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Assembling molecular machines into crystals provides a way to harness their power on large length scales, but the development of a crystal analogue to a molecular motor remains a challenge. The molecule (Z)-5-(anthracen-9-ylmethylene)-3-butylthiazolidine-2,4-dione (C4-ATD) has E and Z isomers with strongly overlapping absorption spectra. This spectroscopic property allows both Z â E and E â Z photoisomerization reactions to be driven by a single light source, and simulations indicate this property can provide a route to robust oscillatory motion. Reprecipitation in an aqueous surfactant enables the growth of single crystal microwires that exhibit continuous mechanical oscillations under a wide range of illumination conditions, including ambient solar irradiation. Molecular crystal motors provide a new approach for transforming continuous light into oscillatory mechanical motion.
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2-Azidoanthracene (2N3-AN) can act as a photochemical source of N2 gas when dissolved in an optically transparent polymer such as poly(methyl methacrylate) (PMMA). Irradiation at 365 or 405 nm of a 150 µm-thick polymer film submerged in water causes the rapid appearance of a surface layer of bubbles. The rapid appearance of surface bubbles cannot be explained by normal diffusion of N2 through the polymer and likely results from internal gas pressure buildup during the reaction. For an azide concentration of 0.1 M and a light intensity of 140 mW/cm2, the yield of gas bubbles is calculated to be approximately 40%. The dynamics of bubble growth depend on the surface morphology, light intensity, and 2N3-AN concentration. A combination of nanoscale surface roughness, high azide concentration, and high light intensity is required to attain the threshold N2 gas density necessary for rapid, high-yield bubble formation. The N2 bubbles adhered to the PMMA surface and survived for days under water. The ability to generate stable gas bubbles "on demand" using light permits the demonstration of photoinduced flotation and patterned bubble arrays.
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RESEARCH QUESTION: Is low serum 25-hydroxyvitamin D (25(OH)D) associated with an increased risk of miscarriage in women who presented with threatened miscarriage to the Early Pregnancy Assessment Clinic (EPAC)? DESIGN: This was a secondary retrospective analysis using archived serum samples from a randomized, double-blind, placebo-controlled trial. Stored serum samples from 371 women presenting to the EPAC with threatened miscarriage during the first trimester were assayed for 25(OH)D by liquid chromatography-mass spectrometry. RESULTS: The overall miscarriage rate was 45/371 (12.1%) in the whole cohort. After grouping vitamin D insufficiency and vitamin D sufficiency together into a 'non-deficient' group and excluding participants who underwent termination of pregnancy, there was no difference in the miscarriage rate between those who were vitamin D deficient compared with those who were not (25/205, 12.2% versus 20/157, 12.7%, P= 0.877, odds ratio 0.951, 95% CI 0.507-1.784). When analysed according to the number of gestational weeks, the miscarriage rate was significantly higher in the vitamin D non-deficient group than the vitamin D-deficient group in women who presented at 6 gestational weeks or earlier (13/33 [39.4%] versus 10/58 [17.2%], P= 0.019), but there were no statistically significant differences between the two groups presenting at later gestations. There was no difference in the vitamin D level in women who had a miscarriage compared with those who had a live birth (48 [37-57] versus 47 [37-58] nmol/l, P= 0.725 median [25th-75th percentile]). CONCLUSIONS: A low serum vitamin D concentration was not associated with an increased risk of miscarriage in women with threatened miscarriage presenting to the EPAC.
