PPARG is dispensable for bovine embryo development up to tubular stages.

Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors (PPARs) mediate the signaling actions of prostaglandins and other lipids and, between them, PPARG has been pointed out to play a relevant role on conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG KO embryos in vitro by two independent gene ablation strategies and assess their developmental ability. In vitro development to Day (D) 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass and trophectoderm cell numbers were similar between WT and KO D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, and embryonic disc formation and hypoblast migration rates were unaffected by the ablation. The development to tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disc was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages.

HH5 Double-Carrier Embryos Fail to Progress through Early Conceptus Elongation.

Massive genotyping in cattle has uncovered several deleterious haplotypes that cause pre-term mortality. Holstein Haplotype 5 (HH5) is a deleterious haplotype present in the Holstein Friesian population that involves the ablation of the Transcription Factor B1 mitochondrial (TFB1M) gene. The developmental stage at which HH5 double-carrier (DC, homozygous) embryos or fetuses die remains unknown and this is a relevant information to estimate the economic losses associated to the inadvertent cross between carriers. To determine if HH5 DC survive to maternal recognition of pregnancy, embryonic day (E)14 embryos were flushed from superovulated carrier cows inseminated with a carrier bull. DC E14 conceptuses were recovered at Mendelian rates but they failed to achieve early elongation, as evidenced by a drastic (>26-fold) reduction in the proliferation of extraembryonic membranes compared with carrier or non-carrier embryos. To assess development at earlier stages, TFB1M knockout (KO) embryos -functionally equivalent to DC embryos- were generated by CRISPR technology and cultured to the blastocyst stage -Day (D)8- and to the early embryonic disc stage -D12-. No significant effect of TFB1M ablation was observed on the differentiation and proliferation of embryonic lineages and relative mtDNA content up to D12. In conclusion, HH5 DC embryos are able to develop to early embryonic disc stage but fail to undergo early conceptus elongation, required for pregnancy recognition.

Efficient and repeatable in vitro fertilization in rabbits.

Rabbits constitute an interesting model to understand gamete interaction and test novel Artificial Reproductive Techniques, but in vitro fertilization (IVF) is particularly problematic in this species. We have conducted a series of experiments to develop a consistent IVF technique. Initially, we checked viability, acrosome integrity, capacitation and motility in ejaculated sperm purified by a density gradient and incubated at different times in three different media: Tyrode's Albumin Lactate Pyruvate (TALP), human tubal fluid (HTF), and Brackett and Oliphant (BO). Total and progressive motility at 10-24 h and linearity from 3 h onwards was significantly higher in BO medium compared to TALP and HTF. Subsequently, cumulus-oocyte complexes (COCs) collected 10 h after induction of ovulation were incubated with sperm in TALP, HTF or BO for 18 h with or without performing sperm pre-incubation for 6 h. Pronuclear formation rate at 18 h was significantly higher in BO compared to other media (∼84 % vs. 17-22 %) and was not improved by pre-incubation. As COCs recovery rate was low at 10 h after induction of ovulation, COCs were collected at 12 h and co-incubated with sperm in BO. Pronuclear formation rate was similar than those obtained in COCs collected at 10 h (∼85 %), and when embryos were allowed to develop in vitro, the protocol yielded high cleavage and blastocyst rates (91 and 59 %, respectively). In conclusion, ejaculated rabbit sperm purified in a density gradient fertilize efficiently COCs collected at 12 h in BO medium.

Effect of arachidonic acid on pre- and post-hatching in vitro bovine embryo development.

