The impact of mode of delivery on parent-infant-bonding and the mediating role of birth experience: a comparison of mothers and fathers within the longitudinal cohort study DREAM.

BACKGROUND: The association between mode of delivery (MOD) and parent-infant-bonding has only been studied in mothers and findings have been inconclusive. The aim of this study was to prospectively investigate how MOD relates to postpartum parent-infant-bonding in both mothers and fathers and whether these associations are mediated by birth experience. METHODS: This study is part of the prospective cohort study "Dresden Study on Parenting, Work, and Mental Health" (DREAM). Our sample comprised N = 1,780 participants who completed quantitative questionnaires during pregnancy as well as 8 weeks and 14 months postpartum. MOD was dummy coded, contrasting spontaneous vaginal delivery against vaginal delivery induced by drugs, operative vaginal delivery, planned, and unplanned cesarean section. Parent-infant bonding and birth experience were assessed using validated scales. A moderated mediation analysis based on ordinary least square (OLS) regression and bootstrapped estimates was conducted, considering relevant confounding variables. RESULTS: Compared to spontaneous vaginal delivery, all categories of MOD predicted more negative birth experiences in both parents. A more positive birth experience predicted stronger parent-infant-bonding at 8 weeks, but not at 14 months postpartum. Mothers who delivered via cesarean section (planned or unplanned) reported stronger parent-infant-bonding at 8 weeks and 14 months postpartum. In fathers, only unplanned cesarean section was associated with stronger parent-infant-bonding at 8 weeks postpartum. At 8 weeks postpartum, birth experience mediated the association between a vaginal delivery induced by drugs and a planned cesarean section and mother-infant-bonding and between a vaginal delivery induced by drugs, an operative vaginal delivery, and planned cesarean section and father-infant-bonding. At 14 months postpartum, birth experience mediated the association between a vaginal delivery induced by drugs, operative vaginal delivery, and planned cesarean section and parent-infant-bonding in both parents. CONCLUSIONS: The results emphasize the importance of the birth experience for parent-infant-bonding in both mothers and fathers. Further research should address the mechanisms by which parents with an unplanned cesarean section establish stronger parent-infant-bonding compared to parents whose baby was delivered via spontaneous vaginal delivery, despite their overall more negative birth experiences.

Psychosocial work stress and parent-child bonding during the COVID-19 pandemic: clarifying the role of parental symptoms of depression and aggressiveness.

BACKGROUND: Parental work stress and impaired mental health seem to have intensified during the current COVID-19 pandemic. Both can have a negative impact on parent-child bonding: psychosocial work stress in the course of a spillover effect from work to family and symptoms of impaired mental health as part of a crossover effect from parent to child. This potentially affects the child's development in the long term. METHOD: This cross-sectional study examined the relationship between psychosocial work stress and parent-child bonding during the early COVID-19 pandemic (May-June 2020). Symptoms of depression and aggressiveness were considered as mediators of this relationship. The sample consisted of employees in Eastern Germany (n = 380; 42.9% mothers, 57.1% fathers), aged 24-55 years, with children aged 0-36 months. RESULTS: In the total sample, an association was only found after adjusting for potential confounders, indicating that higher psychosocial work stress is associated with weaker bonding between the parent and child (ß = 0.148, p = .017, 95% CI [0.566, 5.614]). The separate analyses for mothers and fathers did not reveal a statistically significant relationship between psychosocial work stress and parent-child bonding. In the total sample, the higher the psychosocial work stress was, the higher were the parental symptoms of depression (ß = 0.372, p < .001, 95% CI [3.417, 5.696]) and aggressiveness ß = 0.254, p < .001, 95% CI [1.008, 3.208]). The mental health symptoms in turn were related to weaker parent-child bonding (symptoms of depression ß = 0.320, p < .001, 95% CI [0.345, 0.749]; symptoms of aggressiveness ß = 0.394, p < .001, 95% CI [0.697, 1.287]). The results furthermore suggested that parental mental health symptoms mediate the association between psychosocial work stress and parent-child bonding (symptoms of depression, ab = 2.491, 95% CI [1.472, 3.577] and of aggressiveness, ab = 2.091, 95% CI [1.147, 3.279]). The mediation effect was also found in the separate analyses for the mothers and fathers. DISCUSSION: The results of this study during the early COVID-19 pandemic in Germany highlight the importance of prevention as well as intervention measures in relation to psychosocial work stress that may play a debilitating role in the context of family relationships. In addition, the results suggest that both employers and employees should be made aware of the importance of psychosocial work stress, as it can have a negative impact on mental health, which in turn may have a major influence on family relationships.

