S) mutant mice.The progressive nature of Wallerian degeneration has long been controversial. Conflicting reports that distal stumps of injured axons degenerate anterogradely, retrogradely, or simultaneously are based on statistical observations at discontinuous locations within the nerve, without observing any single axon at two distant points. As axon degeneration is asynchronous, there are clear advantages to longitudinal studies of individual degenerating axons. We recently validated the study of Wallerian degeneration using yellow fluorescent protein (YFP) in a small, representative population of axons, which greatly improves longitudinal imaging. Here, we apply this method to study the progressive nature of Wallerian degeneration in both wild-type and slow Wallerian degeneration  among a majority of fully intact or degenerated axons 37–42 h after transection and 40–44 h after crush injury. Axons exist in this state only transiently, probably for less than one hour. Surprisingly, axons degenerated anterogradely after transection but retrogradely after a crush, but in both cases a sharp boundary separated intact and fragmented regions of individual axons, indicating that Wallerian degeneration progresses as a wave sequentially affecting adjacent regions of the axon. In contrast, most or all WldS axons, once they finally degenerate, do so by a fundamentally different mechanism, indicated by differences in the rate, direction and abruptness of progression, and by different early morphological signs of degeneration. These observations suggest that WldS axons undergo a slow anterograde decay as axonal components are gradually depleted, and do not simply follow the degeneration pathway of wild-type axons at a slower rate.We conclude that Wallerian degeneration progresses rapidly along individual wild-type axons after a heterogeneous latent phase. The speed of progression and its ability to travel in either direction challenges earlier models in which clearance of trophic or regulatory factors by axonal transport triggers degeneration. Wld S) mutant mouse, in which this active process seems to be turned off. Accordingly, this mutant shows a tenfold delay in Wallerian degeneration and synapse breakdown after experimental nerve injury [WldS that arose by spontaneous mutation [WldS mutation on mouse chromosome 4 comprises a stable 85-kb tandem triplication [S mice express a novel chimeric protein (WldS protein) in neuronal nuclei that has full Nmnat 1 activity but seemingly no Ube4b function since the expressed N-terminus lacks ability for multi-ubiquitination within the ubiquitin-proteasome system (UPS) [S protein could be responsible for the phenotype through a nuclear process that has an indirect effect on the axon although recent results foster that the WldS mechanism is likely to involve a gain of function of NAD synthesis [S protein remains a possibility [Wallerian degeneration, the characteristic degeneration sequence of nerve fibres separated from their cell bodies, was described by Waller in 1850 . Followie injury . The dele injury . The undmutation ,10. Genelication ,12 encodem (UPS) ,14, the ynthesis . Howeversibility ,17.S is currently under intense investigation. The neuroprotective mutation alleviates diverse PNS axon disorders, including dysmyelination and dying back neuropathy in P0-/- mutants [pmn mutants [S was reported to attenuate pathology in acute CNS lesions caused by stroke [gad) mice, a CNS axonal spheroid pathology [From a clinical point of view not only traumatic disorders such as nerve, spinal cord or head injury result in Wallerian degeneration  but it i mutants , motor n mutants  and axon mutants -28. Morey stroke , Parkinsy stroke , and in athology . A betteDespite research extending over more than 150 years and its frequent use as tool to detect interneuronal connections in the CNS since the time of Cajal, fundamental issues of Wallerian degeneration remain unresolved and controversial even on a purely morphological level. Among these is the spatiotemporal pattern of Wallerian degeneration along the separated nerve stump. Understanding the exact pattern of spread should provide additional insights into the mechanisms of axon death and may indicate strategies to alter Wallerian degeneration in neurological disease. Shortly after the pioneering investigations of Waller, and in the following decades, there has been much debate as to whether degeneration occurs in an anterograde direction, a retrograde direction or simultaneously along the separated nerve stump axons .4L5L6 radiculotomy. Contrary to these views, Lunn and colleagues [The controversy over the directionality of Wallerian degeneration has arisen partly because appropriate methods to follow axons over considerable distances did not exist until recently but also because the course of Wallerian degeneration varies with many experimental factors. Thus, spatiotemporal evolution of Wallerian degeneration depends on the laboratory animals used ,34-36, olleagues  showed blleagues . In celllleagues . Taking lleagues  tracked lleagues ,53.In view of the contradictions and anomalies in the previous literature, we have reassessed the directionality of Wallerian degeneration using a recently introduced technique to visualize individual fluorescent axons over cm-long distances during degeneration . This waS nerves that could yield important clues for understanding classic Wallerian degeneration. Therefore, we also report a detailed characterisation of injury-induced axon degeneration in slow Wallerian degeneration mutant mice in order to determine whether axons degenerate with a similar spatial evolution to that in wild-type mice, but in "slow motion", or whether the process is fundamentally distinct. We report a series of differences between axon degeneration in wild-type and WldS mice, suggesting that irreversible injury in axons where Wallerian degeneration is blocked eventually leads to a different pathway of degeneration.To our knowledge there have been no reports about the spatiotemporal pattern of the much-delayed axon degeneration in peripheral WldIn preliminary experiments we used conventional light and electron microscopy to investigate whether Wallerian degeneration is progressive in wild-type mouse peripheral nerves. We were never able to find any significant gradients of degeneration along injured nerves that were processed with these traditional methods (data not shown). We then looked for signs of progression in localised observations of degenerating YFP-H nerves because fragmentation of YFP-labelled axons from these mice corresponds to granular disintegration of axoplasm as well as myelin ovoid formation and YFP positive axons represent the whole myelinated axon population . Axonal The failure to observe any gradient of degeneration in the above experiment does not mean that Wallerian degeneration is not progressive. It could propagate so rapidly that it was not detectable by this method, or a gradient might not be detectable because of the considerable statistical noise of the highly asynchronous process. In order to investigate these possibilities we turned to confocal tracing of individual axons in long wholemount YFP-H nerve segments. At 37 h after transection we found 2.0 % of fluorescent axons with extensive proximal fragmentation and intact distal regions indicating an anterograde gradient of Wallerian degeneration in these axons Fig. . Around In contrast, all partially fragmented axons after crush injury at all investigated timepoints were fragmented in distal tibial nerve but not at the proximal end of the distal stump Fig. . Once agSummarising all these quantification results of cut and crush lesions at time points where partially fragmented fibres were observed, on average 94.8 % of all axons were either completely intact or fragmented and 5.2 % showed a gradient of Wallerian degeneration, whose orientation depended on the lesion type.To study further the gradients of axonal fragmentation both after cut and crush injury Fig. , 4 we quConcerning the first question we found that Wallerian degeneration progresses as a wave, with the wave front defining the point to which fragmentation had spread along the axon. In partially fragmented axons separated from the cell body by transection or proximally compressed by crush lesion, axon regions with no features of degeneration abruptly change into segment lengths with marked breakdown within a transition zone of less than one millimeter Fig. , 6. A shIn order to determine whether the anterograde and retrograde fragmentation wave runs at the same velocity along the axon we next estimated the rate of progression. As the average axon length measured was 24 mm and the majority of axons must have entirely fragmented between 41 h and 42 h after transection  Fig. , the minAdd Fig 1.pdf). These data are consistent with the axonal membrane remaining intact after 30 sec nerve crush, unlike that of a transected nerve.We then tested whether our crush lesions interrupted axon continuity as in nerve transection, because a failure to do so could underlie the different direction of propagation in crushed nerves (see Discussion). In fluorescent wholemount preparations of crushed nerve segments we found continuous longitudinal YFP signals across the crush site, and in teased fibre bundles of osmificated nerve segments after crush injury, a majority of fibres remained continuous across the crush site  and subsequent multiple axonal swellings . This feature was far more prominent in crushed WldS axons than in the transection experiment where we observed end bulbs just occasionally. Further distally these swellings disappear with remaining axonal constrictions and breaks  representing incomplete fragmentation. As in transected nerves, distal parts of the crushed WldS axon were free of degeneration signs . Compared to transection lesion, more axons at 15 days remained entirely intact (15.4 %)  while more distal regions of the same axons were again incompletely fragmented  and further distal still lacked any degeneration signs . The proximal end bulbs and localised swellings were larger than at 15 days , possibly due to continued accumulation of retrogradely transported material.We then followed up the earlier EM experiments using crushed nerves from Wldges Fig. . An exam %) Fig. . By 20 dS axons is directional with an exclusively anterograde pattern both after transection and crush injury. This pattern of degeneration is qualitatively different from that in wild-type mice, which shows asynchronous, bidirectional fragmentation and degeneration.In summary, quantification 20 days after crush lesion revealed that all axons showed anterograde gradients of complete or incomplete fragmentation Fig. . Thus, tS axons. In contrast to our observations in wild-type mice, there was no clearly delineated boundary or wave front separating degenerated and fully intact regions. In order to quantify this, we counted the number of axonal constrictions and breaks along the length of YFP positive WldS axons following injury. At all post-lesion time-points randomly chosen axons showed gradually decreasing signs of degeneration (constrictions or interruptions) along their length , giving a maximum velocity of WldS