Live Cell Protein Imaging of Tandem Complemented-GFP11-Tagged Coiled-Coil Domain-Containing Protein-124 Identifies this Factor in G3BP1-Induced Stress-Granules.

Coiled-coil domain-containing 124 protein is a multifunctional RNA-binding factor, and it was previously reported to interact with various biomolecular complexes localized at diverse subcellular locations, such as the ribosome, centrosome, midbody, and nucleoli. We aimed to better characterize the subcellular CCDC124 translocation by labelling this protein with a fluorescent tag, followed by laser scanning confocal microscopy methods. As traditional GFP-tagging of small proteins such as CCDC124 often faces limitations like potential structural perturbations of labeled proteins, and interference of the fluorescent-tag with their endogenous cellular functions, we aimed to label CCDC124 with the smallest possible split-GFP associated protein-tagging system (GFP11/GFP1-10) for better characterization of its subcellular localizations and its translocation dynamics. By recombinant DNA techniques we generated CCDC124-constructs labelled with either single of four tandem copies of GFP11 (GFP11 × 1::CCDC124, GFP11 × 4::CCDC124, or CCDC124::GFP11 × 4). We then cotransfected U2OS cells with these split-GFP constructs (GFP11 × 1(or X4)::CCDC124/GFP1-10) and analyzed subcellular localization of CCDC124 protein by laser scanning confocal microscopy. Tagging CCDC124 with four tandem copies of a 16-amino acid short GFP-derived peptide-tag (GFP11 × 4::CCDC124) allowed better characterization of the subcellular localization of CCDC124 protein in our model human bone osteosarcoma (U2OS) cells. Thus, by this novel methodology we successfully identified GFP11 × 4::CCDC124 molecules in G3BP1-overexpression induced stress-granules by live cell protein imaging for the first time. Our findings propose CCDC124 as a novel component of the stress granule which is a membraneless organelle involved in translational shut-down in response to cellular stress.

Development and functionalization of electrospun fiber coated thin film microextraction devices for rapid mass spectrometric determination of biologically important polar molecules.

Rapid diagnosis of diseases is one of the challenging areas in clinical research. From the analytical chemist's perspective, the main challenges are isolating the compounds from the bio-specimen and lengthy analysis times. In this regard, solid phase microextraction offers a platform to address the abovementioned challenges. Moreover, its sharp tip-thin film geometry, known as coated blade spray (CBS), can enhance the extraction and act as an ionization source in direct mass spectrometric analysis. In this study, a new CBS device specifically designed for polar analytes was prepared and optimized to determine urinary metabolites. For this purpose, polyacrylonitrile (PAN) was selected as a base polymer as it can be electrospun to form a nanofibrous structure, and it can be modified with weak ion exchange moieties to interact with polar analytes. Following the electrospinning of PAN, hydrolysis was optimized, and conditions leading to sufficient extraction enhancement without dissolving the polymer were obtained when probes were treated with 5.0 M of NaOH for 2.5 h. Using the coated blades prepared as explained, the evaluation of various extraction conditions showed that 5 min is sufficient for equilibrium extraction. In addition, the solution's ionic strength and pH significantly affect the extraction. Optimum sorption was obtained at no salt added and pH 7.0 conditions. The CBS-MS optimization showed that 10.0 µL of ACN/MeOH/H2O (40:40:20, v/v/v) with formic acid kept for 15 seconds on the blade before voltage application leads to the highest signal. The limits of quantification of the analytes are between 50 and 100 ng/mL.

A multi-faceted approach to unravel coding and non-coding gene fusions and target chimeric proteins in ataxia.

