Heme oxygenase-1 deficiency triggers exhaustion of hematopoietic stem cells.

While intrinsic changes in aging hematopoietic stem cells (HSCs) are well characterized, it remains unclear how extrinsic factors affect HSC aging. Here, we demonstrate that cells in the niche-endothelial cells (ECs) and CXCL12-abundant reticular cells (CARs)-highly express the heme-degrading enzyme, heme oxygenase 1 (HO-1), but then decrease its expression with age. HO-1-deficient animals (HO-1-/- ) have altered numbers of ECs and CARs that produce less hematopoietic factors. HSCs co-cultured in vitro with HO-1-/- mesenchymal stromal cells expand, but have altered kinetic of growth and differentiation of derived colonies. HSCs from young HO-1-/- animals have reduced quiescence and regenerative potential. Young HO-1-/- HSCs exhibit features of premature exhaustion on the transcriptional and functional level. HO-1+/+ HSCs transplanted into HO-1-/- recipients exhaust their regenerative potential early and do not reconstitute secondary recipients. In turn, transplantation of HO-1-/- HSCs to the HO-1+/+ recipients recovers the regenerative potential of HO-1-/- HSCs and reverses their transcriptional alterations. Thus, HSC-extrinsic activity of HO-1 prevents HSCs from premature exhaustion and may restore the function of aged HSCs.

It is Not (Always) the Mismatch That Beats You-On the Relationship Between Interaction of Early and Recent Life Stress and Emotion Regulation, an fMRI Study.

Stress may impact the ability to effectively regulate emotions. To study the impact of stressful experiences in early and recent life on emotion regulation, we examined the relationship between early life stress, recent stress, and brain activation during cognitive reappraisal. We investigated two regulation goals: the decrease and increase of emotional response to both negative and positive stimuli. Furthermore, two models of stress consequences were examined: the cumulative and match/mismatch models. A total of 83 participants (Mage = 21.66) took part in the study. There was an interaction between cumulative stress and stimuli valence in the cuneus, superior lateral occipital cortex, superior parietal lobule, supramarginal gyrus extending to superior temporal gyrus, and precentral gyrus extending to supplementary motor area. Interaction between mismatched stress index and stimuli valence was found in the left hippocampus, left insula extending to the orbitofrontal cortex and amygdala, and in a cluster including the anterior cingulate cortex, superior frontal gyrus, and frontal pole. Furthermore, there were differences between the effects of cumulative and mismatched stress indices on brain activation during reappraisal of positive but not negative stimuli. Results indicate that cumulative stress and match/mismatch approaches are both useful for explaining brain activation during reappraisal. This finding is important for our understanding of the multifaceted impact of stress on emotion regulation.

Childhood Gender Nonconformity and Recalled Perceived Parental and Peer Acceptance Thereof, Internalized Homophobia, and Psychological Well-Being Outcomes in Heterosexual and Gay Men from Poland.

The link between gender nonconformity and psychopathology may be due in part to negative childhood experiences resulting from other people's reactions to gender nonconformity. The aim of this study was to test whether recalled perceived levels of parental and peer acceptance of childhood gender nonconforming behaviors and play mediate the relationship of childhood gender nonconformity with depression and social anxiety in adulthood. We also tested whether this relationship was moderated by sexual orientation and, among gay men, whether internalized homophobia was an additional mediator. All variables were measured in a large sample of male participants using self-report (n = 449 gay men, age: M = 27.8 years, SD = 6.69; and n = 296 heterosexual men, age: M = 27.4 years, SD = 6.57) in Poland. Gay men reported more childhood gender nonconformity than heterosexual men. The relationship between gender nonconformity and depressive symptoms as well as social anxiety symptoms was significant in both gay and heterosexual men. Among gay men, this relationship was partially mediated by peer but not parental acceptance of the measured aspects of gender nonconformity and internalized homophobia. Among heterosexual men, recalled perceived parental acceptance of gender nonconformity partially mediated the relationship between gender nonconformity and depressive and social anxiety symptoms. Our findings were partially in line with those found in Western European and North American samples. Although the two groups differed in their recalled perceived gender nonconformity, they did not differ in their depression or social anxiety scores. Nevertheless, childhood gender nonconformity may be an indirect risk associated with mental health symptoms, irrespective of sexual orientation. Its higher prevalence among nonheterosexual individuals makes it a particular risk for this group.

Neogenin-1 distinguishes between myeloid-biased and balanced Hoxb5+ mouse long-term hematopoietic stem cells.

