Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

Role of Hydrogen Sulfide in Inflammatory Bowel Disease.

Hydrogen sulfide (H2S), originally known as toxic gas, has now attracted attention as one of the gasotransmitters involved in many reactions in the human body. H2S has been assumed to play a role in the pathogenesis of many chronic diseases, of which the exact pathogenesis remains unknown. One of them is inflammatory bowel disease (IBD), a chronic intestinal disease subclassified as Crohn's disease (CD) and ulcerative colitis (UC). Any change in the amount of H2S seems to be linked to inflammation in this illness. These changes can be brought about by alterations in the microbiota, in the endogenous metabolism of H2S and in the diet. As both too little and too much H2S drive inflammation, a balanced level is needed for intestinal health. The aim of this review is to summarize the available literature published until June 2023 in order to provide an overview of the current knowledge of the connection between H2S and IBD.

Posterior circulation ischaemic stroke diagnosis and management.

This narrative review provides an overview of the posterior circulation and the clinical features of common posterior circulation stroke (PCS) syndromes in the posterior arterial territories and how to distinguish them from mimics. We outline the hyperacute management of patients with suspected PCS with emphasis on how to identify those who are likely to benefit from intervention based on imaging findings. Finally, we review advances in treatment options, including developments in endovascular thrombectomy (EVT) and intravenous thrombolysis (IVT), and the principles of medical management and indications for neurosurgery. Observational and randomised clinical trial data have been equivocal regarding EVT in PCS, but more recent studies strongly support its efficacy. There have been concomitant advances in imaging of posterior stroke to guide optimal patient selection for thrombectomy. Recent evidence suggests that clinicians should have a heightened suspicion of posterior circulation events with the resultant implementation of timely, evidence-based management.

The influence of liver transplantation on the interplay between gut microbiome and bile acid homeostasis in children with biliary atresia.

BACKGROUND: Biliary atresia (BA) causes neonatal cholestasis and rapidly progresses into cirrhosis if left untreated. Kasai portoenterostomy may delay cirrhosis. BA remains among the most common indications for liver transplantation (LT) during childhood. Liver function and gut microbiome are interconnected. Disturbed liver function and enterohepatic signaling influence microbial diversity. We, herein, investigate the impact of LT and reestablishment of bile flow on gut microbiome-bile acid homeostasis in children with BA before (pre, n = 10), 3 months (post3m, n = 12), 12 months (post12m, n = 9), and more than 24 months (post24 + m, n = 12) after LT. METHODS: We analyzed the intestinal microbiome of BA patients before and after LT by 16S-rRNA-sequencing and bioinformatics analyses, and serum primary and secondary bile acid levels. RESULTS: The gut microbiome in BA patients exhibits a markedly reduced alpha diversity in pre (p = 0.015) and post3m group (p = 0.044), and approximated healthy control groups at later timepoints post12m (p = 1.0) and post24 + m (p = 0.74). Beta diversity analysis showed overall community structure similarities of pre and post3m (p = 0.675), but both differed from the post24 + m (p < 0.001). Longitudinal analysis of the composition of the gut microbiome revealed the Klebsiella genus to show increased abundance in the post24 + m group compared with an age-matched control (p = 0.029). Secondary bile acid production increased 2+ years after LT (p = 0.03). Multivariable associations of microbial communities and clinical metadata reveal several significant associations of microbial genera with tacrolimus and mycophenolate mofetil-based immunosuppressive regimens. CONCLUSIONS: In children with BA, the gut microbiome shows strongly reduced diversity before and shortly after LT, and approximates healthy controls at later timepoints. Changes in diversity correlate with altered secondary bile acid synthesis at 2+ years and with the selection of different immunosuppressants.

Developing a metabolic clearance rate framework as a translational analysis approach for hyperpolarized 13C magnetic resonance imaging.