CONTEXT: Arachidonic acid (AA) is the precursor of prostaglandins, which may play autocrine roles during early embryo development. AIMS: To test the developmental effects of addition of AA to pre- and post-hatching culture media on in vitro -produced bovine embryos. METHODS: Pre-hatching effects of AA were tested by culturing bovine zygotes in synthetic oviductal fluid (SOF) supplemented with 100 or 333µM AA. Post-hatching effects of AA were tested by culturing Day 7 blastocysts in N2B27 supplemented with 5, 10, 20 or 100µM AA up to Day 12. KEY RESULTS: Pre-hatching development to blastocyst was completely abrogated at 333µM AA, whereas blastocyst rates and cell numbers were not altered at 100µM AA. Impaired post-hatching development was observed at 100µM AA, whereas no effect on survival rates was noted at 5, 10 and 20µM AA. However, a significant reduction in Day 12 embryo size was observed at 10 and 20µM AA. Hypoblast migration, epiblast survival and formation of embryonic-disc-like structures were unaffected at 5-10µM AA. AA exposure downregulated the genes PTGIS , PPARG , LDHA and SCD in Day 12 embryos. CONCLUSIONS: Pre-hatching embryos are mostly irresponsive to AA, whereas AA was observed to have negative effects during early post-hatching development. IMPLICATIONS: AA does not improve in vitro bovine embryo development and is not required up to early post-hatching stages.

PUF60-related developmental disorder: A case series and phenotypic analysis of 10 additional patients with monoallelic PUF60 variants.

PUF60-related developmental disorder (also referred to as Verheij syndrome), resulting from haploinsufficiency of PUF60, is associated with multiple congenital anomalies affecting a wide range of body systems. These anomalies include ophthalmic coloboma, and congenital anomalies of the heart, kidney, and musculoskeletal system. Behavioral and intellectual difficulties are also observed. While less common than other features associated with PUF60-related developmental disorder, for instance hearing impairment and short stature, identification of specific anomalies such as ophthalmic coloboma can aid with diagnostic identification given the limited spectrum of genes linked with this feature. We describe 10 patients with PUF60 gene variants, bringing the total number reported in the literature, to varying levels of details, to 56 patients. Patients were recruited both via locally based exome sequencing from international sites and from the DDD study in the United Kingdom. Eight of the variants reported were novel PUF60 variants. The addition of a further patient with a reported c449-457del variant to the existing literature highlights this as a recurrent variant. One variant was inherited from an affected parent. This is the first example in the literature of an inherited variant resulting in PUF60-related developmental disorder. Two patients (20%) were reported to have a renal anomaly consistent with 22% of cases in previously reported literature. Two patients received specialist endocrine treatment. More commonly observed were clinical features such as: cardiac anomalies (40%), ocular abnormalities (70%), intellectual disability (60%), and skeletal abnormalities (80%). Facial features did not demonstrate a recognizable gestalt. Of note, but remaining of unclear causality, we describe a single pediatric patient with pineoblastoma. We recommend that stature and pubertal progress should be monitored in PUF60-related developmental disorder with a low threshold for endocrine investigations as hormone therapy may be indicated. Our study reports an inherited case with PUF60-related developmental disorder which has important genetic counseling implications for families.

Ruptured abdominal aortic aneurysm, what does the interventional radiologist and vascular surgeon need from our report?

Abdominal aortic aneurysm is defined as a dilatation of the abdominal aorta greater than 3cm. Its prevalence is between 1 and 1.5 cases per 100 people, constituting an important cause of morbidity and mortality. Rare in women, its frequency increases with age and its most frequent location is between the renal arteries and the aorto-iliac bifurcation. Approximately 5% of cases will involve the visceral branches. It is a silent pathological process whose natural evolution is rupture, which often has a fatal outcome and whose diagnosis is part of the pathology that we will find in emergency radiology. The involvement of the radiologist and the preparation of an accurate diagnostic report, as soon as possible, is essential for decision-making by the team in charge of the patient's surgery.

Pre-hatching exposure to N2B27 medium improves post-hatching development of bovine embryos in vitro.

