Predicting whaler shark presence and interactions with humans in southern Queensland, Australia.

The Queensland Shark Control Program (QSCP) started in 1962 to reduce the number of shark-human incidents by deploying nets and drumlines across the most popular beaches. The program targets large shark species (white, tiger and bull sharks) that are potentially hazardous to bathers. However, this strategy is lethal for other sharks and marine wildlife, including threatened and endangered species. Thus, finding non-lethal strategies is a priority. To better manage shark-human interactions, establishing a better understanding of the factors that drive shark movement is key. Here we used sea surface temperature (SST), rainfall and distance to rivers as environmental variables to predict the presence of whaler sharks in southern Queensland based on 26 years of catch data from the QSCP. We found that SST is positively corelated to sharks caught by drumlines, while rainfall was associated with the number of sharks captured in shark nets. In addition, more sharks were captured by nets and drumlines further away from rivers, and nets captured roughly 10 times more sharks than drumlines over the period of study. In contrast to tiger sharks, the catch data indicate the number of whalers has not declined over the past 26 years. Our findings suggest that environmental variables can be used to predict the movement of large sharks and by incorporating this knowledge into management plans and public education programs, may ultimately reduce shark-human incidents.

Various repair events following CRISPR/Cas9-based mutational correction of an infertility-related mutation in mouse embryos.

PURPOSE: Unpredictable genetic modifications and chromosomal aberrations following CRISPR/Cas9 administration hamper the efficacy of germline editing. Repair events triggered by double-strand DNA breaks (DSBs) besides non-homologous end joining and repair template-driven homology-directed repair have been insufficiently investigated in mouse. In this work, we are the first to investigate the precise repair mechanisms triggered by parental-specific DSB induction in mouse for paternal mutational correction in the context of an infertility-related mutation. METHODS: We aimed to correct a paternal 22-nucleotide deletion in Plcz1, associated with lack of fertilisation in vitro, by administrating CRISPR/Cas9 components during intracytoplasmic injection of Plcz1-null sperm in wild-type oocytes combined with assisted oocyte activation. Through targeted next-generation sequencing, 77 injected embryos and 26 blastomeres from seven injected embryos were investigated. In addition, low-pass whole genome sequencing was successfully performed on 17 injected embryo samples. RESULTS: Repair mechanisms induced by two different CRISPR/Cas9 guide RNA (gRNA) designs were investigated. In 13.73% (7/51; gRNA 1) and 19.05% (4/21; gRNA 2) of the targeted embryos, only the wild-type allele was observed, of which the majority (85.71%; 6/7) showed integrity of the targeted chromosome. Remarkably, for both designs, only in one of these embryos (1/7; gRNA 1 and 1/4; gRNA2) could repair template use be detected. This suggests that alternative repair events have occurred. Next, various genetic events within the same embryo were detected after single-cell analysis of four embryos. CONCLUSION: Our results suggest the occurrence of mosaicism and complex repair events after CRISPR/Cas9 DSB induction where chromosomal integrity is predominantly contained.

Plant growth regulators and mobilization of reserves in imbibition phases of yellow passion fruit.

The production of seedlings of the passion fruit tree, usually, is sexual, and the seeds are not uniform in the seedling emergence, and soaking treatments of seeds can provide faster and more uniform germination. It was aimed to study the action of plant growth regulators and the mobilization of reserves in the stages of soaking of yellow passion fruit seeds. The seeds were soaked for five hours in solutions containing plant growth regulators, in a completely randomized design, in a factorial 8 x 4, with four replications. The first factor corresponds to eight plant growth regulators: T1 - distilled water (control); T2 - 6-benzylaminepurine ​​500 mg L-1; T3 - 4-(3-indolyl) butyric acid 500 mg L-1; T4 - gibberellic acid 500 mg L-1; T5 - spermine 250 mg L-1; T6 - spermine 750 mg L-1; T7 - spermidine 750 mg L-1; T8 - spermidine 1250 mg L-1; and the second factor, to the four soaking times: zero, four, 72 and 120 hours, corresponding, respectively, to the dry seed, and to phases I, II, and III of the imbibition curve. It was evaluated the biochemical composition of seeds (lipids, soluble sugars and starch). The seeds showed accumulation of lipids in phase III; the content of soluble sugars increased in phase I and decreased in phase II. The starch content increased until the phase II and decreased in phase III. Starch is the main reserve in the seeds and the main source of energy used in phase III; soaking the seeds in polyamines generates an accumulation of lipids in the seeds and soaking in plant growth regulators increases the burning of starch.

