Zebrafish is one of the most commonly used model organisms in biomedical, developmental and genetic research. The production of several thousands of transgenic lines is leading to difficulties in maintaining valuable genetic resources as cryopreservation protocols for eggs and embryos are not yet developed. In this study, we utilized testis cryopreservation (through both slow-rate freezing and vitrification) and spermatogonia transplantation as effective methods for long-term storage and line reconstitution in zebrafish. During freezing, utilization of 1.3 M of dimethyl sulfoxide (Me2SO) displayed the highest spermatogonia viability (~60%), while sugar and protein supplementation had no effects. Needle-immersed vitrification also yielded high spermatogonia viability rates (~50%). Both optimal slow-rate freezing and vitrification protocols proved to be reproducible in six tested zebrafish lines after displaying viability rates of >50% in all lines. Both fresh and cryopreserved spermatogonia retained their ability to colonize the recipient gonads after intraperitoneal transplantation of vasa::egfp and actb:yfp spermatogonia into wild-type AB recipient larvae. Colonization rate was significantly higher in dnd-morpholino sterilized recipients than in non-sterilized recipients. Lastly, wild-type recipients produced donor-derived sperm and donor-derived offspring through natural spawning. The method demonstrated in this study can be used for long-term storage of valuable zebrafish genetic resources and for reconstitution of whole zebrafish lines which will greatly improve the current preservation practices.

Many studies correlate changes in human gut microbiome with the onset of various diseases, mostly by 16S rRNA gene sequencing. Setting up the optimal sampling and DNA isolation procedures is crucial for robustness and reproducibility of the results. We performed a systematic comparison of several sampling and DNA isolation kits, quantified their effect on bacterial gDNA quality and the bacterial composition estimates at all taxonomic levels. Sixteen volunteers tested three sampling kits. All samples were consequently processed by two DNA isolation kits. We found that the choice of both stool sampling and DNA isolation kits have an effect on bacterial composition with respect to Gram-positivity, however the isolation kit had a stronger effect than the sampling kit. The proportion of bacteria affected by isolation and sampling kits was larger at higher taxa levels compared to lower taxa levels. The PowerLyzer PowerSoil DNA Isolation Kit outperformed the QIAamp DNA Stool Mini Kit mainly due to better lysis of Gram-positive bacteria while keeping the values of all the other assessed parameters within a reasonable range. The presented effects need to be taken into account when comparing results across multiple studies or computing ratios between Gram-positive and Gram-negative bacteria.

Osteocytes are terminally differentiated osteoblasts embedded in the bone matrix. Evidence indicates that cells in the mesenchymal lineage possess plasticity. However, whether or not osteocytes have the capacity to dedifferentiate back into osteoblasts is unclear. This study aimed to clarify the dedifferentiation potential of osteocytes. Mouse calvarial osteoblasts were isolated and maintained in normal two-dimensional (2D) or collagen gel three-dimensional (3D) cultures. In 2D cultures, osteoblasts exhibited a typical fibroblast-like shape with high Alpl and minimal Sost, Fgf23, and Dmp1 expression and osteoblasts formed mineralised nodules. When these osteoblasts were transferred into 3D cultures, they showed a stellate shape with diminished cytoplasm and numerous long processes and expression of Alpl decreased while Sost, Fgf23, and Dmp1 were significantly increased. These cells were in cell cycle arrest and showed suppressed mineralisation, indicating that they were osteocytes. When these osteocytes were recovered from 3D cultures and cultured two-dimensionally again, they regained adequate cytoplasm and lost the long processes, resulting in a fibroblast-like shape. These cells showed high Alpl and low Sost, Fgf23, and Dmp1 expression with a high mineralisation capability, indicating that they were osteoblasts. This report shows that osteocytes possess the capacity to dedifferentiate back into mature osteoblasts without gene manipulation.