Hyperpolarized carbon-13 magnetic resonance imaging is a promising technique for in vivo metabolic interrogation of alterations between health and disease. This study introduces a formalism for quantifying the metabolic information in hyperpolarized imaging. This study investigated a novel perfusion formalism and metabolic clearance rate (MCR) model in pre-clinical stroke and in the healthy human brain. Simulations showed that the proposed model was robust to perturbations in T1, transmit B1, and kPL. A significant difference in ipsilateral vs contralateral pyruvate derived cerebral blood flow (CBF) was detected in rats (140 ± 2 vs 89 ± 6 mL/100 g/min, p < 0.01, respectively) and pigs (139 ± 12 vs 95 ± 5 mL/100 g/min, p = 0.04, respectively), along with an increase in fractional metabolism (26 ± 5 vs 4 ± 2%, p < 0.01, respectively) in the rodent brain. In addition, a significant increase in ipsilateral vs contralateral MCR (0.034 ± 0.007 vs 0.017 ± 0.02/s, p = 0.03, respectively) and a decrease in mean transit time (31 ± 8 vs 60 ± 2 s, p = 0.04, respectively) was observed in the porcine brain. In conclusion, MCR mapping is a simple and robust approach to the post-processing of hyperpolarized magnetic resonance imaging.

Cerebroprotection in the endovascular era: an update.

Despite advances in clinical diagnosis and increasing numbers of patients eligible for revascularisation, ischaemic stroke remains a significant public health concern accounting for 3.3 million deaths annually. In addition to recanalisation therapy, patient outcomes could be improved through cerebroprotection, but all translational attempts have remained unsuccessful. In this narrative review, we discuss potential reasons for those failures. We then outline the diverse, multicellular effects of ischaemic stroke and the complex temporal sequences of the pathophysiological cascade during and following ischaemia, reperfusion, and recovery. This evidence is linked with findings from prior cerebroprotective trials and interpreted for the modern endovascular era. Future cerebroprotective agents that are multimodal and multicellular, promoting cellular and metabolic health to different targets at time points that are most responsive to treatment, might prove more successful.

Hydrogen Sulfide Metabolizing Enzymes in the Intestinal Mucosa in Pediatric and Adult Inflammatory Bowel Disease.

Hydrogen sulfide (H2S) is a toxic gas that has important regulatory functions. In the colon, H2S can be produced and detoxified endogenously. Both too little and too much H2S exposure are associated with inflammatory bowel disease (IBD), a chronic intestinal disease mainly classified as Crohn's disease (CD) and ulcerative colitis (UC). As the pathogenesis of IBD remains elusive, this study's aim was to investigate potential differences in the expression of H2S-metabolizing enzymes in normal aging and IBD. Intestinal mucosal biopsies of 25 adults and 22 children with IBD along with those of 26 healthy controls were stained immunohistochemically for cystathionine-γ-lyase (CSE), 3-mercapto-sulfurtransferase (3-MST), ethylmalonic encephalopathy 1 protein (ETHE1), sulfide:quinone oxidoreductase (SQOR) and thiosulfate sulfurtransferase (TST). Expression levels were calculated by multiplication of the staining intensity and percentage of positively stained cells. Healthy adults showed an overall trend towards lower expression of H2S-metabolizing enzymes than healthy children. Adults with IBD also tended to have lower expression compared to controls. A similar trend was seen in the enzyme expression of children with IBD compared to controls. These results indicate an age-related decrease in the expression of H2S-metabolizing enzymes and a dysfunctional H2S metabolism in IBD, which was less pronounced in children.

Role of Energy Metabolism and Mitochondrial Function in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic recurring inflammation of the intestine which can be debilitating for those with intractable disease. However, the etiopathogenesis of inflammatory bowel disorders remains to be solved. The hypothesis that mitochondrial dysfunction is a crucial factor in the disease process is being validated by an increasing number of recent studies. Thus mitochondrial alteration in conjunction with previously identified genetic predisposition, changes in the immune response, altered gut microbiota, and environmental factors (eg, diet, smoking, and lifestyle) are all posited to contribute to IBD. The implicated factors seem to affect mitochondrial function or are influenced by mitochondrial dysfunction, which explains many of the hallmarks of the disease. This review summarizes the results of studies reporting links between mitochondria and IBD that were available on PubMed through March 2021. The aim of this review is to give an overview of the current understanding of the role of mitochondria in the pathogenesis of IBD.

We address the effect of energy metabolism and mitochondrial function on the pathogenesis of inflammatory bowel disease. Because many studies on this topic have been published recently, it is important to give an overview of the results of that work.

Expression of Oxidative Phosphorylation Complexes and Mitochondrial Mass in Pediatric and Adult Inflammatory Bowel Disease.