degeneration progression of 11 mm / 5 days = 2.2 mm/day. This is similar to the reported velocity of slow axonal transport (0.1–3.0 mm/day) [S peripheral nerves are summarized schematically in Fig. In the above experiments we noted the gradual change from degenerated regions to intact regions in Wldgth Fig. . This maion Fig. . This qu mm/day) -59. All S mutant mice undergo a fundamentally distinct process rather than simply following the same pathway in slow motion.We have shown that the fragmentation of axons undergoing Wallerian degeneration in a mixed wild-type peripheral nerve is a rapid, asynchronous and progressive process. By using a recently developed method to visualise individual axons over cm-long distances, and by targeting a short critical period during which nearly all axons degenerate, we have made the first observations in vivo of partially fragmented individual axons and thus determined the directionality and the wave-like nature of Wallerian degeneration, as well as estimating its velocity. Furthermore, we have shown that nerves of WldLesioned wild-type axons remain morphologically normal for a latency period of ca. 36–44 hours, which depends on lesion type and individual axonal properties ,44,46,60We have shown that Wallerian degeneration in wild-type nerves is a wave-like process that can travel in either direction along the axon, depending on lesion type. 29 partially fragmented axons were observed, and all showed a sharp boundary between fragmented and non-fragmented zones, such that all axon regions up to the wavefront were degenerated and all regions beyond it remained intact. Fragmentation had reached different points along the nerve in different individual axons Fig. , reflectS protein. The fastest reported components of axonal transport move at around 14–25 mm/h, but it is reasonable to expect that some minor, thus far undetected, components may move faster [We discuss here two models to explain the wave-like propagation of Wallerian degeneration in wild-type nerves: one based on fast axonal transport and the other based on calcium influx . Thus, the proximal end of the distal stump may be less vulnerable than after transection, and fragmentation may begin instead at the distal end because this is the hardest part to supply with everything the axon needs to survive.There are several possible reasons why degeneration may begin proximally in a transected nerve but distally in a crushed nerve. The proximal end of a transected distal stump is especially vulnerable because of the exposure of the axoplasm to the external ionic environment, and because all extra-axonal structures that normally support the axon have been totally disrupted, e.g., blood vessels, Schwann cells, extracellular matrix, perineurium. One of these factors may cause a calcium entry wave to begin at this point. In contrast, intact endoneural blood vessels can be found close to a nerve crush ,78, the S nerves that are incompatible with delayed axon degeneration following a similar mechanism to Wallerian degeneration in wild-type nerves, only slower. The spread of degeneration along WldS nerves is around 100-fold slower, axon degeneration is more synchronous, at least relative to how long it takes to occur, it progresses in a proximal to distal direction in crushed nerves as well as transected nerves, there is a continuous gradient of degeneration along the length of the axon rather than an abrupt change at a boundary, and the first sign of axon degeneration is a constriction rather than a complete interruption. We therefore propose that the ultimate degeneration of axons in WldS mice be termed "slow anterograde axon decay" rather than Wallerian degeneration as summarised schematically in Fig. We observed a series of differences in the pattern of Wallerian degeneration in WldS axons eventually undergo a passive process of atrophy, rather than an active process of self-destruction similar to apoptosis that appears to take place in wild-type axons [S (data not shown). Even the fact that some proteins are synthesised locally in axons [S axons, as it remains unclear which proteins are made there and in what quantities.Based on these differences, we propose that injured Wldpe axons ,15,16,86in axons ,87-89 maS axons: one based on slow axonal transport and the other on a temperature gradient along the limb. The gradual nature of axonal atrophy in WldS makes it difficult to be precise about the rate at which it progresses along the axon, but it is certainly not incompatible with the velocity of slow axonal transport of 0.1–3.0 mm/day [S nerves, leading to localised neurofilament-depleted constrictions and terminal and intermediate swellings containing disorganised neurofilaments [S axons, and additionally report a gradient of such features along the nerve. A net anterograde movement of cytoskeletal proteins could therefore underlie the anterograde gradient of axonal atrophy in WldS axons injured for many days.We discuss two models to explain the slow anterograde progression of degeneration along Wld0 mm/day -58,90. Cilaments ,92. We hS nerve. A decrease in temperature has been shown to delay degeneration both in wild-type and WldS axons after injury [S axons there is a proximal to distal gradient of degeneration regardless of lesion type and the change from intact to degenerated is a gradual one. Thus, a temperature gradient could play a more important role here.Alternatively, a proximal-distal decreasing gradient of temperature along the limb could underlie the observed difference in degeneration rate at different points in the Wldr injury ,74,93-96S is in marked contrast to the presynaptic nerve terminal at the neuromuscular junction, which is the first structure to degenerate in both wild-type and WldS nerves [S heterozygotes . This supports the hypothesis of compartmentalised degeneration mechanisms of axons and synapses [The extremely long survival of distal tibial nerve following injury in WldS nerves ,8,46. Insynapses  and suggWldS gene and the direction of degeneration is also shared [in vivo after nerve lesion or in disease, once methods for in vivo imaging of single axons become more refined and more readily available, e.g., Cell ViZio, Mauna Kea Technologies [Finally, the methods we report here could now be applied to study spontaneous nerve degeneration in 'dying-back' disease. The 'dying-back' model also predicts the transitory existence of partially degenerated axons, but as in Wallerian degeneration such axons have never been directly observed and there is no indication of the speed of 'dying-back' of individual axons ,99. Thero shared . Thus, io shared ,100, wheo shared  and it inologies .S nerves is also progressive, but differences in the topographic pattern and morphology of degeneration indicate a fundamentally different process from that in wild-type nerves. We propose that WldS axons ultimately undergo atrophy, in a passive process similar to that which Wallerian degeneration was once thought to be.In summary, we report the first direct observation of partially degenerated single axons in lesioned nerves undergoing Wallerian degeneration, indicating that Wallerian degeneration propagates in wild-type nerves as a wave whose speed is at least as fast as the highest reported rate of fast axonal transport. It could be faster still in mouse sciatic and tibial nerve. The direction of degeneration is proximal to distal after a cut, but the reverse after a crush. For now the mechanism remains unknown, but these observations will ultimately need to be explained in any comprehensive model of the Wallerian degeneration mechanism. Injury-induced axon degeneration in WldWldS mutation, the transgenic WldS mutation (tg-WldS) [S/YFP-H mice were used purely for reasons of convenience in their breeding. They express similar levels of WldS protein as homozygous natural mutant WldS mice and display a similar retarded time-course of axon degeneration. They additionally express YFP in approximately 3% of myelinated motor and sensory fibres in the PNS, equal to the original YFP-H line obtained from the Jackson Laboratories.Crossbreeding and genotyping of the YFP-H line  and triptg-WldS)  and the tg-WldS) . Triple Six- to 10-week-old mice from the YFP-H line and triple heterozygote mice for the second part of the study were anaesthetised by intraperitoneal injection of Ketanest  and Rompun , sciatic nerves were transected or crushed firmly close to the Foramen intrapiriforme and the wound was closed with a single suture. Complete nerve transection was performed with conventional surgical scissors and crush lesion was achieved with fine watchmaker's forceps  for 30 seconds. The continuity of the sciatic nerve was always preserved after crush lesion as checked in situ. For light and electron microscopy we removed distal ~2.5 centimeter long nerve stumps after 15 (transection only), 20, 25 and 30 days in triple heterozygote mice following intracardial perfusion. The first 2 millimeters of the distal nerve stump were discarded to reduce artifacts, and the next 2 mm from the most proximal and distal end of the peripheral nerve stump was prepared for Durcupan embedding and electron microscopy.For conventional fluorescence microscopy and confocal tracing of individual YFP labelled wild-type axons after 34 h, 37 h, 40 h, 41 h, 42 h and 48 h following cut lesion and 37 h, 40 h, 42 h, 43 h, 44 h and 48 h following crush lesion the operated YFP-H mice were sacrificed by cervical dislocation and nerve segments prepared as follows.S axons from triple heterozygote mice we dissected sciatic-tibial nerve segments after 12.5 d (transection only), 15d, 17.5 d (transection only), 20 d and 22.5 d (transection only). For each investigated time-point 2 – 3 YFP-H or triple heterozygote mice were operated.For confocal tracing of individual YFP labelled WldSciatic nerves of mice from the YFP-H line were crushed as described in the paragraph above and the short nerve segment containing the crush site immediately removed after lesion, freed from surrounding connective tissue and subsequently either prepared for wholemount fluorescence embedding as described previously  or for tAfter sternotomy under deep anaesthesia mice were killed by cardiac puncture and instantly intracardially perfused first with a solution containing 10 000 i.e./l heparin  and 1 % procainhydrochloride in 0.1 M PBS for 30 s and then with fresh half-strength Karnovsky's fixative .Nerve samples for light and electron microscopy were embedded in Durcupan and further processed for examination with a Zeiss Axiophot light microscope and Zeiss EM 902 electron microscope as described previously .The percentage of preserved myelinated axons in proximal and distal ends of peripheral nerve stumps from triple heterozygote mice after transection and crush injury was determined as described previously ,54.S axons running through the excised stumps over their whole length was achieved as described previously [In both YFP-H and triple heterozygote mice after cut or crush lesion the entire nerve distances from the proximal sciatic to the distal tibial nerve were carefully excised, the perineurium and the branch of the commune fibular nerve removed and the remaining ~2.5 centimeter long stumps treated