Enzymatic gold nanoparticles (B-GNPs) have been synthesized using a natural anticancer agent bromelain (a cysteine protease) and these nanoparticles were used to bioconjugate Cisplatin (highly effective against osteosarcoma and lung cancer). Cisplatin bioconjugated bromelain encapsulated gold nanoparticles (B-C-GNPs) were found profoundly potent against same cancers at much lower concentration with minimum side effects due to the synergistic effect of bromelain. The B-C-GNPs have been observed to inhibit the proliferation of osteosarcoma cell lines Saos-2 and MG-63 with IC50 estimation of 4.51 µg/ml and 3.21 µg/ml, respectively, and against small lung cancer cell line A-549 with IC50 2.5 µg/ml which is lower than IC50 of cisplatin against same cell lines. The B-GNPs/B-C-GNPs were characterized by TEM, UV-Visible spectroscopy, Zeta potential and DLS to confirm the production, purity, crystalline nature, stability of nanoemulsion, size and shape distribution. The change in 2D and 3D conformation of bromelain after encapsulation was studied by Circular Dichroism and Fluorometry, respectively. It was found that after encapsulation, a 19.4% loss in secondary structure was observed, but tertiary structure was not altered significantly and this loss improved the anticancer activity. The confirmation of bioconjugation of cisplatin with B-GNPs was done by UV-Visible spectroscopy, TEM, FTIR, 2D 1H NMR DOSY and ICP-MS. Further, it was found that almost ~4 cisplatin molecules bound with each B-GNPs nanoparticle.

The mechanical response of brain tissue closely relates to cerebral blood flow and brain diseases. During intracerebral haemorrhage (ICH), a mass effect occurs during the initial bleeding and results in significant tissue deformation. However, fewer studies have focused on the brain damage mechanisms and treatment approaches associated with mass effects compared to the secondary brain injuries after ICH, which may be a result of the absence of acceptable animal models mimicking a mass effect. Thus, a thermo-sensitive poly (N-isopropylacrylamide) (PNIPAM) hydrogel was synthesized and injected into the rat brain to establish an ICH model for mass effect research. The PNIPAM hydrogel or autologous blood was injected to establish an ICH animal model, and the space-occupying volumes, brain tissue elasticity, brain oedema, neuronal cell death, iron deposition and behavioural recovery were evaluated. The lower critical solution temperature of PNIPAM hydrogel was 32 °C, and the PNIPAM hydrogel had a rough surface with similar topography and pore structure to a blood clot. Furthermore, the ICH model animals who received an injection of PNIPAM and blood produced similar lesion volumes, elasticity changes and mechanically activated ion channel piezo-2 upregulation in brain tissue. Meanwhile, slight iron deposition, neuronal cell death and brain oedema were observed in the PNIPAM hydrogel model compared to the blood model. In addition, the PNIPAM hydrogel showed good biocompatibility and stability in vivo via subcutaneous implantation. Our findings show that PNIPAM hydrogel cerebral infusion can form a mass effect similar to haematoma and minimize the interference of blood, and the establishment of a mass effect ICH model is beneficial for understanding the mechanism of primary brain injury and the role of mass effects in secondary brain damage after ICH.

The upregulation of ELTD1 ([epidermal growth factor (EGF), latrophilin and seven transmembrane domain-containing 1] on chromosome 1) in tumor cells has been reported in several types of cancer and correlates with poor cancer prognosis. However, the role of ELTD1 in glioma progression remains unknown. In this study, we examined ELTD1 expression levels in human glioma cell lines and in sixteen human gliomas of different grades. The molecular effects of ELTD1 in glioma cells were measured using quantitative polymerase chain reaction (qRT-PCR), Western blotting, Cell proliferation assays, Matrigel migration and invasion assays and brain orthotopic xenografts. We found that high expression levels of ELTD1 were positively associated with cancer progression and poor prognosis in human glioma. Mechanistically, ELTD1 activated the JAK/STAT3/HIF-1α signaling axis and p-STAT3 bound with HIF-1α. Taken together, our data provide a plausible mechanism for ELTD1-modulated glioma progression and suggest that ELTD1 may represent a potential therapeutic target in the prevention and therapy of glioma.

Accumulating evidence has confirmed that the expression of sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) is downregulated in heart failure and cardiac allograft rejection. Although many SERCA2a-related genes and proteins involved in the regulation of myocardial Ca2+ fluxes have been explored, its related metabolites remain poorly studied. Our main objective was to identify circulating SERCA2a-related metabolites altered in cardiac allograft rejection and to determine whether these could serve as non-invasive biomarkers. Sixty plasma samples from adult heart transplant were included in a metabolomic analysis. Sphingosine-1 phosphate (S1P), metabolite closely related with SERCA, were increased in patients with cardiac rejection (p < 0.0001). S1P discriminated between patients with and without rejection: normal grafts vs. all rejecting grafts (AUC = 0.911, p < 0.0001), normal grafts vs. Grade 1 R (AUC = 0.819, p < 0.01), Grade 2 R (AUC = 0.911, p < 0.0001), Grade 3 R (AUC = 0.996, p < 0.0001). In addition, we found changes in key enzymes and receptors of S1P pathway analysed on explanted hearts from heart failure patients. This preliminary study reveals that circulating S1P determination could be a novel approach to detect cardiac rejection, showing a robust capability for detection that improves gradually with the severity of rejection. These alterations could be relevant to better understand the involvement of calcium regulation on the pathophysiology of rejection.