Hematopoietic stem cells (HSCs) self-renew and generate all blood cells. Recent studies with single cell transplants and lineage tracing suggest that adult HSCs are diverse in their reconstitution and lineage potentials. However, prospective isolation of these subpopulations has remained challenging. Here, we identify Neogenin-1 (NEO1) as a unique surface marker on a fraction of mouse HSCs labeled with Hoxb5, a specific reporter of long-term HSCs (LT-HSCs). We show that NEO1+Hoxb5+ LT-HSCs expand with age and respond to myeloablative stress in young mice while NEO1-Hoxb5+ LT-HSCs exhibit no significant change in number. Furthermore, NEO1+Hoxb5+ LT-HSCs are more often in the G2/S cell cycle phase compared to NEO1-Hoxb5+ LT-HSCs in both young and old bone marrow. Upon serial transplantation, NEO1+Hoxb5+ LT-HSCs exhibit myeloid-biased differentiation and reduced reconstitution while NEO1-Hoxb5+ LT-HSCs are lineage-balanced and stably reconstitute recipients. Gene expression analysis reveals erythroid and myeloid priming in the NEO1+ fraction and association of quiescence and self-renewal-related transcription factors with NEO1- LT-HSCs. Finally, transplanted NEO1+Hoxb5+ LT-HSCs rarely generate NEO1-Hoxb5+ LT-HSCs while NEO1-Hoxb5+ LT-HSCs repopulate both LT-HSC fractions. This supports a model in which dormant, balanced NEO1-Hoxb5+ LT-HSCs can hierarchically precede active, myeloid-biased NEO1+Hoxb5+ LT-HSCs.

Heme Oxygenase-1 Has a Greater Effect on Melanoma Stem Cell Properties Than the Expression of Melanoma-Initiating Cell Markers.

Melanoma-initiating cells (MICs) contribute to the tumorigenicity and heterogeneity of melanoma. MICs are identified by surface and functional markers and have been shown to display cancer stem cell (CSC) properties. However, the existence of MICs that follow the hierarchical CSC model has been questioned by studies showing that single unselected melanoma cells are highly tumorigenic in xenotransplantation assays. Herein, we characterize cells expressing MIC markers (CD20, CD24, CD133, Sca-1, ABCB1, ABCB5, ALDHhigh) in the B16-F10 murine melanoma cell line. We use flow cytometric phenotyping, single-cell sorting followed by in vitro clonogenic assays, and syngeneic in vivo serial transplantation assays to demonstrate that the expression of MIC markers does not select CSC-like cells in this cell line. Previously, our group showed that heme-degrading enzyme heme oxygenase-1 (HO-1) can be upregulated in melanoma and increase its aggressiveness. Here, we show that HO-1 activity is important for non-adherent growth of melanoma and HO-1 overexpression enhances the vasculogenic mimicry potential, which can be considered protumorigenic activity. However, HO-1 overexpression decreases clone formation in vitro and serial tumor initiation in vivo. Thus, HO-1 plays a dual role in melanoma, improving the progression of growing tumors but reducing the risk of melanoma initiation.

The relationship between early and recent life stress and emotional expression processing: A functional connectivity study.

The aim of this study was to characterize neural activation during the processing of negative facial expressions in a non-clinical group of individuals characterized by two factors: the levels of stress experienced in early life and in adulthood. Two models of stress consequences were investigated: the match/mismatch and cumulative stress models. The match/mismatch model assumes that early adversities may promote optimal coping with similar events in the future through fostering the development of coping strategies. The cumulative stress model assumes that effects of stress are additive, regardless of the timing of the stressors. Previous studies suggested that stress can have both cumulative and match/mismatch effects on brain structure and functioning and, consequently, we hypothesized that effects on brain circuitry would be found for both models. We anticipated effects on the neural circuitry of structures engaged in face perception and emotional processing. Hence, the amygdala, fusiform face area, occipital face area, and posterior superior temporal sulcus were selected as seeds for seed-based functional connectivity analyses. The interaction between early and recent stress was related to alterations during the processing of emotional expressions mainly in to the cerebellum, middle temporal gyrus, and supramarginal gyrus. For cumulative stress levels, such alterations were observed in functional connectivity to the middle temporal gyrus, lateral occipital cortex, precuneus, precentral and postcentral gyri, anterior and posterior cingulate gyri, and Heschl's gyrus. This study adds to the growing body of literature suggesting that both the cumulative and the match/mismatch hypotheses are useful in explaining the effects of stress.

