Bulk lysis procedures alter target cell population counts.

To achieve high-sensitivity cell measurements (<1 in 105 cells) by flow cytometry (FCM), the minimum number of acquired cells must be considered and conventional immunophenotyping protocols fall short of these numbers. The bulk lysis (BL) assay is a standardized erythrocyte lysing approach that allows the analysis of the millions of cells required for high-sensitivity measurable residual disease (MRD) detection. However, this approach has been associated with significant cell loss, along with potential over or underestimates of rare cells when using this method. The aim of this study was to evaluate bulk lysis protocols and compare them with minimal sample perturbation (MSP) protocols, which are reported to better preserve the native cellular state and avoid significant cell loss due to washing steps. To achieve this purpose, we first generated an MRD model by spiking fresh peripheral blood with K562 cells, stably expressing EGFP, at known percentages of EGFP positive cells to leukocytes. Samples were then prepared with BL and MSP protocols and analyzed using FCM. For all percentages of K562 cells established and evaluated, a significant decrease of this population was detected in BL samples compared with MSP samples, even at low K562 cell percentages. Significant decreases for non-necrotic cells were also observed in BL samples relative to MSP samples. In conclusion, the evaluation of the potential effects of BL protocols in obtaining the final count is of great interest, especially for over- or under-estimation of target cells, as in the case of measurable residual disease. Since conventional flow cytometry or minimal sample perturbation assays fall short in obtaining the minimum numbers required to reach high sensitivity measurements, significant efforts may be needed to improve bulk lysis solution reagents.

True volumetric counting of CD34+ cells using flow cytometry.

While the single-platform flow cytometric CD34+ cell counting method is the preferred choice to predict the yield of mobilized peripheral blood stem cells, most flow cytometers lack the ability of hematology counter analyzers to perform volumetric counting. However, one of the problems using reference microbeads is the vanishing counting bead phenomenon. This phenomenon results in a drop in microbeads concentration and reduces the total and relative number of beads in calibration procedures. In the last years, flow cytometers including a volumetric system to quantify cells have been developed and may represent a promising alternative to enumerate CD34+ cells avoiding the use of beads. In this study we have used a direct true volumetric counting of CD34+ cells under continuous flow pump to overcome potential drawbacks with impact in rare cell analysis. To confirm this hypothesis, we have compared the results of CD34+ cell enumeration using non-volumetric vs. volumetric systems with FC500 (Beckman Coulter) and Attune NxT (ThermoFisher) flow cytometers, respectively, in mobilized peripheral blood samples. No statistically significant differences were observed between measurements of CD34+ cells using beads, when the FC500 and Attune NxT absolute counting values were compared, or when CD34+ counts were compared on the Attune NxT, either using or not using beads. Linear regressions to study the relationship between volumetric and non-volumetric CD34+ counts confirmed the accuracy of each method. Bland-Altman test showed agreement between both methods. Our data showed that CD34+ cell enumeration using a volumetric system is comparable with current counting systems. This method represents an alternative with the advantage of the simplification of sample preparation and the reduction of the analysis subjectivity.

Persistence of Chronic Lymphocytic Leukemia Stem-like Populations under Simultaneous In Vitro Treatment with Curcumin, Fludarabine, and Ibrutinib: Implications for Therapy Resistance.

Leukemic stem cells (LSCs) possess similar characteristics to normal hematopoietic stem cells, including self-renewal capacity, quiescence, ability to initiate leukemia, and drug resistance. These cells play a significant role in leukemia relapse, persisting even after apparent remission. LSCs were first described in 1994 by Lapidot et al. Although they have been extensively studied in acute leukemia, more LSC research is still needed in chronic lymphocytic leukemia (CLL) to understand if reduced apoptosis in mature cells should still be considered as the major cause of this disease. Here, we provide new evidence suggesting the existence of stem-like cell populations in CLL, which may help to understand the disease as well as to develop effective treatments. In this study, we identified a potential leukemic stem cell subpopulation using the tetraploid CLL cell line I83. This subpopulation is characterized by diploid cells that were capable of generating the I83 tetraploid population. Furthermore, we adapted a novel flow cytometry analysis protocol to detect CLL subpopulations with stem cell properties in peripheral blood samples and primary cultures from CLL patients. These cells were identified by their co-expression of CD19 and CD5, characteristic markers of CLL cells. As previously described, increased alkaline phosphatase (ALP) activity is indicative of stemness and pluripotency. Moreover, we used this method to investigate the potential synergistic effect of curcumin in combination with fludarabine and ibrutinib to deplete this subpopulation. Our results confirmed the effectiveness of this ALP-based analysis protocol in detecting and monitoring leukemic stem-like cells in CLL. This analysis also identified limitations in eradicating these populations using in vitro testing. Furthermore, our findings demonstrated that curcumin significantly enhanced the effects of fludarabine and ibrutinib on the leukemic fraction, exhibiting synergistic effects (combination drug index, CDI 0.97 and 0.37, respectively). Our results lend support to the existence of potential stem-like populations in CLL cell lines, and to the idea that curcumin could serve as an effective adjuvant in therapies aimed at eliminating these populations and improving treatment efficacy.