Association between maternal employment and the child´s mental health: a systematic review with meta-analysis.

The recent rise in maternal workforce participation has led to more research regarding the role of maternal employment for (early) childhood mental health. This systematic review with meta-analysis covers new evidence on the association of both variables. A systematic literature search was conducted. Studies had to compare children 0-7 years of age on the basis of their mothers' employment status, working amount, employment duration, i.e., how long the mother had been back at work after birth, or timing of return to work. Child mental health was operationalized as behavior problems and prosocial behavior. Narrative and meta-analytic syntheses of evidence were conducted. Maternal employment was associated with more conduct problems but less internalizing behavior problems and anxious/depressed behavior in children; full-time employment was linked to more externalizing behavior problems and more hyperactivity/inattention. Longer employment duration was related to less (internalizing) behavior problems and more prosocial behavior but also more externalizing behavior problems. Narrative syntheses indicated early maternal return to work to be associated with more child externalizing behavior problems and less prosocial behavior. Whether maternal employment is associated with child mental health strongly depends on both variables' operationalization. Especially part-time employment, longer employment duration, and return to work only after the first year postpartum may be beneficial for child mental health. Practical implications pertain to an expanded offer of family leave and the endorsement of maternal employment after the first year postpartum. Here, factors that may buffer the negative associations with full-time employment warrant consideration.

Impact of hospitalisation on behavioural and physiological stress responses associated with cardiovascular risk in visiting family members.

BACKGROUND: The hospitalisation of a patient in intensive care impacts the psychological health of family members, with a high prevalence of anxiety, depression, and post-traumatic stress symptoms reported among families of critically ill patients. Understanding of the behavioural and physiological impact is limited and presents a new area of focus. OBJECTIVES: The objective of this study was to evaluate behavioural and physiological stress responses of visiting family members following hospitalisation of their adult relative. METHODS: Prospective longitudinal evaluation included 40 family members of adult patients with admission to intensive or coronary care in a large tertiary care metropolitan hospital. Assessments were conducted at three timepoints: in-hospital within 1 week of admission and 2 weeks and 3 months post discharge. Assessments included duration and quality of sleep (self-reported and actigraphy measured), physical activity, dietary and alcohol patterns, resting heart rate and blood pressure, and morning blood cortisol and lipid levels. Assessment of a reference group of 40 non-hospital-exposed control participants was also conducted. RESULTS: At the in-hospital assessment, study participants reported lower sleep time, altered 24-h physical activity patterns, reduced dietary and alcohol intake, and higher systolic and diastolic blood pressure than a nonhospitalised reference group. Compared to in-hospital assessment, these altered behavioural and physiological responses improved over time except for systolic blood pressures which remained unchanged at 3 months post family member discharge. CONCLUSION: Hospitalisation is associated with altered behavioural and physiological responses in family members. These findings contribute to understanding of the impact of unexpected hospitalisation on family members' cardiovascular risk factors and provide insights into potential mechanisms for the proposed increased risk during this time. Elevated systolic blood pressure at 3 months post discharge suggests a prolonged cardiovascular stress response in many family members of critical care patients that requires further study, with a focus on contributing and potential modifiable factors.

Dysregulation of oncogenic factors by GFI1B p32: investigation of a novel GFI1B germline mutation.

GFI1B is a transcription factor essential for the regulation of erythropoiesis and megakaryopoiesis, and pathogenic variants have been associated with thrombocytopenia and bleeding. Analysing thrombocytopenic families by whole exome sequencing, we identified a novel GFI1B variant (c.648+5G>A), which causes exon 9 skipping and overexpression of a shorter p32 isoform. We report the clinical data of our patients and critically review the phenotype observed in individuals with different GFI1B variants leading to the same effect on the p32 expression. Since p32 is increased in acute and chronic leukemia cells, we tested the expression level of genes playing a role in various type of cancers, including hematological tumors and found that they are significantly dysregulated, suggesting a potential role for GFI1B in carcinogenesis regulation. Increasing the detection of individuals with GFI1B variants will allow us to better characterize this rare disease and determine whether it is associated with an increased risk of developing malignancies.

The utility of flow cytometric platelet forward scatter as an alternative to mean platelet volume.

