Minimally invasive transforaminal lumbar interbody fusion with unilateral pedicle screw fixation (UNILF): outcomes at 7 years.

PURPOSE: To assess clinical, functional and radiographical results of one-level minimally invasive transforaminal interbody fusion with unilateral pedicle screw fixation (UNILIF) in the treatment of stable lumbar degenerative diseases with a minimum of 5 years of follow-up. MATERIAL AND METHOD: From January 2012 to December 2016, clinical and radiological data of patients with degenerative lumbar disease managed by UNILIF were prospectively collected. Patients with a follow-up that ended before 5 years were excluded. SF-12, Oswestry disability index (ODI) and visual analog scale (VAS) were collected preoperatively, at 2 years and at the last follow-up. A full-spine standing radiograph was performed at each follow-up appointment. RESULTS: Mean operative time was 74.7 (± 19) minutes, mean blood loss was 131.1 (± 207) ml and mean follow-up was 7.5 (± 1.7) years. All functional scores and VAS were significantly improved between the preoperative and the 2 years postoperative. Between the 2 years postoperative and the last follow-up ODI and VAS continued to significantly improved. Fusion rate was 98.6% on radiographic analysis at follow-up. CONCLUSION: UNILIF method is a safe and effective surgical strategy. It provides a durable improvement in functional score over 7 years of follow-up with a stable radiological correction over time.

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles.

BACKGROUND: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. METHODS: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. RESULTS: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. CONCLUSIONS: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS.

Expression of ALS-linked SOD1 Mutation in Motoneurons or Myotubes Induces Differential Effects on Neuromuscular Function In vitro.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that selectively affects upper and lower motoneurons. Dismantlement of the neuromuscular junction (NMJ) is an early pathological hallmark of the disease whose cellular origin remains still debated. We developed an in vitro NMJ model to investigate the differential contribution of motoneurons and muscle cells expressing ALS-causing mutation in the superoxide dismutase 1 (SOD1) to neuromuscular dysfunction. The primary co-culture system allows the formation of functional NMJs and fosters the expression of the ALS-sensitive fast fatigable type II-b myosin heavy chain (MHC) isoform. Expression of SOD1G93A in myotubes does not prevent the formation of a functional NMJ but leads to decreased contraction frequency and lowers the slow type I MHC isoform transcript levels. Expression of SOD1G93A in both motoneurons and myotubes or in motoneurons alone however alters the formation of a functional NMJ. Our results strongly suggest that motoneurons are a major factor involved in the process of NMJ dismantlement in an experimental model of ALS.

Inhibition of neuronal FLT3 receptor tyrosine kinase alleviates peripheral neuropathic pain in mice.

Peripheral neuropathic pain (PNP) is a debilitating and intractable chronic disease, for which sensitization of somatosensory neurons present in dorsal root ganglia that project to the dorsal spinal cord is a key physiopathological process. Here, we show that hematopoietic cells present at the nerve injury site express the cytokine FL, the ligand of fms-like tyrosine kinase 3 receptor (FLT3). FLT3 activation by intra-sciatic nerve injection of FL is sufficient to produce pain hypersensitivity, activate PNP-associated gene expression and generate short-term and long-term sensitization of sensory neurons. Nerve injury-induced PNP symptoms and associated-molecular changes were strongly altered in Flt3-deficient mice or reversed after neuronal FLT3 downregulation in wild-type mice. A first-in-class FLT3 negative allosteric modulator, discovered by structure-based in silico screening, strongly reduced nerve injury-induced sensory hypersensitivity, but had no effect on nociception in non-injured animals. Collectively, our data suggest a new and specific therapeutic approach for PNP.

Postoperative quality-of-life assessment in patients with spine metastases treated with long-segment pedicle-screw fixation.

