Since the emergence of Zika virus (ZIKV), reports of microcephaly have increased considerably in Brazil; however, causality between the viral epidemic and malformations in fetal brains needs further confirmation. We examined the effects of ZIKV infection in human neural stem cells growing as neurospheres and brain organoids. Using immunocytochemistry and electron microscopy, we showed that ZIKV targets human brain cells, reducing their viability and growth as neurospheres and brain organoids. These results suggest that ZIKV abrogates neurogenesis during human brain development. P rimary microcephaly is a severe brain malformation characterized by the reduction of the head circumference. Patients display a heterogeneous range of brain impairments that compromise motor, visual, hearing, and cognitive functions (1).Microcephaly is associated with decreased neuronal production as a consequence of proliferative defects and death of cortical progenitor cells (2). During pregnancy, the primary etiology of microcephaly varies from genetic mutations to external insults. The so-called TORCHS factors (toxoplasmosis, rubella, cytomegalovirus, herpes virus, and syphilis) are the main congenital infections that compromise brain development in utero (3).An increase in the rate of microcephaly in Brazil has been associated with the recent outbreak of Zika virus (ZIKV) (4, 5), a flavivirus that is transmitted by mosquitoes (6) and sexually (7-9). So far, ZIKV has been described in the placenta and amniotic fluid of microcephalic fetuses (10-13) and in the blood of microcephalic newborns (11, 14). ZIKV had also been detected within the brain of a microcephalic fetus (13, 14), and recently, direct evidence has emerged that ZIKV is able to infect and cause the death of neural stem cells (15).We used human induced pluripotent stem (iPS) cells cultured as neural stem cells (NSCs), neurospheres, and brain organoids to explore the consequences of ZIKV infection during neurogenesis and growth with three-dimensional culture models. Human iPS-derived NSCs were exposed to ZIKV [multiplicity of infection (MOI), 0.25 to 0.0025]. After 24 hours, ZIKV was detected in NSCs (Fig. 1, A to D); viral envelope protein was evident in 10.10% (MOI, 0.025) and 21.7% (MOI, 0.25) of cells exposed to ZIKV (Fig. 1E). Viral RNA was also detected in the supernatant of infected NSCs (MOI, 0.0025) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) ( Fig. 1F), providing evidence of productive infection.To investigate the effects of ZIKV during neural differentiation, mock-and ZIKV-infected NSCs were cultured as neurospheres. After 3 days in vitro (DIV), mock-infected NSCs generated round neurospheres. However, ZIKVinfected NSCs generated neurospheres with morphological abnormalities and cell detachment ( Mock-infected neurospheres presented the expected ultrastructural morphology of the nucleus and mitochondria (Fig. 3A). Viral particles were present in ZIKV-infected neurospheres, similar to those observed in murine glial and neuronal cel...

SUMMARY:We provide evidence that Zika virus infects human iPS-derived neural stem cells, causing cell death and reduced growth in neurospheres and cerebral organoids.

This experiment was performed to develop and validate practical techniques for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function in bovine spermatozoa using associations of fluorescent probes. Four protocols of fluorescent probes association were defined: protocol 1: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and rhodamine 123; protocol 2: PI, FITC-PSA and MitoTracker Green FM (MITO); protocol 3: PI, Hoechst 33342 (H342), FITC-PSA and CMXRos; and protocol 4: PI, H342, FITC-PSA and JC-1. Three ejaculates from each of the four bulls (n = 12) were utilized, showing sperm motility >/=80% and abnormal morphology

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BackgroundBirch bark has a long lasting history as a traditional medicinal remedy to accelerate wound healing. Recently, the efficacy of birch bark preparations has also been proven clinically. As active principle pentacyclic triterpenes are generally accepted. Here, we report a comprehensive study on the underlying molecular mechanisms of the wound healing properties of a well-defined birch bark preparation named as TE (triterpene extract) as well as the isolated single triterpenes in human primary keratinocytes and porcine ex-vivo wound healing models.Methodology/Principal FindingsWe show positive wound healing effects of TE and betulin in scratch assay experiments with primary human keratinocytes and in a porcine ex-vivo wound healing model (WHM). Mechanistical studies elucidate that TE and betulin transiently upregulate pro-inflammatory cytokines, chemokines and cyclooxygenase-2 on gene and protein level. For COX-2 and IL-6 this increase of mRNA is due to an mRNA stabilizing effect of TE and betulin, a process in which p38 MAPK and HuR are involved. TE promotes keratinocyte migration, putatively by increasing the formation of actin filopodia, lamellipodia and stress fibers. Detailed analyses show that the TE components betulin, lupeol and erythrodiol exert this effect even in nanomolar concentrations. Targeting the actin cytoskeleton is dependent on the activation of Rho GTPases.Conclusion/SignificanceOur results provide insights to understand the molecular mechanism of the clinically proven wound healing effect of birch bark. TE and betulin address the inflammatory phase of wound healing by transient up-regulation of several pro-inflammatory mediators. Further, they enhance migration of keratinocytes, which is essential in the second phase of wound healing. Our results, together with the clinically proven efficacy, identify birch bark as the first medical plant with a high potential to improve wound healing, a field which urgently needs effective remedies.

Zika virus (ZIKV) has been associated with microcephaly and other brain abnormalities; however, the molecular consequences of ZIKV to human brain development are still not fully understood. Here we describe alterations in human neurospheres derived from induced pluripotent stem (iPS) cells infected with the strain of Zika virus that is circulating in Brazil. Combining proteomics and mRNA transcriptional profiling, over 500 proteins and genes associated with the Brazilian ZIKV infection were found to be differentially expressed. These genes and proteins provide an interactome map, which indicates that ZIKV controls the expression of RNA processing bodies, miRNA biogenesis and splicing factors required for self-replication. It also suggests that impairments in the molecular pathways underpinning cell cycle and neuronal differentiation are caused by ZIKV. These results point to biological mechanisms implicated in brain malformations, which are important to further the understanding of ZIKV infection and can be exploited as therapeutic potential targets to mitigate it.

Since the emergence of Zika virus (ZIKV), reports of microcephaly have increased dramatically in Brazil; however, causality between the widespread epidemic and malformations in fetal brains has not been confirmed. Here, we examine the effects of ZIKV infection in human neural stem cells growing as neurospheres and cerebral organoids. Using immunocytochemistry and electron microscopy, we show that ZIKV targets human brain cells, reducing their viability and growth as neurospheres and cerebral organoids. These results suggest that ZIKV abrogates neurogenesis during human brain development.

Dimethyltryptamines are entheogenic serotonin-like molecules present in traditional Amerindian medicine recently associated with cognitive gains, antidepressant effects, and changes in brain areas related to attention. Legal restrictions and the lack of adequate experimental models have limited the understanding of how such substances impact human brain metabolism. Here we used shotgun mass spectrometry to explore proteomic differences induced by 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) on human cerebral organoids. Out of the 6,728 identified proteins, 934 were found differentially expressed in 5-MeO-DMT-treated cerebral organoids. In silico analysis reinforced previously reported anti-inflammatory actions of 5-MeO-DMT and revealed modulatory effects on proteins associated with long-term potentiation, the formation of dendritic spines, including those involved in cellular protrusion formation, microtubule dynamics, and cytoskeletal reorganization. Our data offer the first insight about molecular alterations caused by 5-MeO-DMT in human cerebral organoids.