The use of mean platelet diameter (MPD) to classify inherited thrombocytopenia (IT) has been demonstrated in several studies. Alternatively, the mean platelet volume (MPV) may be used, but in macrothrombocytopenia this may not be available. We hypothesized that platelet forward scatter (FSC) measurements using flow cytometry may be used for the size-based classification of IT. The study aimed to assess the ability of platelet FSC to measure platelet size and whether it could be used as an alternative to the MPD or MPV.Blood samples were obtained from individuals undergoing investigation for inherited platelet function disorders (IPFD, n = 40) or platelet number disorders (IPND, n = 46). A hematology analyzer was used to obtain MPV and platelet counts, flow cytometry to measure platelet FSC and ImageJ software to measure MPD from stained blood smears. The International Society of Thrombosis and Hemostasis (ISTH) Bleeding Assessment Tool (BAT) was used to calculate bleeding scores.Twenty-nine(63%) of IPND patients had an MPV that could not be reported. A significant correlation to platelet FSC was found to the MPD (p < .0001) and MPV (p < .0001) and an inverse correlation with platelet count (p < .0001). No significant correlation was found between FSC and bleeding history. In conclusion, platelet FSC is an alternative to MPV and may be used in macrothrombocytopenia where the MPV is not recorded.

Building platelet phenotypes: Diaphanous-related formin 1 (DIAPH1)-related disorder.

Variants of the Diaphanous-Related Formin 1 (DIAPH-1) gene have recently been reported causing inherited macrothrombocytopenia. The essential/"diagnostic" characteristics associated with the disorder are emerging; however, robust and complete criteria are not established. Here, we report the first cases of DIAPH1-related disorder in Australia caused by the autosomal dominant gain-of-function DIAPH1 R1213X variant formed by truncation of the protein within the diaphanous auto-regulatory domain (DAD) with loss of regulatory motifs responsible for autoinhibitory interactions within the DIAPH1 protein. We affirm phenotypic changes induced by the DIAPH1 R1213X variant to include macrothrombocytopenia, early-onset progressive sensorineural hearing loss, and mild asymptomatic neutropenia. High-resolution microscopy confirms perturbations of cytoskeletal dynamics caused by the DIAPH1 variant and we extend the repertoire of changes generated by this variant to include alteration of procoagulant platelet formation and possible dental anomalies.

Mental health of working parents during the COVID-19 pandemic: can resilience buffer the impact of psychosocial work stress on depressive symptoms?

BACKGROUND: The COVID-19 pandemic has confronted working parents with an accumulation of stressors regarding changes in work, family, and social life, putting their mental health at risk. Stressors include altered working conditions such as working from home or changes in working hours as well as the difficulty to reconcile work and childcare due to the closure of childcare facilities. The present study examined the relationship of psychosocial work stress (i.e., work-privacy conflict and effort-reward imbalance at work) and depressive symptoms in working parents and whether this association was moderated by individual resilience. METHODS: Data of the present study (n = 452) were collected in Germany between May and June 2020 as part of the DREAMCORONA study. A subsample of working mothers (n = 191) and fathers (n = 261) completed the subscale for work-privacy conflict (WPC) of the Copenhagen Psychosocial Questionnaire, the Effort-Reward Imbalance (ERI) Questionnaire, the Connor-Davidson Resilience Scale (CD-RISC), and the Edinburgh Postnatal Depression Scale (EPDS). Multiple linear regression analyses including moderation were performed, controlling for gender, working hours per week, and a lifetime history of depression as potential confounders. RESULTS: Both WPC (ß = 0.336, p < .001) and ERI (ß = 0.254, p < .001) were significantly associated with depressive symptoms. Resilience moderated the relationship between ERI and depressive symptoms (ß = - 0.101, p = .018), indicating that higher resilience weakened the relationship. However, this effect was not found regarding the relationship between WPC and depressive symptoms (ß = 0.055, p = .167). CONCLUSIONS: The results highlight the need for measures to reduce psychosocial work stressors such as WPC and ERI during the COVID-19 pandemic on the one hand and to promote resilience on the other hand. The findings partially support the potential protective role of resilience buffering the association between psychosocial stress and mental health in working parents. Longitudinal studies are needed to confirm this effect.

Ionotropic glutamate receptors in platelets: opposing effects and a unifying hypothesis.

