Comparison of serial amnioinfusion strategies for isolated early-onset fetal renal anhydramnios.

Introduction The optimal protocol for serial amnioinfusions to maintain amniotic fluid in pregnancies with early onset-fetal renal anhydramnios before 22 weeks is not known. We compared the performance of two different approaches. Methods A secondary analysis was conducted of serial amnioinfusions performed by a single center during the external pilot and feasibility phases of the Renal Agenesis Fetal Therapy (RAFT) trial. During the external pilot, higher amnioinfusion volumes were given less frequently; in the feasibility study, smaller volume amnioinfusions were administered more frequently. Procedural details, complications, and obstetric outcomes were compared between the two groups using Pearson's chi-squared or Fisher's Exact tests for categorical variables and Student's t-tests or Wilcoxon Rank-Sum tests for continuous variables. The adjusted association between procedural details and chorioamniotic separation was obtained through a multivariate repeated measure logistic regression model. Results Eleven participants underwent 159 amnioinfusions (external pilot: three patients, 21 amnioinfusions; feasibility: eight patients, 138 amnioinfusions). External pilot participants had fewer amnioinfusions (7 vs. 19.5 in the feasibility group, p = 0.04), larger amnioinfusion volume (750 vs. 500 mL, p < 0.01), and longer interval between amnioinfusions (6 [4-7] vs. 4 [3-5] days, p < 0.01). In the external pilot, chorioamniotic separation was more common (28.6% vs. 5.8%, p < 0.01), preterm prelabor rupture of membranes (PPROM) occurred sooner after amnioinfusion initiation (28 ± 21.5 vs. 75.6 ± 24.1 days, p = 0.03), and duration of maintained amniotic fluid between first and last amnioinfusion was shorter (38 ± 17.3 vs. 71 ± 19 days, p=0.03), compared to the feasibility group. While delivery gestational age was similar (35.1 ± 1.7 vs. 33.8 ± 1.5 weeks, p=0.21), feasibility participants maintained amniotic fluid longer. Conclusion Small volume serial amnioinfusions performed more frequently maintain normal amniotic fluid volume longer because of delayed occurrence of PPROM.

Does glial lipid dysregulation alter sleep in Alzheimer's and Parkinson's disease?

In this opinion article, we discuss potential connections between sleep disturbances observed in Alzheimer's disease (AD) and Parkinson's disease (PD) and the dysregulation of lipids in the brain. Research using Drosophila has highlighted the role of glial-mediated lipid metabolism in sleep and diurnal rhythms. Relevant to AD, the formation of lipid droplets in glia, which occurs in response to elevated neuronal reactive oxygen species (ROS), is required for sleep. In disease models, this process is disrupted, arguing a connection to sleep dysregulation. Relevant to PD, the degradation of neuronally synthesized glucosylceramides by glia requires glucocerebrosidase (GBA, a PD-associated risk factor) and this regulates sleep. Loss of GBA in glia causes an accumulation of glucosylceramides and neurodegeneration. Overall, research primarily using Drosophila has highlighted how dysregulation of glial lipid metabolism may underlie sleep disturbances in neurodegenerative diseases.

Whole organism snRNA-seq reveals systemic peripheral changes in Alzheimer's Disease fly models.

Peripheral tissues become disrupted in Alzheimer's Disease (AD). However, a comprehensive understanding of how the expression of AD-associated toxic proteins, Aß42 and Tau, in neurons impacts the periphery is lacking. Using Drosophila, a prime model organism for studying aging and neurodegeneration, we generated the Alzheimer's Disease Fly Cell Atlas (AD-FCA): whole-organism single-nucleus transcriptomes of 219 cell types from adult flies neuronally expressing human Aß42 or Tau. In-depth analyses and functional data reveal impacts on peripheral sensory neurons by Aß42 and on various non-neuronal peripheral tissues by Tau, including the gut, fat body, and reproductive system. This novel AD atlas provides valuable insights into potential biomarkers and the intricate interplay between the nervous system and peripheral tissues in response to AD-associated proteins.

Autolysosomal exocytosis of lipids protect neurons from ferroptosis.

