Expression of Retinoid Acid Receptor-Responsive Genes in Rodent Models of Placental Pathology

In humans, retinoic acid receptor responders (RARRES) have been shown to be altered in third trimester placentas complicated by the pathologies preeclampsia (PE) and PE with intrauterine growth restriction (IUGR). Currently, little is known about the role of placental Rarres in rodents. Therefore, we examined the localization and expression of Rarres1 and 2 in placentas obtained from a Wistar rat model of isocaloric maternal protein restriction (E18.5, IUGR-like features) and from an eNOS-knockout mouse model (E15 and E18.5, PE-like features). In both rodent models, Rarres1 and 2 were mainly localized in the placental spongiotrophoblast and giant cells. Their placental expression, as well as the expression of the Rarres2 receptor chemokine-like receptor 1 (CmklR1), was largely unaltered at the examined gestational ages in both animal models. Our results have shown that RARRES1 and 2 may have different expression and roles in human and rodent placentas, thereby underlining immanent limitations of comparative interspecies placentology. Further functional studies are required to elucidate the potential involvement of these proteins in early placentogenesis

Application of next-generation sequencing to Enterobacter hormaechei subspecies analysis during a neonatal intensive care unit outbreak

Introduction: The Enterobacter cloacae complex (ECC) species are potential neonatal pathogens, and ECC strains are among the most commonly encountered Enterobacter spp. associated with nosocomial bloodstream infections. Outbreaks caused by ECC can lead to significant morbidity and mortality in susceptible neonates. At the molecular level, ECC exhibits genomic heterogeneity, with six closely related species and subspecies. Genetic variability poses a challenge in accurately identifying outbreaks by determining the clonality of ECC isolates. This difficulty is further compounded by the limitations of the commonly used molecular typing methods, such as pulsed field gel electrophoresis, which do not provide reliable accuracy in distinguishing between ECC strains and can lead to incorrect conclusions. Next-generation sequencing (NGS) offers superior resolution in determining strain relatedness. Therefore, we investigated the clinical pertinence of incorporating NGS into existing bundle measures to enhance patient management during an outbreak of ECC in a level-3 neonatal intensive care unit (NICU) in Germany. Methods: As the standard of care, all neonates on the NICU received weekly microbiological swabs (nasopharyngeal and rectal) and analysis of endotracheal secretion, where feasible. During the 2.5-month outbreak, colonisation with ECC was detected in n = 10 neonates. The phylogenetic relationship and potential antimicrobial resistance genes as well as mobile genetic elements were identified via bacterial whole-genome sequencing (WGS) using Illumina MiSeq followed by in silico data analysis. Results: Although all ECC isolates exhibited almost identical antimicrobial susceptibility patterns, the WGS data revealed the involvement of four different ECC clones. The isolates could be characterised as Enterobacter hormaechei subspecies steigerwaltii (n = 6, clonal), subsp. hoffmannii (n = 3, two clones) and subsp. oharae (n = 1). Despite the collection of environmental samples, no source of this diffuse outbreak could be identified. A new standardised operating procedure was implemented to enhance the management of neonates colonised with MRGN. This collaborative approach involved both parents and medical professionals and successfully prevented further transmission of ECC. Conclusions: Initially, it was believed that the NICU outbreak was caused by a single ECC clone due to the similarity in antibiotic resistance. However, our findings show that antibiotic susceptibility patterns can be misleading in investigating outbreaks of multi-drug-resistant ECC. In contrast, bacterial WGS accurately identified ECC at the clonal level, which significantly helped to delineate the nature of the observed outbreak