OBJECTIVE In recent decades, progress in the medical management of cancer has been significant, resulting in considerable extension of survival for patients with metastatic disease. This has, in turn, led to increased attention to the optimal surgical management of bone lesions, including metastases to the spine. In addition, there has been a shift in focus toward improving quality of life and reducing hospital stay for these patients, and many minimally invasive techniques have been introduced with the aim of reducing the morbidity associated with more traditional open approaches. The goal of this study was to assess the efficacy of long-segment percutaneous pedicle screw stabilization for the treatment of instability associated with thoracolumbar spine metastases in neurologically intact patients. METHODS This study was a retrospective review of data from a prospective database. The authors analyzed cases in which long-segment percutaneous pedicle screw fixation was performed for the palliative treatment of thoracolumbar spinal instability due to spinal metastases in neurologically intact patients. All of the patients included in the study underwent surgery between January 2014 and May 2015 at the authors' institution. Postoperative radiation therapy was planned within 10 days following the stabilization in all cases. Clinical and radiological follow-up assessments were planned for 3 days, 3 weeks, 6 weeks, 3 months, 6 months, and 1 year after surgery. Outcome was assessed by means of standard postoperative evaluation and oncological and spinal quality of life measures (European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Version 3.0 [EORTC QLQ-C30] and Oswestry Disability Index [ODI], respectively). Moreover, 5 patients were given an activity monitoring device for recording the distance walked daily; preoperative and postoperative daily distances were compared. RESULTS Data from 17 cases were analyzed. There were no complications, and patients showed improvement in pain level and quality of life from the early postoperative period on. The mean ODI score was 62.7 (range 40-84) preoperatively, 35.4 (range 24-59) on postoperative Day 3, and 46.1 (range 30-76) at 3 weeks, 37.6 (range 25-59) at 6 weeks, 34.0 (range 24-59) at 3 months, 39.1 (range 22-64) at 6 months, and 30.0 (range 20-55) at 1 year after screw placement. The mean ODI was significantly improved in the first 45 days (p < 0.001). Improvement was also evident in scores for functional and symptomatic scales of the EORTC QLQ-C30. All patients underwent postoperative radiation therapy within 10 days (mean 7.5). All patients (n = 5) with an activity monitoring device showed improvement in daily walking distance. CONCLUSIONS Less-invasive palliative treatment for advanced spinal metastases is promising as part of a multidisciplinary approach to the care of patients with metastatic disease. The results of this study indicate that percutaneous surgery may allow for rapid improvement in quality of life and walking ability for patients with thoracolumbar instability due to spine metastases. Long-segment percutaneous screw fixation followed by early radiation therapy appears to be a safe and effective treatment option for providing solid and durable stability and improved quality of life for these patients.

Toxoplasma gondii Arginine Methyltransferase 1 (PRMT1) Is Necessary for Centrosome Dynamics during Tachyzoite Cell Division.

UNLABELLED: The arginine methyltransferase family (PRMT) has been implicated in a variety of cellular processes, including signal transduction, epigenetic regulation, and DNA repair pathways. PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. To further define the biological function of the PRMT family, we generated T. gondii mutants lacking PRMT1 (Δprmt1) by deletion of the PRMT1 gene. Δprmt1 parasites exhibit morphological defects during cell division and grow slowly, and this phenotype reverses in the Δprmt::PRMT1mRFP complemented strain. Tagged PRMT1 localizes primarily in the cytoplasm with enrichment at the pericentriolar material, and the strain lacking PRMT1 is unable to segregate progeny accurately. Unlike wild-type and complemented parasites, Δprmt1 parasites have abnormal daughter buds, perturbed centrosome stoichiometry, and loss of synchronous replication. Whole-genome expression profiling demonstrated differences in expression of cell-cycle-regulated genes in the Δprmt1 strain relative to the complemented Δprmt1::PRMT1mRFP and parental wild-type strains, but these changes do not correlate with a specific block in cell cycle. Although PRMT1's primary biological function was previously proposed to be methylation of histones, our studies suggest that PRMT1 plays an important role within the centrosome to ensure the proper replication of the parasite. IMPORTANCE: Apicomplexan parasites include several important pathogens, including Toxoplasma gondii, a major cause of opportunistic infections and congenital birth defects. These parasites divide using a unique form of cell division called endodyogeny that is different from those of most eukaryotes. PRMT1 is a conserved arginine methyltransferase that was thought to regulate gene expression of T. gondii by modifying histone methylation. Using genetic techniques, we show that disruption of PRMT1 affects the parasite's ability to perform accurate cell division. Our studies reveal an unexpected role for arginine methylation in centrosome biology and regulation of parasite replication.

Identifying novel cell cycle proteins in Apicomplexa parasites through co-expression decision analysis.