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Ameaça de Aborto , Primeiro Trimestre da Gravidez , Deficiência de Vitamina D , Vitamina D , Humanos , Feminino , Gravidez , Vitamina D/sangue , Vitamina D/análogos & derivados , Primeiro Trimestre da Gravidez/sangue , Adulto , Estudos Retrospectivos , Ameaça de Aborto/sangue , Ameaça de Aborto/epidemiologia , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/epidemiologia , Aborto Espontâneo/sangue , Aborto Espontâneo/epidemiologia , Método Duplo-CegoRESUMO
PURPOSE: This study aimed to optimize the non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) in the laboratory by comparing two collection timing of the spent culture medium (SCM), two embryo rinsing protocols, and the use of conventional insemination instead of intracytoplasmic sperm injection (ICSI). METHODS: Results of two embryo rinsing methods (one-step vs sequential) and SCM collected on day 5 vs day 6 after retrieval were compared against trophectoderm (TE) biopsies as reference. Results from day 6 SCM in cycles fertilized by conventional insemination were compared with PGT-A using ICSI. RESULTS: The rate of concordance was higher in day 6 samples than in day 5 samples when the sequential method was used, in terms of total concordance (TC; day 6 vs day 5: 85.0% vs 60.0%, p = 0.0228), total concordance with same sex (TCS, 82.5% vs 28,0%, p < 0.0001), and full concordance with same sex (FCS, 62.5% vs 24.0%, p = 0.0025). The sequential method significantly out-performed the one-step method when SCM were collected on day 6 (sequential vs one-step, TC: 85.0% vs 64.5%, p = 0.0449; TCS: 82.5% vs 54.8%, p = 0.0113; FCS: 62.5% vs 25.8%, p = 0.0021). There was no significant difference in niPGT-A results between cycles fertilized by the conventional insemination and ICSI. CONCLUSION: We have shown a higher concordance rate when SCM was collected on day 6 and the embryos were rinsed in a sequential manner. Comparable results of niPGT-A when oocytes were fertilized by conventional insemination or ICSI. These optimization steps are important prior to commencement of a randomized trial in niPGT-A.
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Fertilização in vitro , Diagnóstico Pré-Implantação , Gravidez , Feminino , Masculino , Humanos , Diagnóstico Pré-Implantação/métodos , Sêmen , Testes Genéticos/métodos , Injeções de Esperma Intracitoplásmicas/métodos , Aneuploidia , Blastocisto/patologiaRESUMO
BACKGROUND: A large proportion of Alzheimer's disease (AD) patients have coexisting subcortical vascular dementia (SVaD), a condition referred to as mixed dementia (MixD). Brain imaging features of MixD presumably include those of cerebrovascular disease and AD pathology, but are difficult to characterize due to their heterogeneity. OBJECTIVE: To perform an exploratory analysis of conventional and non-conventional structural magnetic resonance imaging (MRI) abnormalities in MixD and to compare them to those observed in AD and SVaD. METHODS: We conducted a cross-sectional, region-of-interest-based analysis of 1) hyperintense white-matter signal abnormalities (WMSA) on T2-FLAIR and hypointense WMSA on T1-weighted MRI; 2) diffusion tensor imaging; 3) quantitative susceptibility mapping; and 4) effective transverse relaxation rate (R2*) in N = 17 participants (AD:5, SVaD:5, MixD:7). General linear model was used to explore group differences in these brain imaging measures. RESULTS: Model findings suggested imaging characteristics specific to our MixD group, including 1) higher burden of WMSAs on T1-weighted MRI (versus both AD and SVaD); 2) frontal lobar preponderance of WMSAs on both T2-FLAIR and T1-weighted MRI; 3) higher fractional anisotropy values within normal-appear white-matter tissues (versus SVaD, but not AD); and 4) lower R2* values within the T2-FLAIR WMSA areas (versus both AD and SVaD). CONCLUSION: These findings suggest a preliminary picture of the location and type of brain imaging characteristics associated with MixD. Future imaging studies may employ region-specific hypotheses to distinguish MixD more rigorously from AD or SVaD.
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Doença de Alzheimer , Demência Vascular , Demências Mistas , Humanos , Demência Vascular/diagnóstico por imagem , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/patologia , Imagem de Tensor de Difusão , Estudos Transversais , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Imageamento por Ressonância Magnética/métodosRESUMO
Hydrogenation reactions are staple transformations commonly used across scientific fields to synthesise pharmaceuticals, natural products, and various functional materials. However, the vast majority of these reactions require the use of a toxic and costly catalyst leading to unpractical, hazardous and often functionally limited conditions. Herein, we report a new, general, practical, efficient, mild and high-yielding hydrogen-free electrochemical method for the reduction of alkene, alkyne, nitro and azido groups. Finally, this method has been applied to deuterium labelling.
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Nitriles are recurring motifs in bioactive molecules and versatile functional groups in synthetic chemistry. Despite recent progress, direct introduction of a nitrile moiety in heteroarenes remains challenging. Recent developments in electrochemical reactions pave the way to more practical cyanation protocols. However, currently available methods typically require hazardous cyanide sources, expensive mediators, and often suffer from narrow substrate scope and laborious reaction set-up. To address the limitations of current synthetic methods, herein, an effective, sustainable, and scalable procedure for the direct C(sp2 )-H cyanation of aromatic N-heterocycles with a user-friendly flow-electrochemical set-up is reported. Furthermore, high substrate and functional-group tolerance is demonstrated, allowing late-stage functionalization of drug-like scaffolds, such as natural products and pharmaceuticals.