Ionotropic glutamate receptors include α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), kainate receptors (KAR), and N-methyl-D-aspartate receptors (NMDAR). All function as cation channels; AMPAR and KAR are more permeable to sodium and NMDAR to calcium ions. Compared to the brain, receptor assemblies in platelets are unusual, suggesting distinctive functionalities.There is convincing evidence that AMPAR and KAR amplify platelet function and thrombus formation in vitro and in vivo. Transgenic mice lacking GluA1 and GluK2 (AMPAR and KAR subunits, respectively) have longer bleeding times and prolonged time to thrombosis in an arterial model. In humans, rs465566 KAR gene polymorphism associates with altered in vitro platelet responses suggesting enhanced aspirin effect. The NMDAR contribution to platelet function is less well defined. NMDA at low concentrations (≤10 µM) inhibits platelet aggregation and high concentrations (≥100 µM) have no effect. However, open NMDAR channel blockers interfere with platelet activation and aggregation induced by other agonists in vitro; anti-GluN1 antibodies interfere with thrombus formation under high shear rates ex vivo; and rats vaccinated with GluN1 develop iron deficiency anemia suggestive of mild chronic bleeding. In this review, we summarize data on glutamate receptors in platelets and propose a unifying model that reconciles some of the opposing effects observed.

Microfluidic Shrinking Droplet Concentrator for Analyte Detection and Phase Separation of Protein Solutions.

We develop a droplet microfluidic platform to increase the concentration of analytes in solution via reduction of the sample volume under well-defined conditions. This approach improves the detection and quantification of analytes without requiring any a priori information on their structure nor physical chemical properties. Samples are compartmentalized and processed in water-in-oil droplets that are individually stored in cylindrical microwells located on top of a microfluidic channel. The individual droplets shrink over time due to water extraction in the surrounding oil, leading to an increase in the analyte concentration up to 100,000-fold within the droplet. We demonstrate the power of this approach for detection applications by quantifying a broad range of single analytes such as small molecules, proteins, nanoparticles, exosomes, and amyloid fibrils. With this setup, we can measure pM concentrations, corresponding to zeptomole (10-21 mol) amounts encapsulated in individual droplets. We further show that the droplet concentrator device, or DroMiCo, can quantify unlabeled proteins in nM concentrations and analyze multicomponent mixtures when coupled with a prefractionation step. We illustrate this concept by detecting femtomoles (10-15 mol) of soluble protein oligomers prefractionated by size exclusion chromatography. Finally, we apply the DroMiCo to the analysis of phase diagrams of macromolecules, including synthetic polymers and proteins. Specifically, we analyze the liquid-liquid phase separation of an in vitro model of cellular membraneless compartments, composed of a phase separating protein in the presence of defined concentrations of molecular modulators such as RNA and ATP.

The effect of metoprolol and aspirin on cardiovascular risk in bereavement: A randomized controlled trial.

BACKGROUND: Bereavement is associated with an increased risk of cardiovascular disease; however, no reports exist of interventions to reduce risk. In a randomized, double-blind, placebo-controlled trial of 85 recently bereaved participants, we determined whether ß-blocker (metoprolol 25 mg) and aspirin (100 mg) reduce cardiovascular risk markers and anxiety, without adversely affecting bereavement intensity. METHODS: Participants were spouses (n = 73) or parents (n = 12) of deceased from 5 hospitals in Sydney, Australia, 55 females, 30 males, aged 66.1 ±â€¯9.4 years. After assessment within 2 weeks of bereavement, subjects were randomized to 6 weeks of daily treatment or placebo, and the effect evaluated using ANCOVA, adjusted for baseline values (primary analysis). RESULTS: Participants on metoprolol and aspirin had lower levels of home systolic pressure (P = .03), 24-hour average heart rate (P < .001) and anxiety (P = .01) platelet response to arachidonic acid (P < .001) and depression symptoms (P = .046) than placebo with no difference in standard deviation of NN intervals index (SDNNi), von Willebrand Factor antigen, platelet-granulocyte aggregates or bereavement intensity. No significant adverse safety impact was observed. CONCLUSIONS: In early bereavement, low dose metoprolol and aspirin for 6 weeks reduces physiological and psychological surrogate measures of cardiovascular risk. Although further research is needed, results suggest a potential preventive benefit of this approach during heightened cardiovascular risk associated with early bereavement.

A Nanoparticle-Based Assay To Evaluate Surface-Induced Antibody Instability.