for wholemount fluorescence preparation using Vectashield Mounting Medium (Vector Laboratories) as described previously . For roueviously . Single Imaging of restricted transition zones dividing intact and fragmented axon regions in YFP-H nerves was performed under highest possible resolution with a Zeiss LSM 510 META confocal system (LSM Software Release 3.2) coupled to a Zeiss Axiovert 200 microscope.S axons in wholemount peripheral nerve stumps (2–3 nerve preparations for each timepoint) after transection and crush injury were equally traced individually with the BioRad Radiance 2000 laser scanning confocal microscope under high resolution, and divided into two groups: axons without any features of axonal disintegration were counted and assigned to the group "intact". Fibres that showed degeneration signs like constrictions or interruptions of the longitudinal YFP signal at the proximal site were associated into the group "fragmented with anterograde gradient". Means and standard deviations for all experiments were calculated using Microsoft Excel.For the first part of the study focusing on sciatic/tibial nerves from the YFP-H line in each wholemount preparation (2 – 3 nerve preparations per time point) YFP labelled wild-type axons running continuously through the excised peripheral nerve stump after cut and crush injury were traced individually with the laser scanning confocal microscope under high resolution and divided into four groups: axons without any features of axonal disintegration were assigned to the group "intact". Axons that showed fragmentation of the longitudinal YFP signal over the whole length of the fibre were assigned to the group "entirely fragmented". Axons without any sign of fragmentation at one end but clear axon breakdown at the other end were assigned either into the group "fragmented with anterograde gradient" or "fragmented with retrograde gradient" depending whether the fragmentation appeared close to the lesion point or at the most distal point of the tibial nerve segment. For the second part of the study dealing with triple heterozygote mice YFP labelled WldS axons was measured during the tracing process with the confocal laser scanning microscope and the distribution of axonal interruptions (both in wild-type and WldS axons) and/or constrictions (only in WldS axons) was graphed against the distance in mm from the cut or crush point. Means and standard deviations were calculated using Microsoft Excel.The length of degenerated YFP labelled wild-type and WldAll animal experiments were carried out under appropriate German licences: Tierschutzgenehmigung K 13, 11/00 and Anzeige K30/99.MPC (corresponding author) and BB jointly conceived of the design for this study, interpreted the experimental results and wrote the manuscript; BB carried out preliminary light and electron microscopy experiments, confocal imaging of YFP labelled axons, axon quantification and statistical analysis; RA: collaborated on the set up of the experiments, improvement of histological techniques and interpretation of results, performed teased fibre experiments, assisted in writing the manuscript; DW and DSG: provided excellent technical assistance in many parts of the experiments, performed sciatic nerve cut lesions and carried out routine work; KA: contributed to the discussion of the experimental results, preliminary light and electron microscopy work was done in his lab; RRR: contributed to the interpretation of the experiments and writing of the manuscript; confocal microscopy of YFP axons was mostly done in his lab; MPC: performed sciatic nerve crush lesions and partially confocal imaging, supervised all aspects of the study and main work was done in his lab.All authors read and approved the final manuscript.Many nerve fibres are unbroken directly after a 30 second crush as shown in YFP-H wholemount nerves and osmificated teased fibre preparations.A-C: Sciatic nerves crushed for 30 seconds at maximum pressure and then immediately fixed and imaged by osmium staining  or YFP fluorescence (C). D: YFP-H nerve crushed for 5 seconds at maximum pressure. Nerves in (A) and (B) were partially teased apart after staining, leading to accidental breakage of a few fibres. However, the majority of fibres clearly cross the crush site unbroken. YFP signal (identifying the axoplasm) at the crush site in (C-D) is weak, probably due to squeezing of the axoplasm longitudinally out of the crush site, or quenching of fluorescence by the crushed tissue, or both. To compensate, the photographs are deliberately overexposed. There is no sign of YFP positive axoplasm escaping laterally into the extracellular space, as would be expected if the axolemma were broken. Instead, some YFP positive axons clearly cross the crush site unbroken. In (C) the overexposure would prevent any broken axons from being identified, but in (D) the signal from most or all axons fades gradually as the axon enters the crush site, rather than stopping abruptly as one would expect if the axon were broken. Scale bars: 20 μm  and 50 μm .Click here for fileS axons proceeds anterogradely beginning with the formation of end bulbs and axonal swellings at the most proximal end and accompanying proximal axonal atrophy.Degeneration of individual crushed Wld Confocal composite picture showing seven consecutive lengths (from top to bottom in overview) of the proximo-distal course of an individual YFP labelled WldS axon within a peripheral triple heterozygote nerve stump 15 days after crush injury displaying an anterograde gradient of axon degeneration. This axon exhibits an end bulb at the most proximal end (red arrow in overview) and subsequent multiple axonal swellings delimited by constrictions (red asterisks) (inset 1). Further distally these swellings disappear with remaining constrictions (red asterisks) and sporadic breaks (white arrow) (inset 2). Distal parts of the crushed WldS axon are free of degeneration signs (inset 3 and 4). Scale bar: YFP fluorescence has been pseudo-coloured yellow with the applied confocal imaging software (Biorad LaserSharp 2000). 500 μmClick here for fileS axons finally includes complete proximal fragmentation.Anterograde degeneration with formation of massive end bulbs of compressed Wld Confocal composite picture showing six consecutive lengths (from top to bottom in overview) of the proximo-distal course of an individual YFP labelled WldS axon within a peripheral triple heterozygote nerve stump 20 days after crush injury demonstrating a clearer anterograde progression of axon degeneration than in Add Fig 2.pdf. Likewise after 15 days following crush injury this picture shows a massive end bulb at the most proximal end of the distal stump (red arrow in overview) and subsequent multiple axonal swellings and constrictions. Complete axonal fragmentation is evident in inset 1  and gradually gives way to incomplete breakup with axonal constrictions (red asterisks) (inset 2). Distal parts of the crushed WldS axon lack degeneration signs (inset 3 and 4). YFP fluorescence has been pseudo-coloured yellow with the applied confocal imaging software (Biorad LaserSharp 2000). Scale bar: 500 μmClick here for file


RNA-mediated gene silencing has emerged in recent years as an important mechanism for regulating gene expression. Some of the key discoveries have been made in plants Although the word ‘revolution’ should not be used lightly in science, there is no other way to describe the recent explosion in our awareness and understanding of RNA-mediated gene silencing pathways. The central player in RNA-mediated gene silencing is a double-stranded RNA (dsRNA) that is chopped into tiny RNAs by the enzyme Dicer. The tiny RNAs associate with various silencing effector complexes and attach to homologous target sequences (RNA or DNA) by basepairing. Depending on the protein composition of the effector complex and the nature of the target sequence, the outcome can be either mRNA degradation, translational repression, or genome modification, all of which silence gene expression . PresentCaenorhabditis elegans . Unexpectedly, the first transgene complex, which was stably active on its own, often became silenced in the presence of the second . The proNeurospora crassa , sequence-specific DNA methylation that triggered TGS, and RNA-directed DNA methylation. They had also proposed models for PTGS involving dsRNA , small RNeurospora identified common components in the respective silencing pathways (RNAi was discovered in experiments designed to compare the silencing activity of single-stranded RNAs (ssRNAs) (antisense or sense) with their dsRNA hybrid. While only marginal silencing of a target gene was achieved after injecting worms with the individual strands, injection of a sense–antisense mixture resulted in potent and specific silencing . This unpathways . This inpathways . PTGS isDrosophila systems demonstrated that 21–23 nucleotide ‘short interfering'RNAs (siRNAs), derived from cutting longer dsRNA, can guide mRNA cleavage  RNAs corresponding to silenced target genes in transgenic plants . They prcleavage ; identifcleavage ; and idecleavage   moves from cell to cell and through the vascular system to induce silencing at distant sites, was initially detected in plants in 1997  and late in 1998 . One of  in 1998 . A recen in 1998 .Arabidopsis has four Dicer-like (DCL) proteins, in contrast to mammals and worms, whose genomes encode only one Dicer protein (Arabidopsis to include at least three active members. An important goal has been to determine the functions of individual family members. Previous studies in Arabidopsis have shown that DCL1 is needed for processing miRNA precursors important for plant development (PLoS Biology delineates distinct functions for DCL2, DCL3, and RDR2. Nuclear-localized DCL3 acts with RDR2 to generate short RNAs that elicit DNA and histone modifications; DCL2 produces short RNAs active in antiviral defense in the cytoplasm of cells. This study illustrates nicely how RNA silencing components have diversified in plants to carry out specialized functions.Although RNA-mediated gene silencing pathways are evolutionarily conserved, there are various elaborations in different organisms. For example, the plant  protein . The RDRelopment , but notelopment . The papBy identifying small RNAs as agents of gene silencing that act at multiple levels throughout the cell, molecular biologists have created a new paradigm for eukaryotic gene regulation. Plant scientists have figured prominently in RNA-mediated silencing research. Instrumental to their success was the early ability to produce large numbers of transgenic plants, which displayed a rich variety of gene silencing phenomena that were amenable to analysis. The agricultural biotechnology industry provided incentives to find ways to stabilize transgene expression and use transgenic approaches to modulate plant gene expression and to genetically engineer virus resistance. As exemplified by the petunia cosuppression experiments, nonessential plant pigments provide conspicuous visual markers that vividly reveal gene silencing. The history of gene silencing research shows once again that plants offer outstanding experimental systems for elucidating general biological principles.