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Produtos Biológicos , Nitrilas , CianetosRESUMO
Experimental measurements of the thermal effects of the same osmolytes on two different globular proteins, C-reactive protein (CRP) and tumor necrosis factor alpha (TNFα), have shown that quantifying the change in the denaturing temperature leads to some results that are unique to each protein. In order to find osmolyte-dependent parameters that can be applied more consistently from protein to protein, this work considers, instead, the overall free energy change associated with that denaturation using coarse-grained models. This is enabled by using theoretical fluid equations that take into account the exclusion of water and osmolyte from the volume occupied by the protein in both its native and denatured forms. Assuming ideal geometric models of the two protein states whose sizes are based on the protein's surface area in each form, and taking into account the density of the aqueous osmolyte solution, the free energy change due to the change in geometry can be calculated. The overall change in free energy of the system is found from that quantity and other protein- and osmolyte-specific parameters, which are determined using the experimental concentration and temperature results. We find that these fitted parameters accurately reproduce experimental results and also show consistent patterns from protein to protein. We also consider two different model geometries of the denatured protein and find little impact on the use of one or the other. Defining the effects of the osmolyte in terms of free energy also allows for prediction of overall phase change behavior, including cold denaturation.
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Proteínas , Concentração Osmolar , Desnaturação Proteica , Proteínas/metabolismo , Temperatura , TermodinâmicaRESUMO
Intracytoplasmic sperm injection (ICSI) is commonly used to treat severe male factor infertility in assisted reproduction. A small percentage of patients face suboptimal fertilisation rate or even fertilisation failure despite having ICSI. Artificial oocyte activation (AOA) has been proposed as a suitable method to overcome their problem. This is a retrospective cohort analysis of ICSI cycles undergoing AOA. Injected metaphase II oocytes were exposed to either calcium ionophore (A23187) after ICSI or injection of calcium chloride during ICSI followed by incubation with A23187 after ICSI. The previous ICSI cycles of the patients formed the historical control group. Thirty-four AOA cycles were analysed. The normal fertilisation rate (52.1%) was significantly improved in the AOA group. The percentage of failed fertilisation cycles (11.8%) were significantly reduced in the AOA group. The cumulative clinical pregnancy rate (47.1%) and live birth rate (29.4%) were significantly increased when compared to the previous cycles. Subgroup analysis revealed that the performance of the A23187 only protocol and the concomitant injection of calcium chloride protocol were comparable in terms of laboratory parameters and pregnancy outcomes. AOA is an effective method to improve the fertilisation rate and pregnancy outcome of infertile couples with previous fertilisation problem after ICSI.IMPACT STATEMENTWhat is already known on this subject? A failed and low fertilisation rate after ICSI is not uncommon in assisted reproduction. AOA is normally used to improve fertilisation but there are discrepancies in the efficacy of the treatment.What do the results of this study add? AOA improves the fertilisation rate and pregnancy outcomes of couples with suboptimal fertilisation rate and fertilisation failure in previous ICSI cycles. The efficacies of two AOA protocols were comparable. The A23187 only protocol was recommended because of its simplicity.What are the implications of these findings for clinical practice and/or further research? AOA should be considered as a routine procedure for infertile couples with compromised fertilisation rates in previous ICSI cycles.