Protein stability against aggregation represents a major quality attribute for the successful development of biopharmaceuticals. Increasing evidence indicates that the formation of protein aggregates in aqueous solutions is often triggered by interactions between proteins and interfaces. Yet, in contrast to a large number of methods available to test protein bulk properties, high-throughput assays to investigate protein instability at interfaces remain much less developed. Major challenges include the control of the amount and type of surfaces, as well as the presence of synergistic effects between interfaces and hydrodynamic flows. Here, we describe and develop a highly controlled surface-mediated stress assay of protein instability based on polymeric nanoparticles. We show that hydrophobic nanoparticles are remarkably powerful in destabilizing large proteins such as antibodies. We further show that this approach can be implemented on a high-throughput microfluidic platform by compartmentalizing the protein solution in picoliter droplets surrounded by an oil phase. Our method allows the evaluation of protein instability at hydrophobic interfaces in a time scale of minutes and requires amounts of the sample in the order of a few hundred micrograms. We demonstrate that our assay represents a good mimic of air-water interfaces and finds application as a screening tool to optimize protein stability toward surface-induced aggregation. We provide a concrete example by identifying the optimal concentration range of Tween 80 that prevents antibody instability in the presence of interfaces. Overall, our hydrophobic nanoparticle surface-mediated stress assay (HNSSA) represents an attractive tool for accelerated tests of protein instability at interfaces under both stagnant and flow conditions, with implications for the optimization of buffer composition and the selection of stable biotherapeutic candidate molecules during early stage development.

Precarious working conditions and psychosocial work stress act as a risk factor for symptoms of postpartum depression during maternity leave: results from a longitudinal cohort study.

BACKGROUND: The majority of Western women work during their reproductive years, but past research has often neglected the influence of work-related factors on postpartum mental health. Especially postpartum depression (PPD) is an enormous psychological burden for mothers. Therefore, this study aims to investigate the prospective impact of precarious working conditions and psychosocial work stress during pregnancy (such as work-privacy conflict and effort-reward imbalance at the job) on symptoms of maternal PPD. METHODS: In the prospective-longitudinal cohort study DREAM (DResdner Studie zu Elternschaft, Arbeit und Mentaler Gesundheit), N = 587 employed women were questioned about their work during pregnancy and their mental health 8 weeks after delivery. RESULTS: Multiple regression analyses revealed that work-privacy conflict, low reward at work, and precarious working conditions significantly predicted symptoms of PPD, even when controlling for lifetime depression, anxiety, education, parity, and age. CONCLUSION: Our results indicate that psychosocial work stress and precarious working conditions have important implications for maternal peripartum mental health. They might act as prospective risk factors for PPD during the period of maternal leave. Hence, future research should focus on preventative measures targeting work life.

Platelet-Reactive Antibodies in Patients after Ischaemic Stroke-An Epiphenomenon or a Natural Protective Mechanism.

Ischaemic brain damage induces autoimmune responses, including the production of autoantibodies with potential neuroprotective effects. Platelets share unexplained similarities with neurons, and the formation of anti-platelet antibodies has been documented in neurological disorders. The aim of this study was to investigate the presence of anti-platelet antibodies in the peripheral blood of patients after ischaemic stroke and determine any clinical correlations. Using a flow cytometry-based platelet immunofluorescence method, we detected platelet-reactive antibodies in 15 of 48 (31%) stroke patients and two of 50 (4%) controls (p < 0.001). Western blotting revealed heterogeneous reactivities with platelet proteins, some of which overlapped with brain proteins. Stroke patients who carried anti-platelet antibodies presented with larger infarcts and more severe neurological dysfunction, which manifested as higher scores on the National Institutes of Health Stroke Scale (NIHSS; p = 0.009), but they had a greater recovery in the NIHSS by the time of hospital discharge (day 7 ± 2) compared with antibody-negative patients (p = 0.043). Antibodies from stroke sera reacted more strongly with activated platelets (p = 0.031) and inhibited platelet aggregation by up to 30.1 ± 2.8% (p < 0.001), suggesting the potential to interfere with thrombus formation. In conclusion, platelet-reactive antibodies can be found in patients soon after ischaemic stroke and correlate with better short-term outcomes, suggesting a potential novel mechanism limiting thrombosis.

A hydrophobic low-complexity region regulates aggregation of the yeast pyruvate kinase Cdc19 into amyloid-like aggregates in vitro.