Hypothetical proteins comprise roughly half of the predicted gene complement of Toxoplasma gondii and Plasmodium falciparum and represent the largest class of uniquely functioning proteins in these parasites. Following the idea that functional relationships can be informed by the timing of gene expression, we devised a strategy to identify the core set of apicomplexan cell division cycling genes with important roles in parasite division, which includes many uncharacterized proteins. We assembled an expanded list of orthologs from the T. gondii and P. falciparum genome sequences (2781 putative orthologs), compared their mRNA profiles during synchronous replication, and sorted the resulting set of dual cell cycle regulated orthologs (744 total) into protein pairs conserved across many eukaryotic families versus those unique to the Apicomplexa. The analysis identified more than 100 ortholog gene pairs with unknown function in T. gondii and P. falciparum that displayed co-conserved mRNA abundance, dynamics of cyclical expression and similar peak timing that spanned the complete division cycle in each parasite. The unknown cyclical mRNAs encoded a diverse set of proteins with a wide range of mass and showed a remarkable conservation in the internal organization of ordered versus disordered structural domains. A representative sample of cyclical unknown genes (16 total) was epitope tagged in T. gondii tachyzoites yielding the discovery of new protein constituents of the parasite inner membrane complex, key mitotic structures and invasion organelles. These results demonstrate the utility of using gene expression timing and dynamic profile to identify proteins with unique roles in Apicomplexa biology.

Changes induced by peripheral nerve injury in the morphology and nanomechanics of sensory neurons.

Peripheral nerve injury in vivo promotes a regenerative growth in vitro characterized by an improved neurite regrowth. Knowledge of the conditioning injury effects on both morphology and mechanical properties of live sensory neurons could be instrumental to understand the cellular and molecular mechanisms leading to this regenerative growth. In the present study, we use differential interference contrast microscopy, fluorescence microscopy, and atomic force microscopy (AFM) to show that conditioned axotomy, induced by sciatic nerve injury, does not increase somatic size of sensory neurons from adult mice lumbar dorsal root ganglia but promotes the appearance of longer and larger neurites and growth cones. AFM on live neurons is also employed to investigate changes in morphology and membrane mechanical properties of somas of conditioned neurons following sciatic nerve injury. Mechanical analysis of the soma allows distinguishing neurons having a regenerative growth from control ones, although they show similar shapes and sizes.

ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst.

Cellular differentiation leading to formation of the bradyzoite tissue cyst stage is the underlying cause of chronic toxoplasmosis. Consequently, mechanisms responsible for controlling development in the Toxoplasma intermediate life cycle have long been sought. Here, we identified 15 Toxoplasma mRNAs induced in early bradyzoite development that encode proteins with apicomplexan AP2 (ApiAP2) DNA binding domains. Of these 15 mRNAs, the AP2IX-9 mRNA demonstrated the largest expression increase during alkaline-induced differentiation. At the protein level, we found that AP2IX-9 was restricted to the early bradyzoite nucleus and is repressed in tachyzoites and in mature bradyzoites from 30-d infected animals. Conditional overexpression of AP2IX-9 significantly reduced tissue cyst formation and conferred alkaline pH-resistant growth, whereas disruption of the AP2IX-9 gene increased tissue cyst formation, indicating AP2IX-9 operates as a repressor of bradyzoite development. Consistent with a role as a repressor, AP2IX-9 specifically inhibited the expression of bradyzoite mRNAs, including the canonical bradyzoite marker, bradyzoite antigen 1 (BAG1). Using protein binding microarrays, we established the AP2 domain of AP2IX-9 binds a CAGTGT DNA sequence motif and is capable of binding cis-regulatory elements controlling the BAG1 and bradyzoite-specific nucleoside triphosphatase (B-NTPase) promoters. The effect of AP2IX-9 on BAG1 expression was direct because this factor inhibits expression of a firefly luciferase reporter under the control of the BAG1 promoter in vivo, and epitope-tagged AP2IX-9 can be immunoprecipitated with the BAG1 promoter in parasite chromatin. Altogether, these results indicate AP2IX-9 restricts Toxoplasma commitment to develop the mature bradyzoite tissue cyst.

Morphology and nanomechanics of sensory neurons growth cones following peripheral nerve injury.

A prior peripheral nerve injury in vivo, promotes a rapid elongated mode of sensory neurons neurite regrowth in vitro. This in vitro model of conditioned axotomy allows analysis of the cellular and molecular mechanisms leading to an improved neurite re-growth. Our differential interference contrast microscopy and immunocytochemistry results show that conditioned axotomy, induced by sciatic nerve injury, did not increase somatic size of adult lumbar sensory neurons from mice dorsal root ganglia sensory neurons but promoted the appearance of larger neurites and growth cones. Using atomic force microscopy on live neurons, we investigated whether membrane mechanical properties of growth cones of axotomized neurons were modified following sciatic nerve injury. Our data revealed that neurons having a regenerative growth were characterized by softer growth cones, compared to control neurons. The increase of the growth cone membrane elasticity suggests a modification in the ratio and the inner framework of the main structural proteins.