Functional Flow Cytometry to Predict PD-L1 Conformational Changes.

The programmed cell death protein 1/programmed cell death protein ligand 1 (PD-1/PD-L1) axis is one of the most widely recognized targets for cancer immunotherapy. Importantly, PD-L1 conformational changes can hinder target binding when living cells are used. Antibody affinity, equilibrium binding, association and dissociation rates, and other affinity-related constants are fundamental to ensure target saturation. Here, PD-L1 changes in conformation and their potential impact on PD-L1 function and mutation are explored. Specifically, we present detailed flow cytometry procedures to analyze PD-L1 reactivity in myeloid-derived suppressor cells (MDSCs). This approach can also be used to study the contribution of protein conformational changes in living cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for PD-L1+ myeloid-derived suppressor cells detection by flow cytometry Basic Protocol 2: Protocol preparation, sample acquisition, and gating strategy for flow cytometric screening of PD-L1+ myeloid-derived suppressor cells in patients with lung cancer Support Protocol 1: Bioinformatic tools for the analysis of flow cytometric data.

Human Health Risk Assessment of Arsenic and Other Metals in Herbal Products Containing St. John's Wort in the Metropolitan Area of Mexico City.

Consumption of St. John's wort plant is high worldwide due to its various medicinal properties. However, herbal products containing St. John's wort may be contaminated with toxic metals. This is often related to contamination of both water and the atmosphere, lack of proper cultivation methods, and inadequate plant storage conditions, as well as a lack of stricter sanitary supervision. A safety assessment of copper (Cu), lead (Pb), cadmium (Cd) and arsenic (As) content in 23 products containing St. John's wort (pharmaceutical herbal products, food supplements and traditional herbal remedies) sold in the metropolitan area of Mexico City was conducted. The analysis of metals was determined using a graphite-furnace atomic absorption spectrometer. All herbal products were contaminated with Cu, Pb, Cd and As. The pharmaceutical herbal items showed less contamination by metals. The daily human intake (DHI) values for Pb exceeded the permissible limits in the group of traditional herbal remedies. The DHI calculation for As exceeded the permitted intake values for all items in the group of traditional herbal remedies, five food supplements and one pharmaceutical herbal product. The hazard indicator calculation of the non-carcinogenic cumulative risk values for traditional herbal remedies was greater than 1, suggesting a risk to human health.

Interaction of the pre- and postnatal environment in the maternal immune activation model.

Adverse influences during pregnancy are associated with a range of unfavorable outcomes for the developing offspring. Maternal psychosocial stress, exposure to infections and nutritional imbalances are known risk factors for neurodevelopmental derangements and according psychiatric and neurological manifestations later in offspring life. In this context, the maternal immune activation (MIA) model has been extensively used in preclinical research to study how stimulation of the maternal immune system during gestation derails the tightly coordinated sequence of fetal neurodevelopment. The ensuing consequence of MIA for offspring brain structure and function are majorly manifested in behavioral and cognitive abnormalities, phenotypically presenting during the periods of adolescence and adulthood. These observations have been interpreted within the framework of the "double-hit-hypothesis" suggesting that an elevated risk for neurodevelopmental disorders results from an individual being subjected to two adverse environmental influences at distinct periods of life, jointly leading to the emergence of pathology. The early postnatal period, during which the caregiving parent is the major determinant of the newborn´s environment, constitutes a window of vulnerability to external stimuli. Considering that MIA not only affects the developing fetus, but also impinges on the mother´s brain, which is in a state of heightened malleability during pregnancy, the impact of MIA on maternal brain function and behavior postpartum may importantly contribute to the detrimental consequences for her progeny. Here we review current information on the interaction between the prenatal and postnatal maternal environments in the modulation of offspring development and their relevance for the pathophysiology of the MIA model.