Bombyx mori to produce textiles. In contrast, despite the more promising properties of their silk, spiders have not been domesticated for large-scale or even industrial applications, since farming the spiders is not commercially viable due to their highly territorial and cannibalistic nature. Before spider silks can be copied or mimicked, not only the sequence of the underlying proteins but also their functions have to be resolved. Several attempts to recombinantly produce spider silks or spider silk mimics in various expression hosts have been reported previously. A new protein engineering approach, which combines synthetic repetitive silk sequences with authentic silk domains, reveals proteins that closely resemble silk proteins and that can be produced at high yields, which provides a basis for cost-efficient large scale production of spider silk-like proteins.Since thousands of years humans have utilized insect silks for their own benefit and comfort. The most famous example is the use of reeled silkworm silk from  Spiders and silks always go together. Currently there are over 34,000 described species of spiders, all of which have a varied tool kit of task-specific silks with divergent mechanical properties -6. Altho9 N/m2) coupled with a reasonable viscoelasticity . Spiders use dragline silk as a strong yet flexible structural element in the web, providing a framework to which other silks are attached, and as a life line when a spider is dropping off to escape an enemy. Minor ampullate (MI) silk, used for structural reinforcement in construction of the web, has a similar high tensile strength in comparison to major ampullate silk but has little elasticity .Araneus diadematus were chosen as templates for the synthetic constructs. A seamless cloning strategy . Th. Th36]. strategy  allowed gg (sense) and cc (antisense) were used for connecting two modules . Glycine is naturally abundant in spider silk proteins (~30%), therefore modules could be designed to match authentic amino acid sequences. Since the arrangement of the cloning cassette's elements remained unchanged upon cloning, repeat units could be multimerized to generate synthetic genes coding for the repetitive proteins (rep-proteins) (AQ)12 and (QAQ)8 . Therefore, the established bacterial expression system provides the basis for cost-efficient large scale production of spider silk-like proteins.A-values [Sf9 cells  used for producing ADF-4 corresponds to the pH in the spinning dope prior to silk thread assembly [Sf9 cells [adf-4 expressing cells revealed filaments that coiled throughout the cytoplasm, whereby most of the cells contained only one or few filaments. In contrast, Sf9 cells infected with control viruses or the adf-3 encoding virus never produced such filaments. The diameter of the ADF-4 filaments (0.2 – 1.5 μm) was in the range of native dragline silks (1.0 – 4.0 μm), but length of the filaments formed in the Sf9 cells seemed to be constrained by the volume of the cells, making them too short for mechanical force measurements. Strikingly, the purified ADF-4 filaments  in the dope without apparent aggregation or assembly. However, spider silk proteins can rapidly assemble into highly stable fibers when needed. The determination of solubility and self-assembly of recombinant spider silk proteins is therefore important to create commercially available silk fibers. For instance, pH-shifts are involved in natural silk assembly, but the exact function of acidification during spider silk assembly has not yet been determined. It seems likely that negatively charged groups are protonated reducing the net charge of spider silk proteins. Phosphoryl groups of phosphorylated amino acid residues, which have been detected in dragline silk , displayA-values  near theA-values . Interes% w/v in s Figure  showed aPhosphate, like other lyotropic ions, is known to increase the surface tension of water, promoting hydrophobic interactions . In the in vitro several aspects have to be taken into account to gain materials with expected properties.A remaining critical step concerning commercial production of silk fibers is the successful spinning of recombinant proteins into fibers resembling the natural silks in their microstructure and in their mechanical properties, which are outstanding by any measure. Besides the chemical parameters discussed above, several mechanical parameters play important roles in generating silk. To draw silk under natural spinning conditions, spiders attach their dragline to an object with glue from the piriform glands, before drawing the silk out by moving away or by descending and using their weight to draw the silk. It is common practice to take advantage of the drawing process by the forced silking of captive animals to collect silk for experiments. Analysis of the differences between naturally and forcibly spun dragline silk provided evidence for discrepancies in their material properties ,68,69. FBombyx mori [Nephila clavipes was wet-spun [Nephila fibers [Several attempts are reported in the literature and even more have been performed to wet-spin recombinant spider silk proteins. In a first attempt, microfabricated spinnerets were constructed using silicon microfabrication methods -73. Thesbyx mori , before wet-spun . The diawet-spun . Other awet-spun ,74. Theswet-spun . Howevera fibers , but lowa fibers .The future objective might not be to prepare identical copies of natural silk fibers, but rather to capture its key structural and functional features in designs that could be useful for engineering applications Figure . Using "Since the physical and chemical properties of bio-polymers and their assembly processes depend on the amino acid composition of the underlying polypeptide, engineering "synthetic" proteins with specific structural features will create a new class of fibrous proteins. However, to design new biomaterials based on spider silk, all properties of the underlying proteins have to be analyzed and in the best case successfully mimicked . Therefo


RescueMu system. The study provides a large resource of tagged and sequenced maize alleles, as well as some insights into the biology of RescueMu.The authors describe a large-scale transposon-tagging study in maize using the  Mu1 transposon, RescueMu provides strategies for maize gene discovery and mutant phenotypic analysis. 9.92 Mb of gene-enriched sequences next to RescueMu insertion sites were co-assembled with expressed sequence tags and analyzed. Multiple plasmid recoveries identified probable germinal insertions and screening of RescueMu plasmid libraries identified plants containing probable germinal insertions. Although frequently recovered parental insertions and insertion hotspots reduce the efficiency of gene discovery per plasmid, RescueMu targets a large variety of genes and produces knockout mutants.Derived from the maize  MuDR/Mu transposable elements are widely used for mutagenesis and as tags for gene cloning in maize [Mu insertional mutagenesis regulated by MuDR in highly active Mutator lines reflects four features of this transposon family. First, a plant typically has 10-50 copies of the mobile Mu elements [Mu insertion alleles in targeted screens [Mu-induced mutations for a chosen target gene is 10-3-10-5 [bronze1 exon [glossy8 [Mu insertion in specific regions, which may explain the higher frequency of mutable allele recovery for these genes.in maize ,2. The helements , althoug screens -6. In di0-3-10-5 . Interesze1 exon  and the [glossy8  contain Mu elements and the regulatory MuDR element become hypermethylated [Mu element mobility.Somatic mutability, visualized as revertant sectors on a mutant background, is indicative of transposon mobility. By monitoring maintenance of a mutable phenotype, it was established that the Mutator transposon system is subject to abrupt epigenetic silencing, which affects some individuals in most families ,11. A mothylated ,13. WithMu mutagenesis has been exploited in several reverse genetics strategies. The first protocol described used PCR to screen plant DNA samples to find Mu insertions into specific genes using one primer reading out from the conserved Mu terminal inverted repeats (TIRs) and a gene-specific primer [Mu insertions yields a list of tagged genes in each plant [RescueMu, a Mu1 element containing a pBluescript plasmid, to conduct plasmid rescue by transformation of Escherichia coli with total maize DNA samples. To identify insertions in genes of interest, RescueMu plasmids can be screened or the contiguous host genomic DNA can be sequenced using primers permitting selective sequencing from the right or left TIRs of Mu1 [The high efficiency of c primer -17. Altech plant ,19. A ths of Mu1 .RescueMu tagging effort conducted by the Maize Gene Discovery Project. The tagging strategy employed grids of up to 2,304 plants organized into 48 rows and 48 columns. Plasmid rescue was undertaken from individual pools of up to 48 plants per row or column. Genomic sequences next to RescueMu insertion sites were obtained for all the rows and for a subset of columns of six grids. Maize genomic sequences were subsequently assembled into 14,887 unique genomic loci using computational approaches. These loci were analyzed for gene content, the presence of repetitive DNA and correspondence to mapped maize genes and ESTs. Gene models were built by co-assembling the genomic sequence with ESTs and cDNAs by spliced alignment and by ab initio gene prediction. Identified gene models were tentatively classified using gene ontology terms of potential homologs [Here we describe the initial results of a large scale homologs .Mu element behavior have been examined previously using hundreds of tagged alleles or by analyzing the population of Mu elements in particular plants and a few descendants. With single founder individuals for the analyzed tagging grids, we could examine the distribution of new insertion sites of RescueMu in large progeny sets. The contiguous genomic sequences were analyzed to determine if there were insertion hotspots, preferential insertion site motifs, routine generation of the expected 9-base-pair (bp) direct target sequence duplication (TSD) and evidence of pre-meiotic insertion events.Many features of Mu elements, RescueMu exhibits a strong bias for insertion into or near genes, as few insertions were recovered in retrotransposons or other repetitive DNA. In addition, for the set of RescueMu insertions into confirmed genes, a bias for insertions into exons (rather than introns) was observed, consistent with the well-established use of Mutator as a mutagen. The gene-enrichment exhibited by RescueMu was compared against two physical methods of gene enrichment, methyl filtration [0t Cgenome fractionation [Like other ltration  and highionation .Mu1 elements maintain copy number through successive outcrosses, indicating that some type of duplicative transposition occurs [Mu insertion events .In standard Mutator lines, n occurs  in the an occurs . Most nen occurs ,27. Consd callus . Using etrRescueMu. By DNA blot hybridization of individuals within grids B through E, the RescueMu transposition frequencies ranged from 0.1 to 0.26 (Table trRescueMu were identified that had inserted into likely maize genes and generated the diagnostic 9-bp TSD characteristic of Mu transposition (data not shown). There were also events initially scored as transposition by blot hybridization that represented RescueMu rearrangements within the transgene array, and deleted forms of RescueMu were detected by blot hybridization and gel electrophoretic sizing of rescued plasmids (data not shown). Although RescueMu insertion frequency was low, overall Mu movement was very high in these grids; visible, independent seedling mutations were identified in 10.1-28.3% of the selfed progeny . Four plants each with a single trRescueMu were identified by these criteria and crossed to r-g. A DNA blot hybridization screen was conducted on 393 progeny of these four individuals. Seven progeny were identified with two new trRescueMu, seven plants were identified with three events, and 33 plants had a single trRescueMu; the original, parental trRescueMu elements were shown to segregate as Mendelian factors in the populations