Ungulate embryos undergo critical cell differentiation and proliferation events around and after blastocyst hatching. Failures in these processes lead to early pregnancy losses, which generate an important economic impact on farming. Conventional embryo culture media, such as SOF, are unable to support embryo development beyond hatching. In contrast, N2B27 medium supports early post-hatching development, evidencing a swift in embryonic nutritional requirements during this developmental window. Here, we investigate if earlier exposure to N2B27 could improve embryo development after hatching. Embryo culture in N2B27 from day (D) 5, 6 or 7 significantly enhanced complete hypoblast migration (>45 vs. ∼24%) and epiblast development into an embryonic disc (ED)-like structure at D12 (>40 vs. 23%), compared to embryos cultured in SOF up to D9. Culture in N2B27 from D5 significantly increased epiblast and hypoblast cell number in D8 blastocysts, but post-hatching embryos cultured in N2B27 from D5 or 6 frequently showed a disorganized distribution of epiblast cells. In conclusion, bovine embryo culture in N2B27 from D7 onwards improves subsequent post-hatching development. This improved fully in vitro system will be very useful to functionally explore cell differentiation mechanisms and the bases of early pregnancy failures without requiring animal experimentation.

Short-term recovery of soil and pine tree canopy after late prescribed burning in a semi-arid landscape.

Climate change worsening due to global warming and progressive abandonment in rural areas mean that wildfires are increasing in extent and severity terms, and are one of the major disturbances in the Mediterranean Basin. To mitigate these disturbances, preventive management tools need to be used. Fire employment is being implemented, known as prescribed burnings, as forestry actions to change vegetation lines both vertically and horizontally to eliminate forest fuel load continuity. This study aimed to know the ecological effects of late prescribed burning treatments under mixed trees. Prescribed burns were carried out in October 2019 in the municipality of Ayna, Albacete (SE Spain). To assess the short-term (12-month) fire impact on soil, we measured soil respiration with a CO2 flow chamber and analyzed soil physico-chemical properties. We also used minidisc infiltrometers to analyze soil repellency and soil hydraulic conductivity over a 1-year monitoring period. In addition, we evaluated the effects on tree strata by performing chemical nature analyses of fallen needles in Pinus pinaster after prescribed burning. According to our initial hypotheses, the results did not show significant changes in any parameter evaluated during the study period. However, some variables were affected in the first 3 months, needlefall and in microbiological activity, such as variations in soil physico-chemical properties, which can be due to long dry seasons combined with prescribed burning. This study attempts to observe and make known the effects that low-intensity prescribed burning has on soil and needlefall, which are relevant for updating forest management tools.

Post-fire management effects on hillslope-stream sediment connectivity in a Mediterranean forest ecosystem.

Forest fires intensify sediment transport and aggravate local and off-site consequences of soil erosion. This study evaluates the influence of post-fire measures on structural and functional sediment connectivity (SC) in five fire-affected Mediterranean catchments, which include 929 sub-catchments, by using the "aggregated index of connectivity" (AIC) at two temporal scenarios: I) immediately after the fire and before implementing post-fire practices ('Pre-man'), and II) two years after the fire ('Post-man'). The latter includes all the emergency stabilization practices, that are hillslope barriers, check-dams and afforestation. The stream system was set as the target of the computation (STR), to be representative of intense rainfall-runoff events with effective sediment delivery outside the catchments. Output normalization (AICN) allows comparing the results of the five basins between them. The sedimentological analysis is based on specific sediment yield (SSY) -measured at the check-dams installed after the fire -, and this data is used for output evaluation. Stream density and slope variables were the most influential factors on AICN-STR results at the sub-catchment scale. Post-fire hillslope treatments (barriers when built in high densities and afforestation) significantly reduced AICN-STR in comparison with untreated areas in both structural and functional approaches. Despite the presence of hillslope treatments, the higher erosive rainfall conditions resulted in higher AICN-STR values in the Post-man scenario (functional approach). A positive and good correlation was found between the measured SSY and the AICN-STR changes due to the post-fire practices and vegetation recovery, showing the good correspondence of the computation results and the real sediment dynamics of the studied catchments. Overall, AICN demonstrated to be a useful and versatile tool for post-fire management, which needs further research to optimize its applicability.