During oxidative stress neurons release lipids that are internalized by glia. Defects in this coordinated process play an important role in several neurodegenerative diseases. Yet, the mechanisms of lipid release and its consequences on neuronal health are unclear. Here, we demonstrate that lipid-protein particle release by autolysosome exocytosis protects neurons from ferroptosis, a form of cell death driven by lipid peroxidation. We show that during oxidative stress, peroxidated lipids and iron are released from neurons by autolysosomal exocytosis which requires the exocytic machinery VAMP7 and syntaxin 4. We observe membrane-bound lipid-protein particles by TEM and demonstrate that these particles are released from neurons using cryoEM. Failure to release these lipid-protein particles causes lipid hydroperoxide and iron accumulation and sensitizes neurons to ferroptosis. Our results reveal how neurons protect themselves from peroxidated lipids. Given the number of brain pathologies that involve ferroptosis, defects in this pathway likely play a key role in the pathophysiology of neurodegenerative disease.

PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells.

Treatment-resistant glioma stem cells are thought to propagate and drive growth of malignant gliomas, but their markers and our ability to target them specifically are not well understood. We demonstrate that podoplanin (PDPN) expression is an independent prognostic marker in gliomas across multiple independent patient cohorts comprising both high- and low-grade gliomas. Knockdown of PDPN radiosensitized glioma cell lines and glioma-stem-like cells (GSCs). Clonogenic assays and xenograft experiments revealed that PDPN expression was associated with radiotherapy resistance and tumor aggressiveness. We further demonstrate that knockdown of PDPN in GSCs in vivo is sufficient to improve overall survival in an intracranial xenograft mouse model. PDPN therefore identifies a subset of aggressive, treatment-resistant glioma cells responsible for radiation resistance and may serve as a novel therapeutic target.

Recent insights into the role of glia and oxidative stress in Alzheimer's disease gained from Drosophila.

Here, we discuss findings made using Drosophila on Alzheimer's disease (AD) risk and progression. Recent studies have investigated the mechanisms underlying glia-mediated neuroprotection in AD. First, we discuss a novel mechanism of glial lipid droplet formation that occurs in response to elevated reactive oxygen species in neurons. The data suggest that disruptions to this process contribute to AD risk. We further discuss novel mechanistic insights into glia-mediated Aß42-clearance made using the fly. Finally, we highlight work that provides evidence that the aberrant accumulation of reactive oxygen species in AD may not just be a consequence of disease but contribute to disease progression as well. Cumulatively, the discussed studies highlight recent, relevant discoveries in AD made using Drosophila.

Neuronal ROS-induced glial lipid droplet formation is altered by loss of Alzheimer's disease-associated genes.

A growing list of Alzheimer's disease (AD) genetic risk factors is being identified, but the contribution of each variant to disease mechanism remains largely unknown. We have previously shown that elevated levels of reactive oxygen species (ROS) induces lipid synthesis in neurons leading to the sequestration of peroxidated lipids in glial lipid droplets (LD), delaying neurotoxicity. This neuron-to-glia lipid transport is APOD/E-dependent. To identify proteins that modulate these neuroprotective effects, we tested the role of AD risk genes in ROS-induced LD formation and demonstrate that several genes impact neuroprotective LD formation, including homologs of human ABCA1, ABCA7, VLDLR, VPS26, VPS35, AP2A, PICALM, and CD2AP Our data also show that ROS enhances Aß42 phenotypes in flies and mice. Finally, a peptide agonist of ABCA1 restores glial LD formation in a humanized APOE4 fly model, highlighting a potentially therapeutic avenue to prevent ROS-induced neurotoxicity. This study places many AD genetic risk factors in a ROS-induced neuron-to-glia lipid transfer pathway with a critical role in protecting against neurotoxicity.

TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.

Evaluation of the Pubocervical Fascia With 3-Dimensional Endovaginal Ultrasonography and Correlation With Intraoperative Findings During Robotic Sacrocervicopexy.