screened (data not shown). The 14 plants with two or three new trRescueMu were each crossed by an anthocyanin tester and also crossed multiple times as pollen parents to tester lines to generate sufficient progeny to construct one grid from each founder plant. Inexplicably, in sampling seedling progeny from each outcross ear, some lineages had very few new trRescueMu. The lines with the highest transposition frequencies had two trRescueMu and were used in grids G through J; DNA blot hybridization analysis of 30-200 grid plants was used to estimate transposition frequencies within each grid, which ranged from 0.38 to 0.66 . The resulting library plates contain 56-96 individual row and column libraries representing the diversity of germinal trRescueMu and a sampling of somatic events present in the harvested leaf tissue (each well in a library plate is a pool of 20-48 plants from a row or column). The parental RescueMu insertion sites inherited from the grid founder(s) are present in every library.As shown schematically in Figure RescueMu shows a strong bias for insertion into genes [trRescueMu insertion sites , with a range of 0.4-15 kb (data not shown). To accommodate the large size of some plasmids, a PCR template preparation protocol was devised to amplify genomic inserts of up to 16 kb for high-throughput sequencing [RescueMu insertion sites. Plasmids from all rows plus several columns of a grid were sequenced, with a routine yield of 80-92% success. A subset of plasmids could not be bidirectionally sequenced, because they lacked the TIRs at one or both ends. Deleted forms of trRescueMu were detected in several percent of the individuals surveyed by DNA blot hybridization  for each grid, and 59%  were recovered multiple times . In addition, a total of 24,875 parental plasmids were transmitted from the founder plants. The percentage of parental plasmids within each grid varied from 17% for grid G to 61% for grid P. Some grids had more parentals than other grids and some parental plasmids were preferentially sequenced for unknown reasons. The parental insertion sites include the two or three known parental sites that each segregated into 50% of the progeny. Somatic sectors in the tassel or ear of the parental plant that generated plasmids found in multiple individuals within the grid are analyzed in a later section.Previous analysis of ecovered . Out of trRescueMu, equivalent to a frequency of 0.68 new insertions per plant. Using a Poisson model based on this transposition frequency for an individual grid ). By PCR screening of library plates containing the immortalized row and column plasmids, plants containing a specific insertion event can be verified .RescueMu insertions occur in diverse gene types. Illustrating the utility of Mu tagging, insertions are found in housekeeping genes, such as actin, as well as in regulatory genes such as those for transcription factors and protein kinases. Using the database of mapped maize genes and expressed sequence tags (ESTs) [RescueMu insertions are identified in genes on all 10 maize chromosomes [Mu insertions using DNA blot hybridization that established that these elements insert throughout the genome and do not show a measurable bias for insertion locally [RescueMu insertion sites that match maize ESTs correspond to genes of unknown function, suggesting the discovery of novel genes.As shown in Figure s (ESTs) , RescueMomosomes . These dRescueMu insertion sites identified in six grids (multiple insertions into a gene from the same grid being counted only once because the majority are the same insertion event), 88% represent single instances of transposon insertion locations. There were 596 instances of a specific genomic sequence having two or more RescueMu insertion events. If the maize genome contains 50,000 distinct genes that are targets of Mu insertional mutagenesis, then far fewer cases of duplicate recovery would be expected by chance alone, given the number of events analyzed (p < 0.001); therefore, RescueMu exhibits some preference for particular genes.Of the 14,887 RescueMu insertion within particular genes, data were compared between grids with independent founder individuals. As summarized in Table RescueMu insertion sites were found in just one grid. This was true for both probable germinal insertion events (plasmids found two or more times within a grid) as well as for singlet sites . The 10% of insertion sites found in two or more grids represent independent recovery of a RescueMu insertion into the same locus.To determine if there were 'hotspots' for et al. [Mu insertions at glossy8 were in the 5' untranslated region, with 15 insertions at the same base; similarly, the beginning of exon 2 within bronze1 is the most frequent site of Mu insertion in that gene [In addition to the computational comparison in which an overlap of 50 bases (95% identity) was scored as insertion into the same gene, over 730 insertion sites were examined manually for 250 cases of genes with insertions from more than one grid. Of these insertion sites, 80% were at different locations within the same locus; we found 85 cases of insertions within a 1-10 bp region and 67 cases of insertions at the same base. Previously, Dietrich RescueMu contig from the Genomic Survey Sequencing (GSS) section of GenBank, ZM_RM_GSStuc03-10-31.4765 [RescueMu insertion, with six plasmids sequenced from row 42 of grid G and one each from grids H, I, and M. Insertion sites were identical across the grids. Sequences generated to both the left and right of the RescueMu element were aligned as demonstrated in Figure RescueMu insertion site was recovered multiple times in grid G, a heritable insertion may exist. After PCR screening of grid G plasmid libraries, summarized in Figure RescueMu insertion site, total leaf DNA was extracted from selfed seed of this plant, namely G 42-22, obtained from the Maize Genetics Cooperation Stock Center. PCR screening of the DNA  or a single missing or additional base. Given that all sequences were single pass but of high average quality , we consider that 9-bp TSDs exist in virtually all trRescueMu insertion sites. A few cases showed anomalies in the TSDs, which probably reflect rearrangements near RescueMu.Because a 9-bp TSD is characteristic of  Figures , 3; notesequence ,35; thesMu based on smaller data sets [RescueMu insertion regions [et al. [RescueMu targeting is at least partially dependent on sequence features. In addition, we have compared the profiles derived independently from insertion sites within confirmed exons, introns and uncharacterized regions, respectively, and found the same base preferences in all three sets (data not shown).Several groups have reported weak consensus insertion site preferences for ata sets ,18,20. W regions . The pro [et al. , showingRescueMu insertion sites that do not match an EST or gene in another species represent introns or other non-coding genic regions. On the basis of the gene structure annotated by maize EST matching, we have located 968 TSD sites within genes. Of these, 849 are inside exons. To check if RescueMu has a preference for insertion into exons , a standard binomial test with normal approximation was performed. On the basis of the matching to ESTs, the lengths of all exons and introns observed from all RescueMu contigs were counted as 2,182,954 bp and 439,403 bp, respectively. Assuming that RescueMu does not have a preference to insert into exons (null hypothesis), the probability of observing an exon insertion event is proportional to the length of exons . The probability of observing at least 849 exon insertion events was calculated . This result suggests that RescueMu has some preference to target exon regions within genes.Of 14,887 genomic loci, 62% matched maize or other plant EST/cDNAs. As more genomic sequence becomes available that can be assembled with ESTs to annotate the non-coding portions of maize genes, it will be interesting to determine if the RescueMu GSS sequences were scanned and masked for repetitive elements as collected in The Institute for Genomic Research (TIGR) Cereal Repeat Database [0t Cselection (HC) using the same repeat-masking criteria [RescueMu, MF and HC, respectively.As outlined in Materials and methods, the Database . The repcriteria . The perRescueMu shows similar repeat content as the physical enrichment methods. However, after we assembled the RescueMu GSS sequences to remove redundancy, only about 3% of the RescueMu loci are composed of repetitive DNA  from TIGR [RescueMu and HC have similar bias against repetitive DNA, superior to the MF bias. It should be noted, however, that the MF and HC GSS sequencing has generated, on average, much longer contigs than RescueMu .Therefore, on the nucleotide level, rom TIGR  and applRescueMu insertions were found in either the approximately 10,000 copies of the 9.1 kb 28S + 18S rRNA genes [In addition, only 0.4% of the NA genes  comprisiRescueMu loci match strongly to maize or other plant ESTs or appear to encode proteins with high similarity to known proteins. In addition, about another 5% of the loci were predicted to be genic regions with high stringency by ab initio gene prediction programs. As a control, we matched ESTs to contigs assembled from unfiltered (random) maize GSS sequences [RescueMu contigs contain more than threefold enrichment of genic regions than random sequencing. This is consistent with our expectation that RescueMu preferentially inserts into genes. It is worth pointing out that plant EST collections contain ESTs from repetitive elements. Although we masked contigs using the annotated TIGR repeat database [RescueMu results, because these authors used much higher stringency for the EST spliced alignments with the BLAT program [RescueMu data, which tolerates less sequence matching without compromising gene structure prediction accuracy [RescueMu, MF, and HC are very similar (data not shown).Also shown in Table equences . From abdatabase , it is pdatabase . However program , requiriaccuracy . The reset al. [RescueMu by comparing the respective sequence sets to rice gene models. They concluded that unique gene discovery is most efficient with MF at a sequencing depth when EST sampling saturates. However, their reported low gene discovery rate for RescueMu does not reflect the RescueMu insertion bias, because their dataset included all sequences deposited in GenBank. That is, they did not remove the redundancy resulting from multiple sequencing of parental insertions.Palmer et al.  evaluateRescueMu insertion sites were found in two or more row libraries  from female transmission. Given that the founder plants produced copious pollen, there is a low probability that two grains carrying the same meiotic insertion will both result in seed; therefore, the same RescueMu insertion site found in two rows should usually be from a pre-meiotic transposition event. For all events found in three or more rows, the insertion event must be pre-meiotic.To calculate the extent and timing of pre-meiotic sectors, the sequenced plasmids from grids G, H, I, K, M and P were classified as occurring in a single row or in multiple rows. The development of the tassel and ear must be considered when evaluating these data. An insertion event that occurs during meiosis can be represented in two haploid cells. During microgametophyte (haploid plant) ontogeny, both of these cells survive, resulting in two pollen grains with the same event. In contrast, only one megagametophyte develops after megaspore meiosis; therefore, female meiotic and subsequent events in the haploid megagametophyte are always represented in just one progeny plant. Most grid plants resulted from male transmission of RescueMu insertion event very early in the somatic development of the inflorescence or in the apical meristems. The majority of trRescueMu insertion sites are found in only one row . The nuRescueMu insertion sites. Only about 8% of the RescueMu insertion sites match repetitive elements and few insertions (0.4%) were recovered in genes transcribed by RNA polymerase I or III. These results suggest that a chromatin component associated with polymerase II transcription units or the absence of a structure in other classes of genes is