Ataxia represents a heterogeneous group of neurodegenerative disorders characterized by a loss of balance and coordination, often resulting from mutations in genes vital for cerebellar function and maintenance. Recent advances in genomics have identified gene fusion events as critical contributors to various cancers and neurodegenerative diseases. However, their role in ataxia pathogenesis remains largely unexplored. Our study Hdelved into this possibility by analyzing RNA sequencing data from 1443 diverse samples, including cell and mouse models, patient samples, and healthy controls. We identified 7067 novel gene fusions, potentially pivotal in disease onset. These fusions, notably in-frame, could produce chimeric proteins, disrupt gene regulation, or introduce new functions. We observed conservation of specific amino acids at fusion breakpoints and identified potential aggregate formations in fusion proteins, known to contribute to ataxia. Through AI-based protein structure prediction, we identified topological changes in three high-confidence fusion proteins-TEN1-ACOX1, PEX14-NMNAT1, and ITPR1-GRID2-which could potentially alter their functions. Subsequent virtual drug screening identified several molecules and peptides with high-affinity binding to fusion sites. Molecular dynamics simulations confirmed the stability of these protein-ligand complexes at fusion breakpoints. Additionally, we explored the role of non-coding RNA fusions as miRNA sponges. One such fusion, RP11-547P4-FLJ33910, showed strong interaction with hsa-miR-504-5p, potentially acting as its sponge. This interaction correlated with the upregulation of hsa-miR-504-5p target genes, some previously linked to ataxia. In conclusion, our study unveils new aspects of gene fusions in ataxia, suggesting their significant role in pathogenesis and opening avenues for targeted therapeutic interventions.Communicated by Ramaswamy H. Sarma.

Proteomics analysis identifies the ribosome associated coiled-coil domain-containing protein-124 as a novel interaction partner of nucleophosmin-1.

BACKGROUND INFORMATION: Coiled-coil domain-containing protein-124 (Ccdc124) is a conserved eukaryotic ribosome-associated RNA-binding protein which is involved in resuming ribosome activity after stress-related translational shutdown. Ccdc124 protein is also detected at cellular localizations devoid of ribosomes, such as the centrosome, or the cytokinetic midbody, but its translation-independent cellular function is currently unknown. RESULTS: By using an unbiased LC-MS/MS-based proteomics approach in human embryonic kidney (HEK293) cells, we identified novel Ccdc124 partners and mapped the cellular organization of interacting proteins, a subset of which are known to be involved in nucleoli biogenesis and function. We then identified a novel interaction between the cancer-associated multifunctional nucleolar marker nucleophosmin (Npm1) and Ccdc124, and we characterized this interaction both in HEK293 (human embryonic kidney) and U2OS (osteosarcoma) cells. As expected, in both types of cells, Npm1 and Ccdc124 proteins colocalized within the nucleolus when assayed by immunocytochemical methods, or by monitoring the localization of green fluorescent protein-tagged Ccdc124. CONCLUSIONS: The nucleolar localization of Ccdc124 was impaired when Npm1 translocates from the nucleolus to the nucleoplasm in response to treatment with the DNA-intercalator and Topo2 inhibitor chemotherapeutic drug doxorubicin. Npm1 is critically involved in maintaining genomic stability by mediating various DNA-repair pathways, and over-expression of Npm1 or specific NPM1 mutations have been previously associated with proliferative diseases, such as acute myelogenous leukemia, anaplastic large-cell lymphoma, and solid cancers originating from different tissues. SIGNIFICANCE: Identification of Ccdc124 as a novel interaction partner of Nmp1 within the frame of molecular mechanisms involving nucleolar stress-sensing and DNA-damage response is expected to provide novel insights into the biology of cancers associated with aberrations in NPM1.

Evaluation of fetal myocardial performance index in gestational diabetes mellitus.

This study aimed to compare fetal myocardial performance index (MPI) between fetuses of pregnant women with gestational diabetes mellitus (GDM) and healthy controls and to evaluate the relationship between MPI and maternal glucose levels. This was a prospective study of 90 pregnant women, including 50 pregnancies with GDM (27 pregnancies with insulin-regulated GDM and 23 pregnancies with diet-regulated GDM) and 40 healthy controls. Isovolumetric contraction time (ICT) + isovolumetric relaxation time (IRT)/ejection time (ET) were used to calculate the MPI (MPI = [ICT + IRT]/ET). Fetal MPI, PR interval, E/A ratio, maternal plasma glucose levels on the day of MPI measurement, and neonatal outcomes were compared. The fetal left-MPI was significantly higher in the GDM group than healthy controls (0.43 ± 0.04 vs. 0.40 ± 0.06, p = 0.007). The best cut-off level for MPI was >0.41 to predict adverse perinatal outcomes (sensitivity: 70%, specificity: 68%, area under the curve: 0.715, 95% confidence interval: 0.5143-0.8205, p < 0.001). The fetal MPI values showed no correlation with maternal plasma fasting, postprandial glucose, and hemoglobin A1c (HbA1c) levels. Reduced E/A ratio, higher neonatal intensive care unit admissions, and the need for cesarean delivery were detected in the GDM group. Fetal MPI is impaired in women with GDM, and the need for insulin therapy is associated with higher MPI values and adverse neonatal outcomes. Fetal MPI can help detect fetuses with potential adverse outcome risks, owing to impaired fetal cardiac function.