Role of gestational age and maternal biological factors in early term neonatal morbidity.

BACKGROUND: The morbidity of early-term newborns (ETNBs) is associated with the immaturity of their organs and maternal biological factors (MBF). In this study, we determined the relationship between MBF and early-term birth. In addition, we assessed the role of gestational age (GA) and MBF in the morbidity of ETNBs compared with full-term newborns (FTNBs). METHODS: This retrospective cohort included ETNBs and FTNBs. The frequency of morbidities was compared between groups stratified by GA with the X2 test or Fisher's exact test. The association of MBF with GA and morbidity was calculated using binomial regression models between the variables that correlated with the morbidity of the ETNBs using Spearman's correlation. A significance level of 5% was estimated for all analyses. RESULTS: The probability of morbidity at birth for ETNBs was 1.9-fold higher than for FTNBs (37.5% vs. 19.9%), as they required more admission to the neonatal unit and more days of hospitalization; the most frequent pathology was jaundice. The MBF associated with early term birth were hypertensive disorders of pregnancy (aRR = 1.4, 95% confidence interval (CI): 1.3-1.6), intrauterine growth restriction (aRR = 1.5, 95% CI: 1.3-1.6), and chronic hypertension (aRR = 1.6, 95% CI: 1.4-1.8). No association was found between MBF and morbidity at 37 and 38 weeks. CONCLUSIONS: The morbidity among ETNBs is related to physiological immaturity. The adverse MBF favor a hostile intrauterine environment, which affects fetal and neonatal well-being.

INTRODUCCIÓN: La morbilidad de los recién nacidos a término temprano (RNTT) se asocia con la inmadurez de sus órganos y factores biológicos maternos (FBM). En este estudio se determinó la relación entre FBM y el nacimiento a término temprano. Además, se evaluó el papel de la edad gestacional (EG) y los FBM en la morbilidad de los RNTT comparados con los recién nacidos a término completo (RNTC). MÉTODOS: Este estudio de cohorte retrospectivo incluyó RNTT y RNTC. La frecuencia de morbilidades se comparó entre grupos estratificados por EG con la prueba de X2 o la prueba exacta de Fisher. La asociación de FBM con EG y morbilidad se calculó mediante modelos de regresión binomial entre variables correlacionadas con morbilidad de ETNB mediante la correlación de Spearman. Se estimó un nivel de significación del 5% para todos los análisis. RESULTADOS: Los RNTT presentaron una probabilidad 1.9 veces mayor de morbilidad al nacer comparado con los RNTC (37.5% vs 19.9%), ya que requirieron mayor admisión a la unidad neonatal y más días de hospitalización; la patología más frecuente fue la ictericia. Los FBM asociados con el nacimiento a término temprano fueron los: trastornos hipertensivos gestacionales, restricción del crecimiento intrauterino e hipertensión crónica. No se encontró asociación entre factores biológicos maternos y la morbilidad a las 37 y 38 semanas. CONCLUSIONES: La morbilidad del RNTT se relaciona con la inmadurez fisiológica. Los FBM adversos favorecen un medio intrauterino hostil afectando el bienestar fetal y neonatal.