important in targeting RescueMu and other Mu elements to maize genes. Similarly, recombination during meiosis and transcription per se is targeted to genes. It is likely that the parasitic Mu elements exploit an element of host gene packaging that evolved for other reasons to facilitate transposition into genes.Given that the maize genome comprises approximately 70% retrotransposons and other highly repetitive sequences, including around 10,000 copies of the rRNA genes , these cRescueMu is close to methyl filtration and high 0t Cfractionation. The probable germinal insertion class defines a collection of mutations of enormous potential for the phenotypic characterization of maize with specifically disrupted functions. However, the low cost of template production is a distinct advantage of both physical enrichment methods compared to the high cost of designing, sampling and self-pollinating tagging grids. Current levels of sample sequencing from the physical enrichment templates highlight the desired redundancy of the RescueMu method, which is important for distinguishing somatic from germinal insertions at individual loci. The physical enrichment methods are considerably below one times coverage of the transcriptome of around 250 Mb; hence the current efficiency of generating novel sequence (the likelihood that the next clone sequenced is new) is much higher with these methods than with RescueMu.The biological specificity for maize genes exhibited by RescueMu insertion site data, several parameters of Mu transposition behavior were investigated. We confirm that a 9-bp TSD is characteristic of virtually all Mu insertion sites. We confirm that a small percentage of trRescueMu suffer deletions, including loss of a TIR, as noted in previous studies of Mu1 [Mu insertion sites [RescueMu insertion sites is consistent with the previously proposed motifs. A bias exists for G+C-rich sequence, reflecting the composition of maize exons. We confirm that there are hotspots for Mu insertion, identified by finding identical trRescueMu insertion sites in independent grids. A few loci were recovered in four or more of the six grids analyzed, and many more in two  or three (233 genes) grids. There is no strong DNA consensus motif at these hotspots, and we consider it more likely that a specific DNA structure or a protein associated with genes establishes conditions for efficient Mu insertion at particular sites. It is important to note that active transcription is not a requirement for Mu element insertion; otherwise Mu would preferentially insert into genes active late in floral development and in gametophytes.Using the s of Mu1 . Throughon sites , consenstrRescueMu insertion sites represent a mixture of non-heritable somatic insertions present in leaves, germinal insertions in single grid individuals, insertion events in pre-germinal sectors within flowers, and parental elements. Parental elements identified in a grid founder plant segregated 1:1 in the progeny as expected. In addition, some insertion events were found in three or more grid rows, and hence in three or more individuals, and must be pre-meiotic transposition events in the founder. This class represented 10.2%  of all the likely germinal insertions identified  defining predictable insertion locations. RescueMu properties confirm attributes established with smaller populations of standard Mu elements.A public resource of transposon-tagged maize alleles was constructed and evaluated. RescueMu contains all of Mu1 plus a 400-bp segment of Sinorhizobium meliloti and pBluescript (Stratagene), as described previously by Raizada et al. [RescueMu was obtained in this study using PCR primers to amplify overlapping sections of the element [RescueMu transposon was placed in the 5' untranslated region of a 35S:Lc expression plasmid where it blocked expression [Lc is a member of the R family of transcriptional regulators of the anthocyanin pathway [r-r/r-g, A1, Bz1, Bz2) hybrid background were crossed to r-g testers and subsequently with r-g Mutator lines containing multiple copies of MuDR to visualize RescueMu somatic excision as red anthocyanin sectors in an otherwise white aleurone. The tagging populations used here were developed by screening for transposition of RescueMu from the original, complex transgene arrays to diverse genomic locations. Using DNA blot hybridization, these once-transposed RescueMu (trRescueMu) were closely monitored for subsequent transposition, and lines were monitored for Mu1 and/or MuDR methylation in the TIRs, a sign of incipient Mutator silencing. Details of line development and evaluation, including DNA blot hybridization methods, will be presented elsewhere. The anthocyanin tester lines  were in inbreds W23, K55, A188, or hybrid combinations of these lines. Some RescueMu lines used in tagging grids were crossed to inbreds A619 or B73, which are both r-g, A1, Bz1, Bz2. Grid backgrounds are presented in detail at [a et al. . The com element  for bidipression . Lc is a pathway . Transgeetail at .BamHI plus BglII digestion of the genomic DNA preparations. These libraries were immortalized in library plates available from the project [RescueMu; although the primers were selective for one TIR, there was some cross-priming. All grid rows plus several columns were sequenced. Three 96-well plates were normally sequenced for each row or column to obtain sequence information for a desired minimum of 200 plasmids; additional sequencing reactions were conducted if necessary. Matches of row and column sequences are designated as probable germinal insertions, because they represent an insertion site present in two leaves of that plant (designated by its row and column address); when only row sequences were available from a particular plasmid, probable germinal insertions were designated after recovery of the same sequence two or more times. Plasmid types recovered just once are a mixture of heritable and strictly somatic insertions. Parental RescueMu insertion sites present in a grid founder plant segregated in the grid progeny, and these insertion sites were expected to be found in all rows and columns. In some cases, particular parental plasmids were over-represented in the sequenced plasmid population. To reduce their contribution and increase recovery of new insertion sites, a rare-cutting restriction enzyme site was identified in the parental plasmid and the corresponding enzyme was included in the genomic DNA preparation to bias against recovery of that parental plasmid.Detailed protocols are presented at , and a s project . Plated RescueMu insertion hotspot Six gene primers plus one RescueMu left readout primer were used in this study:PCR screening of a library plate to quantify a 1. 5'-TTGGGAGGTTGAAGGTAAAGACAT-3'2. 5'-GTGCTG GATTGGTTACTCCG-3'3. 5'-CGATGATTCTAGTTGAGCGTCTG-3'4. 5'-ACTCGCACCAACATGAATACC-3'5. 5'-GTTTCCGAGGACGCAGAGG-3'6. 5'-AGCGCCAGGGCCAGGGGATTC-3'RescueMu)L. 5'-CAT TTC GTC GAA TCC CCT TCC-3' , AGATCT (BglII), or a GGATCT (BglII/BamHI) or AGATCC (BamHI/BglII) motif. The two restriction digestion sites represent a true ligation site of sequence that was non-contiguous in the maize genome, but the post-restriction site sequences can unambiguously be assigned to the right or the left of RescueMu. On the other hand, the GGATCT or AGATCC motif could be contiguous genomic sequence or could have been generated during the ligation step of the plasmid rescue. Consequently, assignment of the position of the sequence beyond the GGATCT or AGATCC motif is ambiguous. If the RescueMu insertion site matched EST sequence across and beyond the GGATCT or AGATCC motif, the post-ligation sequence could be properly assigned  per plasmid.As shown in Figure nsertion , the seq GenBank , for com program  to deterRescueMu GSS sequences deposited at GenBank were screened for vector sequences against the UniVec database at the National Center for Biotechnology Information (NCBI) [RescueMu-transformed plant contains the parental RescueMu elements that were recovered at a high frequency during sequencing (from every sequenced row or column). Because our goal is to analyze the gene discovery by newly inserted RescueMu , we decided to filter out the parental sequences as much as possible. We used Vmatch to cluster near-identical left and right sequences for each grid. A parental cluster contains sequences from nearly all the row or column sequences. A total of 59,069 parental sequences were identified and were excluded from the subsequent assembly. All the non-parental sequences were first preassembled for each plasmid using the left and right 9-bp TSD overlap. The merged GSSs were first clustered by PaCE [RescueMu insertion sites. For the particular case where TSDs were not recovered during sequencing, the left and right sequences could not be assembled together, even though they were from the same plasmid. Therefore, a Perl script was developed to conduct single-linkage clustering based on clone-pair constraints to assemble the GSS to the same 'genomic loci' if they were derived from the same plasmid clone.The 131,364 n (NCBI)  using thn (NCBI)  using thn (NCBI)  with the by PaCE   was used in combination with the TIGR cereal repeat database . The contigs were also scanned from tRNA genes by tRANscan-SE program [To be successful as a gene-discovery tool, the transposon insertions must be predominantly into the genic regions of the maize genome. To quantify the potential enrichment of the  program  with itsab initio approaches have been used to detect gene structures of the GSS contigs. For the similarity-based approach, GeneSeqer [ab initio prediction, GENSCAN [Both similarity-based and eneSeqer  programseneSeqer . For the GENSCAN   was derived from the sequences and their reverse-complement orientation determined using the Expectation Maximization Algorithm [For the analysis of lgorithm .The GSS seqeunces present in each tentative unique contig (TUCs) were extracted from  and assiRescueMu flanking sequences where established. Instructions on how to obtain seed of grid plants is also provided.Grid plants were self-pollinated unless male or female-sterile. The resulting F1 families were evaluated by inspection of ears and kernels, at weekly intervals for five weeks after germination in a sand bench in a greenhouse, and at weekly intervals throughout the life cycle in the field. Phenotypes observed were recorded and are assembled into a searchable database at . Unique RescueMu (Additional data file The following additional data are available with the online version of this article: a table listing the internal primers used in sequencing RescueMuA table listing the internal primers used in sequencing Click here for additional data fileSupplementary material for this paper, including details of methodsClick here for additional data file