Spheroid-plug model as a tool to study tumor development, angiogenesis, and heterogeneity in vivo.

Subcutaneous injection of the tumor cell suspension is a simple and commonly used tool for studying tumor development in vivo. However, subcutaneous models poorly resemble tumor complexity due to the fast growth not reflecting the natural course. Here, we describe an application of the new spheroid-plug model to combine the simplicity of subcutaneous injection with improved resemblance to natural tumor progression. Spheroid-plug model relies on in vitro formation of tumor spheroids, followed by injection of single tumor spheroid subcutaneously in Matrigel matrix. In spheroid-plug model, tumors grow slower in comparison to tumors formed by injection of cell suspension as assessed by 3D ultrasonography (USG) and in vivo bioluminescence measurements. The slower tumor growth rate in spheroid-plug model is accompanied by reduced necrosis. The spheroid-plug model ensures increased and more stable vascularization of tumor than classical subcutaneous tumor model as demonstrated by 3D USG Power Doppler examination. Flow cytometry analysis showed that tumors formed from spheroids have enhanced infiltration of endothelial cells as well as hematopoietic and progenitor cells with stem cell phenotype (c-Kit(+) and Sca-1(+)). They also contain more tumor cells expressing cancer stem cell marker CXCR4. Here, we show that spheroid-plug model allows investigating efficiency of anticancer drugs. Treatment of spheroid-plug tumors with known antiangiogenic agent axitinib decreased their size and viability. The antiangiogenic activity of axitinib was higher in spheroid-plug model than in classical model. Our results indicate that spheroid-plug model imitates natural tumor growth and can become a valuable tool for cancer research.

miR-378 influences muscle satellite cells and enhances adipogenic potential of fibro-adipogenic progenitors but does not affect muscle regeneration in the glycerol-induced injury model.

Skeletal muscle regeneration relies on the reciprocal interaction between many types of cells. Regenerative capacity may be altered in different disorders. In our study, we investigated whether the deletion of miR-378a (miR-378) affects muscle regeneration. We subjected 6-week-old wild-type (WT) and miR-378 knockout (miR-378-/-) animals to the glycerol-induced muscle injury and performed analyses in various time-points. In miR-378-/- animals, an elevated abundance of muscle satellite cells (mSCs) on day 3 was found. Furthermore, fibro-adipogenic progenitors (FAPs) isolated from the muscle of miR-378-/- mice exhibited enhanced adipogenic potential. At the same time, lack of miR-378 did not affect inflammation, fibrosis, adipose tissue deposition, centrally nucleated fiber count, muscle fiber size, FAP abundance, and muscle contractility at any time point analyzed. To conclude, our study revealed that miR-378 deletion influences the abundance of mSCs and the adipogenic potential of FAPs, but does not affect overall regeneration upon acute, glycerol-induced muscle injury.

Slow-cycling murine melanoma cells display plasticity and enhanced tumorigenicity in syngeneic transplantation assay.

Slow-cycling cancer cells (SCC) contribute to the aggressiveness of many cancers, and their invasiveness and chemoresistance pose a great therapeutic challenge. However, in melanoma, their tumor-initiating abilities are not fully understood. In this study, we used the syngeneic transplantation assay to investigate the tumor-initiating properties of melanoma SCC in the physiologically relevant in vivo settings. For this we used B16-F10 murine melanoma cell line where we identified a small fraction of SCC. We found that, unlike human melanoma, the murine melanoma SCC were not marked by the high expression of the epigenetic enzyme Jarid1b. At the same time, their slow-cycling phenotype was a temporary state, similar to what was described in human melanoma. Progeny of SCC had slightly increased doxorubicin resistance and altered expression of melanogenesis genes, independent of the expression of cancer stem cell markers. Single-cell expansion of SCC revealed delayed growth and reduced clone formation when compared to non-SCC, which was further confirmed by an in vitro limiting dilution assay. Finally, syngeneic transplantation of 10 cells in vivo established that SCC were able to initiate growth in primary recipients and continue growth in the serial transplantation assay, suggesting their self-renewal nature. Together, our study highlights the high plasticity and tumorigenicity of murine melanoma SCC and suggests their role in melanoma aggressiveness.

New data on the validity of the Fazio Laterality Inventory.

