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d- chiro-Inositol (DCI), an isomer of inositol, possesses antioxidative and endothelial protective properties. Possibly due to a deficiency of insulin mediators, polycystic ovary syndrome (PCOS) is often associated with insulin resistance (IR) and hyperinsulinemia, likely responsible for an elevated production of reactive oxygen species. We investigated oxidative-related alterations of inositol in the blood of women with PCOS before and after treatment with DCI. A total of 38 normal-weight PCOS women were investigated before and after DCI administration (500 mg/day for 12 weeks; n = 38) by evaluating serum testosterone, serum androstenedione, fasting serum insulin, fasting serum glucose, and parameters of IR. From the blood, we determined biomarkers of oxidative stress: superoxide anion radicals, hydrogen peroxide, nitric oxide, and the index of lipid peroxidation. The activity of catalase and superoxide dismutase and the reduced glutathione (GSH) content in the hemolysate were also assessed. Data showed that PCOS patients' plasma underwent oxidative stress, as indicated by the higher level of prooxidants and reduced cytosolic GSH content. DCI treatment significantly improved the metabolic parameters. Also, serum values of testosterone were reduced. In conclusion, PCOS patients suffer from a systemic oxidative stress that induces endothelial dysfunction. Treatment with DCI is effective in reducing hormonal, metabolic, and oxidative abnormalities in PCOS patients by improving IR.

Supplementary Data from KAT6A and ENL Form an Epigenetic Transcriptional Control Module to Drive Critical Leukemogenic Gene-Expression Programs

Epigenetic programs are dysregulated in acute myeloid leukemia (AML) and help enforce an oncogenic state of differentiation arrest. To identify key epigenetic regulators of AML cell fate, we performed a differentiation-focused CRISPR screen in AML cells. This screen identified the histone acetyltransferase KAT6A as a novel regulator of myeloid differentiation that drives critical leukemogenic gene-expression programs. We show that KAT6A is the initiator of a newly described transcriptional control module in which KAT6A-catalyzed promoter H3K9ac is bound by the acetyl-lysine reader ENL, which in turn cooperates with a network of chromatin factors to induce transcriptional elongation. Inhibition of KAT6A has strong anti-AML phenotypes in vitro and in vivo, suggesting that KAT6A small-molecule inhibitors could be of high therapeutic interest for mono-therapy or combinatorial differentiation-based treatment of AML.Significance:AML is a poor-prognosis disease characterized by differentiation blockade. Through a cell-fate CRISPR screen, we identified KAT6A as a novel regulator of AML cell differentiation. Mechanistically, KAT6A cooperates with ENL in a “writer–reader” epigenetic transcriptional control module. These results uncover a new epigenetic dependency and therapeutic opportunity in AML.This article is highlighted in the In This Issue feature, p. 587

An impaired neutrophil response to pathogenic fungi puts patients at risk for fungal infections with a high risk of morbidity and mortality. Acquired neutrophil dysfunction in the setting of iatrogenic immune modulators can include the inhibition of critical kinases such as spleen tyrosine kinase (Syk). In this study, we used an established system of conditionally immortalized mouse neutrophil progenitors to investigate the ability to augment Syk-deficient neutrophil function against Candida albicans with TLR agonist signaling. LPS, a known immunomodulatory molecule derived from Gram-negative bacteria, was capable of rescuing effector functions of Syk-deficient neutrophils, which are known to have poor fungicidal activity against Candida species. LPS priming of Syk-deficient mouse neutrophils demonstrates partial rescue of fungicidal activity, including phagocytosis, degranulation, and neutrophil swarming, but not reactive oxygen species production against C. albicans, in part due to c-Fos activation. Similarly, LPS priming of human neutrophils rescues fungicidal activity in the presence of pharmacologic inhibition of Syk and Bruton’s tyrosine kinase (Btk), both critical kinases in the innate immune response to fungi. In vivo, neutropenic mice were reconstituted with wild-type or Syk-deficient neutrophils and challenged i.p. with C. albicans. In this model, LPS improved wild-type neutrophil homing to the fungal challenge, although Syk-deficient neutrophils did not persist in vivo, speaking to its crucial role on in vivo persistence. Taken together, we identify TLR signaling as an alternate activation pathway capable of partially restoring neutrophil effector function against Candida in a Syk-independent manner.