Maturation and harvest time of Ateleia glazioveana Baill. seeds.

Ateleia glazioveana Baill. is a pioneer, rustic and can be used for forest recovery. This work aimed to study the process of physiological maturation of this species. The research was carried out in the city of Alegre - ES, the trees were identified in the floral anthesis and accompanied during the filling of the fruits and development of the seeds until the complete maturation. The fruits were harvested at the following stages 7, 14, 21, 28, 35 and 42 days after anthesis, and characterized according to: morphometry, moisture, fresh and dry mass of fruits and seeds, germination, germination speed index, shoot and root length and dry mass of seedlings. The regression equations were adjusted for the main characteristics analyzed as a function of the harvest period. The point of physiological maturity of timbó occurred at 42 days after anthesis.

Multivariate analysis of agronomic, physicochemical, and physiological characters of passion fruit hybrids cultivated at different environments.

Simultaneous analysis studies of several agronomic traits in cultivated plants make it possible to identify phenotypic and genotypic differences due to environmental variations, such as altitude. Therefore, the objective was to evaluate, through multivariate analysis of agronomic, physicochemical and physiological characters, passion fruit hybrids cultivated in different environments. The hybrids used were Gigante Amarelo, Rubi do Cerrado and Sol do Cerrado, cultivated in the southern region of Espírito Santo in four municipalities/environments: Marataízes (41 m), Jerônimo Monteiro (104 m), Alegre (711 m), and Ibitirama (1016 m). The agronomic characters of the plants, the physical-chemical characteristics of the fruits and the physical, biochemical and physiological qualities of the seeds were analyzed. The Singh method was used to determine the most important differentiating characters between hybrids growing in different environments. Based on these characters, a dissimilarity matrix was generated and a principal coordinate analysis was performed. It was observed that the pulp yield was influenced by altitude. The three hybrids showed greater performance in terms of agronomic characters at altitude (41 m) than at altitude (104 m). The Sol do Cerrado hybrid showed high performance in the physical-chemical characteristics of the fruits at altitude (104 m).

Phylogenetic diversity and structure in moist and dry upland forests in the semi-arid region of Brazil.

Understanding the factors influencing variation in the diversity and structure of rich biological communities (e.g., Neotropical upland forests) is essential in the context of climate change. In this study, we examine how environmental filters (temperature, precipitation, and elevation) and distinct habitats (moist upland forests - MUF and dry upland forests - DHF) influence the phylogenetic diversity and structure of 54 tree communities (28 MHF and 26 DHF). We used the standardized effect size (ses) of the metrics phylogenetic diversity (ses.PD), mean pairwise distance (ses.MPD), and mean nearest neighbor distance (ses.MNTD) to quantify changes in tree community diversity and structure. Then, we assessed the relationships of the phylogenetic metrics with the environmental filters as predictors using generalized linear models (GLMs). Our results indicate that increasing temperature negatively affects the phylogenetic indices analyzed, leading to less diverse and more clustered communities. In contrast, increasing precipitation and elevation showed a significant positive relationship with the analyzed indices, directing communities towards greater phylogenetic diversity and random or overdispersed structure. Our findings also reveal that phylogenetic diversity and structure vary with habitat type. For example, while MUFs exhibit higher phylogenetic diversity and random structure, DUFs display lower phylogenetic diversity and clustered structure. In conclusion, our results suggest that the phylogenetic patterns exhibited by upland communities in the semiarid region are strongly related to climatic conditions and the habitat in which they are found. Therefore, if the predicted temperature increases and precipitation decreases in climate change scenarios for the semi-arid region materialize, these communities may face significant biodiversity loss.

Phenylpropiolic acid isolated in cryogenic nitrogen and xenon matrices: NIR and UV-induced study.

Phenylpropiolic acid (C6H5C≡CCOOH, PPA) isolated in nitrogen and xenon cryogenic matrices was studied by infrared spectroscopy. The experimental studies were complemented by a series of quantum chemical calculations carried out at the density functional theory (B3LYP) and MP2 levels of theory (with different basis sets). The calculations predicted the existence of two planar PPA conformers, differing in the arrangement of the carboxylic group. The higher-energy trans-PPA conformer has a negligible population in the gas phase at room temperature and was prepared in situ in the N2 cryomatrix through vibrationally-induced rotamerization of the lower-energy cis-PPA conformer, achieved using selective narrowband infrared excitation of the OH stretching coordinate of the latter species. Broadband UV (λ > 235 nm) irradiation of matrix-isolated cis-PPA was also undertaken, leading to the observation of cis-PPA → trans-PPA isomerization. No other UV-induced photoreactions were observed. The in situ generated trans-PPA conformer was found to decay back to cis-PPA in the dark by tunneling, and its lifetimes under different experimental conditions were determined. The assignment of the infrared spectra of both conformers is presented, considerably extending the vibrational information available on this molecule.