The Fazio Laterality Inventory (FLI) is a recent measure of handedness. Although initially validated, there is still a lack of studies assessing its psychometric properties in samples outside the USA. The present study explores the validity of the Polish adaptation of the FLI. We used data gathered from a convenience sample of 727 participants. They completed the FLI and the Edinburgh Handedness Inventory to establish concurrent validity. Confirmatory factor analysis was used to investigate the factor structure of the FLI. In addition, an Item Response Theory (IRT) model for continuous item scores was also used to identify the discrimination and difficulty parameters of the FLI items. The Polish version of the FLI was characterized by good reliability indices and has high concurrent validity with the Edinburgh Handedness Inventory. We identified a bi-factorial structure for the questionnaire. The IRT analyses showed that the FLI items have good discrimination and difficulty parameters. Our study provides new insights into the properties of the Fazio Laterality Inventory.

Brain activation during cognitive reappraisal depending on regulation goals and stimulus valence.

Neural bases of cognitive reappraisal may depend on the direction of regulation (up- or downregulation) and stimulus valence (positive or negative). This study aimed to examine this using a cognitive reappraisal task and conjunction analysis on a relatively large sample of 83 individuals. We identified regions in which activations were common for all these types of emotion regulation. We also investigated differences in brain activation between the 'decrease' and 'increase' emotional response conditions, and between the regulation of negative and positive emotions. The common activation across conditions involved mainly the prefrontal and temporal regions. Decreasing emotions was associated with stronger involvement of the dorsolateral prefrontal cortex, while increasing with activation of the amygdala and hippocampus. Regulation of negative emotions involved stronger activation of the lateral occipital cortex, while regulation of positive emotions involved stronger activation of the anterior cingulate cortex extending to the medial prefrontal cortex. This study adds to previous findings, not only by doing a conjunction analysis on both emotional valences and regulation goals, but also doing this in a bigger sample size. Results suggest that reappraisal is not a uniform process and may have different neural bases depending on regulation goals and stimulus valence.

Myths Concerning Sexual Violence Toward Women in Poland, Hungary, and Norway in the Context of System Justification Theory: The Role of Beliefs in the Biological Origins of Gender Differences and Ambivalent Sexism.

The purpose of this study was to compare attitudes toward rape in Poland, Hungary, and Norway. Based on system justification theory, we examined whether country of origin predicts levels of rape myth acceptance, beliefs in the biological origins of gender differences, and ambivalent sexism. There is also some evidence that beliefs in the biological origins of gender differences predict rape myth among Polish students and that this relationship is mediated by hostile sexism. The current study aimed to test whether this model can be applied to other countries. Participants (N = 266) were from Poland, Hungary, and Norway. The study was conducted online. The dependent and independent variables were measured with questionnaires. Polish participants had significantly higher levels of rape myth acceptance, beliefs in the biological origins of gender differences, and ambivalent sexism than Norwegian and Hungarian participants. Our proposed model was confirmed: belief in the biological origins of gender differences was associated with rape myth acceptance, with hostile sexism as a mediator. Benevolent sexism also turned out to be a mediator between beliefs in the biological origins of gender differences and rape myth acceptance. The mediational model of the relationship between beliefs in the biological origins of gender differences and rape myth acceptance can be applied to all three countries. This result suggests that anti-rape educational interventions should take into account the role of culture and society in the construction of gender differences.

Proximity Ligation Assay Detection of Protein-DNA Interactions-Is There a Link between Heme Oxygenase-1 and G-quadruplexes?

G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1-/- mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4-protein colocalizations can be quantitatively analyzed using PLA, even in rare cells.

Male sexual orientation, gender nonconformity, and neural activity during mental rotations: an fMRI study.

The cross-sex shift hypothesis predicts that gay men should perform more like heterosexual women on important neurocognitive tasks on which men score higher than women, such as mental rotation. Studies also suggest sex differences exist in the neural correlates of mental rotation. However, no studies have taken sexual orientation into account or considered within-group variation attributable to recalled gender nonconformity (a developmental trait reliably associated with human nonheterosexuality). We quantified the neural correlates of mental rotation by comparing two groups of gay men, gender conforming (n = 23) and gender nonconforming (n = 23), to gender conforming heterosexual men (n = 22) and women (n = 22). We observed a sex difference between heterosexual men and women in the premotor cortex/supplementary motor cortex and left medial superior frontal gyrus. We also observed a sex difference as well as a cross-sex shift in gay men who recalled being gender nonconforming as children in the right superior frontal gyrus, right angular gyrus, right amygdala/parahippocampal gyrus, and bilaterally in the middle temporal gyrus and precuneus. Thus, cross-sex shifts may be associated with underlying developmental factors which are associated with sexual orientation (such as gender nonconformity). The results also suggest that gay men should not be studied as a homogenous group.