We dissected the genetic basis of total spikelet number (TSN) along with other traits, viz. spike length (SL) and flowering time (FT) in a panel of 518 elite European winter wheat varieties. Genome-wide association studies (GWAS) based on 39,908 SNP markers revealed highly significant quantitative trait loci (QTL) for TSN on chromosomes 2D, 7A, and 7B, for SL on 5A, and FT on 2D, with 2D-QTL being the functional marker for the gene Ppd-D1. The physical region of the 7A-QTL for TSN revealed the presence of a wheat ortholog (TaAPO-A1) to APO1-a rice gene that positively controls the spikelet number on the panicles. Interspecific analyses of the TaAPO-A1 orthologs showed that it is a highly conserved gene important for floral development and present in a wide range of terrestrial plants. Intraspecific studies of the TaAPO-A1 across wheat genotypes revealed a polymorphism in the conserved F-box domain, defining two haplotypes. A KASP marker developed on the polymorphic site showed a highly significant association of TaAPO-A1 with TSN, explaining 23.2% of the total genotypic variance. Also, the TaAPO-A1 alleles showed weak but significant differences for SL and grain yield. Our results demonstrate the importance of wheat sequence resources to identify candidate genes for important traits based on genetic analyses.

We performed a pilot randomised study to assess the feasibility and radiation exposure of a new computed tomography (CT) protocol that allows screening of both coronary artery disease (CAD) and lung cancer. Current or former heavy smokers at high lung cancer risk with indication to cardiac CT for suspected or known CAD were randomised to undergo concomitant CT evaluation of either cardiac or thoracic area or cardiac CT only. Out of 129 subjects deemed eligible for the study, 110 agreed to participate and were randomised to simultaneous cardiac and lung CT (Gr.A; n = 55) or cardiac CT only (Gr.B; n = 55). The feasibility (i.e. adequate visualization of coronary artery segments) was noninferior with simultaneous cardiac and lung CT compared with the standard cardiac CT (870 of 889 segments [97%] in Gr.A vs 878/890 segments [99%] in Gr.B; mean difference 2.0% [90% confidence interval: -0.3% to 4.1%]). The safety (i.e. effective radiation dose) of the concomitant cardiac and lung CT protocol was noninferior to the standard cardiac CT (1.5 [95% confidence intervals: 1.2-1.7] vs. 1.4 [95% confidence intervals: 1.1-1.6] mSv; mean difference 0.1 mSv [90% confidence interval: -0.2 to 0.3 mSv]). In the two groups, a total of 25 significant (>70%) coronary stenoses were found at cardiac CT (9/55 cases of Gr.A vs 11/55 cases of Gr.B). Pulmonary nodules >2 mm were detected in 7 of the 55 Gr.A subjects. This pilot randomised study shows that concomitant CAD and lung cancer screening by means of a new CT protocol is both feasible and safe, thus allowing a comprehensive evaluation of both cardiac and thoracic regions during one CT scanning only. (ClinicalTrials.gov Identifier: NCT03727958).

3D bioprinting has been evolving as an important strategy for the fabrication of engineered tissues for clinical, diagnostic, and research applications. A major advantage of bioprinting is the ability to recapitulate the patient-specific tissue macro-architecture using cellular bioinks. The effectiveness of bioprinting can be significantly enhanced by incorporating the ability to preferentially organize cellular constituents within 3D constructs to mimic the intrinsic micro-architectural characteristics of native tissues. Accordingly, this work focuses on a new non-contact and label-free approach called ultrasound-assisted bioprinting (UAB) that utilizes acoustophoresis principle to align cells within bioprinted constructs. We describe the underlying process physics and develop and validate computational models to determine the effects of ultrasound process parameters (excitation mode, excitation time, frequency, voltage amplitude) on the relevant temperature, pressure distribution, and alignment time characteristics. Using knowledge from the computational models, we experimentally investigate the effect of selected process parameters (frequency, voltage amplitude) on the critical quality attributes (cellular strand width, inter-strand spacing, and viability) of MG63 cells in alginate as a model bioink system. Finally, we demonstrate the UAB of bilayered constructs with parallel (0°-0°) and orthogonal (0°-90°) cellular alignment across layers. Results of this work highlight the key interplay between the UAB process design and characteristics of aligned cellular constructs, and represent an important next step in our ability to create biomimetic engineered tissues.