Cemiplimab in Advanced Cutaneous Squamous Cell Carcinoma: Real-World Experience in a Monographic Oncology Center. / [Artículo traducido] Cemiplimab en el carcinoma de células escamosas cutáneo avanzado: experiencia del mundo real en un centro oncológico monográfico.

Management of advanced cSCC is challenging, and many available systemic medications have modest efficacy. Cemiplimab has demonstrated efficacy in the treatment of advanced cSCC in clinical trials, but real-world data are still limited. With the objective of evaluating the efficacy of cemiplimab in a real-world clinical setting, we conducted a prospective observational study of 13 patients with advanced cSCC. Six patients (46%) had locally advanced disease, while 7 (54%) had metastatic disease. A total of 8 patients (62%) responded to cemiplimab. Five (38%) showed a partial response, while 3 (23%) showed a complete response. Four patients with an initial partial response presented subsequent disease progression during follow-up. Six patients (46%) developed AEs, most of which were mild (G1). PFS was 5.9 months, with a median follow-up was 9 months. In conclusion, cemiplimab demonstrated its utility in the treatment of advanced cSCC, with acceptable response rates, a remarkable number of complete responses, and a very good safety profile.

Cemiplimab in Advanced Cutaneous Squamous Cell Carcinoma: Real-World Experience in a Monographic Oncology Center.

Management of advanced cSCC is challenging, and many available systemic medications have modest efficacy. Cemiplimab has demonstrated efficacy in the treatment of advanced cSCC in clinical trials, but real-world data are still limited. With the objective of evaluating the efficacy of cemiplimab in a real-world clinical setting, we conducted a prospective observational study of 13 patients with advanced cSCC. Six patients (46%) had locally advanced disease, while 7 (54%) had metastatic disease. A total of 8 patients (62%) responded to cemiplimab. Five (38%) showed a partial response, while 3 (23%) showed a complete response. Four patients with an initial partial response presented subsequent disease progression during follow-up. Six patients (46%) developed AEs, most of which were mild (G1). PFS was 5.9 months, with a median follow-up was 9 months. In conclusion, cemiplimab demonstrated its utility in the treatment of advanced cSCC, with acceptable response rates, a remarkable number of complete responses, and a very good safety profile.

Indications and results of sentinel lymph node biopsy in breast cancer: Changes observed in 2018 compared to 2012.

BACKGROUND AND OBJECTIVE: Over the last 15 years, breast cancer (BC) treatment has undergone numerous changes, which have also affected the indications for Sentinel Lymph Node Biopsy (SLNB) as well as the procedures depending on its outcome. The aim of this study is to compare the results of the SLNB carried out at our Center during 2012, when we stopped performing an axillary lymph node dissection (ALND) after the finding of a micrometastasis, with those conducted in 2018, when we started applying Z011 criteria. MATERIALS AND METHODS: We have performed a comparative retrospective observational study, including cN0 BC patients that underwent a SLNB in 2012 versus those that underwent this procedure in 2018. RESULTS: 174 patients from 2012 and 165 from 2018 were studied. We found significant differences between the two groups: in 2018 there were fewer lobular invasive cancers (14 vs 28) (P < .05), a smaller mean pathological size (P < .001), a higher proportion of HER2 and triple negative tumors (28 vs 49) (P < .01) and, finally, an increase in use of neoadjuvant treatments (42.0% vs 6.7%). Regarding axillary involvement, we observed a decrease in both the presence of a positive sentinel node (24.1% in 2018 vs 42.4% in 2012) (P < .0001) as well as in the proportion of ALND performed (12.6% in 2018 vs 21.2% in 2012) and the presence of positive non sentinel nodes after a ALND (59.1% vs74.3%) (ns) CONCLUSION: Although the 2018 cohort that underwent SLNB had more aggressive tumors, there were fewer positive SN and ALND performed. This is probably due to a more accurate radiological diagnosis with ultrasound, that enables to detect cN1 cases before surgery, and to the increasing use of neoadjuvant treatments that may downstage the axilla.