OBJECTIVES: To evaluate the pubocervical fascia (PF) in patients with pelvic organ prolapse (POP) using 3-dimensonal endovaginal ultrasonography (EVUS) and to correlate the PF appearance with both pelvic examination and intraoperative findings during ultrasonographic robotic-assisted laparoscopic sacrocervicopexy and pubocervical fascia reconstruction (u-RALS-PFR). METHODS: A retrospective analysis was performed in 120 women with symptomatic POP. Preoperative evaluation was done using EVUS. We identified areas of PF weakness based on pelvic examination as hypoechoic and hyperechoic defects (HHD) between the bladder and vagina. Study measurements included distance from the HHD to the pubic symphysis, HHD to the bladder neck, HHD to the posterior bladder wall, and hypoechoic-hyperechoic area. We correlated these metrics with the respective POP-Q stages and findings during u-RALS-PFR. RESULTS: Using the quantitative measures during EVUS, we found a significant association between mean HHD (2.7 cm) and POP-Q stage III, and between HHD and number of plications performed during surgery. The larger the HHD, the more severe the POP-Q stage of the anterior compartment of the vaginal wall; thus, more plications were performed on the PF (7-12 plications) during robotic sacrocervicopexy, and consequently the anterior arm of the Y-mesh was significantly trimmed (6-8 cm). CONCLUSION: HHD obtained by EVUS was associated with severe POP-Q stage III and seemed to correlate with the number of plications during robotic sacrocervicopexy. Performing these plications on the PF significantly decreased the length of the anterior vaginal mesh needed for the procedure. These findings may open new applications for preoperative ultrasonography in evaluation and treatment of patients with apical and anterior POP.

Ultrasonography and robotic-assisted laparoscopic sacrocervicopexy with pubocervical fascia reconstruction: comparison with standard technique.

The objective of this study was to evaluate our technique of ultrasonography and robotic-assisted sacrocervicopexy with pubocervical fascia reconstruction (u-RALS-PFR) versus standard robotic-assisted laparoscopic sacrocervicopexy (s-RALS) in the treatment of patients with symptomatic apical/anterior vaginal prolapse. A retrospective analysis was done using the data in two community hospitals. Thirty women presented with symptomatic vaginal apical prolapse and desired minimally invasive surgery (video): (a) standard robotic-assisted laparoscopic sacrocervicopexy (s-RALS) (n = 15) or (b) ultrasound and robotic-assisted sacrocervicopexy with pubocervical fascia reconstruction (u-RALS-PFR) (n = 15) were eligible to participate. All participants underwent a standardized evaluation, including a structured urogynecologic history and physical examination with pelvic organ prolapse quantitative staging. There was longer operating room time in the u-RALS-PFR group compared with the s-RALS group (average difference 35 min); however, sacral promontory dissection time was less in the u-RALS-PFR (average difference of 15 min). The anterior/posterior vaginal dissection and mesh tensioning time was longer in the u-RALS-PFR, as expected. There was only one surgical and anatomic failure (7%) in the s-RALS group after 6 months of surgery (POP Q = Aa + 1, Ba0, Ap-2, Bp-3, C-7). Our technique of ultrasonography and pubocervical fascia reconstruction during RALS appears to be feasible and safe. It aims to improve anterior and apical support, minimize the use of mesh and improve visualization during surgery. u-RALS-PFR approach will add some additional time during surgery but may provide better outcomes.

New Roles for Canonical Transcription Factors in Repeat Expansion Diseases.

The presence of microsatellite repeat expansions within genes is associated with >30 neurological diseases. Of interest, (GGGGCC)>30-repeats within C9orf72 are associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). These expansions can be 100s to 1000s of units long. Thus, it is perplexing how RNA-polymerase II (RNAPII) can successfully transcribe them. Recent investigations focusing on GGGGCC-transcription have identified specific, canonical complexes that may promote RNAPII-transcription at these GC-rich microsatellites: the DSIF complex and PAF1C. These complexes may be important for resolving the unique secondary structures formed by GGGGCC-DNA during transcription. Importantly, this process can produce potentially toxic repeat-containing RNA that can encode potentially toxic peptides, impacting neuron function and health. Understanding how transcription of these repeats occurs has implications for therapeutics in multiple diseases.

Repeat-associated non-AUG (RAN) translation mechanisms are running into focus for GGGGCC-repeat associated ALS/FTD.

Many human diseases are associated with the expansion of repeat sequences within the genes. It has become clear that expressed disease transcripts bearing such long repeats can undergo translation, even in the absence of a canonical AUG start codon. Termed "RAN translation" for repeat associated non-AUG translation, this process is becoming increasingly prominent as a contributor to these disorders. Here we discuss mechanisms and variables that impact translation of the repeat sequences associated with the C9orf72 gene. Expansions of a G4C2 repeat within intron 1 of this gene are associated with the motor neuron disease ALS and dementia FTD, which comprise a clinical and pathological spectrum. RAN translation of G4C2 repeat expansions has been studied in cells in culture (ex vivo) and in the fly in vivo. Cellular states that lead to RAN translation, like stress, may be critical contributors to disease progression. Greater elucidation of the mechanisms that impact this process and the factors contributing will lead to greater understanding of the repeat expansion diseases, to the potential development of novel approaches to therapeutics, and to a greater understanding of how these players impact biological processes in the absence of disease.