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Infertilidade Masculina , Injeções de Esperma Intracitoplásmicas , Calcimicina/uso terapêutico , Cloreto de Cálcio , Feminino , Fertilização in vitro/métodos , Humanos , Infertilidade Masculina/terapia , Masculino , Oócitos/fisiologia , Gravidez , Taxa de Gravidez , Estudos Retrospectivos , Injeções de Esperma Intracitoplásmicas/métodosRESUMO
Electrosynthetic organic chemistry is an old discipline that takes its root in Faraday's seminal works. The field has a rich history and is in the midst of a renaissance, due to the growing impetus of the chemical community to develop greener, more economical, and more efficient synthetic methodologies. Indeed, electrosynthesis relies on one of the greenest and cheapest reagents in the world: the electron itself. In this Account, the recent developments in the use of carboxylic acid derivatives in electrosynthesis are summarized. Until lately, the fate of the monoelectronic reduction of aromatic esters in nonprotic solvents remained unclear. Recent investigations have shown that aromatic esters are reduced and form surprisingly long-lived radical anions. Under the right conditions, these radical anions decompose into the corresponding carboxylates and alkyl radicals. These principles have been used to develop a novel electrochemical alcohol deoxygenation reaction using aromatic esters as stable and versatile radical precursors. In contrast to esters, the electrochemistry of carboxylic acids has been intensively studied. Pioneering works by Faraday and Kolbe in the late 1800s have revealed that the anodic oxidation of carboxylic acids leads to a radical decarboxylation. Interestingly, radical recombination is observed due to the very high concentration of radicals in the vicinity of the electrode. Such radical recombination is rarely observed during classical homogeneous radical reactions. The "Kolbe" reaction and its carbocationic variation have been intensively used across the fields due to their versatility. As we will develop in this Account, almost two hundred years after its discovery, the anodic decarboxylation of carboxylic acids is still relevant to modern organic chemists. For instance, we will examine how the non-decarboxylate Kolbe reaction of aromatic acids forms aroyloxy radicals and how oxycarbonyl radicals could be generated from hemioxalates. Finally, the carbocationic variant of the Kolbe reaction, known as the Hofer-Moest reaction, will be examined in the context of two newly developed reactions: a green MOM-type ether formation and the use of malonic acid derivatives as carbonyl synthons. Electrosynthesis is a powerful synthetic tool. Even if it might still be underutilized at the moment, there is little doubt that it will become one of the "classic" methods to activate small organic molecules in a very near future.
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The hybrid bidentate 1-(2-pyridyl)benzotriazole (pyb) ligand was introduced into 3d transition metal catalysis. Specifically, [CuII (OTf)2 (pyb)2 ]â 2 CH3 CN (1) enables the synthesis of a wide range of propargylamines by the A3 coupling reaction at room temperature in the absence of additives. Experimental and high-level theoretical calculations suggest that the bridging N atom of the ligand imposes exclusive trans coordination at Cu and allows ligand rotation, while the N atom of the pyridine group modulates charge distribution and flux, and thus orchestrates structural and electronic precatalyst control permitting alkyne binding with simultaneous activation of the C-H bond via a transient CuI species.
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RESEARCH QUESTION: Is PIEZO-intracytoplasmic sperm injection (ICSI) coupled with a new novel operational fluid (perfluoro-n-octane) superior to standard ICSI? DESIGN: A cohort of patients (nâ¯=â¯69) undertaking microinjection were recruited between January and November 2019 and were then prospectively case-matched. Patients required six or more mature oocytes for inclusion in the study. PIEZO-ICSI uses high-speed microinjection drilling to penetrate the zona and oolemma and deposit the spermatozoa into the cytoplasm, compared with the traditional 'cutting' action of ICSI. The primary outcome was fertilization, with secondary outcomes including oocyte degeneration, abnormal fertilization, embryo cryopreservation and embryo utilization. RESULTS: PIEZO-ICSI resulted in significantly higher fertilization rates (80.5 ± 2.4% vs 65.8 ± 2.3%, P < 0.0001) and lower oocyte degeneration rates (4.4 ± 1.3% vs 8.6 ± 1.2%, Pâ¯=â¯0.019) and abnormal fertilization rates (2.9 ± 1.1% vs 7.4 ± 1.1%; Pâ¯=â¯0.003) compared with standard ICSI. This improvement in fertilization was of most benefit in patients aged ≥38 years. This increase in fertilization increased the number of good quality embryos that were available for cryopreservation/transfer (3.8 ± 0.2 vs 3.1 ± 0.2; P = 0.038), such that patients on average had one extra usable embryo per cycle compared with standard ICSI. There were no differences to Day 5 embryo development or clinical pregnancy from fresh embryo transfer (57.1% PIEZO-ICSI vs 60.0% ICSI) between microinjection methods, although pregnancy outcomes were underpowered. CONCLUSIONS: PIEZO-ICSI significantly increased fertilization rates, thereby increasing the number of embryos available for cryopreservation compared with standard ICSI. Further prospective studies assessing cumulative pregnancy rates are warranted.