Role of serostatus in pediatric neuromyelitis optica spectrum disorders: A nationwide multicentric study.

BACKGROUND: Neuromyelitis optica spectrum disorders (NMOSD) are immune-mediated inflammatory disorders of the central nervous system (CNS) mostly presenting as optic neuritis and acute myelitis. NMOSD can be associated with seropositivity for aquaporin 4 antibody (AQP4 IgG), myelin oligodendrocyte glycoprotein antibody (MOG IgG), or can be seronegative for both. In this study, we retrospectively examined our seropositive and seronegative pediatric NMOSD patients. METHOD: Data were collected from all participating centres nationwide. Patients diagnosed with NMOSD were divided into three subgroups according to serology: AQP4 IgG NMOSD, MOG IgG NMOSD, and double seronegative (DN) NMOSD. Patients with at least six months of follow-up were compared statistically. RESULTS: The study included 45 patients, 29 female and 16 male (ratio:1.8), mean age 15.16 ± 4.93 (range 5.5-27) years. Age at onset, clinical manifestations, and cerebrospinal fluid findings were similar between AQP4 IgG NMOSD (n = 17), MOG IgG NMOSD (n = 10), and DN NMOSD (n = 18) groups. A polyphasic course was more frequent in the AQP4 IgG and MOG IgG NMOSD groups than DN NMOSD (p = 0.007). The annualized relapse rate and rate of disability were similar between groups. Most common types of disability were related to optic pathway and spinal cord involvement. Rituximab in AQP4 IgG NMOSD, intravenous immunoglobulin in MOG IgG NMOSD, and azathioprine in DN NMOSD were usually preferred for maintenance treatment. CONCLUSION: In our series with a considerable number of double seronegatives, the three major serological groups of NMOSD were indistinguishable based on clinical and laboratory findings at initial presentation. Their outcome is similar in terms of disability, but seropositive patients should be more closely followed-up for relapses.

An experimental study on the environmental impact of hydrogen and natural gas blend burning.

In this study, an experimental investigation is conducted to analyze the effect of adding hydrogen into natural gas on emissions and the burning performance of the obtained blends. Natural gas alone and natural gas-hydrogen blends are burned in identical gas stoves, and the emitted CO, CO2, and NOx are measured. The base case with natural gas only is compared with the natural gas and hydrogen blends (including hydrogen additions of 10%, 20% and 30% volumetrically). The experimental results show that the combustion efficiency increases from 39.32% to 44.4% by enhancing the hydrogen blending ratio from 0 to 0.3. While CO2 and CO emissions are reduced with rising the hydrogen ratio in the blend, NOx emissions have a fluctuating trend. Moreover, a life cycle analysis is performed to determine the environmental impact of the considered blending scenarios. With the blending ratio of 0.3 hydrogen by volume, global warming potential decreases from 6.233 to 6.123 kg CO2 equivalents per kg blend, and acidification potential reduces from 0.0507 to 0.04928 kg SO2 equivalents per kg blend in comparison with natural gas. On the other hand, human toxicity, abiotic depletion, and ozone depletion potentials per kg blend show slight augmentation from 5.30 to 5.52 kg 1,4-dichlorobenzene (DCB) eq., 0.0000107 to 0.00005921 kg SB eq., and 3.17 × 10-8 to 5.38 × 10-8 kg CFC-11 eq., respectively.

Probable association between mRNA COVID-19 vaccine and opsoclonus-myoclonus-ataxia syndrome.

Opsoclonus-myoclonus-ataxia syndrome (OMAS) in children is most often of paraneoplastic origin, but it can also result from infectious processes, toxic and metabolic disorders, and organic events that cause damage to the brainstem or cerebellum. Post-vaccination OMAS has also been reported. We report the case of a 15-year-old girl who developed OMAS 24 hours after her first dose of mRNA COVID-19 (BioNTech) vaccine.