Drosophila Ref1/ALYREF regulates transcription and toxicity associated with ALS/FTD disease etiologies.

RNA-binding proteins (RBPs) are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the underlying disease mechanisms remain unclear. In an unbiased screen in Drosophila for RBPs that genetically interact with TDP-43, we found that downregulation of the mRNA export factor Ref1 (fly orthologue to human ALYREF) mitigated TDP-43 induced toxicity. Further, Ref1 depletion also reduced toxicity caused by expression of the C9orf72 GGGGCC repeat expansion. Ref1 knockdown lowered the mRNA levels for these related disease genes and reduced the encoded proteins with no effect on a wild-type Tau disease transgene or a control transgene. Interestingly, expression of TDP-43 or the GGGGCC repeat expansion increased endogenous Ref1 mRNA levels in the fly brain. Further, the human orthologue ALYREF was upregulated by immunohistochemistry in ALS motor neurons, with the strongest upregulation occurring in ALS cases harboring the GGGGCC expansion in C9orf72. These data support ALYREF as a contributor to ALS/FTD and highlight its downregulation as a potential therapeutic target that may affect co-existing disease etiologies.

Toxic expanded GGGGCC repeat transcription is mediated by the PAF1 complex in C9orf72-associated FTD.

An expanded GGGGCC hexanucleotide of more than 30 repeats (termed (G4C2)30+) within C9orf72 is the most prominent mutation in familial frontotemporal degeneration (FTD) and amyotrophic lateral sclerosis (ALS) (termed C9+). Through an unbiased large-scale screen of (G4C2)49-expressing Drosophila we identify the CDC73/PAF1 complex (PAF1C), a transcriptional regulator of RNA polymerase II, as a suppressor of G4C2-associated toxicity when knocked-down. Depletion of PAF1C reduces RNA and GR dipeptide production from (G4C2)30+ transgenes. Notably, in Drosophila, the PAF1C components Paf1 and Leo1 appear to be selective for the transcription of long, toxic repeat expansions, but not shorter, nontoxic expansions. In yeast, PAF1C components regulate the expression of both sense and antisense repeats. PAF1C is upregulated following (G4C2)30+ expression in flies and mice. In humans, PAF1 is also upregulated in C9+-derived cells, and its heterodimer partner, LEO1, binds C9+ repeat chromatin. In C9+ FTD, PAF1 and LEO1 are upregulated and their expression positively correlates with the expression of repeat-containing C9orf72 transcripts. These data indicate that PAF1C activity is an important factor for transcription of the long, toxic repeat in C9+ FTD.

eIF4B and eIF4H mediate GR production from expanded G4C2 in a Drosophila model for C9orf72-associated ALS.

The discovery of an expanded (GGGGCC)n repeat (termed G4C2) within the first intron of C9orf72 in familial ALS/FTD has led to a number of studies showing that the aberrant expression of G4C2 RNA can produce toxic dipeptides through repeat-associated non-AUG (RAN-) translation. To reveal canonical translation factors that impact this process, an unbiased loss-of-function screen was performed in a G4C2 fly model that maintained the upstream intronic sequence of the human gene and contained a GFP tag in the GR reading frame. 11 of 48 translation factors were identified that impact production of the GR-GFP protein. Further investigations into two of these, eIF4B and eIF4H, revealed that downregulation of these factors reduced toxicity caused by the expression of expanded G4C2 and reduced production of toxic GR dipeptides from G4C2 transcripts. In patient-derived cells and in post-mortem tissue from ALS/FTD patients, eIF4H was found to be downregulated in cases harboring the G4C2 mutation compared to patients lacking the mutation and healthy individuals. Overall, these data define eIF4B and eIF4H as disease modifiers whose activity is important for RAN-translation of the GR peptide from G4C2-transcripts.

Robotic-assisted laparoscopic apical suspension: description of the spiral technique.