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Fertilização/fisiologia , Taxa de Gravidez , Injeções de Esperma Intracitoplásmicas/métodos , Adulto , Austrália/epidemiologia , Estudos de Coortes , Feminino , Fertilização in vitro/métodos , Humanos , Infertilidade/epidemiologia , Infertilidade/terapia , Masculino , Idade Materna , Gravidez , Resultado da Gravidez/epidemiologia , Injeções de Esperma Intracitoplásmicas/normas , Padrão de CuidadoRESUMO
A library of glycoforms of human interleukin 6 (IL-6) comprising complex and mannosidic N-glycans was generated by semisynthesis. The three segments were connected by sequential native chemical ligation followed by two-step refolding. The central glycopeptide segments were assembled by pseudoproline-assisted Lansbury aspartylation and subsequent enzymatic elongation of complex N-glycans. Nine IL-6 glycoforms were synthesized, seven of which were evaluated for in vivo plasma clearance in rats and compared to non-glycosylated recombinant IL-6 from E. coli. Each IL-6 glycoform was tested in three animals and reproducibly showed individual serum clearances depending on the structure of the N-glycan. The clearance rates were atypical, since the 2,6-sialylated glycoforms of IL-6 cleared faster than the corresponding asialo IL-6 with terminal galactoses. Compared to non-glycosylated IL-6 the plasma clearance of IL-6 glycoforms was delayed in the presence of larger and multibranched N-glycans in most cases.
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Glicopeptídeos/metabolismo , Interleucina-6/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Galactose/metabolismo , Glicopeptídeos/sangue , Glicopeptídeos/genética , Glicosilação , Humanos , Interleucina-6/sangue , Interleucina-6/genética , Interleucina-6/farmacologia , Camundongos , Ácido N-Acetilneuramínico/metabolismo , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The titratable acidity, alkalinity, and carboxylate content are fundamental properties required for the understanding of aqueous chemical systems. Here, we present a set of new methods that allow these properties to be determined directly by 1H NMR without the labor, cost, and sample quantity associated with running separate potentiometric or conductometric titrations. Our methods require only the measurement of the pH-sensitive 1H chemical shifts of indicator molecules and do not require the tedious titration of reagents into a sample. To determine the titratable acidity, an excess of 2-methylimidazole (2MI) is added to a sample and the quantity of protons absorbed by 2MI is determined from its 1H chemical shifts. The titratable alkalinity of a sample can be similarly determined using acetic acid. To determine the concentration of deprotonated carboxylates, a sample is acidified with HCl, and the quantity of H+ absorbed is determined from the 1H chemical shift of methylphosphonic acid. We validate our methods by demonstrating the measurement of the acidity of fruit-flavored drinks, the alkalinity of tap water, and the carboxylate content of nanocellulose dispersions.
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Guidelines recommend annual dermatology screening after solid organ transplantation to facilitate early detection of keratinocyte carcinoma (nonmelanoma skin cancer), the most common posttransplant malignancy. There are limited data on adherence levels and barriers to screening. We conducted a cross-sectional survey of 477 physicians and nurses providing posttransplant care in Canada. The questionnaire asked about skin cancer screening and education practices, including the perceived importance and barriers. Whereas care providers viewed skin cancer screening as important for adult patients (median rating of 10/10, interquartile range 8-10), only 53% ensured annual screening for white adult transplant recipients. Having a screening policy in place (adjusted odds ratio 6.78, 95% confidence interval 3.12-14.74) and a dermatologist present at the transplant center (adjusted odds ratio 2.19, 95% confidence interval 1.03-4.67) were independently associated with higher adherence. Long wait times, lack of specialized transplant dermatologists, long travel distances, and insufficient priority were cited as the most common barriers for access to dermatologic care. Skin cancer education was provided to patients by over three quarters of care providers. Given the self-reported lack of adherence to annual skin cancer screening, there is need to develop, evaluate, and implement interventions that improve screening rates and skin cancer outcomes.
