Gradient lithography of engineered proteins to fabricate 2D and 3D cell culture microenvironments

Spatial patterning of proteins is a valuable technique for many biological applications and is the prevailing tool for defining microenvironments for cells in culture, a required procedure in developmental biology and tissue engineering research. However, it is still challenging to achieve protein patterns that closely mimic native microenvironments, such as gradient protein distributions with desirable mechanical properties. By combining projection dynamic mask lithography and protein engineering with non-canonical photosensitive amino acids, we demonstrate a simple, scalable strategy to fabricate any user-defined 2D or 3D stable gradient pattern with complex geometries from an artificial extracellular matrix (aECM) protein. We show that the elastic modulus and chemical nature of the gradient profile are biocompatible and allow useful applications in cell biological research

Weak Glycolipid Binding of a Microdomain-Tracer Peptide Correlates with Aggregation and Slow Diffusion on Cell Membranes

10.1371/journal.pone.0051222PLoS ONE712

Mammalian BTBD12 (SLX4) Protects against Genomic Instability during Mammalian Spermatogenesis

The mammalian ortholog of yeast Slx4, BTBD12, is an ATM substrate that functions as a scaffold for various DNA repair activities. Mutations of human BTBD12 have been reported in a new sub-type of Fanconi anemia patients. Recent studies have implicated the fly and worm orthologs, MUS312 and HIM-18, in the regulation of meiotic crossovers arising from double-strand break (DSB) initiating events and also in genome stability prior to meiosis. Using a Btbd12 mutant mouse, we analyzed the role of BTBD12 in mammalian gametogenesis. BTBD12 localizes to pre-meiotic spermatogonia and to meiotic spermatocytes in wildtype males. Btbd12 mutant mice have less than 15% normal spermatozoa and are subfertile. Loss of BTBD12 during embryogenesis results in impaired primordial germ cell proliferation and increased apoptosis, which reduces the spermatogonial pool in the early postnatal testis. During prophase I, DSBs initiate normally in Btbd12 mutant animals. However, DSB repair is delayed or impeded, resulting in persistent γH2AX and RAD51, and the choice of repair pathway may be altered, resulting in elevated MLH1/MLH3 focus numbers at pachynema. The result is an increase in apoptosis through prophase I and beyond. Unlike yeast Slx4, therefore, BTBD12 appears to function in meiotic prophase I, possibly during the recombination events that lead to the production of crossovers. In line with its expected regulation by ATM kinase, BTBD12 protein is reduced in the testis of Atm−/− males, and Btbd12 mutant mice exhibit increased genomic instability in the form of elevated blood cell micronucleus formation similar to that seen in Atm−/− males. Taken together, these data indicate that BTBD12 functions throughout gametogenesis to maintain genome stability, possibly by co-ordinating repair processes and/or by linking DNA repair events to the cell cycle via ATM

Growth of a human mammary tumor cell line is blocked by galangin, a naturally occurring bioflavonoid, and is accompanied by down-regulation of cyclins D3, E, and A

INTRODUCTION: This study was designed to determine if and how a non-toxic, naturally occurring bioflavonoid, galangin, affects proliferation of human mammary tumor cells. Our previous studies demonstrated that, in other cell types, galangin is a potent inhibitor of the aryl hydrocarbon receptor (AhR), an environmental carcinogen-responsive transcription factor implicated in mammary tumor initiation and growth control. Because some current breast cancer therapeutics are ineffective in estrogen receptor (ER) negative tumors and since the AhR may be involved in breast cancer proliferation, the effects of galangin on the proliferation of an ER(-), AhR(high )line, Hs578T, were studied. METHODS: AhR expression and function in the presence or absence of galangin, a second AhR inhibitor, α-naphthoflavone (α-NF), an AhR agonist, indole-3-carbinol, and a transfected AhR repressor-encoding plasmid (FhAhRR) were studied in Hs578T cells by western blotting for nuclear (for instance, constitutively activated) AhR and by transfection of an AhR-driven reporter construct, pGudLuc. The effects of these agents on cell proliferation were studied by (3)H-thymidine incorporation and by flow cytometry. The effects on cyclins implicated in mammary tumorigenesis were evaluated by western blotting. RESULTS: Hs578T cells were shown to express high levels of constitutively active AhR. Constitutive and environmental chemical-induced AhR activity was profoundly suppressed by galangin as was cell proliferation. However, the failure of α-NF or FhAhRR transfection to block proliferation indicated that galangin-mediated AhR inhibition was either insufficient or unrelated to its ability to significantly block cell proliferation at therapeutically relevant doses (IC(50 )= 11 μM). Galangin inhibited transition of cells from the G(0)/G(1 )to the S phases of cell growth, likely through the nearly total elimination of cyclin D3. Expression of cyclins A and E was also suppressed. CONCLUSION: Galangin is a strong inhibitor of Hs578T cell proliferation that likely mediates this effect through a relatively unique mechanism, suppression of cyclin D3, and not through the AhR. The results suggest that this non-toxic bioflavonoid may be useful as a chemotherapeutic, particularly in combination with agents that target other components of the tumor cell cycle and in situations where estrogen receptor-specific therapeutics are ineffective

Predisposition to Cancer Caused by Genetic and Functional Defects of Mammalian Atad5

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5+/m) mice that were haploinsuffficient for Atad5. Atad5+/m mice displayed high levels of genomic instability in vivo, and Atad5+/m mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5+/m mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5+/m mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis

Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples

Morbidity and mortality after anaesthesia in early life: results of the European prospective multicentre observational study, neonate and children audit of anaesthesia practice in Europe (NECTARINE)

Background: Neonates and infants requiring anaesthesia are at risk of physiological instability and complications, but triggers for peri-anaesthetic interventions and associations with subsequent outcome are unknown. Methods: This prospective, observational study recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. The primary aim was to identify thresholds of pre-determined physiological variables that triggered a medical intervention. The secondary aims were to evaluate morbidities, mortality at 30 and 90 days, or both, and associations with critical events. Results: Infants (n=5609) born at mean (standard deviation [SD]) 36.2 (4.4) weeks postmenstrual age (35.7% preterm) underwent 6542 procedures within 63 (48) days of birth. Critical event(s) requiring intervention occurred in 35.2% of cases, mainly hypotension (&gt;30% decrease in blood pressure) or reduced oxygenation (SpO2 &lt;85%). Postmenstrual age influenced the incidence and thresholds for intervention. Risk of critical events was increased by prior neonatal medical conditions, congenital anomalies, or both (relative risk [RR]=1.16; 95% confidence interval [CI], 1.04–1.28) and in those requiring preoperative intensive support (RR=1.27; 95% CI, 1.15–1.41). Additional complications occurred in 16.3% of patients by 30 days, and overall 90-day mortality was 3.2% (95% CI, 2.7–3.7%). Co-occurrence of intraoperative hypotension, hypoxaemia, and anaemia was associated with increased risk of morbidity (RR=3.56; 95% CI, 1.64–7.71) and mortality (RR=19.80; 95% CI, 5.87–66.7). Conclusions: Variability in physiological thresholds that triggered an intervention, and the impact of poor tissue oxygenation on patient's outcome, highlight the need for more standardised perioperative management guidelines for neonates and infants. Clinical trial registration: NCT02350348

Difficult tracheal intubation in neonates and infants. NEonate and Children audiT of Anaesthesia pRactice IN Europe (NECTARINE): a prospective European multicentre observational study

Background: Neonates and infants are susceptible to hypoxaemia in the perioperative period. The aim of this study was to analyse interventions related to anaesthesia tracheal intubations in this European cohort and identify their clinical consequences. Methods: We performed a secondary analysis of tracheal intubations of the European multicentre observational trial (NEonate and Children audiT of Anaesthesia pRactice IN Europe [NECTARINE]) in neonates and small infants with difficult tracheal intubation. The primary endpoint was the incidence of difficult intubation and the related complications. The secondary endpoints were the risk factors for severe hypoxaemia attributed to difficult airway management, and 30 and 90 day outcomes. Results: Tracheal intubation was planned in 4683 procedures. Difficult tracheal intubation, defined as two failed attempts of direct laryngoscopy, occurred in 266 children (271 procedures) with an incidence (95% confidence interval [CI]) of 5.8% (95% CI, 5.1e6.5). Bradycardia occurred in 8% of the cases with difficult intubation, whereas a significant decrease in oxygen saturation (SpO2&lt;90% for 60 s) was reported in 40%. No associated risk factors could be identified among comorbidities, surgical, or anaesthesia management. Using propensity scoring to adjust for confounders, difficult anaesthesia tracheal intubation did not lead to an increase in 30 and 90 day morbidity or mortality. Conclusions: The results of the present study demonstrate a high incidence of difficult tracheal intubation in children less than 60 weeks post-conceptual age commonly resulting in severe hypoxaemia. Reassuringly, the morbidity and mortality at 30 and 90 days was not increased by the occurrence of a difficult intubation event. Clinical trial registration: NCT02350348

Morbidity and mortality after anaesthesia in early life: results of the European prospective multicentre observational study, neonate and children audit of anaesthesia practice in Europe (NECTARINE)

BACKGROUND: Neonates and infants requiring anaesthesia are at risk of physiological instability and complications, but triggers for peri-anaesthetic interventions and associations with subsequent outcome are unknown. METHODS: This prospective, observational study recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. The primary aim was to identify thresholds of pre-determined physiological variables that triggered a medical intervention. The secondary aims were to evaluate morbidities, mortality at 30 and 90 days, or both, and associations with critical events. RESULTS: Infants (n=5609) born at mean (standard deviation [sd]) 36.2 (4.4) weeks postmenstrual age (35.7% preterm) underwent 6542 procedures within 63 (48) days of birth. Critical event(s) requiring intervention occurred in 35.2% of cases, mainly hypotension (>30% decrease in blood pressure) or reduced oxygenation (SpO2 <85%). Postmenstrual age influenced the incidence and thresholds for intervention. Risk of critical events was increased by prior neonatal medical conditions, congenital anomalies, or both (relative risk [RR]=1.16; 95% confidence interval [CI], 1.04–1.28) and in those requiring preoperative intensive support (RR=1.27; 95% CI, 1.15–1.41). Additional complications occurred in 16.3% of patients by 30 days, and overall 90-day mortality was 3.2% (95% CI, 2.7–3.7%). Co-occurrence of intraoperative hypotension, hypoxaemia, and anaemia was associated with increased risk of morbidity (RR=3.56; 95% CI, 1.64–7.71) and mortality (RR=19.80; 95% CI, 5.87–66.7). CONCLUSIONS: Variability in physiological thresholds that triggered an intervention, and the impact of poor tissue oxygenation on patient's outcome, highlight the need for more standardised perioperative management guidelines for neonates and infants