SIRT1 regulates Mxd1 during malignant melanoma progression

In a murine melanoma model, malignant transformation promoted by a sustained stress condition was causally related to increased levels of reactive oxygen species resulting in DNA damage and massive epigenetic alterations. Since the chromatin modifier Sirtuin-1 (SIRT1) is a protein attracted to double-stranded DNA break (DSB) sites and can recruit other components of the epigenetic machinery, we aimed to define the role of SIRT1 in melanomagenesis through our melanoma model. The DNA damage marker, gamma H2AX was found increased in melanocytes after 24 hours of deadhesion, accompanied by increased SIRT1 expression and decreased levels of its target, H4K16ac. Moreover, SIRT1 started to be associated to DNMT3B during the stress condition, and this complex was maintained along malignant progression. Mxd1 was identified by ChIP-seq among the DNA sequences differentially associated with SIRT1 during deadhesion and was shown to be a common target of both, SIRT1 and DNMT3B. In addition, Mxd1 was found downregulated from pre-malignant melanocytes to metastatic melanoma cells. Treatment with DNMT inhibitor 5AzaCdR reversed the Mxd1 expression. Sirt1 stable silencing increased Mxd1 mRNA expression and led to down-regulation of MYC targets, such as Cdkn1a, Bcl2 and Psen2, whose upregulation is associated with human melanoma aggressiveness and poor prognosis. We demonstrated a novel role of the stress responsive protein SIRT1 in malignant transformation of melanocytes associated with deadhesion. Mxd1 was identified as a new SIRT1 target gene. SIRT1 promoted Mxd1 silencing, which led to increased activity of MYC oncogene contributing to melanoma progression.FAPESP [2011/0166-38, 2011/12306-1, 2014/13663-0, 2015/07925-5, 2016/06488-3]DAAD [PKZ A/12/79134]FAPESP/BAYLAT [2012/51300-7]Univ Fed Sao Paulo UNIFESP, Dept Pharmacol, Ontogeny & Epigenet Lab, Sao Paulo, SP, BrazilUniv Sao Paulo, Ribeirao Preto Med Sch, Dept Genet, Ribeirao Preto, SP, BrazilFriedrich Alexander Univ Erlangen Nurnberg FAU, Inst Pathol, Expt Tumorpathol, Erlangen, GermanyFriedrich Alexander Univ Erlangen Nurnberg FAU, Dept Pediat & Adolescent Med, Erlangen, GermanyUniv Fed Sao Paulo UNIFESP, Dept Pharmacol, Ontogeny & Epigenet Lab, Sao Paulo, SP, BrazilFAPESP [2011/0166-38, 2011/12306-1, 2014/13663-0, 2015/07925-5, 2016/06488-3]DAAD [PKZ A/12/79134]FAPESP/BAYLAT [2012/51300-7]Web of Scienc

Thymoquinone-induced conformational changes of PAK1 interrupt prosurvival MEK-ERK signaling in colorectal cancer

Background Thymoquinone (TQ) was shown to reduce tumor growth in several cancer models both in vitro and in vivo. So far only a few targets of TQ, including protein kinases have been identified. Considering that kinases are promising candidates for targeted anticancer therapy, we studied the complex kinase network regulated by TQ. Methods Novel kinase targets influenced by TQ were revealed by in silico analysis of peptide array data obtained from TQ-treated HCT116wt cells. Western blotting and kinase activity assays were used to determine changes in kinase expression patterns in colorectal cancer cells (HCT116wt, DLD-1, HT29). To study the viability/apoptotic effects of combining the PAK1 inhibitor IPA-3 and TQ, crystal violet assay and AnnexinV/PI staining were employed. Interactions between PAK1 and ERK1/2 were investigated by co-immunoprecipitation and modeled by docking studies. Transfection with different PAK1 mutants unraveled the role of TQ-induced changes in PAK1 phosphorylation and TQ´s effects on PAK1 scaffold function. Results Of the 104 proteins identified, 50 were upregulated ≥2 fold by TQ and included molecules in the AKT-MEK-ERK1/2 pathway. Oncogenic PAK1 emerged as an interesting TQ target. Time-dependent changes in two PAK1 phosphorylation sites generated a specific kinase profile with early increase in pPAKThr212 followed by late increase in pPAKThr423. TQ induced an increase of pERK1/2 and triggered the early formation of an ERK1/2-PAK1 complex. Modeling confirmed that TQ binds in the vicinity of Thr212 accompanied by conformational changes in ERK2-PAK1 binding. Transfecting the cells with the non-phosphorylatable mutant T212A revealed an increase of pPAKThr423 and enhanced apoptosis. Likewise, an increase in apoptosis was observed in cells transfected with both the kinase-dead K299R mutant and PAK1 siRNA. Using structural modeling we suggest that TQ interferes also with the kinase domain consequently disturbing its interaction with pPAKThr423, finally inhibiting MEK-ERK1/2 signaling and disrupting its prosurvival function. pERK1/2 loss was also validated in vivo. Conclusions Our study shows for the first time that the small molecule TQ directly binds to PAK1 changing its conformation and scaffold function. Because TQ affects the central RAF/MEK/ERK1/2 pathway, the combination of TQ with targeted therapies is worth considering for future anticancer treatments

Survey in radiation oncology departments in Germany, Austria, and Switzerland: state of digitalization by 2023.