Characterization of ovarian tissue oocytes from transgender men reveals poor calcium release and embryo development, which might be overcome by spindle transfer.

STUDY QUESTION: Can spindle transfer (ST) overcome inferior embryonic development of in vitro matured ovarian tissue oocytes (OTO-IVM) originating from testosterone-treated transgender men? SUMMARY ANSWER: ST shows some potential to overcome the embryo developmental arrest observed in OTO-IVM oocytes from transgender men. WHAT IS KNOWN ALREADY: OTO-IVM is being applied as a complementary approach to increase the number of oocytes/embryos available for fertility preservation during ovarian tissue cryopreservation in cancer patients. OTO-IVM has also been proposed for transgender men, although the potential of their oocytes remains poorly investigated. Currently, only one study has examined the ability of OTO-IVM oocytes originating from transgender men to support embryo development, and that study has shown that they exhibit poor potential. STUDY DESIGN, SIZE, DURATION: Both ovaries from 18 transgender men undergoing oophorectomy were collected for the purposes of this study, from November 2020 to September 2022. The patients did not wish to cryopreserve their tissue for fertility preservation and donated their ovaries for research. All patients were having testosterone treatment at the time of oophorectomy and some of them were also having menses inhibition treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sibling ovaries were collected in either cold or warm medium, to identify the most optimal collection temperature. Cumulus oocyte complexes (COCs) from each condition were isolated from the ovarian tissue and matured in vitro for 48 h. The quality of OTO-IVM oocytes was assessed by calcium pattern releasing ability, embryo developmental competence following ICSI, and staining for mitochondrial membrane potential. In vitro matured metaphase I (MI) oocytes, germinal vesicle (GV) oocytes, and in vivo matured oocytes with aggregates of smooth endoplasmic reticulum (SERa) were donated from ovarian stimulated women undergoing infertility treatment and these served as Control oocytes for the study groups. ST was applied to overcome poor oocyte quality. Specifically, enucleated mature Control oocytes served as cytoplasmic recipients of the OTO-IVM spindles from the transgender men. Embryos derived from the different groups were scored and analysed by shallow whole genome sequencing for copy number variations (CNVs). MAIN RESULTS AND THE ROLE OF CHANCE: In total, 331 COCs were collected in the cold condition (OTO-Cold) and 282 were collected in the warm condition (OTO-Warm) from transgender men. The maturation rate was close to 54% for OTO-Cold and 57% for OTO-Warm oocytes. Control oocytes showed a calcium releasing ability of 2.30 AU (n = 39), significantly higher than OTO-Cold (1.47 AU, P = 0.046) oocytes (n = 33) and OTO-Warm (1.03 AU, P = 0.036) oocytes (n = 31); both values of calcium release were similar between the two collection temperatures. Mitochondrial membrane potential did not reveal major differences between Control, OTO-Warm, and OTO-Cold oocytes (P = 0.417). Following ICSI, 59/70 (84.2%) of Control oocytes were fertilized, which was significantly higher compared to 19/47 (40.4%) of OTO-Cold (P < 0.01) and 24/48 (50%) of OTO-Warm oocytes (P < 0.01). In total, 15/59 (25.4%) blastocysts were formed on Day 5 in the Control group, significantly higher than 0/19 (0%) from the OTO-Cold (P = 0.014) and 1/24 (4.1%) in OTO-Warm oocytes (P = 0.026). Application of ST rescued the poor embryo development, by increasing the Day 5 blastocyst rate from 0% (0/19) to 20.6% (6/29) (P = 0.034), similar to that in the ICSI-Control group (25.4%, 15/59). A normal genetic profile was observed in 72.7% (8/11) of OTO-Cold, 72.7% (8/11) of OTO-Warm and 64.7% (11/17) of Control Day 3-Day 5 embryos. After ST was applied for OTO-IVM oocytes, 41.1% (7/17) of the embryos displayed normal genetic patterns, compared to 57.1% (4/7) among ST-Control Day 3-Day 5 embryos. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Due to the limited access to human oocytes and ovarian tissue, our results should be interpreted with some caution, as only a limited number of human oocytes and embryos could be investigated. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study, clearly indicate that OTO-IVM oocytes originating from transgender patients are of inferior quality, which questions their use for fertility preservation. The poor quality is likely to be related to cytoplasmic factors, supported by the increased blastocyst numbers following application of ST. Future research on OTO-IVM from transgender men should focus on the cytoplasmic content of oocytes or supplementation of media with factors that promote cytoplasmic maturation. A more detailed study on the effect of the length of testosterone treatment is also currently missing for more concrete guidelines and guidance on the fertility options of transgender men. Furthermore, our study suggests a potentially beneficial role of experimental ST in overcoming poor embryo development related to cytoplasmic quality. STUDY FUNDING/COMPETING INTEREST(S): A.C. is a holder of FWO grants (1S80220N and 1S80222N). A.B. is a holder of an FWO grant (1298722N). B.H. and A.V.S. have been awarded with a special BOF (Bijzonder Onderzoeksfonds), GOA (Geconcerteerde onderzoeksacties) and 2018000504 (GOA030-18 BOF) funding. B.H. has additional grants from FWO-Vlaanderen (Flemish Fund for Scientific Research, G051516N and G1507816N) and Ghent University Special Research Fund (Bijzonder Onderzoeksfonds, BOF funding (BOF/STA/202109/005)), and has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Conductive carbon paint: An efficient way to electrodeposit metallic nanowires by using porous membranes.