Peripheral paralysis of facial nerve in the newly-born can be congenital and developed. In clinical sense, paralysis of facial nerve is characterised by paralysis of mimic face muscles that are controlled by a facial nerve. A female newly-born, delivered by caesarean section was clinically diagnosed weakness on the right side of the face. Thirteen days after the birth the newly-born was examined by a physiatrist for the first time due to the weakness of the right facial side. During the first year of life a severe congenital lesion of facial nerve was diagnosed. Rehabilitation treatments were administered during the first year of life, with partial clinical improvement. The seriousness of facial nerve lesion hasa significant influence on the degree of recovery. It is very important to identify the type of lesion by using efficient technology, since it is the only way to provide early and adequate therapy

Epigenetic programs are dysregulated in acute myeloid leukemia (AML) and help enforce an oncogenic state of differentiation arrest. To identify key epigenetic regulators of AML cell fate, we performed a differentiation-focused CRISPR screen in AML cells. This screen identified the histone acetyltransferase KAT6A as a novel regulator of myeloid differentiation that drives critical leukemogenic gene-expression programs. We show that KAT6A is the initiator of a newly described transcriptional control module in which KAT6A-catalyzed promoter H3K9ac is bound by the acetyl-lysine reader ENL, which in turn cooperates with a network of chromatin factors to induce transcriptional elongation. Inhibition of KAT6A has strong anti-AML phenotypes in vitro and in vivo, suggesting that KAT6A small-molecule inhibitors could be of high therapeutic interest for mono-therapy or combinatorial differentiation-based treatment of AML. Significance: AML is a poor-prognosis disease characterized by differentiation blockade. Through a cell-fate CRISPR screen, we identified KAT6A as a novel regulator of AML cell differentiation. Mechanistically, KAT6A cooperates with ENL in a “writer–reader” epigenetic transcriptional control module. These results uncover a new epigenetic dependency and therapeutic opportunity in AML. This article is highlighted in the In This Issue feature, p. 587

The two-dimensional systems of first order nonlinear differential equations (S1) x? = p(t)y?, y? = q(t)x? and (S2) x? + p(t)y? = 0, y? + q(t)x? = 0 are analyzed using the theory of rapid variation. This approach allows us to prove that all strongly increasing solutions of system (S1) (and, respectively, all strongly decreasing solutions of system (S2) ) are rapidly varying functions under the assumption that p and q are rapidly varying. Also, the asymptotic equivalence relations for these solutions are given.

Tissue function depends on proper cellular organization. While the properties of individual cells are increasingly being deciphered using powerful single-cell sequencing technologies, understanding their spatial organization and temporal evolution remains a major challenge. Here, we present Image-seq, a technology that provides single-cell transcriptional data on cells that are isolated from specific spatial locations under image guidance, thus preserving the spatial information of the target cells. It is compatible with in situ and in vivo imaging and can document the temporal and dynamic history of the cells being analyzed. Cell samples are isolated from intact tissue and processed with state-of-the-art library preparation protocols. The technique therefore combines spatial information with highly sensitive RNA sequencing readouts from individual, intact cells. We have used both high-throughput, droplet-based sequencing, as well as SMARTseq-v4 library preparation to demonstrate its application to bone marrow and leukemia biology. We discovered that DPP4 is a highly upregulated gene during early AML progression and that it marks a more proliferative subpopulation which is confined to specific bone marrow microenvironments. Furthermore, Image-seq’s ability to isolate viable, intact cells should make it compatible with a range of downstream single-cell analysis tools including multi-omics protocols.