PURPOSE The aim of this work was to assess the current state of digitalization in radiation oncology departments in Germany, Austria, and Switzerland. METHODS A comprehensive survey was conducted in a digital format, consisting of 53 questions that covered various aspects of digitalization including patient workflow, departmental organization, radiotherapy planning, and employee-related aspects. RESULTS Overall, 120 forms were eligible for evaluation. Participants were mainly physicians or medical physicists responsible for digitalization aspects in their departments. Nearly 70% of the institutions used electronic patient records, with 50% being completely paperless. However, the use of smartphone apps for electronic patient reported outcomes (ePROMs) and digital health applications (DIGA) was limited (9% and 4.9%, respectively). In total, 70.8% of the radio-oncology departments had interfaces with diagnostic departments, and 36% had digital interchanges with other clinics. Communication with external partners was realized mainly through fax (72%), e‑mails (55%), postal letters (63%), or other digital exchange formats (28%). Almost half of the institutions (49%) had dedicated IT staff for their operations. CONCLUSION To the best of our knowledge, this survey is the first of its kind conducted in German-speaking radiation oncology departments within the medical field. The findings suggest that there is a varied level of digitalization implementation within these departments, with certain areas exhibiting lower rates of digitalization that could benefit from targeted improvement initiatives

Influence of Low Protein Diet-Induced Fetal Growth Restriction on the Neuroplacental Corticosterone Axis in the Rat

Objectives: Placental steroid metabolism is linked to the fetal hypothalamus-pituitary-adrenal axis. Intrauterine growth restriction (IUGR) might alter this cross-talk and lead to maternal stress, in turn contributing to the pathogenesis of anxiety-related disorders of the offspring, which might be mediated by fetal overexposure to, or a reduced local enzymatic protection against maternal glucocorticoids. So far, direct evidence of altered levels of circulating/local glucocorticoids is scarce. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) allows quantitative endocrine assessment of blood and tissue. Using a rat model of maternal protein restriction (low protein [LP] vs. normal protein [NP]) to induce IUGR, we analyzed fetal and maternal steroid levels via LC-MS/MS along with the local expression of 11beta-hydroxysteroid-dehydrogenase (Hsd11b).Methods: Pregnant Wistar dams were fed a low protein (8%, LP; IUGR) or an isocaloric normal protein diet (17%, NP; controls). At E18.5, the expression of Hsd11b1 and 2 was determined by RT-PCR in fetal placenta and brain. Steroid profiling of maternal and fetal whole blood, fetal brain, and placenta was performed via LC-MS/MS.Results: In animals with LP-induced reduced body (p < 0.001) and placental weights (p < 0.05) we did not observe any difference in the expressional Hsd11b1/2-ratio in brain or placenta. Moreover, LP diet did not alter corticosterone (Cort) or 11-dehydrocorticosterone (DH-Cort) levels in dams, while fetal whole blood levels of Cort were significantly lower in the LP group (p < 0.001) and concomitantly in LP brain (p = 0.003) and LP placenta (p = 0.002). Maternal and fetal progesterone levels (whole blood and tissue) were not influenced by LP diet.Conclusion: Various rat models of intrauterine stress show profound alterations in placental Hsd11b2 gatekeeper function and fetal overexposure to corticosterone. In contrast, LP diet in our model induced IUGR without altering maternal steroid levels or placental enzymatic glucocorticoid barrier function. In fact, IUGR offspring showed significantly reduced levels of circulating and local corticosterone. Thus, our LP model might not represent a genuine model of intrauterine stress. Hypothetically, the observed changes might reflect a fetal attempt to maintain anabolic conditions in the light of protein restriction to sustain regular brain development. This may contribute to fetal origins of later neurodevelopmental sequelae

Sex differences in long-term kidney fibrosis following neonatal nephron loss during ongoing nephrogenesis