A Critical Study on the Potential of Solid-Phase Fluorescence Spectroscopy (SPFS) in Quantitative Analysis: Effect of Solid Diluent and Sampling Method on Signal Quality.

BACKGROUND: Solid-phase fluorescence spectroscopy (SPFS) is a very useful non-destructive technique for directly analyzing samples in solid form without the use of solvents. However, due to the so-called inner-filter effect, it is sometimes necessary to dilute solid samples using non-fluorescent solids as diluents. OBJECTIVE: This study aimed to explore the potential of SPFS in the quantitative analysis of fluorescent species based on: (1) the type of solid diluent; and (2) the sampling method used in the SPFS analysis. METHODS: Four different solids were used as solid diluents in the preparation of standard mixtures having different concentrations of rhodamine b and fluorescein as model compounds. Standard mixtures of model compounds were sampled by two different methods called: (1) the powder-cell method; and (2) the adhesive tape method. LOQ and calibration sensitivity calculated from the calibration graphs were used to assess the measurement performance. The usability of SPFS in real-sample analyses was also evaluated in detail. RESULTS: Among the solid diluents studied, the best results were obtained with sodium carbonate. The powder-cell method yielded a significant advantage over the adhesive tape method. The lowest LOQs for rhodamine b and fluorescein were obtained by sodium carbonate and the powder-cell method as 0.06 and 0.11 mg/kg, respectively. The results of real-sample analyses were verified using conventional liquid-phase fluorescence spectroscopy (LPFS). CONCLUSION: Solid-diluent type and sampling method were found to affect the performance of the SPFS technique. A combination of sodium carbonate and the powder-cell method gave the best results. According to the t-test, no difference was observed between the means obtained by SPFS and LPFS techniques in real-sample analyses. HIGHLIGHTS: In SPFS, toxic organic solvents and difficult sample preparation steps are not required. This makes the method advantageous over conventional fluorescence analyses performed in the liquid phase.

Warmth and competence perceptions of key protagonists are associated with containment measures during the COVID-19 pandemic: Evidence from 35 countries.

It is crucial to understand why people comply with measures to contain viruses and their effects during pandemics. We provide evidence from 35 countries (Ntotal = 12,553) from 6 continents during the COVID-19 pandemic (between 2021 and 2022) obtained via cross-sectional surveys that the social perception of key protagonists on two basic dimensions-warmth and competence-plays a crucial role in shaping pandemic-related behaviors. Firstly, when asked in an open question format, heads of state, physicians, and protest movements were universally identified as key protagonists across countries. Secondly, multiple-group confirmatory factor analyses revealed that warmth and competence perceptions of these and other protagonists differed significantly within and between countries. Thirdly, internal meta-analyses showed that warmth and competence perceptions of heads of state, physicians, and protest movements were associated with support and opposition intentions, containment and prevention behaviors, as well as vaccination uptake. Our results have important implications for designing effective interventions to motivate desirable health outcomes and coping with future health crises and other global challenges.

Proteome effects of genome-wide single gene perturbations.

Protein abundance is controlled at the transcriptional, translational and post-translational levels, and its regulatory principles are starting to emerge. Investigating these principles requires large-scale proteomics data and cannot just be done with transcriptional outcomes that are commonly used as a proxy for protein abundance. Here, we determine proteome changes resulting from the individual knockout of 3308 nonessential genes in the yeast Schizosaccharomyces pombe. We use similarity clustering of global proteome changes to infer gene functionality that can be extended to other species, such as humans or baker's yeast. Furthermore, we analyze a selected set of deletion mutants by paired transcriptome and proteome measurements and show that upregulation of proteins under stable transcript expression utilizes optimal codons.

Doxorubicin induces prolonged DNA damage signal in cells overexpressing DEK isoform-2.