Control of cellular identity involves coordination of developmental programs with environmental factors such as nutrient availability, suggesting that modulating aspects of metabolism could enable therapeutically relevant changes in cell fate. We show that nucleotide depletion facilitates gene expression changes towards a new cell fate by perturbing DNA replication in models of acute myeloid leukemia, a cancer characterized by a differentiation blockade. This transition starts in S phase and is independent of replication stress signaling and DNA damage signaling pathways. Moreover, it occurs despite sustained oncogene-driven expression of the progenitor program and is accompanied by limited changes in chromatin accessibility. Altering lineage-determining transcription factor expression redirects cell fate progression towards an alternate fate upon replication stress, suggesting that perturbing DNA replication allows cells to mobilize primed maturation programs. Our work, along with other findings in diverse systems, suggests a conserved mechanism by which metabolic changes can orchestrate cell fate transitions.

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The association of cervical pregnancy and gestational trophoblastic disease is extreme rare, but it is obvious that such combination is associated with devastating effects on future fertility. We present a case of the patient with cervical molar pregnancy, who was admitted due to hemorrhagic shock caused by profuse metrorrhagia at 7th gestational week. Slightly enlarged uterine corpus, with bulky cervix and normal adnexa were found on laparotomy. Total abdominal hysterectomy with the conservation of one ovary was performed. Pathohistological examination confirmed cervical molar pregnancy. In the case of an ectopic, molar or ectopic molar pregnancy, the early visit to gynecologist would give the opportunity to plan the treatment and to preserve the uterus for further pregnancies.

The structural capacity of buildings to guarantee acceptable safety levels during and after earthquakes has significant influence on community and urban resilience. The paper presents the implementation in the Geographic Information System (GIS) of the data related to the expected seismic damage scenarios for some existing residential mixed masonry-reinforced concrete buildings located in Lisbon area. At city scale, such kind of representation can effectively support the seismic risk management including the design of mitigation policies. In fact, the spatial representation of results provides clear and global overview of the area under analysis, understandable not only to scientist and engineers, but also to the city councils and population. Damage scenario are defined on basis on fragility curves computed analytically through a nonlinear static approach by focusing the attention only to the in-plane global response and considering also the aggregate effect. The application to Lisbon city shows that the expected level of the damage consequent to the code action is high for the structural typology under investigation.

Background/Aim. Craniosynostosis is a condition that occurs intrauterine or develops in the infant period, and represents premature fusion of cranial sutures. This fusion of sutures limits the normal cranium development and leads to disorder in the phase of rapid growth and development of the brain. Creation of craniosynostoses is associated with an increased incidence of developmental delay during the breastfeeding period. Craniosynostoses are treated by surgery. The role of a physiatrist is to postoperatively assess psychomotor development and implement habilitation treatment. The aim of this study was to determine distribution of the type of craniosynostoses according to the age and gender of patients, effectiveness of habilitation treatment and to estimate the somatosensory evoked potential in the preoperative and postoperative period in children who underwent craniosynostosis surgery in the first year of life. Methods. The study was designed as a retrospective research. The data were collected from medical records of 51 children with craniosynostoses and delay in psychomotor development who underwent surgical intervention. The children included in this study, during follow-up, were involved in the habilitation treatment. Results. An early diagnosis and surgical intervention had a favorable effect on the development of motor function in children with craniosynostoses. The importance of stimulation treatment in the postoperative period was also proved for achievement of an adequate degree of motor development in children in relation to age. The results of our study confirmed the results obtained in previously published studies that the children who did not undergo surgery and start with the habilitation treatment immediately after it, had delay in psychomotor development of moderate degree. Conclusion. Habilitation treatment significantly reduced the deviations in psychomotor development of children with craniosynostoses if it started immediately after the surgical procedure.