INTRODUCTION: Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), is a multifactorial intestinal disorder but its precise etiology remains elusive. As the cells of the intestinal mucosa have high energy demands, mitochondria may play a role in IBD pathogenesis. The present study is aimed at evaluating the expression levels of mitochondrial oxidative phosphorylation (OXPHOS) complexes in IBD. Material and Methods. 286 intestinal biopsy samples from the terminal ileum, ascending colon, and rectum from 124 probands (34 CD, 33 UC, and 57 controls) were stained immunohistochemically for all five OXPHOS complexes and the voltage-dependent anion-selective channel 1 protein (VDAC1 or porin). Expression levels were compared in multivariate models including disease stage (CD and UC compared to controls) and age (pediatric/adult). RESULTS: Analysis of the terminal ileum of CD patients revealed a significant reduction of complex II compared to controls, and a trend to lower levels was evident for VDAC1 and the other OXPHOS complexes except complex III. A similar pattern was found in the rectum of UC patients: VDAC1, complex I, complex II, and complex IV were all significantly reduced, and complex III and V showed a trend to lower levels. Reductions were more prominent in older patients compared to pediatric patients and more marked in UC than CD. CONCLUSION: A reduced mitochondrial mass is present in UC and CD compared to controls. This is potentially a result of alterations of mitochondrial biogenesis or mitophagy. Reductions were more pronounced in older patients compared to pediatric patients, and more prominent in UC than CD. Complex I and II are more severely compromised than the other OXPHOS complexes. This has potential therapeutic implications, since treatments boosting biogenesis or influencing mitophagy could be beneficial for IBD treatment. Additionally, substances specifically stimulating complex I activity should be tested in IBD treatment.

Human Skin Permeation Enhancement Using PLGA Nanoparticles Is Mediated by Local pH Changes.

The steady improvement and optimization of transdermal permeation is a constant and challenging pharmaceutical task. In this study the influence of poly(lactide-co-glycolide) (PLGA) nanoparticles on the dermal permeation of the anti-inflammatory drug flufenamic acid (FFA) was investigated. For this aim, different vehicles under non-buffered and buffered conditions and different skin models (human heat separated epidermis and reconstructed human epidermis equivalents) were tested. Permeation experiments were performed using static Franz diffusion cells under infinite dosing conditions. Already the presence of drug-free nanoparticles increased drug permeation across the skin. Drug permeation was even enhanced when applying drug-loaded nanoparticles. In contrast, buffered vehicles with different pH values (pH 5.4-7.4) revealed the influence of the pH on the permeation of FFA. The change of the surrounding pH of the biodegradable nanoparticulate system was demonstrated and visualized using pH-sensitive fluorescent probes. While a potential contribution of hair follicles could be ruled out, our data suggest that the enhanced permeation of FFA through human skin in the presence of PLGA nanoparticles is mediated by a locally decreased pH during hydrolytic degradation of this polymer. This hypothesis is supported by the observation that skin permeation of the weak base caffeine was not affected.

Age-Related Deterioration of Mitochondrial Function in the Intestine.

Aging is an important and inevitable biological process in human life, associated with the onset of chronic disease and death. The mechanisms behind aging remain unclear. However, changes in mitochondrial function and structure, including reduced activity of the mitochondrial respiratory chain and increased production of reactive oxygen species-thus oxidative damage-are believed to play a major role. Mitochondria are the main source of cellular energy, producing adenosine triphosphate (ATP) via oxidative phosphorylation. Accumulation of damaged cellular components reduces a body's capacity to preserve tissue homeostasis and affects biological aging and all age-related chronic conditions. This includes the onset and progression of classic degenerative diseases such as cardiovascular disease, kidney failure, neurodegenerative diseases, and cancer. Clinical manifestations of intestinal disorders, such as mucosal barrier dysfunction, intestinal dysmotility, and chronic obstipation, are highly prevalent in the elderly population and have been shown to be associated with an age-dependent decline of mitochondrial function. This review summarizes our current understanding of the role of mitochondrial dysfunction in intestinal aging.

Rapamycin Induces an eNOS (Endothelial Nitric Oxide Synthase) Dependent Increase in Brain Collateral Perfusion in Wistar and Spontaneously Hypertensive Rats.