Scalp-recorded electroencephalographic (EEG) signals produced by partial synchronization of cortical field activity mix locally synchronous electrical activities of many cortical areas. Analysis of event-related EEG signals typically assumes that poststimulus potentials emerge out of a flat baseline. Signals associated with a particular type of cognitive event are then assessed by averaging data from each scalp channel across trials, producing averaged event-related potentials (ERPs). ERP averaging, however, filters out much of the information about cortical dynamics available in the unaveraged data trials. Here, we studied the dynamics of cortical electrical activity while subjects detected and manually responded to visual targets, viewing signals retained in ERP averages not as responses of an otherwise silent system but as resulting from event-related alterations in ongoing EEG processes. We applied infomax independent component analysis to parse the dynamics of the unaveraged 31-channel EEG signals into maximally independent processes, then clustered the resulting processes across subjects by similarities in their scalp maps and activity power spectra, identifying nine classes of EEG processes with distinct spatial distributions and event-related dynamics. Coupled two-cycle postmotor theta bursts followed button presses in frontal midline and somatomotor clusters, while the broad postmotor “P300” positivity summed distinct contributions from several classes of frontal, parietal, and occipital processes. The observed event-related changes in local field activities, within and between cortical areas, may serve to modulate the strength of spike-based communication between cortical areas to update attention, expectancy, memory, and motor preparation during and after target recognition and speeded responding. A new analysis considers EEG signals, not as average responses, but as individual event-related perturbations in ongoing dynamical brain processes The waking brain updates and fulfills intentions through brain processes that operate within and across multiple brain areas to integrate perception, association, and action. Fulfillment of intentions is facilitated by features of these and other processes that support informed anticipation of and selective attention to events and their probable consequences. The dynamics of ongoing electroencephalographic (EEG) activity recorded from the human scalp differ markedly with state of attention and intention , yet mosThe adequacy of time-domain ERP averaging for modeling macroscopic brain dynamics also depends on the assumption that the cortical sources of EEG activity contributing to and not contributing to average ERP waveforms are somehow distinct. The scalp topographies of unaveraged EEG and averaged ERP data may, however, be quite similar , stronglThe above considerations suggest that event-related EEG dynamics may be better modeled as coordinated event-related perturbations in the statistics of multiple, intermittently active local field processes. Since the volume-conducted scalp projections of such processes generally overlap, they cannot be separately identified in the scalp-recorded data. An alternate approach we adopt here is to separate the contributions of local field processes by using distinct differences in their time courses .Following stimuli belonging to an anticipated but infrequently presented category, the averaged ERP is dominated by a broad vertex-positive peak often called P300 after its earliest appearance in auditory responses . ResultsThe independent component analysis (ICA) method provides a complete decomposition of single-trial (or continuous) EEG data, separating the data into distinct information sources. As the results presented below show, the amount of information about cortical dynamics provided by this method is large. Here, we detail for the first time dynamics occurring within single trials of the classes of maximally independent EEG processes whose event-related activities contribute to 31-channel visual target responses recorded during a test of spatial selective visual attention .To orient readers used to analyses of the raw scalp-channel data, we first present conventional ERP results at selected channels. As we previously reported, in these experiments performance level was high; more than 95% of targets were followed by a button press within the allotted response window (150–1000 ms). Mean subject-median reaction time (RT) was 352 ms. The average ERP time locked to onset of the target stimulus followed by a subject button press contained the expected late positive complex or P300 positivity following early stimulus-locked peaks conventionally termed P1, N1, P2, and N2 A. The scIn the grand average of the same epochs, each time-locked to the subject response B, the eaAt frontocentral channel Fz, however, the late positivity in the stimulus-locked grand average C, bottomp < 0.01) for 14 of the 15 subjects even after the subject-mean ERP was subtracted from each trial (data not shown).A concurrent but weaker theta power increase near 6 Hz B was maxConventionally, characterizing the sources of ERP  or eventWhile component IC1 accounted for the largest part of the P300 peak in the upper trial, in the lower trial the same IC1 process showed mixed low alpha and beta activity with a smaller postmotor response positivity. Note that the positive postresponse contribution of IC1 in this trial (thin blue trace) was sometimes larger than the observed positivity in the whole-channel data (thick black trace). At these times, some of the other 24 components contributed negative potentials to the signal at this scalp channel, partially canceling the IC1 positivity in the recorded data. Thus ICA, applied to the continuous or concatenated single-trial data, may actually recover more of the actual projected signals than are available in the single-channel data.Cluster analysis, applied to the normalized scalp topographies and power spectra of all 465 components from the 15 subjects see , identifThe location of the equivalent dipole for a radially oriented cortical source patch  is typically deeper than the cortical patch itself . The equThe spatial standard deviations of the other component cluster dipoles were smaller. Their equivalent dipoles indicate the respective dominant cortical regions of their source domains. Though the mean cluster scalp map for the central occipital alpha cluster (Cα) could be fit satisfactorily by a single equivalent dipole located in the central occiput, for several of the cluster components a better model of the component scalp map was obtained from a symmetric dipole pair in left and right pericalcarine cortex (data not shown).Mean dynamics properties of nine nonartifact component clusters are summarized in Two component clusters made distinct contributions to the late positive complex of the target ERP. After subtracting the larger back-projected scalp-data contributions of components accounting for blinks and saccadic eye movements, the response-locked ERP at both periocular channels contained a broad, approximately 2-μV positive-going scalp potential peaking on average 39 ms before the recorded button press. The P3f cluster-mean response-locked positivity began near 150 ms, consistent with direct neurophysiological evidence that by 150 ms after stimulus onset, visual information is spread throughout the brain by a complex web of afferent and efferent connections . SubtracThe far-frontal (P3f) component cluster response appears similar to the 280-ms “P2a” peak noted in responses to  visual “oddball” stimuli by The significance of the stimulus-locked P300 (or P3b) peak over central parietal cortex has long been debated. Our results clearly show E that inIn the RT-sorted FM cluster ERP image C, the twThough the equivalent dipole locations of components in the two midline clusters were somewhat overlapping see , their mThe theta burst appears to resemble other reported FM EEG activity patterns: theta bursts or trains (fmθ) appearing during mental concentration  and brieThe scalp map of the CM cluster E resemblThe left and right mu rhythm component clusters . Onset of the stimulus-locked ERP of the P3b cluster at about the same time was soon followed by the far-frontal P3f cluster onset  poststimulus time windows following nontarget and target stimuli in these experiments . The majThe group mean equivalent dipole locations shown in Production of scalp EEG signals requires partial synchronies in local field activity extending across the relatively large (2-cm or more) domains of neuropile . These mTraditionally, averaging event-locked EEG data from single scalp channels to form an ERP is assumed to reject, by random phase cancellation, “background” EEG rhythms whose statistics are tacitly assumed to be unaffected by experimental events. To test the effect of the average ERP on the observed spectral perturbations, we computed the cluster ERSPs again after removing the component mean ERP from each trial (data not shown). All the effects shown in Note that many of the cluster ERSPs –9 share At and after the button press, a mean frontocentral theta power increase appeared in all 15 subjects' data. It was partially phase coherent in four of the component clusters and was not eliminated by removing the subject-mean ERP from each trial. Local bursts of theta-band activity are widely distributed on the human cortex  and assoFollowing nontarget stimuli in this experiment, FM components exhibited weak “theta ringing”  not accompanied by increased theta power . Here, fThe postmotor theta bursts seen here following correct speeded responses very likely are tightly linked to the ERP feature with strong theta-band energy that follows highly speeded manual (or foot) responses in the Erickson flanker task . Luu andIn this speeded response paradigm at least, the ERP P300 positivity was nearly strictly time-locked to and predominantly followed the motor response. The P300 positivity was, here, indeed a late positive complex of potentials generated in several brain areas, confirming results of invasive recording  and clinAs in our previous single-trial analysis of some of these data , partialThe central posterior alpha-ringing feature of the stimulus-locked ERP can be parsimoniously described as arising through alpha phase resetting. The P300/P3b feature, on the other hand, might better conform to the usual conception of an ERP as measuring evoked response activity added to ongoing EEG activity. Note in both induced and evoked aspects.The variety of these ERP features suggests that assuming a strict dichotomy between evoked and phase-reset activities is unproductive. Rather, each ERP feature may be usefully and better characterized as summing event-related perturbations of various sorts in the ongoing activities of one or more localized cortical EEG processes. To produce a reproducible peak or peaks in the average ERP, these perturbations should involve some degree of  phase locking of the contributing process activities to the time-locking events in one or more frequency regions. At very low (near-DC) frequencies, event-related “phase locking” implies event-related “sign locking.” For example, the P300 would never appear in ERP averages at all if it were negative-going in half the trials. Similarly, there need be no strong dichotomy between evoked phenomena that involve partial phase locking and so contribute to average ERPs, and induced phenomena that may involve changes in spectral amplitudes but do not show phase locking and thus do not contribute to average ERPs. Rather, it is important to realize that most event-related EEG dynamics have The results presented here confirm that extensive, complex, and flexible information concerning links between cognitive processes and macroscopic brain dynamics are available in noninvasive high-density EEG data. Availability of more comprehensive analysis techniques, such as that introduced here, should make EEG  data analysis of increasing interest both to cognitive neuroscientists and to neurophysiologists, as event-related EEG dynamic models complement observations of slow-changing hemodynamics while greatly expanding the restricted spatial information available from single- and multineuron spike recordings.ERPs were recorded from subjects who attended to randomized sequences of filled disks appearing briefly inside one of five empty squares that were constantly displayed 0.8 cm above a central fixation cross see  followinFifteen right-handed volunteers  with normal or corrected-to-normal vision participated in the experiment. Subjects were instructed to maintain fixation on the central cross while responding only to stimuli presented in the green-colored (attended) square. Subjects were required to press a thumb button held in their right hand as quickly as possible following stimuli presented in the attended location see . Thirty EEG data were collected from 29 scalp electrodes mounted in a standard electrode cap  at locations based on a modified International 10–20 System, and from two periocular electrodes placed below the right eye and at the left outer canthus. All channels were referenced to the right mastoid with input impedance less than 5kΩ. Data were sampled at 512 Hz with an analog pass band of 0.01–50 Hz. To further minimize line noise artifacts, responses were digitally low-pass filtered below 40 Hz prior to analysis. Trials containing electrooculographic potentials larger than 70 μV or amplifier blocking were rejected, and brain responses to stimuli presented at each location in each attention condition were stored separately. Responses to