Metaphor Processing Dysfunctions in Schizophrenia Patients With and Without Substance Use Disorders.

BACKGROUND: Patients with schizophrenia have difficulties comprehending metaphors, which significantly impedes communication. However, this topic has not been thoroughly studied in people with a dual diagnosis. On this basis, we formulated two research aims: a) to compare the ability to comprehend metaphors in schizophrenia patients without (SZ) and with substance use disorder (SZ-SUD) and b) to determine the relationship between the processing of metaphorical content and the severity of psychopathological symptoms in both clinical groups. METHODS: A total of 40 individuals with SZ and 40 individuals with SZ-SUD took part in the study. The control group was composed of 40 individuals without a psychiatric or neurological diagnosis. Four subtests from the Right Hemisphere Language Battery (Picture Metaphor Test, Written Metaphor Test, Picture Metaphor Explanation Test, Written Metaphor Explanation Test) were used to measure the ability to understand and explain metaphors. RESULTS: Both groups of individuals with schizophrenia (SZ and SZ-SUD) scored lower than individuals from the control group on all tests of metaphor processing. However, no differences were observed between the two clinical groups. SZ-SUD patients had better results for Picture Metaphor Explanation than for Written Metaphor Explanation. Negative symptoms were found to be significant predictors of difficulties with understanding and explaining metaphors. CONCLUSION: Individuals with schizophrenia, regardless of their substance use disorder (SUD) status, exhibit impaired metaphorical content processing. SUD in schizophrenia is not associated with significant impairments in understanding and explaining metaphorical content. Moreover, impairments in processing metaphorical content are associated with more severe negative symptoms of schizophrenia.

Proteomic analysis of young and old mouse hematopoietic stem cells and their progenitors reveals post-transcriptional regulation in stem cells.

The balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for a healthy blood supply; imbalances underlie hematological diseases. The importance of HSCs and their progenitors have led to their extensive characterization at genomic and transcriptomic levels. However, the proteomics of hematopoiesis remains incompletely understood. Here we report a proteomics resource from mass spectrometry of mouse young adult and old adult mouse HSCs, multipotent progenitors and oligopotent progenitors; 12 cell types in total. We validated differential protein levels, including confirmation that Dnmt3a protein levels are undetected in young adult mouse HSCs until forced into cycle. Additionally, through integrating proteomics and RNA-sequencing datasets, we identified a subset of genes with apparent post-transcriptional repression in young adult mouse HSCs. In summary, we report proteomic coverage of young and old mouse HSCs and progenitors, with broader implications for understanding mechanisms for stem cell maintenance, niche interactions and fate determination.

Cobalt protoporphyrin IX increases endogenous G-CSF and mobilizes HSC and granulocytes to the blood.

Granulocyte colony-stimulating factor (G-CSF) is used in clinical practice to mobilize cells from the bone marrow to the blood; however, it is not always effective. We show that cobalt protoporphyrin IX (CoPP) increases plasma concentrations of G-CSF, IL-6, and MCP-1 in mice, triggering the mobilization of granulocytes and hematopoietic stem and progenitor cells (HSPC). Compared with recombinant G-CSF, CoPP mobilizes higher number of HSPC and mature granulocytes. In contrast to G-CSF, CoPP does not increase the number of circulating T cells. Transplantation of CoPP-mobilized peripheral blood mononuclear cells (PBMC) results in higher chimerism and faster hematopoietic reconstitution than transplantation of PBMC mobilized by G-CSF. Although CoPP is used to activate Nrf2/HO-1 axis, the observed effects are Nrf2/HO-1 independent. Concluding, CoPP increases expression of mobilization-related cytokines and has superior mobilizing efficiency compared with recombinant G-CSF. This observation could lead to the development of new strategies for the treatment of neutropenia and HSPC transplantation.

Measuring ATP Concentration in a Small Number of Murine Hematopoietic Stem Cells.

The metabolism of quiescent adult stem cells differs from the metabolism of differentiated cells. The metabolic processes are tightly regulated and their alterations disturb function of stem cells. One of the indicators of metabolic status of cells is the ATP level. While the method of measuring the ATP levels has been known for many years, estimating ATP levels in small population of defined stem cells isolated directly from the tissue has remained challenging. Here, we show our method of measuring the ATP levels in hematopoietic stem cells sorted from murine bone marrow. We used magnetic sorting as well as cell sorter and adopted the commonly used bioluminescence-based detection kits in described protocol. Our strategy allows to measure ATP levels in 1000 highly purified HSC.