BACKGROUND AND PURPOSE: Rapamycin is a clinically approved mammalian target of rapamycin inhibitor that has been shown to be neuroprotective in animal models of stroke. However, the mechanism of rapamycin-induced neuroprotection is still being explored. Our aims were to determine if rapamycin improved leptomeningeal collateral perfusion, to determine if this is through eNOS (endothelial nitric oxide synthase)-mediated vessel dilation and to determine if rapamycin increases immediate postreperfusion blood flow. METHODS: Wistar and spontaneously hypertensive rats (≈14 weeks old, n=22 and n=15, respectively) were subjected to ischemia by middle cerebral artery occlusion (90 and 120 minutes, respectively) with or without treatment with rapamycin at 30-minute poststroke. Changes in middle cerebral artery and collateral perfusion territories were measured by dual-site laser Doppler. Reactivity to rapamycin was studied using isolated and pressurized leptomeningeal anastomoses. Brain injury was measured histologically or with triphenyltetrazolium chloride staining. RESULTS: In Wistar rats, rapamycin increased collateral perfusion (43±17%), increased reperfusion cerebral blood flow (16±8%) and significantly reduced infarct volume (35±6 versus 63±8 mm3, P<0.05). Rapamycin dilated leptomeningeal anastomoses by 80±9%, which was abolished by nitric oxide synthase inhibition. In spontaneously hypertensive rats, rapamycin increased collateral perfusion by 32±25%, reperfusion cerebral blood flow by 44±16%, without reducing acute infarct volume 2 hours postreperfusion. Reperfusion cerebral blood flow was a stronger predictor of brain damage than collateral perfusion in both Wistar and spontaneously hypertensive rats. CONCLUSIONS: Rapamycin increased collateral perfusion and reperfusion cerebral blood flow in both Wistar and comorbid spontaneously hypertensive rats that appeared to be mediated by enhancing eNOS activation. These findings suggest that rapamycin may be an effective acute therapy for increasing collateral flow and as an adjunct therapy to thrombolysis or thrombectomy to improve reperfusion blood flow.

Changes in the expression of oxidative phosphorylation complexes in the aging intestinal mucosa.

OBJECTIVE: Mitochondria produce cellular energy via oxidative phosphorylation (OXPHOS), mediated by respiratory chain complexes I to IV and ATP synthase (complex V). Mitochondrial respiratory complexes have been shown to decline with age in several tissues. As the intestinal epithelium is a tissue with a high energy demand, the aim of the present study was to establish whether the expression profile of OXPHOS subunits in the intestinal mucosa changes during the aging process. DESIGN: Biopsies of intestinal mucosa with no evidence of endoscopic or histomorphologic abnormalities, taken from 55 patients (mean age 42 years, age range 4-82 years; 62% female), were divided into four age groups (4-19, 20-39, 40-59, ≥60 years). Sections from different intestinal segments (terminal ileum, ascending colon, and sigmoid colon/rectum) were stained immunohistochemically (IHC) for subunits of OXPHOS complexes I-V and the voltage-dependent anion-selective channel 1 protein (VDAC1, porin), a marker of mitochondrial mass. Scores for IHC staining were determined by multiplication of the staining intensity and the percentage of positive cells. In addition, the numbers of intestinal crypts staining positive, partly positive, and negative were assessed. RESULTS: The average protein expression levels of OXPHOS subunits increased continuously from childhood onward, peaked in persons aged 20 to 59 years, and declined thereafter. This was seen for complexes II to V in the terminal ileum, complexes I to V in the ascending colon, and complexes I to IV in the sigmoid colon/rectum. Across all age groups, no effect of age on expression of the porin subunit VDAC1 was detected. The number of complex I- and IV-negative crypts in different intestinal segments increased with age. CONCLUSION: The protein expression levels of OXPHOS complexes increases from childhood onward and declines in elderly individuals, while the numbers of crypts with partial or complete loss of expression of complexes I and IV increase continuously with age. These data suggest that the continued reductions in the levels of mitochondrial OXPHOS complexes in crypts might be compensated in adulthood, but that, ultimately, reduced expression levels occur in persons aged 60 years and older. These findings raise two important questions: first, can the process of aging could be delayed through (pharmacological) intervention of mitochondrial pathways, and second, pathophysiologically, are these findings associated with disorders of the intestinal mucosa, e.g. inflammation?

Self-Reported Headaches in Patients with Unruptured Intracranial Aneurysms Treated with the Pipeline Embolization Device.