Cells form stress granules (SGs) upon stress stimuli to protect sensitive proteins and RNA from degradation. In the yeast Saccharomyces cerevisiae, specific stresses such as nutrient starvation and heat-shock trigger recruitment of the yeast pyruvate kinase Cdc19 into SGs. This RNA-binding protein was shown to form amyloid-like aggregates that are physiologically reversible and essential for cell cycle restart after stress. Cellular Cdc19 exists in an equilibrium between a homotetramer and monomer state. Here, we show that Cdc19 aggregation in vitro is governed by protein quaternary structure, and we investigate the physical-chemical basis of Cdc19's assembly properties. Equilibrium shift toward the monomer state exposes a hydrophobic low-complexity region (LCR), which is prone to induce intermolecular interactions with surrounding proteins. We further demonstrate that hydrophobic/hydrophilic interfaces can trigger Cdc19 aggregation in vitro Moreover, we performed in vitro biophysical analyses to compare Cdc19 aggregates with fibrils produced by two known dysfunctional amyloidogenic peptides. We show that the Cdc19 aggregates share several structural features with pathological amyloids formed by human insulin and the Alzheimer's disease-associated Aß42 peptide, particularly secondary ß-sheet structure, thermodynamic stability, and staining by the thioflavin T dye. However, Cdc19 aggregates could not seed aggregation. These results indicate that Cdc19 adopts an amyloid-like structure in vitro that is regulated by the exposure of a hydrophobic LCR in its monomeric form. Together, our results highlight striking structural similarities between functional and dysfunctional amyloids and reveal the crucial role of hydrophobic/hydrophilic interfaces in regulating Cdc19 aggregation.

Diagnosis and management of heparin-induced thrombocytopenia: a consensus statement from the Thrombosis and Haemostasis Society of Australia and New Zealand HIT Writing Group.

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder that occurs following the administration of heparin and is caused by antibodies to platelet factor 4 and heparin. Diagnosis of HIT is essential to guide treatment strategies using non-heparin anticoagulants and to avoid unwanted and potential fatal thromboembolic complications. This consensus statement, formulated by members of the Thrombosis and Haemostasis Society of Australia and New Zealand, provides an update on HIT pathogenesis and guidance on the diagnosis and management of patients with suspected or confirmed HIT. MAIN RECOMMENDATIONS: A 4Ts score is recommended for all patients with suspected HIT prior to laboratory testing. Further laboratory testing with a screening immunoassay or confirmatory functional assay is not recommended in individuals with a low 4Ts score. However, if there are missing or unreliable clinical data, then laboratory testing should be performed. A positive functional assay result confirms the diagnosis of HIT and should be performed to confirm a positive immunoassay result. Heparin exposure must be ceased in patients with suspected or confirmed HIT and initial treatment with a non-heparin alternative instituted. Non-heparin anticoagulants (danaparoid, argatroban, fondaparinux and bivalirudin) used to treat HIT should be given in therapeutic rather than prophylactic doses. Direct oral anticoagulants may be used in place of warfarin after patients with HIT have responded to alternative parenteral anticoagulants with platelet count recovery. CHANGES IN MANAGEMENT AS A RESULT OF THIS STATEMENT: These are the first Australasian recommendations for diagnosis and management of HIT, with a focus on locally available diagnostic assays and therapeutic options. The importance of examining both clinical and laboratory data in considering a diagnosis of HIT cannot be overstated.

Dynamics of Synthetic Membraneless Organelles in Microfluidic Droplets.

Cells can form membraneless organelles by liquid-liquid phase separation. As these organelles are highly dynamic, it is crucial to understand the kinetics of these phase transitions. Here, we use droplet-based microfluidics to mix reagents by chaotic advection and observe nucleation, growth, and coarsening in volumes comparable to cells (pL) and on timescales of seconds. We apply this platform to analyze the dynamics of synthetic organelles formed by the DEAD-box ATPase Dhh1 and RNA, which are associated with the formation of processing bodies in yeast. We show that the timescale of phase separation decreases linearly as the volume of the compartment increases. Moreover, the synthetic organelles coarsen into one single droplet via gravity-induced coalescence, which can be arrested by introducing a hydrogel matrix that mimics the cytoskeleton. This approach is an attractive platform to investigate the dynamics of compartmentalization in artificial cells.

Diagnosis and treatment of MYH9-RD in an Australasian cohort with thrombocytopenia.