Role of gestational age and maternal biological factors in early term neonatal morbidity / Rol de la edad gestacional y factores biológicos maternos en la morbilidad del neonato a término temprano

Abstract Background: The morbidity of early-term newborns (ETNBs) is associated with the immaturity of their organs and maternal biological factors (MBF). In this study, we determined the relationship between MBF and early-term birth. In addition, we assessed the role of gestational age (GA) and MBF in the morbidity of ETNBs compared with full-term newborns (FTNBs). Methods: This retrospective cohort included ETNBs and FTNBs. The frequency of morbidities was compared between groups stratified by GA with the X2 test or Fisher's exact test. The association of MBF with GA and morbidity was calculated using binomial regression models between the variables that correlated with the morbidity of the ETNBs using Spearman's correlation. A significance level of 5% was estimated for all analyses. Results: The probability of morbidity at birth for ETNBs was 1.9-fold higher than for FTNBs (37.5% vs. 19.9%), as they required more admission to the neonatal unit and more days of hospitalization; the most frequent pathology was jaundice. The MBF associated with early term birth were hypertensive disorders of pregnancy (aRR = 1.4, 95% confidence interval (CI): 1.3-1.6), intrauterine growth restriction (aRR = 1.5, 95% CI: 1.3-1.6), and chronic hypertension (aRR = 1.6, 95% CI: 1.4-1.8). No association was found between MBF and morbidity at 37 and 38 weeks. Conclusions: The morbidity among ETNBs is related to physiological immaturity. The adverse MBF favor a hostile intrauterine environment, which affects fetal and neonatal well-being.

Resumen Introducción: La morbilidad de los recién nacidos a término temprano (RNTT) se asocia con la inmadurez de sus órganos y factores biológicos maternos (FBM). En este estudio se determinó la relación entre FBM y el nacimiento a término temprano. Además, se evaluó el papel de la edad gestacional (EG) y los FBM en la morbilidad de los RNTT comparados con los recién nacidos a término completo (RNTC). Métodos: Este estudio de cohorte retrospectivo incluyó RNTT y RNTC. La frecuencia de morbilidades se comparó entre grupos estratificados por EG con la prueba de X2 o la prueba exacta de Fisher. La asociación de FBM con EG y morbilidad se calculó mediante modelos de regresión binomial entre variables correlacionadas con morbilidad de ETNB mediante la correlación de Spearman. Se estimó un nivel de significación del 5% para todos los análisis. Resultados: Los RNTT presentaron una probabilidad 1.9 veces mayor de morbilidad al nacer comparado con los RNTC (37.5% vs 19.9%), ya que requirieron mayor admisión a la unidad neonatal y más días de hospitalización; la patología más frecuente fue la ictericia. Los FBM asociados con el nacimiento a término temprano fueron los: trastornos hipertensivos gestacionales, restricción del crecimiento intrauterino e hipertensión crónica. No se encontró asociación entre factores biológicos maternos y la morbilidad a las 37 y 38 semanas. Conclusiones: La morbilidad del RNTT se relaciona con la inmadurez fisiológica. Los FBM adversos favorecen un medio intrauterino hostil afectando el bienestar fetal y neonatal.

Thallium-induced DNA damage, genetic, and epigenetic alterations.

Thallium (Tl) is a toxic heavy metal responsible for noxious effects in living organisms. As a pollutant, Tl can be found in the environment at high concentrations, especially in industrial areas. Systemic toxicity induced by this toxic metal can affect cell metabolism, including redox alterations, mitochondrial dysfunction, and activation of apoptotic signaling pathways. Recent focus on Tl toxicity has been devoted to the characterization of its effects at the nuclear level, with emphasis on DNA, which, in turn, may be responsible for cytogenetic damage, mutations, and epigenetic changes. In this work, we review and discuss past and recent evidence on the toxic effects of Tl at the systemic level and its effects on DNA. We also address Tl's role in cancer and its control.

Fast-screening flow cytometry method for detecting nanoplastics in human peripheral blood.

Plastic pollution is a global problem. Animals and humans can ingest and inhale plastic particles, with uncertain health consequences. Nanoplastics (NPs) are particles ranging from 1 nm to 1000 nm that result from the erosion or breakage of larger plastic debris, and can be highly polydisperse in physical properties and heterogeneous in composition. Potential effects of NPs exposure may be associated with alterations in the xenobiotic metabolism, nutrients absorption, energy metabolism, cytotoxicity, and behavior. In humans, no data on NPs absorptions has been reported previously. Given that their detection relies significantly on environmental exposure, we have prospectively studied the presence of NPs in human peripheral blood (PB). Specifically, we have used fluorescence techniques and nanocytometry, together with the staining of the lipophilic dye Nile Red (NR), to demonstrate that NPs can be accurately detected using flow cytometry.•Potential effects of nanoplastics exposure.•Fluorescence techniques and nanocytometry.•Accurate detection using flow cytometry.