The present work demonstrates that conductive carbon paint, used for sample preparation in electron microscopy, can be a more straightforward and as-effective substitute for the metallic layer deposition usually used for the electrodeposition of metallic nanowires within porous membranes. AFM images demonstrated the good surface quality of the carbon layer. Raman spectroscopy confirmed the high crystallinity of carbon and high density of π-electrons. The electrical conductivity of the carbon layer was estimated using the linear sweep voltammetry technique. This new cathode was employed to grow continuous (Ni) and composition-modulated (Ni/Cu) nanowires within alumina templates, starting from aqueous solutions of Ni2+ and Cu2+ mixed salts. The obtention of metallic copper and nickel, and their separation can be readily observed by scanning electron microscopy and elemental mapping by EDS.

Acute and chronic effects of the organophosphate malathion on the pancreatic α and ß cell viability, cell structure, and voltage-gated K+ currents.

Studies indicate that the pesticide malathion may have a role in diabetes. Herein, we determined the effects of different concentrations of malathion on survival, ultrastructure, and electrophysiologic islet cell parameters. Acutely, high concentrations of malathion (0.5 or 1 mM) increased cell death in rat islet cells, while low concentrations (0.1 mM) caused signs of cell damage in pancreatic α and ß cells. Exposure of RINm5F cells to malathion for 24 or 48 h confirmed the reduction in ß-cell viability at lower concentrations (0.001-100 µM). Chronic exposure of mouse pancreatic α and ß cells to 3 nM of malathion led to increased voltage-gated K+ (Kv) currents in α-cells. Our findings show a time and concentration dependency for the malathion effect on the reduction of islet cell viability and indicate that pancreatic α cells are more sensitive to malathion effects on Kv currents and cell death.

Inserción intrapleural involuntaria de un catéter epidural en cirugía torácica: ¿está fuera de juego la analgesia regional o existe otra salida? / Unintended intrapleural insertion of an epidural catheter in thoracic surgery: Regional analgesia game over, or is there another way out?

En cirugía torácica, el control optimizado del dolor es esencial para prevenir disfunciones de la mecánica cardiorrespiratoria. La anestesia epidural y el bloqueo paravertebral son las técnicas analgésicas más populares. La inserción intrapleural involuntaria de un catéter epidural es una complicación infrecuente. Nuestro informe presenta un caso de un paciente sometido a resección de tumor pulmonar mediante cirugía toracoscópica asistida por video. Existió dificultad para la inserción del catéter debido a la obesidad del paciente, pero tras la inducción de la anestesia no se necesitó analgesia intravenosa adicional tras la inyección epidural. Se requirió convertir la cirugía en toracotomía, con identificación intrapleural del catéter epidural. Al finalizar la cirugía los cirujanos reorientaron el catéter en el espacio paravertebral, con confirmación de ausencia de fuga tras la inyección de anestesia local a través del catéter. En el periodo postoperatorio el control del dolor fue eficaz y sin complicaciones. Se trata de un caso exitoso que muestra que cuando encontramos complicaciones imprevistas podemos buscar soluciones alternativas para proporcionar a nuestro paciente el mejor tratamiento.(AU)