Service-Learning, Movies, and Infectious Diseases: Implementation of an Active Educational Program in Microbiology as a Tool for Engagement in Social Justice.

Service-Learning is an educational methodology that allows student learning while addressing community needs. A program in microbiology and infectious diseases was implemented in Universidad Complutense de Madrid, Spain. University lecturers, clinical microbiologists, doctorate students, and undergraduates from several Bachelor Degrees and courses worked in an interdisciplinary team along with social institutions that attend disadvantaged persons. Using commercial movies that deal with infectious diseases, the students learn clinical microbiology, prepare divulgation materials, visit social centers to accompany, and help others to know about illnesses and prevention. The program was developed through two academic years and involved 58 voluntary students, 13 teachers and tutors, and 4 social entities as community partners. Postsurvey evaluation of the program revealed a highly satisfactory achievement of goals: acquiring scientific and personal competencies by university students, including critical analysis and science diffusion, solving problems or collaborative team working, and contributing, together with the tutors, to the social responsibility of the university.

Characterization of inflammatory bowel disease in Colombia: Results of a national register. / Caracterización de la enfermedad inflamatoria intestinal en Colombia: resultados de un registro nacional.

AIM: To determine the clinical, sociodemographic, and treatment characteristics of inflammatory bowel disease (IBD) in a Colombian population register. METHODS: A descriptive, analytic, observational, cross-sectional, multicenter study on patients with IBD from 17 hospital centers in 9 Colombian cities was conducted. RESULTS: A total of 2,291 patients with IBD were documented, 1,813 (79.1%) of whom presented with ulcerative colitis (UC), 456 (19.9%) with Crohn's disease (CD), and 22 with IBD unclassified (0.9%). The UC/CD ratio was 3.9:1. A total of 18.5% of the patients with UC and 47.3% with CD received biologic therapy. Patients with extensive UC had greater biologic therapy use (OR = 2.78, 95% CI: 2.10-3.65, p = 0.000), a higher surgery rate (OR = 5.4, 95% CI: 3.5-8.3, p = 0.000), and greater frequency of hospitalization (OR = 4.34, 95% CI: 3.47-5.44, p = 0.000). Patients with severe UC had greater biologic therapy use (OR = 5.04, 95% CI: 3.75-6.78, p = 0.000), a higher surgery rate (OR = 8.64, 95% CI: 5.4-13.78, p = 0.000), and greater frequency of hospitalization (OR = 28.45, 95% CI: 19.9-40.7, p = 0.000). CD patients with inflammatory disease behavior (B1) presented with a lower frequency of hospitalization (OR = 0.12, 95% CI: 0.07-0.19, p = 0.000), a lower surgery rate (OR = 0.08, 95% CI: 0.043-0.15, p = 0.000), and less biologic therapy use (OR = 0.26, 95% CI: 0.17-0.41, p = 0.000). CONCLUSION: In Colombia, there is a predominance of UC over CD (3.9:1), as occurs in other Latin American countries. Patients with extensive UC, severe UC, or CD with noninflammatory disease behavior (B2, B3) have a worse prognosis.

Assessment of saliva and oral candidiasis levels 12, 24 and 36 months after radiotherapy in patients with head and neck cancer.