INTRODUCTION: The objective of this study was to analyze patients' perceptions of their headaches both before and after Pipeline flow diversion treatment of their unruptured intracranial aneurysms, with the goal of identifying prognostic factors associated with headache patterns to improve predictions of overall outcomes. METHODS: We retrospectively identified 133 patients treated with the Pipeline embolization device (PED) between January 1, 2014, and December 31, 2016, at an academic institution in the United States. Patients with at least 6 months of clinical follow-up, who had completed a validated telephone survey assessing their headache perceptions before and after treatment of their UIAs, were included. RESULTS: Sixty patients (57.7%) responded to the questionnaire. Median aneurysm size was 6.3 mm. Thirty-two patients (53.3%) presented with headaches before treatment, which had a median intensity of 7 out of 10 (range, 3-10). On postprocedural evaluation, 8 patients (25%) had complete resolution of their headaches. Of the 24 (75%) patients with persistent headaches, the frequency and severity of the headaches decreased or remained the same in most (58.3%) patients. Eleven (18.3%) patients had new-onset headaches. This study found an association between aneurysm size and a reduction in headache frequency and severity after PED treatment. No significant association was found between headache persistence, severity, or frequency and patient demographics, aneurysm characteristics, and other comorbidities. CONCLUSION: An overall improvement of headaches in patients with UIAs who underwent treatment with the PED was observed, particularly in patients harboring large aneurysms and who had presented with daily headaches.

Defining a Clopidogrel Response Cut-Off Value Using Light Transmission Aggregometry Before Pipeline Embolization of Unruptured Intracranial Aneurysms.

BACKGROUND: The choice of appropriate antiplatelet therapy before the Pipeline Embolization Device (PED) placement is usually guided by platelet function testing such as light transmission aggregometry (LTA). In this study, we aimed to define the optimal threshold LTA value for clopidogrel responsiveness to predict the risk of postprocedural thromboembolic complications and to help guide appropriate antiplatelet regimen. METHODS: A prospectively maintained database at an academic neurosurgical center in the United States was retrospectively analyzed from 2014 to 2017 to identify patients with unruptured intracranial aneurysms treated with the PED. Clinical and radiographic data were analyzed to identify thromboembolic complications in the context of platelet function testing performed by LTA. RESULTS: A total of 95 procedures were performed for PED placement to treat 110 unruptured intracranial aneurysms. Thromboembolic complications were encountered in 4 (4.2%) of these patients. After stratifying the complication rate based on the maximal extent of platelet aggregation after administration of an exogenous platelet agonist, a marked increase in thromboembolic events was observed in patients with LTA values greater than 50%. When LTA was dichotomized based on this value, patients with an LTA value less than 50% had a thromboembolic complication rate of 1.3% (1/80), compared with 20% (3/15) for those with LTA values ≥50% (P = 0.001). CONCLUSIONS: We observed the greatest increase in the rate of thromboembolic complications with LTA values of ≥50%. This can serve as an appropriate cut-off value for determining the clopidogrel response in patients undergoing endovascular treatment with the PED.

Neurosurgical Resident Error: A Survey of U.S. Neurosurgery Residency Training Program Directors' Perceptions.

BACKGROUND: Efforts to address resident errors and to enhance patient safety have included systemic reforms, such as the Accreditation Council for Graduate Medical Education's (ACGME's) mandated duty-hour restrictions, and specialty-specific initiatives such as the neurosurgery Milestone Project. However, there is currently little data describing the basis for these errors or outlining trends in neurosurgical resident error. METHODS: An online questionnaire was distributed to program directors of 108 U.S. neurosurgery residency training programs to assess the frequency, most common forms and causes of resident error, the resulting patient outcomes, and the steps taken by residency programs to address these errors. RESULTS: Thirty-one (28.7%) responses were received. Procedural/surgical error was the most commonly observed type of error. Transient injury and no injury to the patient were perceived to be the 2 most frequent outcomes. Inexperience or resident mistake despite adequate training were cited as the most common causes of error. Twenty-three (74.2%) respondents stated that a lower post graduate year level correlated with an increased incidence of errors. There was a trend toward an association between an increased number of residents within a program and the number of errors attributable to a lack of supervision (r = 0.36; P = 0.06). Most (93.5%) program directors do not believe that mandated duty-hour restrictions reduce error frequency. CONCLUSIONS: Program directors believe that procedural error is the most commonly observed form of error, with post graduate year level believed to be an important predictor of error frequency. The perceived utility of systemic reforms that aim to reduce the incidence of resident error remains unclear.

Somatic retrotransposition is infrequent in glioblastomas.