MicroRNAs (miRNA) modulate gene expression through feed-back and forward loops. Previous studies identified miRNAs that regulate transcription factors, including Peroxisome Proliferator Activated Receptor Gamma (PPARG), in adipocytes, but whether they influence adipogenesis via such regulatory loops remain elusive. Here we predicted and validated a novel feed-forward loop regulating adipogenesis and involved miR-27a/b-3p, PPARG and Secretory Carrier Membrane Protein 3 (SCAMP3). In this loop, expression of both PPARG and SCAMP3 was independently suppressed by miR-27a/b-3p overexpression. Knockdown of PPARG downregulated SCAMP3 expression at the late phase of adipogenesis, whereas reduction of SCAMP3 mRNA levels increased PPARG expression at early phase in differentiation. The latter was accompanied with upregulation of adipocyte-enriched genes, including ADIPOQ and FABP4, suggesting an anti-adipogenic role for SCAMP3. PPARG and SCAMP3 exhibited opposite behaviors regarding correlations with clinical phenotypes, including body mass index, body fat mass, adipocyte size, lipolytic and lipogenic capacity, and secretion of pro-inflammatory cytokines. While adipose PPARG expression was associated with more favorable metabolic phenotypes, SCAMP3 expression was linked to increased fat mass and insulin resistance. Together, we identified a feed-forward loop through which miR-27a/b-3p, PPARG and SCAMP3 cooperatively fine tune the regulation of adipogenesis, which potentially may impact whole body metabolism.

Reactive microgliosis is an important pathological component of neuroinflammation and has been implicated in a wide range of brain diseases including brain tumors, multiple sclerosis, Parkinson's disease, Alzheimer's disease, and schizophrenia. Mapping reactive microglia in-vivo is often performed with PET scanning whose resolution, cost, and availability prevent its widespread use. The advent of diffusion compartment imaging (DCI) to probe tissue microstructure in vivo holds promise to map reactive microglia using MRI scanners. But this potential has never been demonstrated. In this paper, we performed longitudinal DCI in rats that underwent dorsal root axotomy triggering Wallerian degeneration of axons-a pathological process which reliably activates microglia. After the last DCI at 51 days, rats were sacrificed and histology with Iba-1 immunostaining for microglia was performed. The fraction of extra-axonal restricted diffusion from DCI was found to follow the expected temporal dynamics of reactive microgliosis. Furthermore, a strong and significant correlation between this parameter and histological measurement of microglial density was observed. These findings strongly suggest that extra-axonal restricted diffusion is an in-vivo marker of reactive microglia. They pave the way for MRI-based microglial mapping which may be important to characterize the pathogenesis of neurological and psychiatric diseases.

Coherent Raman microscopy has become a powerful tool in label-free, non-destructive and fast cell imaging. Here we apply high spectral resolution multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy in the high wavenumber region to the study of the cell cycle. We show that heterochromatin - the condensed state of chromatin - can be visualised by means of the vibrational signature of proteins taking part in its condensation. Thus, we are able to identify chromosomes and their movement during mitosis, as well as structures like nucleoli and nuclear border in interphase. Furthermore, the specific organization of the endoplasmic reticulum during mitosis is highlighted. Finally, we stress that MCARS can reveal the biochemical impact of the fixative method at the cellular level. Beyond the study of the cell cycle, this work introduces a label-free imaging approach that enables the visualization of cellular processes where chromatin undergoes rearrangements.