INTRODUCTION: Hyposalivation is a serious complication during radiotherapy (RT) and it is one of the major risk factors for the presence of candidiasis. The aim of this study was to evaluate the salivary hypofunction during the different stages of RT, analysing its connection with the presence of candidiasis. MATERIAL AND METHODS: A retrospective study was performed in 83 patients who had been diagnosed with head and neck tumours and who were undergoing RT treatment. Their salivary function was clinically analysed throughout the course of the RT treatment (before, during and after treatment) by means of the whole saliva test (WST), both unstimulated (WST-I) and stimulated (WST-II), and its relationship with candidiasis was evaluated using culture-based methods. RESULTS: The WST-I before RT was 37.24±17.36mm and the WST-II was 60.70±30.98mm, with 47% of patients testing positive for candidiasis. The prevalence of candidiasis increased up to 55.8% during RT and it returned to similar pre-RT levels at the end of treatment (45.2%). A statistical significant relationship was found between low WST-I and candidiasis in the 1st (13.58 vs 20.78mm), 3rd (18.06 vs 24.36mm), 6th (16.83 vs 24.5) and 12th (16 vs 28.74mm) months after RT; and this relationship was also detected for WST-II in the 1st (24.73 vs 41.26mm) and 3rd (27.71 vs 39.91mm) months after RT. Female sex was identified as an independent associated risk factor for mild hyposalivation before RT (OR=6.50, CI: 95% 1.77-23.93, p=0.005) and glandular hypofunction (OR=3.01, CI: 95% 1.12-8.10, p=0.029). DISCUSSION: There is a clear relation between hyposalivation and the presence of candidiasis during and after RT. Larger studies must be performed in order to further elucidate this effect.

Burning season and vegetation coverage influenced the community-level physiological profile of Mediterranean mixed-mesogean pine forest soils.

Knowledge of forest soil ecology is necessary to assess vulnerability to disturbances, such as wildfires, and improve its microbial diversity and functional value. Soil microbiota play an important role in forest soil processes and are a key driver of postfire recovery, but they are very vulnerable to heat. According to future scenarios for climate and land-use change, fire regimes will undergo transformations in semiarid terrestrial ecosystems, mainly in the Mediterranean Basin. To develop tools for forest management in fire-prone areas, i.e., fire prevention, we assessed the impact of prescribed burnings on soil microorganisms in Mediterranean mixed pine forests. We hypothesised that low severity fire burns would not influence the functional diversity of soil microorganisms, although the burning season could influence that response due to seasonal variations in its vulnerability. We used the Biolog EcoPlate System to record soil biological indicators and assess the effect of the prescribed burning season (early or late season) on bacterial communities, including the soil-plant interphase. The soil microbiome response differed significantly according to vegetation coverage but prescribed burning season was not directly related. Burning increased the proportions of soil organic matter and soil organic carbon, and also promoted cation-exchange capacity and total phosphorus, which were higher following spring burns. Microbial richness and the Shannon-Weaver diversity index both showed a positive correlation with vegetation cover. However, microbial richness was triggered after burning uncovered patches of vegetation. We also noted differences in the usage pattern for the six substrate groups defined in our study: the use of carboxylic acids, amino acids and carbohydrates was higher in unburned plots and those subject to late burns, whereas amino acids did not predominate in early burn plots.

El espacio muerto (VD/VT) como predictor de éxito en la extubación de pacientes postquirúrgicos de cirugía cardiovascular pediátrica / Dead space (VD/VT) as a predictor of successful extubation of patients after pediatric cardiovascular surgery

Introducción: La ventilación mecánica (VM) forma parte de la recuperación postoperatoria (PO) de niños con cirugía de cardiopatía congénita, pero su uso no está exento de riesgos. El fracaso de extubación (FE) se ha asociado con internaciones prolongadas, aumento de complicaciones y mortalidad. El objetivo es determinar un valor de Vd/Vt predictor de extubación exitosa (EE). Material y métodos: estudio de cohorte prospectivo y observacional realizado del 1 de Enero al 31 de Diciembre de 2016 en niños menores de 6 meses cursando PO de cirugía cardiovascular con circulación extracorpórea (CEC) con requerimientos de VM por más de 48 horas. En los mismo se analizó el éxito o fracaso de extubación. Previo a la extubación se registraron valores de mecánica respiratoria; Vd/Vt, CO2 espiratoria final, Vt/kg, etc. Otras variables registradas: edad, sexo, peso, requerimiento de VM antes de la cirugía, fisiología de ventrículo único, duración de VM, complicaciones, duración de la internación y mortalidad. Las variables continuas se describieron como mediana y rango intercuartilo (25-75) y se compararon con prueba de Wilcoxon, las categóricas como proporciones o porcentajes y se analizaron con chi2 . Se efectuó un análisis bivariado con diferentes puntos de corte de Vd/Vt pre extubación para realizar un análisis de sensibilidad del valor predictivo de EE. Resultados: Se evaluó Vd/Vt en 67 pacientes, tres se eliminaron por parálisis cordal (1) y parálisis del diafragma (2). Mediana de edad 23 días (10-55), peso 3.2 Kg (2.89- 3.88), días de VM 5 (3-7), días de internación 15 (2- 128), mortalidad 7,8%. Se extubaron con éxito 76% de los pacientes (50/64). Las características demográficas de los pacientes, la mecánica respiratoria, gases de sangre arterial y EtCO2 no tuvieron asociación significativa con EE. Un Vd/Vt pre extubación < 0,53 se asoció con EE. Conclusión: En la población estudiada un valor de Vd/Vt <0,53 se asoció con EE. Los pacientes con ventrículo único presentaron mayor FE.(AU)