In thoracic surgery, optimized pain control is crucial to prevent dysfunction in cardiorespiratory mechanics. Epidural anesthesia and paravertebral block are the most popular techniques for analgesia. Unintended intrapleural insertion of an epidural catheter is a rare complication. Our report presents a case of a patient submitted to pulmonary tumor resection by video-assisted thoracoscopic surgery. There was difficulty in epidural insertion related to patient's obesity, but after general anesthesia induction, no additional intravenous analgesia was needed after epidural injection. Surgery required conversion to thoracotomy, with intrapleural identification of epidural catheter. At the end of surgery, surgeons reoriented catheter to paravertebral space, with leak absence confirmation after local anesthetic injection through the catheter. In postoperative period, pain control was efficient, with no complications. It was a successful case that shows that when we find unexpected complications, we can look for alternative solutions to give our patient the best treatment.(AU)

Unintended intrapleural insertion of an epidural catheter in thoracic surgery: regional analgesia game over, or is there another way out?

In thoracic surgery, optimized pain control is crucial to prevent dysfunction in cardiorespiratory mechanics. Epidural anesthesia (EA) and paravertebral block (PVB) are the most popular techniques for analgesia. Unintended intrapleural insertion of an epidural catheter is a rare complication. Our report presents a case of a patient submitted to pulmonary tumor resection by video-assisted thoracoscopic surgery (VATS). There was difficulty in epidural insertion related to patient's obesity, but after general anesthesia induction, no additional intravenous analgesia was needed after epidural injection. Surgery required conversion to thoracotomy, with intrapleural identification of epidural catheter. At the end of surgery, surgeons reoriented catheter to paravertebral space, with leak absence confirmation after local anesthetic injection through the catheter. In postoperative period, pain control was efficient, with no complications. It was a successful case that shows that when we find unexpected complications, we can look for alternative solutions to give our patient the best treatment.