Abstract Background Clinical studies suggest that female sex plays a protective role in the development and progression of kidney disease. Recent experimental studies indicate that in male rats early nephron loss under ongoing nephrogenesis is accompanied by severe long-term sequelae. In humans, nephron formation occurs mainly in the third trimester, ceasing with 36 weeks of gestation. Due to perinatal complications, preterm infants delivered during this vulnerable period may undergo acute nephron loss. In rats nephrogenesis persists until postnatal day 10, reflecting the situation of human preterms with persisting nephrogenesis. In our animal model of neonatal uninephrectomy, female and male rats were uninephrectomized at day 1 of life. Hypothesizing sex-dependent differences, long-term renal outcome was assessed after 1 year. Results In both sexes, neonatal uninephrectomy was not followed by arterial hypertension at 1 year of age. Compensatory weight gain and glomerular hypertrophy of the remaining kidney occurred in uninephrectomized female and male animals. Selected markers of interstitial inflammation and fibrosis were regulated sex-dependently. The expression of monocyte chemoattractant protein-1 was increased in females, while tubulointerstitial infiltration by M1 macrophages was significantly higher in males after neonatal uninephrectomy. Neonatally uninephrectomized male rats had more glomerulosclerosis and podocyte damage compared to females, which was assessed by a semiquantitative score and desmin staining. RT-PCR revealed that after neonatal uninephrectomy in the remaining contralateral kidney of female rats the expression of candidate genes of renal development and function, i.e., wt-1, nephrin, synaptopodin, gdnf, and itga8 was higher than in males. Conclusions Based on these observations we conclude that female sex is protective in the long-term response of the kidney to acute nephron loss under active nephrogenesis

Expression of the Alpha8 Integrin Chain Facilitates Phagocytosis by Renal Mesangial Cells

Background/Aims: Healing of mesangioproliferative glomerulonephritis involves degradation of excess extracellular matrix, resolution of hypercellularity by apoptosis and phagocytosis of apoptotic cells. Integrin receptors participate in the regulation of phagocytosis. In mice deficient for alpha8 integrin (Itga8-/-) healing of glomerulonephritis is delayed. As Itga8 is abundant in mesangial cells (MC) which are non-professional phagocytes, we hypothesized that Itga8 facilitates phagocytosis of apoptotic cells and matrix components by MC. Methods: MC were isolated from wild type (WT) and Itga8-/- mice. Latex beads were coated with matrix components. Apoptosis was induced by cisplatin in macrophages and in DiI-stained MC. After coincubation of latex beads or apoptotic cells with MC, the phagocytosis rate was detected in WT and Itga8-/- MC via fluorescence microscopy and FACS analysis. Results: Itga8-/- MC showed reduced phagocytosis of matrix-coated beads and apoptotic cells compared to WT MC. Reduction of stress fibers was observed in Itga8-/- compared to WT MC. Inhibition of cytoskeletal reorganization by inhibition of Rac1 or ROCK during phagocytosis significantly decreased the rate of phagocytosis by WT MC but not by Itga8-/- MC. Conclusion: The expression of Itga8 facilitates phagocytosis in MC, likely mediated by Itga8-cytoskeleton interactions. An impairment of MC phagocytosis might thus contribute to a delayed glomerular regeneration in Itga8-/- mice

Postoperative pain in small-for-gestational age infants after hernia repair, orchidopexy and urethral reconstruction surgery: A pilot study

Background: Small-for-gestational-age (SGA) birth bears an enhanced risk of developing hypertension, obesity, insulin resistance and mental health disorders in later life as a consequence of adaptive processes in utero. Only a small number of studies on pain perception in SGA infants exist. These are indicative of a blunted stress response to pain in SGA newborns. Aim: We initiated a pilot study investigating differences in postoperative pain perception between SGA and appropriate-for-gestational-age (AGA) infants. Methods: Pain and alertness levels of 10 formerly SGA and 14 AGA infants at the age 0.5-2 years were evaluated by the FLACC scale, Steward and Aldrete Scores following hernia repair, reconstructive surgery of hypospadia and orchidopexy. In addition, the postoperative consumption of non-steroidal anti-inflammatory drugs was compared between SGA and AGA. Results: Postoperative pain and alertness levels were not significantly different in SGA and AGA children. We did not observe significant group differences regarding the consumption of non-steroidal anti-inflammatory drugs. Conclusion: While previous studies were suggestive of a suppressed stress response to pain in SGA newborns, these findings did not fully translate into an altered response to pain beyond the newborn age. Further studies in a larger cohort seem necessary to verify this finding