Introduction: Mechanical ventilation (MV) is part of postoperative (PO) recovery of children with congenital heart disease surgery, but is not without risks. Extubation failure (EF) has been associated with prolonged hospital stays and increased complication and mortality rates. The goal is to determine the value of Vd/Vt as a predictor of successful extubation (SE). Material and methods: A prospective and observational cohort study was conducted from January 1 to December 31, 2016, in children under 6 months of age undergoing cardiovascular surgery with extracorporeal circulation (ECC) and requiring MV for more than 48 hours. Intubation success or failure was evaluated. Prior to extubation, respiratory mechanics values, such as Vd/Vt, final expiratory CO2, and Vt/kg, were recorded. Other variables, including age, sex, weight, VM requirement before surgery, single ventricle physiology, VM duration, complications, length of hospital stay, and mortality were also recorded. Continuous variables were described as median and interquartile range (25-75) and compared with the Wilcoxon test. Categorical variables were described as proportions or percentages and analyzed with chi2. Bivariate analysis was performed with different pre-extubation Vd/Vt cut-off points to analyze the sensitivity of the predictive value for SE. Results: Vd/Vt was evaluated in 67 patients; three were excluded because of vocal fold (1) and diaphragm paralysis (2). Median age was 23 days (10-55), weight 3.2 Kg (2.89- 3.88), days on MV 5 (3-7), length of hospital stay 15 (2- 128), and mortality rate 7.8%. Overall, 76% of patients (50/64) were successfully extubated. Patient demographics, respiratory mechanics, arterial blood gases, and EtCO2 were not significantly associated with SE. A pre-extubation Vd/ Vt < 0.53 was associated with SE. Conclusion: In this series of patients, a Vd/Vt value of <0.53 was associated with SE. EF was increased in patients with a single ventricle (AU)

Development of two-photon polymerised scaffolds for optical interrogation and neurite guidance of human iPSC-derived cortical neuronal networks.

Recent progress in the field of human induced pluripotent stem cells (iPSCs) has led to the efficient production of human neuronal cell models for in vitro study. This has the potential to enable the understanding of live human cellular and network function which is otherwise not possible. However, a major challenge is the generation of reproducible neural networks together with the ability to interrogate and record at the single cell level. A promising aid is the use of biomaterial scaffolds that would enable the development and guidance of neuronal networks in physiologically relevant architectures and dimensionality. The optimal scaffold material would need to be precisely fabricated with submicron resolution, be optically transparent, and biocompatible. Two-photon polymerisation (2PP) enables precise microfabrication of three-dimensional structures. In this study, we report the identification of two biomaterials that support the growth and differentiation of human iPSC-derived neural progenitors into functional neuronal networks. Furthermore, these materials can be patterned to induce alignment of neuronal processes and enable the optical interrogation of individual cells. 2PP scaffolds with tailored topographies therefore provide an effective method of producing defined in vitro human neural networks for application in influencing neurite guidance and complex network activity.

RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos.

Targeted knock-in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)-assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR-assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS-1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single-stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS-1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS-1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS-1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.