MYH9-related disorders (MYH9-RDs) caused by mutation of the MYH9 gene which encodes non-muscle myosin heavy-chain-IIA (NMMHC-IIA), an important motor protein in hemopoietic cells, are the most commonly encountered cause of inherited macrothrombocytopenia. Despite distinguishing features including an autosomal dominant mode of inheritance, giant platelets on the peripheral blood film accompanied by leucocytes with cytoplasmic inclusion bodies (döhle-like bodies), these disorders remain generally under-recognized and often misdiagnosed as immune thrombocytopenia (ITP). This may result in inappropriate treatment with corticosteroids, immunosupressants and in some cases, splenectomy. We explored the efficacy of next generation sequencing (NGS) with a candidate gene panel to establish the aetiology of thrombocytopenia for individuals who had been referred to our center from hematologists in the Australasian region in whom the cause of thrombocytopenia was suspected to be secondary to an inherited condition but which remained uncharacterized despite phenotypic investigations. Pathogenic MYH9 variants were detected in 15 (15/121, 12.4%) individuals and the pathogenecity of a novel variant of uncertain significance was confirmed in a further two related individuals following immunofluorescence (IF) staining performed in our laboratory. Concerningly, only one (1/17) individual diagnosed with MYH9-RD had been referred with this as a presumptive diagnosis, in all other cases (16/17, 94.1%), a diagnosis was not suspected by referring clinicians, indicating a lack of awareness or a failing of our diagnostic approach to these conditions. We examined the mean platelet diameter (MPD) measurements as a means to better identify and quantify platelet size. MPDs in cases with MYH9-RDs were significantly larger than controls (p < 0.001) and in 91% were greater than a previously suggested threshold for platelets in cases of ITP. In addition, we undertook IF staining in a proportion of cases and confirm that this test and/or NGS are satisfactory diagnostic tests. We propose that fewer cases of MYH9-RDs would be missed if diagnostic algorithms prioritized IF and/or NGS in cases of thrombocytopenia associated with giant platelets, even if döhle-like bodies are not appreciated on the peripheral blood film. Finally, our report describes the long-term use of a thrombopoietin agonist in a case of MYH9-RD that had previously been diagnosed as ITP, and demonstrates that treatment with these agents may be possible, and is well tolerated, in this group of patients.

Paris-Trousseau thrombocytopenia is phenocopied by the autosomal recessive inheritance of a DNA-binding domain mutation in FLI1.

Hemizygous deletion of a variable region on chromosome 11q containing FLI1 causes an inherited platelet-related bleeding disorder in Paris-Trousseau thrombocytopenia and Jacobsen syndrome. These multisystem disorders are also characterized by heart anomalies, changes in facial structure, and intellectual disability. We have identified a consanguineous family with autosomal recessive inheritance of a bleeding disorder that mimics Paris-Trousseau thrombocytopenia but has no other features of the 11q23 deletion syndrome. Affected individuals in this family have moderate thrombocytopenia; absent collagen-induced platelet aggregation; and large, fused α-granules in 1% to 5% of circulating platelets. This phenotype was caused by a FLI1 homozygous c.970C>T-point mutation that predicts an arginine-to-tryptophan substitution in the conserved ETS DNA-binding domain of FLI1. This mutation caused a transcription defect at the promoter of known FLI1 target genes GP6, GP9, and ITGA2B, as measured by luciferase assay in HEK293 cells, and decreased the expression of these target proteins in affected members of the family as measured by Western blotting of platelet lysates. This kindred suggests abnormalities in FLI1 as causative of Paris-Trousseau thrombocytopenia and confirms the important role of FLI1 in normal platelet development.

Microarray-based MALDI-TOF mass spectrometry enables monitoring of monoclonal antibody production in batch and perfusion cell cultures.

Cell culture process monitoring in monoclonal antibody (mAb) production is essential for efficient process development and process optimization. Currently employed online, at line and offline methods for monitoring productivity as well as process reproducibility have their individual strengths and limitations. Here, we describe a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based on a microarray for mass spectrometry (MAMS) technology to rapidly monitor a broad panel of analytes, including metabolites and proteins directly from the unpurified cell supernatant or from host cell culture lysates. The antibody titer is determined from the intact antibody mass spectra signal intensity relative to an internal protein standard spiked into the supernatant. The method allows a semi-quantitative determination of light and heavy chains. Intracellular mass profiles for metabolites and proteins can be used to track cellular growth and cell productivity.