DEK has a short isoform (DEK isoform-2; DEK2) that lacks amino acid residues between 49-82. The full-length DEK (DEK isoform-1; DEK1) is ubiquitously expressed and plays a role in different cellular processes but whether DEK2 is involved in these processes remains elusive. We stably overexpressed DEK2 in human bone marrow stromal cell line HS-27A, in which endogenous DEKs were intact or suppressed via short hairpin RNA (sh-RNA). We have found that contrary to ectopic DEK1, DEK2 locates in the nucleus and nucleolus, causes persistent γH2AX signal upon doxorubicin treatment, and couldn't functionally compensate for the loss of DEK1. In addition, DEK2 overexpressing cells were more sensitive to doxorubicin than DEK1-cells. Expressions of DEK1 and DEK2 in cell lines and primary tumors exhibit tissue specificity. DEK1 is upregulated in cancers of the colon, liver, and lung compared to normal tissues while both DEK1 and DEK2 are downregulated in subsets of kidney, prostate, and thyroid carcinomas. Interestingly, only DEK2 was downregulated in a subset of breast tumors suggesting that DEK2 can be modulated differently than DEK1 in specific cancers. In summary, our findings show distinct expression patterns and subcellular location and suggest non-overlapping functions between the two DEK isoforms.

Effect of placenta previa on umbilical cord hematocrit value.

OBJECTIVE: Placenta previa is one of the causes of neonatal anemia. This condition is mainly explained by antenatal hemorrhage and incision of the anteriorly located placenta during cesarean section. However, the mechanism of neonatal anemia in placenta previa has not been extensively studied or well elucidated. This study investigates whether placenta previa is associated with lower hematocrit levels in newborns with no antenatal hemorrhage and placental incision. KEY FINDINGS: This prospective study investigated 47 patients with previa and 43 control patients who gave birth with a cesarean section at 34-38 weeks of gestation. Blood samples were obtained from the fetal end of the umbilical vein. The mean umbilical cord hematocrit value was 49.3% in the control patients and 46.7% in the patients with previa, and the difference was statistically significant (p = .029). No significant association was observed between hematocrit value and birth weight, gestational age, newborn gender, placenta position, or preoperative maternal hemoglobin level. CONCLUSION: The study findings reveal that even if not complicated by antepartum or intrapartum hemorrhage, placenta previa may be associated with lower hematocrit values in newborns. Although in none of the cases, the umbilical cord hematocrit value was not as low as to be defined as anemia, this effect of previa on newborns should be considered because of the importance of iron status.

Dimerization underlies the aggregation propensity of intrinsically disordered coiled-coil domain-containing 124.

Coiled-coil domain-containing 124 (CCDC124) is a recently discovered ribosome-binding protein conserved in eukaryotes. CCDC124 has regulatory functions on the mediation of reversible ribosomal hibernation and translational recovery by direct attachment to large subunit ribosomal protein uL5, 25S rRNA backbone, and tRNA-binding P/A-site major groove. Moreover, it independently mediates cell division and cellular stress response by facilitating cytokinetic abscission and disulfide stress-dependent transcriptional regulation, respectively. However, the structural characterization and intracellular physiological status of CCDC124 remain unknown. In this study, we employed advanced in silico protein modeling and characterization tools to generate a native-like tertiary structure of CCDC124 and examine the disorder, low sequence complexity, and aggregation propensities, as well as high-order dimeric/oligomeric states. Subsequently, dimerization of CCDC124 was investigated with co-immunoprecipitation (CO-IP) analysis, immunostaining, and a recent live-cell protein-protein interaction method, bimolecular fluorescence complementation (BiFC). Results revealed CCDC124 as a highly disordered protein consisting of low complexity regions at the N-terminus and an aggregation sequence (151-IAVLSV-156) located in the middle region. Molecular docking and post-docking binding free energy analyses highlighted a potential involvement of V153 residue on the generation of high-order dimeric/oligomeric structures. Co-IP, immunostaining, and BiFC analyses were used to further confirm the dimeric state of CCDC124 predominantly localized at the cytoplasm. In conclusion, our findings revealed in silico structural characterization and in vivo subcellular physiological state of CCDC124, suggesting low-complexity regions located at the N-terminus of disordered CCDC124 may regulate the formation of aggregates or high-order dimeric/oligomeric states.

Facile modification of polycaprolactone nanofibers with egg white protein.