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Dermatologia/normas , Detecção Precoce de Câncer , Transplante de Órgãos , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/prevenção & controle , Adulto , Canadá , Criança , Estudos Transversais , Feminino , Humanos , Masculino , Enfermeiras e Enfermeiros , Cooperação do Paciente , Educação de Pacientes como Assunto , Médicos , Período Pós-Operatório , Fatores de Risco , Neoplasias Cutâneas/complicações , Inquéritos e Questionários , Transplantados , Resultado do TratamentoRESUMO
Human spermatozoa can fertilize an oocyte only after post-testicular maturation and capacitation. These processes involve dynamic modification and reorganization of the sperm plasma membrane, which allow them to bind to the zona pellucida (ZP) of the oocyte. Defective sperm-ZP binding is one of the major causes of male subfertility. Galectin-3 is a secretory lectin in human seminal plasma well known for its action on cell adhesion. The aim of this study was to determine the role of galectin-3 in spermatozoa-ZP interaction and its association with fertilization rate in clinical assisted reproduction. Our studies revealed that the acrosomal region of ejaculated and capacitated spermatozoa possess strong galectin-3 immunoreactivity, which is much stronger than that of epididymal spermatozoa. Expression of galectin-3 can also be detected on seminal plasma-derived extracellular vesicles (EVs) and can be transferred to the sperm surface. Blocking of sperm surface galectin-3 function by antibody or carbohydrate substrate reduced the ZP-binding capacity of spermatozoa. Purified galectin-3 is capable of binding to ZP, indicating that galectin-3 may serve as a cross-linking bridge between ZP glycans and sperm surface glycoproteins. Galectin-3 levels in seminal plasma-derived EVs were positively associated with fertilization rates. These results suggest that galectin-3 in EVs is transferred to the sperm surface during post-testicular maturation and plays a crucial role in spermatozoa-ZP binding after capacitation. Reduced galectin-3 expression in seminal plasma-derived EVs may be a cause behind a low fertilization rate. Further studies with more clinical samples are required to confirm the relationship between galectin-3 levels and IVF outcomes.
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Fertilização/fisiologia , Galectina 3/metabolismo , Zona Pelúcida/metabolismo , Reação Acrossômica/genética , Reação Acrossômica/fisiologia , Adesão Celular/fisiologia , Fertilização/genética , Galectina 3/genética , Humanos , Masculino , Oócitos/metabolismo , Sêmen/metabolismo , Capacitação Espermática/fisiologia , Interações Espermatozoide-Óvulo/genética , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/metabolismoRESUMO
STUDY QUESTION: Does glycodelin-A (GdA) induce conversion of human peripheral blood CD16-CD56bright natural killer (NK) cells to decidual NK (dNK) cells to facilitate placentation? SUMMARY ANSWER: GdA binds to blood CD16-CD56bright NK cells via its sialylated glycans and converts them to a dNK-like cells, which in turn regulate endothelial cell angiogenesis and trophoblast invasion via vascular endothelial growth factor (VEGF) and insulin-like growth factor-binding protein 1 (IGFBP-1) secretion, respectively. WHAT IS KNOWN ALREADY: dNK cells are the most abundant leucocyte population in the decidua. These cells express CD16-CD56bright phenotype. Peripheral blood CD16-CD56bright NK cells and hematopoietic precursors have been suggested to be capable of differentiating towards dNK cells upon exposure to the decidual microenvironment. These cells regulate trophoblast invasion during spiral arteries remodelling and mediate homoeostasis and functions of the endothelial cells. GdA is an abundant glycoprotein in the human decidua with peak expression between the 6th and 12th week of gestation, suggesting a role in early pregnancy. Indeed, GdA interacts with and modulates functions and differentiation of trophoblast and immune cells in the human feto-maternal interface. Aberrant GdA expression during pregnancy is associated with unexplained infertility, pregnancy loss and pre-eclampsia. STUDY DESIGN, SIZE, DURATION: CD16+CD56dim, CD16-CD56bright and dNK cells were isolated from human peripheral blood and decidua tissue, respectively, by immuno-magnetic beads or fluorescence-activated cell sorting. Human extravillous trophoblasts were isolated from first trimester placental tissue after termination of pregnancy. Biological activities of the cells were studied after treatment with GdA at a physiological dose of 5 µg/mL. GdA was purified from human amniotic fluid by immuno-affinity chromatography. PARTICIPANTS/MATERIALS, SETTING, METHODS: Expression of VEGF, CD9, CD49a, CD151 and CD158a in the cells were determined by flow cytometry. Angiogenic proteins in the spent media of NK cells were determined by cytokine array and ELISA. Blocking antibodies were used to study the functions of the identified angiogenic proteins. Endothelial cell angiogenesis was determined by tube formation and trans-well migration assays. Cell invasion and migration were determined by trans-well invasion/migration assay. Binding of normal and de-sialylated GdA, and expression of L-selectin and siglec-7 on the NK