Laser-treated Nucleus pulposus as an innovative model of intervertebral disc degeneration.

This study describes an innovative experimentally induced model of intervertebral disc degeneration. This innovative approach is based on the induction of extracellular matrix disorders in the intervertebral disc (IVD) using a diode laser. For this study, 15 one-year-old and five 30-month-old New Zealand White rabbits were used. Two procedures were tested to trigger IVD degeneration: needle aspiration (reference technique) and a laser approach. The IVD degeneration process was assessed 20, 40, 60, 90 and 120 days after surgery by X-ray radiography (IVD height), magnetic resonance imaging (MRI) (T2 intensity of IVD signal) and histological analysis using modified Boos' scoring. Our data indicate that a marked IVD degeneration was found compared with sham-operated animals regardless of the procedure tested. A significant decrease in disc height on X-ray radiographs was first demonstrated. In addition, MRI disc signals were significantly reduced in both groups. Finally, a statistically significant increase in Boos' scoring was found in both laser and aspiration-induced IVD degeneration. Interestingly, IVD degeneration induced by laser treatment was more progressive compared with aspiration. Moreover, the histological results indicated that laser-induced disc degeneration was quite similar to that obtained during the natural aging process as observed in 30-month-old rabbits. Our study describes the consistency of this innovative experimentally-induced animal model of IVD degeneration. The radiological, MRI and histological data confirm its relevance. The histological examination indicates that IVD degeneration induced by laser treatment is comparable to the degenerative process observed during the onset of spontaneous IVD degeneration. This model could be a useful tool to help us validate biomaterial-assisted, cell-based, regenerative medicine strategies for the prevention and treatment of IVD degeneration.

KCC3-dependent chloride extrusion in adult sensory neurons.

The cation-Cl(-) cotransporters participate to neuronal Cl(-) balance and are responsible for the post-natal Cl(-) switch in central neurons. In the adult peripheral nervous system, it is not well established whether a Cl(-) transition occurs during maturation. We investigated the contribution of cation-Cl(-) cotransporters in the Cl(-) handling of sensory neurons derived from the dorsal root ganglia (DRG) of neonatal mice (postnatal days 1-6) and adult mice. Gramicidin-perforated patch-clamp recordings in wild-type neurons revealed that Cl(-) accumulated to very high values in P1-6 sensory neurons and decreased in adulthood. In post-natal sensory neurons, quantitative RT-PCR showed that NKCC1, KCC1 and KCC3 had a higher transcript expression level compared to KCC2 and KCC4. NKCC1 was the main cation-Cl(-) cotransporter controlling Cl(-) accumulation at this developmental stage. In adulthood, the KCC3 transcript was produced in larger amounts than the other cation-Cl(-) cotransporter transcripts and RT-PCR shows larger expression of the shorter KCC3a isoform in adult DRG. Pharmacological inhibitors of cation-Cl(-) cotransporters and the use of KCC3(-/-) mice demonstrated that NKCC1 sustained Cl(-) accumulation in the majority of adult sensory neurons while KCC3 contributed to Cl(-) extrusion in a subset of these neurons. Beta-galactosidase detection in adult KCC3(-/-) DRG showed that KCC3 transcripts were present in all adult sensory neurons suggesting a KCC3 isoform specific regulation of Cl(-) handling. The contribution of KCC3 to Cl(-) extrusion in a subset of sensory neurons indicates that KCC3 could play a major role in GABAergic/glycinergic transmission.

Neurotrophin-4 modulates the mechanotransducer Cav3.2 T-type calcium current in mice down-hair neurons.