Accurate identification of cell doublet profiles: Comparison of light scattering with fluorescence measurement techniques.

Doublet discrimination is usually based on pulse analysis of light scatter parameters. A combination of two pulse parameters (Area, A; Height, H; or Width, W) can be used to discriminate a pulse originated in a single cell from a pulse originated from cells stuck together. Fluorescence signals can be also used to discriminate aggregates, being essential to identify cells in the G2/M phase from doublets in the G0/G1 phase in cell cycle/DNA applications. The most used method combines FSC-A versus FSC-H, whereas other strategies combine FSC-H versus FSC-W, SSC-H versus SSC-A and SSC-H versus SSC-W. However, when studying activated or proliferating cells, scatter discrimination can be difficult. In this study, we have compared the use of light scattering with fluorescence measurement techniques for successful doublet discrimination for single cells. Effective use of FSC and SSC height, area and width are commonly used to eliminate aggregates. However, fluorescence-based methods using viable DNA stains provide a good compromise between performance and accurate manual gating methods, especially for highly concentrated cell products and pathological specimens. Viable DNA dyes, such as Vybrant™ DyeCycle™ Violet stain or Hoechst 33342, can be used to detect nucleated cells in blood and in bone marrow, or to discriminate cell aggregates and debris based on no-lyse no-wash assays, where scatter degradation is a dominant component of the measured data, which increases with event rate.

Comparison of visual performance between two aspheric monofocal intraocular lens models.

CLINICAL RELEVANCE: It is important to distinguish between visual acuity, optical quality and quality of vision when outcomes obtained with intraocular lenses are evaluated. These parameters, that includeobjective and subjective tests, should be assessed to obtain results that are not biased. BACKGROUND: To assess the difference in visual and optical quality between two monofocal intraocular lens models. METHODS: : This was a prospective, parallel and randomised clinical study conducted at Miranza IOA, a private clinic in Madrid, Spain. Sixty patients were implanted bilaterally, 30 per group, with two aspheric IOLs with induced spherical aberration of -0.27 µm for Group A and -0.20 µm for Group B. Visual outcomes obtained at 1 and 3 months after surgery included both uncorrected (UCVA) and corrected monocular distance visual acuity (DCVA), objective scattering index (OSI), modulation transfer function (MTF) cut-off, Strehl Ratio (SR), contrast sensitivity defocus curve (CSDC), intraocular lens spherical aberration (SA), and longitudinal chromatic aberration of the eye. Activity limitations in daily life were assessed using CatQuest-9SF questionnaire. RESULTS: There were statistically significant differences for DCVA (0.04 LogMAR; p = .008) and SR (0.03; p = .003) between groups. Outcomes related to CSDC showed statistically significant differences for vergences between -0.50 D and +1.00 D (3 mm pupil) and for vergences of 0.00 D and +0.50 D (4.5 mm pupil) between groups. Overall, Group A showed better results regarding visual and optical quality, including a lower longitudinal chromatic aberration result in comparison to Group B. Patient satisfaction evaluated with CatQuest-9SF showed that Group A achieved better outcomes, although the differences were statistically significant only for the 'Reading text on television' item (p = 0.027). CONCLUSIONS: Both intraocular lens models showed excellent quantity of vision, optical and visual quality as well as high patient satisfaction. Despite this, the the Group A model provided slightly better outcomes than the Group B model.

Erythrocyte lysing solutions have a detrimental effect in flow cytometric dendritic cell detection.

Flow cytometry (FCM) enumeration of peripheral blood dendritic cells (PBDCs) is a minimally invasive procedure extremely useful for immunological studies. Numbers of PBDCs vary depending on age, lifestyle, or in pathologies like cancer, leukemia or immunodeficiencies. Conventional methods for PBDC identification by FCM involve red blood cell lysis using either formaldehyde or ammonium chloride-based solutions. This specific procedure has been widely reported to cause a detrimental effect as well as an artifactual detection of target populations. Alternatively, minimal sample perturbation assays that avoid the use of erythrolytic solutions with centrifugation steps and preserve the native cellular state are simpler and more robust than conventional methods. In this study, we aimed to evaluate how conventional FCM assays can alter dendritic cell (DC) counting when compared with minimal sample perturbation protocols, in terms of absolute cell counting, percentage and stain index (SI) of PBDC subsets. We evaluated the use of three different erythrolytic solutions (CyLyse, OptiLyse C, and Pharm Lyse) on a series of n = 20 peripheral blood specimens for conventional and plasmacytoid DCs detection as well as for leukocyte and basophil detection. Our results showed a significant reduction of leukocytes and specifically, of DCs and basophils in terms of absolute number when using erythrolytic solutions. In conclusion, our study shows that PBDC counting is heavily affected when lysing solutions are used, indicating that these stellate-shaped populations appear to be more labile.