Synthetic polymers remain to be a major choice for scaffold fabrication due to their structural stability and mechanical strength. However, the lack of functional moieties limits their application for cell-based therapies which necessitate modification and functionalization. Blending synthetic polymers with natural components is a simple and effective way to achieve the desired biological properties for a scaffold. Herein, nanofibrous mats made of polycaprolactone (PCL) and egg white protein (EWP) blend were developed and further evaluated for use as a scaffold for tissue engineering applications. Homogeneous distribution of EWP was achieved throughout the nanofibrous mats, as shown by immunohistochemistry. ATR-FTIR analysis and contact angle measurements have further confirmed the presence of EWP on the surface of the samples. The swelling test showed that PCL/EWP nanofibers have higher water uptake than PCL nanofibrous mats. Also, EWP addition on the nanofibrous mats resulted in an increase in the tensile strength and Young's modulus of the mats, indicating that the presence of protein can greatly enhance the mechanical properties of the mats. A significantly higher, more uniform, and dispersed cell spreading was observed on days 7 and 14 than that on neat PCL mats, demonstrating the importance of providing the required cues for cell homing by the availability of EWP. Hence, EWP is shown to be a simple and low-cost source for the functionalization of PCL nanofibrous mats. EWP is, therefore, a facile candidate to enhance cellular interactions of synthetic polymers for a wide range of tissue engineering applications.

Is GDF-15 level associated with gestational diabetes mellitus and adverse perinatal outcomes?

OBJECTIVE: Growth differentiation factor-15 (GDF-15), the new member of transforming growth factor (TGF)-beta family, is released as a response of oxidative stress, inflammation and tissue injury. We aimed to determine GDF-15 levels in patients with Gestational Diabetes Mellitus (GDM) and the relation between GDF-15 and adverse perinatal outcomes. MATERIALS AND METHODS: Forty pregnant women with GDM (receiving diet and insulin therapy) and forty healthy pregnant women as control group participated in this current study. GDF- 15 levels were analyzed by enzyme-linked immunosorbent assess kit. RESULTS: The median serum GDF-15 level was measured higher in patients with GDM, and it was statistically meaningful (p: 0.000). Logistic regression analysis indicated that with the increase of GDF-15 level, the risk of GDM diseases increases as well. (P: 0.001, OR = 1.009; 95% CI = 1.003-1.014). There were no differences between GDF-15 levels and perinatal outcomes. CONCLUSION: We concluded that higher GDF-15 levels are related to GDM in the third trimester. The optimal GDF-15 cut-off value was measured as 326 pg/ml for the diagnosis of GDM with 70% sensitivity and 60% specificity in our study. Further studies are needed to show the significance of GDF-15 as a biomarker for the disease.

Possums-based parental education for infant sleep: cued care resulting in sustained breastfeeding.

For infants and their families, sleep consolidation is important in maturing neural and circadian rhythms, and in family dynamics. The Possums Infant Sleep Program is a cued care approach to infant sleep, responding to infant cues in a flexible manner, dialing down the infant's sympathetic nervous system. The current study evaluated the effect of the Possums program on infant sleep and breastfeeding in infants (6-12 months) from a well-child outpatient clinic in Turkey, with the program intervention group (n = 91) compared with usual care (n = 92). In total, 157 mother-infant dyads completed the study. Infant sleep and breastfeeding rates were assessed at baseline and after 3 months. Nocturnal wakefulness, daytime sleep duration, naps, and night wakening decreased in both groups. Nocturnal sleep duration and the longest stretch of time the child was asleep during the night increased significantly in both groups without any change in total sleep duration. Night wakening was significantly lower and nocturnal sleep duration was significantly higher in the intervention group. However, mixed effects model analyses indicated no significant differences between the groups on any of the sleep outcomes after adjusting for confounders. Despite this, breastfeeding rates were significantly higher in the intervention group compared with those in the usual care group at follow-up.Conclusion: The Possum infant sleep program provided equivalent positive results on sleep parameters compared to usual care while advocating a more cued response. The critical difference was evident in sustained breastfeeding. What is Known: • Responsive sleep programs produce sleep consolidation, by responding to the infant's cues without ignoring, and then gradually reducing parental interaction. • Breastfeeding to sleep may be considered an undesirable sleep association in some infant sleep interventions. What is New: • The Possums Infant Sleep Program provided equivalent positive results to usual care while advocating a more cued response. • The critical difference was in sustaining breastfeeding, and the program was associated with better breastfeeding rates.