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

STUDY QUESTION: What is the role of transcriptional-enhanced associate (TEA) domain family member 4 (TEAD4) in trophectoderm (TE) differentiation during human embryo preimplantation development in comparison to mouse? SUMMARY ANSWER: TEAD4 regulates TE lineage differentiation in the human preimplantation embryo acting upstream of caudal-type homeobox protein 2 (CDX2), but in contrast to the mouse in a GATA-binding protein 3 (GATA3)-independent manner. WHAT IS KNOWN ALREADY: Tead4 is one of the earliest transcription factors expressed during mouse embryo preimplantation development and is required for the expression of TE-associated genes. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination, have shown that Tead4-targeted embryos have compromised development and expression of the TE-specific Cdx2 and Gata3 is downregulated. Cdx2 and Gata3 act in parallel pathways downstream of Tead4 to induce successful TE differentiation. Downstream loss of Cdx2 expression, compromises TE differentiation and subsequent blastocoel formation and leads to the ectopic expression of inner cell mass (ICM) genes, including POU Class 5 homeobox 1 (Pou5f1) and SRY-box transcription factor (Sox2). Cdx2 is a more potent regulator of TE fate in mouse as loss of Cdx2 expression induces more severe phenotypes compared with loss of Gata3 expression. The role of TEAD4 and its downstream effectors during human preimplantation embryo development has not been investigated yet. STUDY DESIGN, SIZE, DURATION: The clustered regularly interspaced short palindromic repeats-clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (CRISPR-Cas9) system was first introduced in pronuclei (PN)-stage mouse zygotes aiming to identify a guide RNA (gRNA), yielding high editing efficiency and effective disruption of the Tead4 locus. Three guides were tested (gRNA1-3), each time targeting a distinct region of Exon 2 of Tead4. The effects of targeting on developmental capacity were studied in Tead4-targeted embryos (n = 164-summarized data from gRNA1-3) and were compared with two control groups; sham-injected embryos (n = 26) and non-injected media-control embryos (n = 51). The editing efficiency was determined by next-generation sequencing (NGS). In total, n = 55 (summarized data from gRNA1-3) targeted mouse embryos were analysed by NGS. Immunofluorescence analysis to confirm successful targeting by gRNA1 was performed in Tead4-targeted embryos, and non-injected media-control embryos. The downregulation of secondary TE-associated markers Cdx2 and Gata3 was used as an indirect confirmation of successful Tead4-targeting (previously shown to be expressed downstream of Tead4). Additional groups of gRNA1 Tead4-targeted (n = 45) and media control (n = 36) embryos were cultured for an extended period of 8.5 days, to further assess the developmental capacity of the Tead4-targeted group to develop beyond implantation stages. Following the mouse investigation, human metaphase-II (MII) oocytes obtained by IVM were microinjected with gRNA-Cas9 during ICSI (n = 74) to target TEAD4 or used as media-control (n = 33). The editing efficiency was successfully assessed in n = 25 TEAD4-targeted human embryos. Finally, immunofluorescence analysis for TEAD4, CDX2, GATA3 and the ICM marker SOX2 was performed in TEAD4-targeted (n = 10) and non-injected media-control embryos (n = 29). PARTICIPANTS/MATERIALS, SETTING, METHODS: A ribonucleoprotein complex consisting of a gRNA-Cas9 mixture, designed to target Exon 2 of Tead4/TEAD4, was microinjected in mouse PN stage zygotes or human IVM MII oocytes along with sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. In mouse, an additional group of Tead4-targeted and media-control embryos was cultured in vitro for an extended period of 8.5 days. Embryonic development and morphology were assessed daily, during culture in vitro of mouse and human embryos and was followed by a detailed scoring at late blastocyst stage. Targeting efficiency following gRNA-Cas9 introduction was assessed via immunostaining and NGS analysis. MAIN RESULTS AND THE ROLE OF CHANCE: NGS analysis of the Tead4-targeted locus revealed very high editing efficiencies for all three guides, with 100% of the mouse embryos (55 out of 55) carrying genetic modifications resulting from CRISPR-Cas9 genome editing. More specifically, 65.22% (15 out 23) of the PN zygotes microinjected with gRNA1-Cas9, which exhibited the highest efficiency, carried exclusively mutated alleles. The developmental capacity of targeted embryos was significantly reduced (data from gRNA1), as 44.17% of the embryos arrested at the morula stage (2.5 days post coitum), coincident with the initiation of TE lineage differentiation, compared with 8.51% in control and 12.50% in sham control groups. High-quality blastocyst formation rates (Grade 3) were 8.97% in the gRNA1-targeted group, compared with 87.23% in the media-control and 87.50% in the sham group. Immunofluorescence analysis in targeted embryos confirmed downregulation of Tead4, Cdx2, and Gata3 expression, which resulted from successful targeting of the Tead4 locus. Tead4-targeted mouse embryos stained positive for the ICM markers Pou5f1 and Sox2, indicating that expression of ICM lineage markers is not affected. Tead4-targeted embryos were able to cavitate and form a blastocoel without being able to hatch. Extended embryo culture following zona pellucida removal, revealed that the targeted embryos can attach and form egg-cylinder-like structures in the absence of trophoblast giant cells. In human embryos, Exon 2 of TEAD4 was successfully targeted by CRISPR-Cas9 (n = 74). In total, 25 embryos from various developmental stages were analysed by NGS and 96.00% (24 out of 25) of the embryos carried genetic modifications because of gRNA-Cas9 editing. In the subgroup of the 24 edited embryos, 17 (70.83%) carried only mutant alleles and 11 out of these 17 (64.70%) carried exclusively frameshift mutations. Six out of 11 embryos reached the blastocyst stage. In contrast to mice, human-targeted embryos formed blastocysts at a rate (25.00%) that did not differ significantly from the control group (23.81%). However, blastocyst morphology and TE quality were significantly compromised following TEAD4-targeting, showing grade C TE scores, with TE containing very few cells. Immunofluorescence analysis of TEAD4-targeted embryos (n = 10) confirmed successful editing by the complete absence of TEAD4 and its downstream TE marker CDX2, but the embryos generated retained expression of GATA3, which is in contrast to what we have observed and has previously been reported in mouse. In this regard, our results indicate that GATA3 acts in parallel with TEAD4/CDX2 towards TE differentiation in human. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: CRISPR-Cas9 germline genome editing, in some cases, induces mosaic genotypes. These genotypes are a result of inefficient and delayed editing, and complicate the phenotypic analysis and developmental assessment of the injected embryos. We cannot exclude the possibility that the observed differences between mouse and human are the result of variable effects triggered by the culture conditions, which were however similar for both mouse and human embryos in this study. Furthermore, this study utilized human oocytes obtained by IVM, which may not fully recapitulate the developmental behaviour of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Elucidation of the evolutionary conservation of molecular mechanisms that regulate the differentiation and formation of the trophoblast lineage can give us fundamental insights into early implantation failure, which accounts for ∼15% of human conceptions. STUDY FUNDING/COMPETING INTEREST(S): The research was funded by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01) and Ghent University (BOF.BAS.2018.0018.01). G.C. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 11L8822N). A.B. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 1298722 N). We further thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Nitric oxide regulates adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells.