Mitochondria provide energy for cellular function. We examine daily changing patterns of mitochondrial function and metabolism in Drosophila in vivo in terms of their complex (I-IV) activity, ATP production, glycolysis, and whole fly respiration in the morning, afternoon and night. Complex activity and respiration showed significant and unexpected variation, peaking in the afternoon. However, ATP levels by contrast are >40% greater in the morning and lowest at night when glycolysis peaks. Complex activity modulation was at the protein level with no evidence for differential transcription over the day. Timing differences between increased ATP production and peaks of complex activity may result from more efficient ATP production early in the day leaving complex activity with spare capacity. Optical stimulation of mitochondria is only possible in the mornings when there is such spare capacity. These results provide first evidence of shifts in cellular energy capacity at the organism level. Understanding their translation may be significant to the chosen timing of energy demanding interventions to improve function and health.

Extracorporeal life support (ECLS) is a world-famous life-saving method. Until now, changes in arterial wave properties due to ECLS have remained unexamined. In this study, we determined the effects of ECLS on arterial wave properties and ventricular/arterial coupling in male Wistar rats with the measured aortic pressure alone. Ascending aortic pressure signals were measured before ECLS and at 30, 60, and 90 min after weaned off. The aortic pressure signal then calculated by fourth-order derivative to obtain an assumed triangular flow wave. The ratio of mean systolic pressure to mean diastolic pressure (Pms/Pmd), a parameter for evaluating the matching condition between myocardial oxygen demand and supply, was significantly higher after ECLS. The magnitude of forward pressure (|Pf|) augmented by ECLS prevailed over the backward pressure (|Pb|), leading to a decline in wave reflection factor. Pms/Pmd was positively linearly correlated with |Pf| (Pms/Pmd = 0.9177 + 0.0078 × |Pf|, r = 0.8677; P < 0.0001). These findings suggest that |Pf| was a predominant factor responsible for the mismatch between the myocardial oxygen demand and supply in rats after ECLS phase of experiment.

The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.

B cells are important in the pathogenesis of many, and perhaps all, immune-mediated diseases. Each B cell expresses a single B cell receptor (BCR)1, and the diverse range of BCRs expressed by the total B cell population of an individual is termed the 'BCR repertoire'. Our understanding of the BCR repertoire in the context of immune-mediated diseases is incomplete, and defining this could provide new insights into pathogenesis and therapy. Here, we compared the BCR repertoire in systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Crohn's disease, Behçet's disease, eosinophilic granulomatosis with polyangiitis, and immunoglobulin A (IgA) vasculitis by analysing BCR clonality, use of immunoglobulin heavy-chain variable region (IGHV) genes and-in particular-isotype use. An increase in clonality in systemic lupus erythematosus and Crohn's disease that was dominated by the IgA isotype, together with skewed use of the IGHV genes in these and other diseases, suggested a microbial contribution to pathogenesis. Different immunosuppressive treatments had specific and distinct effects on the repertoire; B cells that persisted after treatment with rituximab were predominately isotype-switched and clonally expanded, whereas the inverse was true for B cells that persisted after treatment with mycophenolate mofetil. Our comparative analysis of the BCR repertoire in immune-mediated disease reveals a complex B cell architecture, providing a platform for understanding pathological mechanisms and designing treatment strategies.

Juglone with naphthoquinone structure has medicinal properties and its anticarcinogenic and antioxidant effects have been proven. In this research, the cytotoxic and apoptosis effects of juglone and Pterocarya fraxinifolia (PF) methanolic extract on human prostate cancer cells were studied.

Alcohol consumption is capable of producing free radicals and inducing disturbance in body antioxidant. Falcaria vulgaris (F. vulgaris) is a vegetable and it has beneficial antioxidant effects.

Influenza A has caused several deadly pandemics throughout human history. The virus is often resistant to developed treatments because of its genetic drift or shift property. Broad-spectrum antibodies show a promising potential to overcome the resistance of influenza viruses. In silico studies on broad-reactive antibodies and their interactions with hemagglutinins might shed light on the rational design of a universal vaccine. In this study, 11 broad-spectrum antibodies (or antigen-binding fragments) and 14 hemagglutinins of H3N2 and H5N1 strains were docked and analyzed to provide information about the construction of the scaffold for using universal antibodies against the influenza A virus. Antigen-binding fragments that have high number of appearances in the top 3 within each H3 and H5 subtypes were chosen for protein-protein interaction analysis. The results show that while the hydrogen bond is important for Ab/Fab binding to H3, the H5-Ab/Fab system may need cation-pi interaction for a strong interaction.