BACKGROUND: Gliomas are the most common primary brain tumors in adults. We sought to understand the roles of endogenous transposable elements in these malignancies by identifying evidence of somatic retrotransposition in glioblastomas (GBM). We performed transposon insertion profiling of the active subfamily of Long INterspersed Element-1 (LINE-1) elements by deep sequencing (TIPseq) on genomic DNA of low passage oncosphere cell lines derived from 7 primary GBM biopsies, 3 secondary GBM tissue samples, and matched normal intravenous blood samples from the same individuals. RESULTS: We found and PCR validated one somatically acquired tumor-specific insertion in a case of secondary GBM. No LINE-1 insertions present in primary GBM oncosphere cultures were missing from corresponding blood samples. However, several copies of the element (11) were found in genomic DNA from blood and not in the oncosphere cultures. SNP 6.0 microarray analysis revealed deletions or loss of heterozygosity in the tumor genomes over the intervals corresponding to these LINE-1 insertions. CONCLUSIONS: These findings indicate that LINE-1 retrotransposon can act as an infrequent insertional mutagen in secondary GBM, but that retrotransposition is uncommon in these central nervous system tumors as compared to other neoplasias.

Structure and properties of the esterase from non-LTR retrotransposons suggest a role for lipids in retrotransposition.

Non-LTR retrotransposons are mobile genetic elements and play a major role in eukaryotic genome evolution and disease. Similar to retroviruses they encode a reverse transcriptase, but their genomic integration mechanism is fundamentally different, and they lack homologs of the retroviral nucleocapsid-forming protein Gag. Instead, their first open reading frames encode distinct multi-domain proteins (ORF1ps) presumed to package the retrotransposon-encoded RNA into ribonucleoprotein particles (RNPs). The mechanistic roles of ORF1ps are poorly understood, particularly of ORF1ps that appear to harbor an enzymatic function in the form of an SGNH-type lipolytic acetylesterase. We determined the crystal structures of the coiled coil and esterase domains of the ORF1p from the Danio rerio ZfL2-1 element. We demonstrate a dimerization of the coiled coil and a hydrolytic activity of the esterase. Furthermore, the esterase binds negatively charged phospholipids and liposomes, but not oligo-(A) RNA. Unexpectedly, the esterase can split into two dynamic half-domains, suited to engulf long fatty acid substrates extending from the active site. These properties indicate a role for lipids and membranes in non-LTR retrotransposition. We speculate that Gag-like membrane targeting properties of ORF1ps could play a role in RNP assembly and in membrane-dependent transport or localization processes.

Dimerization and direct membrane interaction of Nup53 contribute to nuclear pore complex assembly.

Nuclear pore complexes (NPCs) fuse the two membranes of the nuclear envelope (NE) to a pore, connecting cytoplasm and nucleoplasm and allowing exchange of macromolecules between these compartments. Most NPC proteins do not contain integral membrane domains and thus it is largely unclear how NPCs are embedded and anchored in the NE. Here, we show that the evolutionary conserved nuclear pore protein Nup53 binds independently of other proteins to membranes, a property that is crucial for NPC assembly and conserved between yeast and vertebrates. The vertebrate protein comprises two membrane binding sites, of which the C-terminal domain has membrane deforming capabilities, and is specifically required for de novo NPC assembly and insertion into the intact NE during interphase. Dimerization of Nup53 contributes to its membrane interaction and is crucial for its function in NPC assembly.

Mobile interspersed repeats are major structural variants in the human genome.

Characterizing structural variants in the human genome is of great importance, but a genome wide analysis to detect interspersed repeats has not been done. Thus, the degree to which mobile DNAs contribute to genetic diversity, heritable disease, and oncogenesis remains speculative. We perform transposon insertion profiling by microarray (TIP-chip) to map human L1(Ta) retrotransposons (LINE-1 s) genome-wide. This identified numerous novel human L1(Ta) insertional polymorphisms with highly variant allelic frequencies. We also explored TIP-chip's usefulness to identify candidate alleles associated with different phenotypes in clinical cohorts. Our data suggest that the occurrence of new insertions is twice as high as previously estimated, and that these repeats are under-recognized as sources of human genomic and phenotypic diversity. We have just begun to probe the universe of human L1(Ta) polymorphisms, and as TIP-chip is applied to other insertions such as Alu SINEs, it will expand the catalog of genomic variants even further.