Anoikis is a type of apoptosis that occurs in response to the loss of adhesion to the extracellular matrix (ECM). Anoikis resistance is a critical mechanism in cancer and contributes to tumor metastasis. Nitric oxide (NO) is frequently upregulated in the tumor area and is considered an important player in cancer metastasis. The aim of this study was to evaluate the effect of NO on adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells. Here, we report that anoikis-resistant endothelial cells overexpress endothelial nitric oxide synthase. The inhibition of NO release in anoikis-resistant endothelial cells was able to decrease adhesiveness to fibronectin, laminin, and collagen IV. This was accompanied by an increase in cell invasiveness and migration. Furthermore, anoikis-resistant cell lines displayed a decrease in fibronectin and collagen IV protein expression after L-NAME treatment. These alterations in adhesiveness and invasiveness were the consequence of MMP-2 up-regulation observed after NO release inhibition. The decrease in NO levels was able to down-regulate the activating transcription factor 3 (ATF3) protein expression. ATF3 represses MMP-2 gene expression by antagonizing p53-dependent trans-activation of the MMP-2 promoter. We speculate that the increased release of NO by anoikis-resistant endothelial cells acted as a response to restrict the MMP-2 action, interfering in MMP-2 gene expression via ATF3 regulation. The up-regulation of nitric oxide by anoikis-resistant endothelial cells is an important response to restrict tumorigenic behavior. Without this mechanism, invasiveness and migration potential would be even higher, as shown after L-NAME treatment.

Exosomal glypican-1 discriminates pancreatic ductal adenocarcinoma from chronic pancreatitis.

BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) diagnosis can be difficult in a chronic pancreatitis (CP) background, especially in its mass forming presentation. We aimed to assess the accuracy of glypican-1-positive circulating exosomes (GPC1+crExos) to distinguish PDAC from CP versus the state-of-the-art CA 19-9 biomarker. METHODS: This was a unicentric prospective cohort. Endoscopic ultrasound with fine-needle aspiration or biopsy and blood tests (GPC1+crExos and serum CA 19-9) were performed. RESULTS: The cohort comprised 60 PDAC and 29 CP (7 of which mass forming - MF) patients. Median levels of GPC1+crExos were significantly higher in PDAC (99.7%) versus CP (28.4%; p<0.0001) with an AUROC of 0.96 with 98.3% sensitivity and 86.2% specificity for a cut-off of 45.0% (p<0.0001); this outperforms CA 19-9 AUROC of 0.82 with 78.3% sensitivity and 65.5% specificity at a cut-off of 37 U/mL (p<0.0001). The superiority of% GPC1+crExos over CA 19-99 in differentiating PDAC from CP was observed in both early (stage I) and advanced tumors (stages II-IV). CONCLUSION: Levels of GPC1+crExos coupled to beads enable differential diagnosis between PDAC and CP including its mass-forming presentation.

Nitric oxide regulates adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells

Anoikis is a type of apoptosis that occurs in response to the loss of adhesion to the extracellular matrix (ECM). Anoikis resistance is a critical mechanism in cancer and contributes to tumor metastasis. Nitric oxide (NO) is frequently upregulated in the tumor area and is considered an important player in cancer metastasis. The aim of this study was to evaluate the effect of NO on adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells. Here, we report that anoikis-resistant endothelial cells overexpress endothelial nitric oxide synthase. The inhibition of NO release in anoikis-resistant endothelial cells was able to decrease adhesiveness to fibronectin, laminin, and collagen IV. This was accompanied by an increase in cell invasiveness and migration. Furthermore, anoikis-resistant cell lines displayed a decrease in fibronectin and collagen IV protein expression after L-NAME treatment. These alterations in adhesiveness and invasiveness were the consequence of MMP-2 up-regulation observed after NO release inhibition. The decrease in NO levels was able to down-regulate the activating transcription factor 3 (ATF3) protein expression. ATF3 represses MMP-2 gene expression by antagonizing p53-dependent trans-activation of the MMP-2 promoter. We speculate that the increased release of NO by anoikis-resistant endothelial cells acted as a response to restrict the MMP-2 action, interfering in MMP-2 gene expression via ATF3 regulation. The up-regulation of nitric oxide by anoikis-resistant endothelial cells is an important response to restrict tumorigenic behavior. Without this mechanism, invasiveness and migration potential would be even higher, as shown after L-NAME treatment.