Point load actions may have a significant impact on deflections of tensile membrane structures. Research presented in this paper is aimed at exploring the effects of point load actions on membrane forces of tensile membrane structures. Therefore, three different parameters of the structure were varied, and the membrane forces resulting from point loads were monitored. Variable parameters are the height of the model, the intensity of prestressing forces, and the orientation of the membrane material. The research was done on models in specialized software. In order to evaluate the significance of point load effects, membrane forces were compared to those under snow load. The results of the research show the layout of membrane forces under point load and dependence of maximal membrane forces on the varied parameters. Specific sets of analyzed parameters lead to significant values of maximal membrane forces under point load action.

Barth syndrome is an inherited X-linked disorder that leads to cardiomyopathy, skeletal myopathy, and neutropenia. These symptoms result from the loss of function of the enzyme TAFAZZIN, a transacylase located in the inner mitochondrial membrane that is responsible for the final steps of cardiolipin production. The link between defective cardiolipin maturation and neutropenia remains unclear. To address potential mechanisms of neutropenia, we examined myeloid progenitor development within the fetal liver of TAFAZZIN knockout (KO) animals as well as within the adult bone marrow of wild-type recipients transplanted with TAFAZZIN-KO hematopoietic stem cells. We also used the ER-Hoxb8 system (estrogen receptor fused to Hoxb8) of conditional immortalization to establish a new murine model system for the ex vivo study of TAFAZZIN-deficient neutrophils. The TAFAZZIN-KO cells demonstrated the expected dramatic differences in cardiolipin maturation that result from a lack of TAFAZZIN enzyme activity. Contrary to our hypothesis, we did not identify any significant differences in neutrophil development or neutrophil function across a variety of assays including phagocytosis and the production of cytokines or reactive oxygen species. However, transcriptomic analysis of the TAFAZZIN-deficient neutrophil progenitors demonstrated an upregulation of markers of endoplasmic reticulum stress and confirmatory testing demonstrated that the TAFAZZIN-deficient cells had increased sensitivity to certain ER stress-mediated and non-ER stress-mediated triggers of apoptosis. Although the link between increased sensitivity to apoptosis and the variably penetrant neutropenia phenotype seen in some patients with Barth syndrome remains to be clarified, our studies and new model system set a foundation for further investigation.

Tissue function depends on cellular organization. While the properties of individual cells are increasingly being deciphered using powerful single-cell sequencing technologies, understanding their spatial organization and temporal evolution remains a major challenge. Here, we present Image-seq, a technology that provides single-cell transcriptional data on cells that are isolated from specific spatial locations under image guidance, thus preserving the spatial information of the target cells. It is compatible with in situ and in vivo imaging and can document the temporal and dynamic history of the cells being analyzed. Cell samples are isolated from intact tissue and processed with state-of-the-art library preparation protocols. The technique therefore combines spatial information with highly sensitive RNA sequencing readouts from individual, intact cells. We have used both high-throughput, droplet-based sequencing as well as SMARTseq-v4 library preparation to demonstrate its application to bone marrow and leukemia biology. We discovered that DPP4 is a highly upregulated gene during early progression of acute myeloid leukemia and that it marks a more proliferative subpopulation that is confined to specific bone marrow microenvironments. Furthermore, the ability of Image-seq to isolate viable, intact cells should make it compatible with a range of downstream single-cell analysis tools including multi-omics protocols.

Childhood medulloblastoma and high-risk neuroblastoma frequently present with segmental gain of chromosome 17q corresponding to aggressive tumors and poor patient prognosis. Located within the 17q-gained chromosomal segments is PPM1D at chromosome 17q23.2. PPM1D encodes a serine/threonine phosphatase, WIP1, that is a negative regulator of p53 activity as well as key proteins involved in cell cycle control, DNA repair and apoptosis. Here, we show that the level of PPM1D expression correlates with chromosome 17q gain in medulloblastoma and neuroblastoma cells, and both medulloblastoma and neuroblastoma cells are highly dependent on PPM1D expression for survival. Comparison of different inhibitors of WIP1 showed that SL-176 was the most potent compound inhibiting medulloblastoma and neuroblastoma growth and had similar or more potent effects on cell survival than the MDM2 inhibitor Nutlin-3 or the p53 activator RITA. SL-176 monotherapy significantly suppressed the growth of established medulloblastoma and neuroblastoma xenografts in nude mice. These results suggest that the development of clinically applicable compounds inhibiting the activity of WIP1 is of importance since PPM1D activating mutations, genetic gain or amplifications and/or overexpression of WIP1 are frequently detected in several different cancers.