This video's objective was to describe our spiral technique and surgical steps of robotic-assisted laparoscopic apical suspension (RALAS) in the treatment of patients with symptomatic apical vaginal prolapse. A 70-year-old Caucasian woman, gravida 3, para 2 had symptomatic pelvic organ prolapse (POP) apical/anterior stage III. At pelvic ultrasound evaluation, the uterus was small and normal appearing of adnexa bilaterally. She failed pessaries and is sexually active. The most relevant complaints were vaginal bulging and pressure. She denied urinary incontinence. During the surgery, we used (1) 3-0, V-Loc™ (Covidien) and we reinforced these absorbable sutures with (2) 2-0, GORE-TEX® Suture (Gore Medical). The Si da Vinci Surgical System was used with 4 arms and 5 trocars configuration, docked on the patient's left side. On the right/left apical support, we used V-Loc and Gore-Tex and these provided the initial 2 points suspension on the uterosacral ligaments (USL). We like to attach the left to the right USL. We then developed the space between the bladder and vagina and proceed with a plication of the pubocervical fascia with V-loc sutures. Two anterior apical support sutures were taken from the vagina to the transversalis fascia on the anterior abdominal wall and then hid behind the bladder peritoneum. The tension of these sutures was maintained with Hem-o-lock (TeleFlex) and LAPRA-TY (Ethicon). With the spiral technique, we secured these sutures through aponeurosis of abdominal muscle inside-outside-inside using a Carter-Thomason (Cooper Surgical) laparoscopic port closure system. This technique may provide a better long-term support for the anterior apical compartment.

ABT-888 restores sensitivity in temozolomide resistant glioma cells and xenografts.

BACKGROUND: Temozolomide (TMZ) is active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced. However, even in responsive cases, its beneficial effect is undermined by the emergence of drug resistance. Here, we tested whether inhibition of poly (ADP-ribose) polymerase-1 and -2 (PARP) enhanced the effectiveness of TMZ. METHODS: Using patient derived brain tumor initiating cells (BTICs) and orthotopic xenografts as models of newly diagnosed and recurrent high-grade glioma, we assessed the effects of TMZ, ABT-888, and the combination of TMZ and ABT-888 on the viability of BTICs and survival of tumor-bearing mice. We also studied DNA damage repair, checkpoint protein phosphorylation, and DNA replication in mismatch repair (MMR) deficient cells treated with TMZ and TMZ plus ABT-888. RESULTS: Cells and xenografts derived from newly diagnosed MGMT methylated high-grade gliomas were sensitive to TMZ while those derived from unmethylated and recurrent gliomas were typically resistant. ABT-888 had no effect on the viability of BTICs or tumor bearing mice, but co-treatment with TMZ restored sensitivity in resistant cells and xenografts from newly diagnosed unmethylated gliomas and recurrent gliomas with MSH6 mutations. In contrast, the addition of ABT-888 to TMZ had little sensitizing effect on cells and xenografts derived from newly diagnosed methylated gliomas. In a model of acquired TMZ resistance mediated by loss of MMR gene MSH6, re-sensitization to TMZ by ABT-888 was accompanied by persistent DNA strand breaks, re-engagement of checkpoint kinase signaling, and interruption of DNA synthesis. CONCLUSION: In laboratory models, the addition of ABT-888 to TMZ overcame resistance to TMZ.

Dipeptide repeat proteins activate a heat shock response found in C9ORF72-ALS/FTLD patients.

A hexanucleotide (GGGGCC) repeat expansion in C9ORF72 is the most common genetic contributor to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Reduced expression of the C9ORF72 gene product has been proposed as a potential contributor to disease pathogenesis. Additionally, repetitive RNAs and dipeptide repeat proteins (DPRs), such as poly-GR, can be produced by this hexanucleotide expansion that disrupt a number of cellular processes, potentially contributing to neural degeneration. To better discern which of these mechanisms leads to disease-associated changes in patient brains, we analyzed gene expression data generated from the cortex and cerebellum. We found that transcripts encoding heat shock proteins (HSPs) regulated by the HSF1 transcription factor were significantly induced in C9ORF72-ALS/FTLD patients relative to both sporadic ALS/FTLD cases and controls. Treatment of human neurons with chemically synthesized DPRs was sufficient to activate a similar transcriptional response. Expression of GGGGCC repeats and also poly-GR in the brains of Drosophila lead to the upregulation of HSF1 and the same highly-conserved HSPs. Additionally, HSF1 was a modifier of poly-GR toxicity in Drosophila. Our results suggest that the expression of DPRs are associated with upregulation of HSF1 and activation of a heat shock response in C9ORF72-ALS/FTLD.