Impact of red blood cell lysing on rare event analysis.

The challenges associated with analyzing rare cells are dependent on a series of factors, which usually require large numbers of cells per sample for successful resolution. Among these is determining the minimum number of total events needed to be acquired as defined by the expected frequency of the target cell population. The choice of markers that identify the target population, as well as the event rate and the number of aborted events/second, will also determine the statistically significant detection of rare cell events. Sample preparation is another important but often overlooked factor in rare cell analysis, and in this study we examine Poisson theory and methods to determine the effect of sample manipulation on rare cell detection. After verifying the applicability of this theory, we have evaluated the potential impact of red cell lysis on rare cell analysis, and how cell rarity can be underestimated or overestimated based on erythrolytic sensitivity or resistance of healthy leukocytes and pathological rare cells.

An accessory prefrontal cortex-thalamus circuit sculpts maternal behavior in virgin female mice.

The ability to care for the young is innate and readily displayed by postpartum females after delivery to ensure offspring survival. Upon pup exposure, rodent virgin (nulliparous) females also develop parental behavior that over time becomes displayed at levels equivalent to parenting mothers. Although maternal behavior in postpartum females and the associated neurocircuits are well characterized, the neural mechanisms underlying the acquisition of maternal behavior without prior experience remain poorly understood. Here, we show that the development of maternal care behavior in response to first-time pup exposure in virgin females is initiated by the activation of the anterior cingulate cortex (ACC). ACC activity is dependent on feedback excitation by Vglut2+ /Galanin+ neurons of the centrolateral nucleus of the thalamus (CL), with their activity sufficient to display parenting behaviors. Accordingly, acute bidirectional chemogenetic manipulation of neuronal activity in the ACC facilitates or impairs the attainment of maternal behavior, exclusively in virgin females. These results reveal an ACC-CL neurocircuit as an accessory loop in virgin females for the initiation of maternal care upon first-time exposure to pups.

Zygotic genome activation by the totipotency pioneer factor Nr5a2.

Life begins with a switch in genetic control from the maternal to the embryonic genome during zygotic genome activation (ZGA). Despite its importance, the essential regulators of ZGA remain largely unknown in mammals. On the basis of de novo motif searches, we identified the orphan nuclear receptor Nr5a2 as a key activator of major ZGA in mouse two-cell embryos. Nr5a2 is required for progression beyond the two-cell stage. It binds to its motif within SINE B1/Alu retrotransposable elements found in cis-regulatory regions of ZGA genes. Chemical inhibition suggests that 72% of ZGA genes are regulated by Nr5a2 and potentially other orphan nuclear receptors. Nr5a2 promotes chromatin accessibility during ZGA and binds nucleosomal DNA in vitro. We conclude that Nr5a2 is an essential pioneer factor that regulates ZGA.

The metabolic regulator USF-1 is involved in the control of affective behaviour in mice.