Background Transgenic animal models are crucial for the study of gene function and disease, and are widely utilized in basic biological research, agriculture and pharma industries. Since the current methods for generating transgenic animals result in the random integration of the transgene under study, the phenotype may be compromised due to disruption of known genes or regulatory regions. Unfortunately, most of the tools that predict transgene insertion sites from high-throughput data are not publicly available or not properly maintained. Results We implemented TC-hunter, Transgene-Construct hunter, an open tool that identifies transgene insertion sites and provides simple reports and visualization aids. It relies on common tools used in the analysis of high-throughput data and makes use of chimeric reads and discordant read pairs to identify and support the transgenic insertion site. To demonstrate its applicability, we applied TC-hunter to four transgenic mice samples harboring the human PPM1D gene, a model used in the study of malignant tumor development. We identified the transgenic insertion site in each sample and experimentally validated them with Touchdown-polymerase chain reaction followed by Sanger sequencing. Conclusions TC-hunter is an accessible bioinformatics tool that can automatically identify transgene insertion sites from DNA sequencing data with high sensitivity (98%) and precision (92.45%). TC-hunter is a valuable tool that can aid in evaluating any potential phenotypic complications due to the random integration of the transgene and can be accessed at https://github.com/bcfgothenburg/SSF.

Simple Summary Medulloblastoma and neuroblastoma are childhood tumors of the central nervous system or the peripheral nervous system, respectively. These are the most common and deadly tumors of childhood. A common genetic feature of medulloblastoma and neuroblastoma is frequent segmental gain or amplification of chromosome 17q. Located on chromosome 17q23.2 is PPM1D which encodes WIP1, a phosphatase that acts as a regulator of p53 and DNA repair. Overexpression of WIP1 correlates with poor patient prognosis. We investigated the effects of genetic or pharmacologic inhibition of WIP1 activity and found that medulloblastoma and neuroblastoma cells were strongly dependent on WIP1 expression for survival. We also tested a number of small molecule inhibitors of WIP1 and show that SL-176 was the most effective compound suppressing the growth of medulloblastoma and neuroblastoma in vitro and in vivo. Abstract Childhood medulloblastoma and high-risk neuroblastoma frequently present with segmental gain of chromosome 17q corresponding to aggressive tumors and poor patient prognosis. Located within the 17q-gained chromosomal segments is PPM1D at chromosome 17q23.2. PPM1D encodes a serine/threonine phosphatase, WIP1, that is a negative regulator of p53 activity as well as key proteins involved in cell cycle control, DNA repair and apoptosis. Here, we show that the level of PPM1D expression correlates with chromosome 17q gain in medulloblastoma and neuroblastoma cells, and both medulloblastoma and neuroblastoma cells are highly dependent on PPM1D expression for survival. Comparison of different inhibitors of WIP1 showed that SL-176 was the most potent compound inhibiting medulloblastoma and neuroblastoma growth and had similar or more potent effects on cell survival than the MDM2 inhibitor Nutlin-3 or the p53 activator RITA. SL-176 monotherapy significantly suppressed the growth of established medulloblastoma and neuroblastoma xenografts in nude mice. These results suggest that the development of clinically applicable compounds inhibiting the activity of WIP1 is of importance since PPM1D activating mutations, genetic gain or amplifications and/or overexpression of WIP1 are frequently detected in several different cancers.