The T-type Ca2+ channel Cav3.2 is expressed in nociceptive and mechanosensitive sensory neurons. The mechanosensitive D-hair (down-hair) neurons, which innervate hair follicles, are characterized by a large-amplitude Cav3.2 T-current involved in the amplification of slow-moving stimuli. The molecules and signalling pathways that regulate T-current expression in mechanoreceptors are unknown. In the present study, we investigated the effects of NT-4 (neurotrophin-4) on Cav3.2 T-current expression in D-hair neurons in vitro. Interruption of the supply of NT-4 with peripheral nerve axotomy induced a non-transcriptional decrease in the T-current amplitude of fluorogold-labelled axotomized sensory neurons. The T-current amplitude was restored by incubation with NT-4. Deletion of NT-4 through genetic ablation resulted in a similar selective loss of the large-amplitude T-current in NT-4-/- sensory neurons, which was rescued by the addition of NT-4. NT-4 had no effect on the T-current in Cav3.2-/- D-hair neurons. Neither the biophysical properties of the T-current nor the transcript expression of Cav3.2 were modified by NT-4. Pharmacological screening of signalling pathways activated under the high-affinity NT-4 receptor TrkB (tropomyosin receptor kinase B) identified a role for PI3K (phosphoinositide 3-kinase) in the potentiation of T-current. The results of the present study demonstrate the post-transcriptional up-regulation of the Cav3.2 T-current through TrkB activation and identify NT-4 as a target-derived factor that regulates the mechanosensitive function of D-hair neurons through expression of the T-current.

A systematic screen to discover and analyze apicoplast proteins identifies a conserved and essential protein import factor.

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes--a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.

An autocrine neuronal interleukin-6 loop mediates chloride accumulation and NKCC1 phosphorylation in axotomized sensory neurons.

The cation-chloride cotransporter NKCC1 plays a fundamental role in the central and peripheral nervous systems by setting the value of intracellular chloride concentration. Following peripheral nerve injury, NKCC1 phosphorylation-induced chloride accumulation contributes to neurite regrowth of sensory neurons. However, the molecules and signaling pathways that regulate NKCC1 activity remain to be identified. Functional analysis of cotransporter activity revealed that inhibition of endogenously produced cytokine interleukin-6 (IL-6), with anti-mouse IL-6 antibody or in IL-6⁻/⁻ mice, prevented chloride accumulation in a subset of axotomized neurons. Nerve injury upregulated the transcript and protein levels of IL-6 receptor in myelinated, TrkB-positive sensory neurons of murine lumbar dorsal root ganglia. Expression of phospho-NKCC1 was observed mainly in sensory neurons expressing IL-6 receptor and was absent from IL-6⁻/⁻ dorsal root ganglia. The use of IL-6 receptor blocking-function antibody or soluble IL-6 receptor, together with pharmacological inhibition of Janus kinase, confirmed the role of neuronal IL-6 signaling in chloride accumulation and neurite growth of a subset of axotomized sensory neurons. Cell-specific expression of interleukin-6 receptor under pathophysiological conditions is therefore a cellular response by which IL-6 contributes to nerve regeneration through neuronal NKCC1 phosphorylation and chloride accumulation.

Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii.

BACKGROUND: Apicomplexan parasites replicate by varied and unusual processes where the typically eukaryotic expansion of cellular components and chromosome cycle are coordinated with the biosynthesis of parasite-specific structures essential for transmission. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the global cell cycle transcriptome of the tachyzoite stage of Toxoplasma gondii. In dividing tachyzoites, more than a third of the mRNAs exhibit significant cyclical profiles whose timing correlates with biosynthetic events that unfold during daughter parasite formation. These 2,833 mRNAs have a bimodal organization with peak expression occurring in one of two transcriptional waves that are bounded by the transition into S phase and cell cycle exit following cytokinesis. The G1-subtranscriptome is enriched for genes required for basal biosynthetic and metabolic functions, similar to most eukaryotes, while the S/M-subtranscriptome is characterized by the uniquely apicomplexan requirements of parasite maturation, development of specialized organelles, and egress of infectious daughter cells. Two dozen AP2 transcription factors form a series through the tachyzoite cycle with successive sharp peaks of protein expression in the same timeframes as their mRNA patterns, indicating that the mechanisms responsible for the timing of protein delivery might be mediated by AP2 domains with different promoter recognition specificities. CONCLUSION/SIGNIFICANCE: Underlying each of the major events in apicomplexan cell cycles, and many more subordinate actions, are dynamic changes in parasite gene expression. The mechanisms responsible for cyclical gene expression timing are likely crucial to the efficiency of parasite replication and may provide new avenues for interfering with parasite growth.

ICP0 antagonizes ICP4-dependent silencing of the herpes simplex virus ICP0 gene.