Epidemiological studies indicate a bidirectional association between metabolic disturbances, including obesity and related pathological states, and mood disorders, most prominently major depression. However, the biological mechanisms mediating the comorbid relationship between the deranged metabolic and mood states remain incompletely understood. Here, we tested the hypothesis that the enhanced activation of brown fat tissue (BAT), known to beneficially regulate obesity and accompanying dysfunctional metabolic states, is also paralleled by an alteration of affective behaviour. We used upstream stimulatory factor 1 (USF-1) knock-out (KO) mice as a genetic model of constitutively activated BAT and positive cardiometabolic traits and found a reduction of depression-like and anxiety-like behaviours associated with USF-1 deficiency. Surgical removal of interscapular BAT did not impact the behavioural phenotype of USF-1 KO mice. Further, the absence of USF-1 did not lead to alterations of adult hippocampal neural progenitor cell proliferation, differentiation, or survival. RNA-seq analysis characterised the molecular signature of USF-1 deficiency in the hippocampus and revealed a significant increase in the expression of several members of the X-linked lymphocyte-regulated (xlr) genes, including xlr3b and xlr4b. Xlr genes are the mouse orthologues of the human FAM9 gene family and are implicated in the regulation of dendritic branching, dendritic spine number and morphology. The transcriptional changes were associated with morphological alterations in hippocampal neurons, manifested in reduced dendritic length and complexity in USF-1 KO mice. Collectively these data suggest that the metabolic regulator USF-1 is involved in the control of affective behaviour in mice and that this modulation of mood states is unrelated to USF-1-dependent BAT activation, but reflected in structural changes in the brain.

Gestational immune activation disrupts hypothalamic neurocircuits of maternal care behavior.

Immune activation is one of the most common complications during pregnancy, predominantly evoked by viral infections. Nevertheless, how immune activation affects mother-offspring relationships postpartum remains unknown. Here, by using the polyinosinic-polycytidylic acid (Poly I:C) model of gestational infection we show that viral-like immune activation at mid-gestation persistently changes hypothalamic neurocircuit parameters in mouse dams and, consequently, is adverse to parenting behavior. Poly I:C-exposed dams favor non-pup-directed exploratory behavior at the expense of pup retrieval. These behavioral deficits are underlain by dendrite pruning and lesser immediate early gene activation in Galanin (Gal)+ neurons with dam-specific transcriptional signatures that reside in the medial preoptic area (mPOA). Reduced activation of an exclusively inhibitory contingent of these distal-projecting Gal+ neurons allows for increased feed-forward inhibition onto putative dopaminergic neurons in the ventral tegmental area (VTA) in Poly I:C-exposed dams. Notably, destabilized VTA output specifically accompanies post-pup retrieval epochs. We suggest that gestational immunogenic insults bias both threat processing and reward perception, manifesting as disfavored infant caregiving.

Flow-cytometry-based protocols for human blood/marrow immunophenotyping with minimal sample perturbation.

This protocol provides instructions to improve flow cytometry analysis of marrow/peripheral blood cells by avoiding erythrolytic solutions, density gradients, and washing steps. We describe two basic approaches for identifying cell surface antigens with minimal sample perturbation, which have been successfully used to identify healthy and pathologically rare cells. The greatest advantage of these approaches is that they minimize the unwanted effect caused by sample preparation, allowing for improved study of live cells at the point of analysis. For complete details on the use and execution of this protocol, please refer to Petriz et al. (2018).

A systematic review of providers' experiences of facilitating group antenatal care.

BACKGROUND: Group antenatal care is a rapidly expanding alternative antenatal care delivery model. Research has shown it to be a safe and effective care model for women, but less is known about the perspectives of the providers leading this care. This systematic review examined published literature that considered health care professionals' experiences of facilitating group antenatal care. METHODS: Systematic searches were conducted in seven databases (Cinahl, Medline, Psychinfo, Embase, Ovid Emcare, Global Health and MIDRS) in April 2020. Qualitative or mixed methods studies with a significant qualitative component were eligible for inclusion if they included a focus on the experiences of health care providers who had facilitated group antenatal care. Prisma screening guidelines were followed and study quality was critically appraised by three independent reviewers. The findings were synthesised thematically. RESULTS: Nineteen papers from nine countries were included. Three main themes emerged within provider experiences of group antenatal care. The first theme, 'Giving women the care providers feel they want and need', addresses richer use of time, more personal care, more support, and continuity of care. The second theme, 'Building skills and relationships', highlights autonomy, role development and hierarchy dissolution. The final theme, 'Value proposition of group antenatal care', discusses provider investment and workload. CONCLUSIONS: Health care providers' experience of delivering group antenatal care was positive overall. Opportunities to deliver high-quality care that benefits women and allows providers to develop their professional role were appreciated. Questions about the providers' perspectives on workload, task shifting, and the structural changes needed to support the sustainability of group antenatal care warrant further exploration.