rolling process with different roller and stage speeds. Large-scale shear was generated only at the location where the BCP film was in contact with both the roller and stage, effectively applying shear stress to a large area of the BCP film within a short time. The repeated application of this shear-rolling process can achieve a higher level of unidirectional alignment. Our aligned BCP vertical lamellae were used to fabricate a high-aspect-ratio sub-10-nm-wide metallic nanowire array via dry/wet processes. In addition, shear-rolling with chemoepitaxy patterns can achieve higher orientational order and lower defectivity.

Comprehensive characterization of migration profiles of murine cerebral cortical neurons during development using FlashTag labeling.

In the mammalian cerebral neocortex, different regions have different cytoarchitecture, neuronal birthdates, and functions. In most regions, neuronal migratory profiles are speculated similar based on observations using thymidine analogs. Few reports have investigated regional migratory differences from mitosis at the ventricular surface. In this study, we applied FlashTag technology, in which dyes are injected intraventricularly, to describe migratory profiles. We revealed a mediolateral regional difference in the migratory profiles of neurons that is dependent on developmental stage; for example, neurons labeled at embryonic day 12.5-15.5 reached their destination earlier dorsomedially than dorsolaterally, even where there were underlying ventricular surfaces, reflecting sojourning below the subplate. This difference was hardly recapitulated by thymidine analogs, which visualize neurogenic gradients, suggesting a biological significance different from the neurogenic gradient. These observations advance our understanding of cortical development and the power of FlashTag in studying migration and are thus resources for future neurodevelopmental studies.

Nationwide Comparison of Surgical and Oncologic Outcomes in Endometrial Cancer Patients Undergoing Robotic, Laparoscopic, and Open Surgery: A Population-Based Cohort Study.

PURPOSE: Population-based comparisons between minimally invasive surgery (MIS) (robotic surgery [RS] and laparoscopic surgery [LS]) and open surgery (OS) for managing endometrial cancer are lacking. This study aimed to compare surgical and oncologic outcomes between endometrial cancer patients who underwent surgical staging via MIS or OS. MATERIALS AND METHODS: A population-based retrospective cohort study was performed using claims data from the Korean National Health Insurance database from January 2012 to December 2016. All patients who underwent hysterectomy under diagnosis of endometrial cancer were identified. Patients were classified into RS, LS, and OS groups. Operative and oncologic outcomes were compared among the three groups after adjustments for age group, risk group (adjuvant therapy status), modified Charlson comorbidity index, income level, insurance type, and index year using propensity scores obtained via the inverse probability of treatment weighted method. RESULTS: After adjustment, 5,065 patients (RS, n=315; LS, n=3,248; OS, n=1,503) were analyzed. Patient demographics were comparable. Hospital stay, postoperative complications, and cost were more favorable in the RS and LS groups than in the OS group (all p < 0.001). Five-year overall survival was significantly longer in the RS and LS groups than in the OS group (94.8%, 91.9%, and 86.9%, respectively; p < 0.001). Moreover, the survival benefit of RS was shown in the subgroup analysis of low-risk endometrial cancer patients. CONCLUSION: Our study provides further evidence for the RS being a safe surgical alternative to the LS and OS, especially in low-risk endometrial cancer patients, offering surgical and oncologic outcomes equivalent to other surgical approaches.