cells were analysed by flow cytometry. The association between GdA and L-selectin on NK cells was confirmed by immunoprecipitation. Extracellular signal-regulated protein kinases (ERK) activation was determined by Western blotting and functional assays. MAIN RESULTS AND THE ROLE OF CHANCE: GdA treatment enhanced the expression of dNK cell markers CD9 and CD49a and the production of the functional dNK secretory product VEGF in the peripheral blood CD16-CD56bright NK cells. The spent media of GdA-treated CD16-CD56bright NK cells promoted tube formation of human umbilical vein endothelial cells and invasiveness of trophoblasts. These stimulatory effects were mediated by the stimulatory activities of GdA on an ERK-activation dependent production of VEGF and IGFBP-1 by the NK cells. GdA had a stronger binding affinity to the CD16-CD56bright NK cells as compared to the CD16+CD56dim NK cells. This GdA-NK cell interaction was reduced by de-sialylation. GdA interacted with L-selectin, expressed only in the CD16-CD56bright NK cells, but not in the CD16+CD56dim NK cells. Anti-L-selectin functional blocking antibody suppressed the binding and biological activities of GdA on the NK cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Some of the above findings are based on a small sample size of peripheral blood CD16-CD56bright NK cells. These results need to be confirmed with human primary dNK cells. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study on the biological role of GdA on conversion of CD16-CD56bright NK cells to dNK-like cells. Further investigation on the glycosylation and functions of GdA will enhance our understanding on human placentation and placenta-associated complications with altered NK cell biology. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Hong Kong Research Grant Council Grant 17122415, Sanming Project of Medicine in Shenzhen, the Finnish Cancer Foundation, Sigrid Jusélius Foundation and the Finnish Society of Clinical Chemistry. The authors have no competing interests to declare.
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Antígeno CD56/metabolismo , Decídua/citologia , Decídua/metabolismo , Glicodelina/farmacologia , Células Matadoras Naturais/metabolismo , Fenótipo , Receptores de IgG/metabolismo , Líquido Amniótico/química , Doadores de Sangue , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Proteínas Ligadas por GPI/metabolismo , Glicodelina/isolamento & purificação , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Selectina L/metabolismo , Neovascularização Fisiológica , Gravidez , Primeiro Trimestre da Gravidez , Transdução de Sinais/efeitos dos fármacos , Trofoblastos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
In vivo, keratocytes are surrounded by aligned type I collagen fibrils that are organized into lamellae. A growing body of literature suggests that the unique topography of the corneal stroma is an important regulator of keratocyte behavior. In this study we describe a microfluidic method to deposit aligned fibrils of type I collagen onto glass coverslips. This high-throughput method allowed for the simultaneous coating of up to eight substrates with aligned collagen fibrils. When these substrates were integrated into a PDMS microwell culture system they provided a platform for high-resolution imaging of keratocyte behavior. Through the use of wide-field fluorescence and differential interference contrast microscopy, we observed that the density of collagen fibrils deposited was dependent upon both the perfusion shear rate of collagen and the time of perfusion. In contrast, a similar degree of fibril alignment was observed over a range of shear rates. When primary normal rabbit keratocytes (NRK) were seeded on substrates with a high density of aligned collagen fibrils and cultured in the presence of platelet derived growth factor (PDGF) the keratocytes displayed an elongated cell body that was co-aligned with the underlying collagen fibrils. In contrast, when NRK were cultured on substrates with a low density of aligned collagen fibrils, the cells showed no preferential orientation. These results suggest that this simple and inexpensive method can provide a general platform to study how simultaneous exposure to topographical and soluble cues influence cell behavior.
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Colágeno/metabolismo , Ceratócitos da Córnea/citologia , Ceratócitos da Córnea/metabolismo , Dispositivos Lab-On-A-Chip , Animais , Fenômenos Biomecânicos , Coelhos , Resistência ao CisalhamentoRESUMO
A new electrochemical methodology has been developed for the generation of oxycarbonyl radicals under mild and green conditions from readily available hemioxalate salts. Mono- and multi-functionalised γ-butyrolactones were synthesised through exo-cyclisation of these oxycarbonyl radicals with an alkene, followed by the sp3 -sp3 capture of the newly formed carbon-centred radical. The synthesis of functionalised valerolactone derivatives was also achieved, demonstrating the versatility of the newly developed methodology. This represents a viable synthetic route towards pharmaceutically important fragments and further demonstrates the practicality of electrosynthesis as a green and economical method to activate small organic molecules.