Silicon as an attenuator of drought stress in plants of Oryza sativa L. treated with dietholate / Silício como atenuador de estresse hídrico em plantas de Oryza sativa L. tratadas com dietholate

Abstract Silicon (Si) is an element that can improve the growth and development of rice plants in water-deficient environments because it is an enzymatic stimulant, signaling for production of antioxidant compounds. Therefore, the aim of this study was to examine the relationship between water deficiency and the effect of Si on two rice cultivars whose seeds were treated with dietholate. The experimental design was fully randomized with three replicates, and treatments were organized in a 3x2x2x4 factorial arrangement: three water soil conditions (50% and 100% of soil water retention capacity (WRC) and complete submergence in a water blade of 5.0 cm); two cultivars (IRGA 424 RI and Guri INTA CL); two sources of Si (sodium metasilicate and potassium metasilicate); and four rates of Si (0; 4.0; 8.0 and 16 g L-1). Chlorophyll a and b, leaf area and shoot and root dry weight increased at higher rates of Si under the three soil water regimes. There was an increase in superoxide dismutase and guaiacol peroxidase enzyme activity in the cultivars at higher rates of Si, reducing lipid peroxidation caused by water deficiency. Therefore, Si did indeed attenuate water deficiency stress in rice plants emerging from seeds treated with dietholate.

Resumo O silício (Si) é um elemento que pode proporcionar melhor crescimento e desenvolvimento às plantas de arroz cultivadas em ambientes com deficiência hídrica, por ser um estimulador enzimático, promovendo sinalização para produção de compostos antioxidantes. Assim, o objetivo deste trabalho foi estudar a relação entre deficiência hídrica e o efeito do Si em duas cultivares de arroz tratadas com dietholate. O delineamento experimental utilizado foi inteiramente casualizado, com três repetições e os tratamentos arranjados em esquema fatorial 3x2x2x4, sendo os fatores: três condições hídricas do solo (50%, 100% da capacidade de retenção de água no solo (CRA) e lâmina d'água de 5,0 cm), duas cultivares (IRGA 424 RI e Guri INTA CL), duas fontes de Si (metassilicato de sódio e metassilicato de potássio) e quatro doses de Si (0; 4,0; 8,0 e 16 g L-1). O índice de clorofila a e b, a área foliar e o teor de massa seca da parte aérea e raiz aumentaram com o aumento das doses de Si nas três condições hídricas do solo. Houve um aumento na atividade das enzimas superóxido dismutase e guaiacol peroxidase nas cultivares estudadas à medida que as doses de Si aumentaram, diminuindo a peroxidação de lipídios, causada pela restrição hídrica. Portanto, o Si atenua o estresse por déficit hídrico em plantas de arroz emergentes de sementes tratadas com dietholate.

Bistability in the synchronization of identical neurons.

We investigate the role of bistability in the synchronization of a network of identical bursting neurons coupled through an generic electrical mean-field scheme. These neurons can exhibit distinct multistable states and, in particular, bistable behavior is observed when their sodium conductance is varied. With this, we consider three different initialization compositions: (i) the whole network is in the same periodic state; (ii) half of the network periodic, half chaotic; (iii) half periodic, and half in a different periodic state. We show that (i) and (ii) reach phase synchronization (PS) for all coupling strengths, while for (iii) small coupling regimes do not induce PS, and instead, there is a coexistence of different frequencies. For stronger coupling, case (iii) synchronizes, but after (i) and (ii). Since PS requires all neurons being in the same state (same frequencies), these different behaviors are governed by transitions between the states. We find that, during these transitions, (ii) and (iii) have transient chimera states and that (iii) has breathing chimeras. By studying the stability of each state, we explain the observed transitions. Therefore, bistability of neurons can play a major role in the synchronization of generic networks, with the simple initialization of the system being capable of drastically changing its asymptotic space.

Phase-locking intermittency induced by dynamical heterogeneity in networks of thermosensitive neurons.

In this work, we study the phase synchronization of a neural network and explore how the heterogeneity in the neurons' dynamics can lead their phases to intermittently phase-lock and unlock. The neurons are connected through chemical excitatory connections in a sparse random topology, feel no noise or external inputs, and have identical parameters except for different in-degrees. They follow a modification of the Hodgkin-Huxley model, which adds details like temperature dependence, and can burst either periodically or chaotically when uncoupled. Coupling makes them chaotic in all cases but each individual mode leads to different transitions to phase synchronization in the networks due to increasing synaptic strength. In almost all cases, neurons' inter-burst intervals differ among themselves, which indicates their dynamical heterogeneity and leads to their intermittent phase-locking. We argue then that this behavior occurs here because of their chaotic dynamics and their differing initial conditions. We also investigate how this intermittency affects the formation of clusters of neurons in the network and show that the clusters' compositions change at a rate following the degree of intermittency. Finally, we discuss how these results relate to studies in the neuroscience literature, especially regarding metastability.