ICP0 is a regulatory protein that plays a critical role in the replication-latency balance of herpes simplex virus (HSV). Absence of ICP0 renders HSV prone to establish quiescent infections, and thus cellular repressor(s) are believed to silence HSV mRNA synthesis when ICP0 fails to accumulate. To date, an ICP0-antagonized repressor has not been identified that restricts HSV mRNA synthesis by more than 2-fold. We report the unexpected discovery that HSV's major transcriptional regulator, ICP4, meets the criteria of a bona fide ICP0-antagonized repressor of viral mRNA synthesis. Our study began when we noted a repressive activity that restricted ICP0 mRNA synthesis by up to 30-fold in the absence of ICP0. When ICP0 accumulated, the repressor only restricted ICP0 mRNA synthesis by 3-fold. ICP4 proved to be necessary and sufficient to repress ICP0 mRNA synthesis, and did so in an ICP4-binding-site-dependent manner. ICP4 co-immunoprecipitated with FLAG-tagged ICP0; thus, a physical interaction likely explains how ICP0 antagonizes ICP4's capacity to silence the ICP0 gene. These findings suggest that ICP0 mRNA synthesis is differentially regulated in HSV-infected cells by the virus-encoded repressor activity embedded in ICP4, and a virus-encoded antirepressor, ICP0. Bacteriophage lambda relies on a similar repression-antirepression regulatory scheme to "decide" whether a given infection will be productive or silent. Therefore, our findings appear to add to the growing list of inexplicable similarities that point to a common evolutionary ancestry between the herpesviruses and tailed bacteriophage.

Cytoprotective Nrf2 pathway is induced in chronically txnrd 1-deficient hepatocytes.

BACKGROUND: Metabolically active cells require robust mechanisms to combat oxidative stress. The cytoplasmic thioredoxin reductase/thioredoxin (Txnrd1/Txn1) system maintains reduced protein dithiols and provides electrons to some cellular reductases, including peroxiredoxins. PRINCIPAL FINDINGS: Here we generated mice in which the txnrd1 gene, encoding Txnrd1, was specifically disrupted in all parenchymal hepatocytes. Txnrd1-deficient livers exhibited a transcriptome response in which 56 mRNAs were induced and 12 were repressed. Based on the global hybridization profile, this represented only 0.3% of the liver transcriptome. Since most liver mRNAs were unaffected, compensatory responses were evidently effective. Nuclear pre-mRNA levels indicated the response was transcriptional. Twenty-one of the induced genes contained known antioxidant response elements (AREs), which are binding sites for the oxidative and chemical stress-induced transcription factor Nrf2. Txnrd1-deficient livers showed increased accumulation of nuclear Nrf2 protein and chromatin immunoprecipitation on the endogenous nqo1 and aox1 promoters in fibroblasts indicated that Txnrd1 ablation triggered in vivo assembly of Nrf2 on each. CONCLUSIONS: Chronic deletion of Txnrd1 results in induction of the Nrf2 pathway, which contributes to an effective compensatory response.

Effects of thioredoxin reductase-1 deletion on embryogenesis and transcriptome.

Thioredoxin reductases (Txnrd) maintain intracellular redox homeostasis in most organisms. Metazoan Txnrds also participate in signal transduction. Mouse embryos homozygous for a targeted null mutation of the txnrd1 gene, encoding the cytosolic thioredoxin reductase, were viable at embryonic day 8.5 (E8.5) but not at E9.5. Histology revealed that txnrd1-/- cells were capable of proliferation and differentiation; however, mutant embryos were smaller than wild-type littermates and failed to gastrulate. In situ marker gene analyses indicated that primitive streak mesoderm did not form. Microarray analyses on E7.5 txnrd-/- and txnrd+/+ littermates showed similar mRNA levels for peroxiredoxins, glutathione reductases, mitochondrial Txnrd2, and most markers of cell proliferation. Conversely, mRNAs encoding sulfiredoxin, IGF-binding protein 1, carbonyl reductase 3, glutamate cysteine ligase, glutathione S-transferases, and metallothioneins were more abundant in mutants. Many gene expression responses mirrored those in thioredoxin reductase 1-null yeast; however, mice exhibited a novel response within the peroxiredoxin catalytic cycle. Thus, whereas yeast induce peroxiredoxin mRNAs in response to thioredoxin reductase disruption, mice induced sulfiredoxin mRNA. In summary, Txnrd1 was required for correct patterning of the early embryo and progression to later development. Conserved responses to Txnrd1 disruption likely allowed proliferation and limited differentiation of the mutant embryo cells.