target stimuli were analyzed only when  subjects responded 150–1000 ms after target onset. The few targets followed by no such button press were not considered in the present analysis.unaveraged data. Systematic application of ICA to unaveraged data from these experiments was first demonstrated for non-target stimulus trials  ICA models of EEG activity are possible e.g., . It remaAs first demonstrated by simulations , when trhttp://sccn.ucsd.edu/eeglab) was applied to sets of 400 to 600 1-s trials  time-locked from −200 ms before to +800 ms after onsets of target stimuli presented at any of the five stimulus locations  component maps and mean activity log spectra from the 15 subjects were clustered by applying a modified Mahalanobis distance measure  to vectoTo examine stimulus- and response-induced changes in the EEG spectrum, we computed ERSP transforms  for eachp < 0.01. To construct between-subject mean ERSPs, we used binomial statistics to select a significance cutoff based on the minimum number of subjects required to have significant differences (in the same direction) from baseline at a given time/frequency point (p < 0.0001). ERSP transforms of the data were computed at each channel and then for each clustered data component. To test for partial phase locking  between EEG processes and the occurrence of experimental events across trials, we used intertrial phase coherence  of oherence .p < 0.01 (uncorrected) between pairs of independent components (from the same subject) included in each pair of independent component clusters (defined below). To exclude the possibility that the observed phase linkages arose only from common phase locking of the portion of the single-trial data constituting the ERP, we also subtracted the concurrent mean ERP from each trial before computing phase coherence. Functions to compute and plot the time/frequency measures used here are also available in the EEGLAB toolbox.To test the presence of nonrandom phase relationships (possibly including fixed delays) between activities in different  independent components, we performed event-related phase coherence analysis , again wBoth simple anatomic considerations and observed results of ICA decomposition suggest that cortical EEG sources may be usefully modeled as patches of cortex with partially synchronous local field activities. In brief, the high local coupling density of both excitatory and, particularly, inhibitory neurons in cortex means that local field potentials sufficiently synchronous to create measurable EEG signals should tend to extend through a compact spatial domain—roughly speaking, a patch of cortex of unspecified extent. Through volume conduction, partially synchronous activities within cortical source patches produce far-field potentials throughout the brain and on the scalp. The distribution of the scalp electrical field produced by such a source patch is nearly identical to that of a small dipolar potential element whose geometry is like a tiny flashlight battery oriented perpendicular to the cortical surface. This “battery” is termed the equivalent dipole for the cortical source.http://sccn.ucsd.edu/eeglab/dipfit.html). Some bilaterally symmetric component maps were better fit with symmetric dual-dipole models. The successful fits of single-dipole models for many of the clustered components is compatible with their generation within a single, compact patch of cortex, while bilateral dual-dipole models are compatible with tightly coupled oscillatory activity (without net phase delay) in two bilaterally symmetric cortical patches densely connected via corpus callosum or common subcortical drive.Here, we used a relatively simple and well-known method for fitting the positions and orientations of equivalent dipoles in a four-shell spherical head model for each component . To reduce the time required to process the 485 component maps, we used a version (3.0) of the BESA software that allowed batch processing to fit single-dipole models to each component scalp map. , normalized, and averaged. These cluster mean maps were then fit with single-dipole models to roughly illustrate the regional distinctions between the sources of the component clusters. Finally, the cluster-mean dipoles and event-related time/frequency information measured by ERSP, intertrial phase coherence,and event-related phase coherence analysis of all the single trials were visualized together in three dimensions using an animated display developed by


Perinatal hypoxia-ischemia remains the most common cause of acute neonatal brain injury and is associated with a high death rate and long-term neurological abnormalities such as memory and cognitive deficits and dyskinesia. Hypoxia-ischemia triggers an inflammatory cascade in the brain that is amplified by the activation of immune cells and the influx of peripheral immune cells into the brain parenchyma in response to cellular injury. Thus, acute cerebral hypoxic-ischemic inflammation is a major contributor to the pathogenesis of newborn hypoxic-ischemic brain injury. Lactate is a glycolysis end product that can regulate inflammation through histone lactylation, a unique posttranslational modification that was identified in recent studies. The purpose of this review is to outline the recent improvements in our understanding of microglia-mediated hypoxic-ischemic inflammation and to further discuss how histone lactylation regulates inflammation by affecting macrophage activation. These findings may suggest that epigenetic reprogramming-associated lactate input is linked to disease outcomes such as acute neonatal brain injury pathogenesis and the therapeutic effects of drugs and other strategies in relieving neonatal hypoxic-ischemic brain injury. Therefore, improving our knowledge of the reciprocal relationships between histone lactylation and inflammation could lead to the development of new immunomodulatory therapies for brain damage in newborns. Approximately four million newborns worldwide have died as a result of perinatal hypoxia. Hypoxic-ischemic encephalopathy (HIE) affects between 0.5 and 2/1000 full-term neonates in developed countries and is one of the most frequently documented causes of neonatal death and poor neurodevelopmental outcomes . PerinatInflammation has long been thought to play a role in the pathogenesis of cerebral HI injury . HypoxiaPrevious studies have reported that extracellular lactate can stimulate histone lactylation and that histone Kla (lysine lactylation) levels are increased by glucose (from which most intracellular lactate is derived) in a dose-dependent fashion . In accoLactate is a glycolytic product and a substantial energy source produced after hypoxia. However, glycolysis inhibitors can diminish lysine lactylation by depleting lactate, and mitochondrial inhibitors or hypoxia can amplify lysine lactylation by enhancing lactate production, indicating that both exogenous and endogenous lactate contribute to lysine lactylation . In addivia the TCA cycle as an energy supply is lower in the developing brain, glucose metabolism via the pentose phosphate pathway is enhanced in adults and provides materials necessary for nucleotide synthesis, as well as lipid biosynthesis and glutathione reduction  wrapped around the DNA double helix . PositivLactylation was proposed as a new histone acylation modification in 2019 . Mass spBoth the lysine acetyltransferase (KAT) enzyme P300 and lactyl-CoA are required for enzymatic lysine lactylation . Despitevia the thioesterase glyoxalase I (GLO1) to produce LGSH, which is hydrolyzed to glutathione and D-lactate by GLO2. Recently, studies have shown that transferring a nonenzymatic lactyl group from LGSH to protein lysine residues results in a “LactoylLys” alteration in proteins, which can be blocked by GLO2 and affects glycolytic activity (Lysine acylation has been demonstrated to result from a nonenzymatic reaction with lactoylglutathione (LGSH), in addition to the enzyme-dependent transfer of a lactyl group from lactyl-CoA to lysine residues . The glyactivity . Furtheractivity .Microglia are derived from a type of erythromyeloid progenitor , and conNeonatal microglia are slightly more active under normal conditions than adult microglia and express higher levels of MHC-II and costimulatory markers, including CD86 and CD40. Postnatal microglia also respond differently to macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in terms of proliferation and are less reliant on astrocytes for expansion than other cells . MicroglMicroglia are the first cells in the developing brain to respond to HI damage. They have a ramified shape and extend and retract their cellular processes to monitor the brain microenvironment continuously under healthy conditions. When microglia are activated, they transform into amoeboid-like cells with immunological functions, including the ability to produce cytokines and matrix metalloproteinases, phagocytic ability, and the ability to present antigens . ActivatMacrophages suppress inflammation by modifying tissue and eliminating cellular detritus. These cells are classified into two subtypes: proinflammatory (M1) macrophages and anti-inflammatory (M2) macrophages. During energy failure, microglia tend to be polarized toward the M1 phenotype after hypoxia-ischemia, and M1 microglia release proinflammatory cytokines, complement factors, proteases, and excitotoxic amino acids, which have toxic effects on neurons and glia in the surrounding area . FurtherMicroglia have also been implicated in the alleviation of inflammation and reparative processes following acute HI brain damage. During the delayed phase after newborn stroke, phagocytosis of debris by activated microglia is crucial for repairing the injured brain . In neonvia the putative histone lactylation writer protein p300 (Lactate inhibits signaling pathways and alters histones to decrease inflammatory macrophage activation and promote homeostatic M2-like polarization . Toll-liein p300 . In addiein p300 . RNA seqein p300 . Since Aein p300 . These fein p300 . HoweverMacrophage phenotypic transformation is a dynamic process resulting from a changing environment . H3K18laLactate and lactylation may have different roles under different pathophysiological conditions. More research is needed to determine the specific temporal relationship between the lactate level and histone lactylation and other interconnected metabolite-epigenetic interactions. The mechanisms controlling lactate concentrations in microdomains within the nucleus, the histone lactylation concentration needed to drive epigenetic changes, and the mechanisms by which fluctuations in lactate levels control the expression of specific genes are all unknown . It willSeveral medications that target histone posttranslational modifications have been clinically tested and have shown significant therapeutic effects in a variety of disorders. For example, drugs that reduce histone methylation by suppressing histone methyltransferases, those that reduce histone deacetylation by suppressing HDACs, and those that increase histone crotonylation are all beneficial in kidney injury . SimilarAccording to a large body of evidence, brain inflammation is a crucial contributor to HIE pathogenesis and is involved in both early and late brain injury following HI, as well as the repair and recovery of brain tissues. Following energy deprivation, several immune cells and chemical mediators of inflammation, such as cytokines, chemokines, and adhesion molecules, are implicated in the brain-immune interaction. If acute inflammation is not appropriately addressed, it can progress to chronic inflammation, which can harm the developing brain and make it more vulnerable to HI injury.Previous studies on lactate are at the vanguard of efforts to delineate the processes by which nutritional cues are integrated with metabolite variations and promote epigenetic programming . The mec


We report a 79-year-old patient who had aortic valve replacement (AVR) using a porcine aortic root. Due to degeneration of the porcine aortic valve, he required reoperation during which a heavily calcified porcine root and aortic annulus prevented insertion of any traditional bioprosthesis. AVR was achieved using a sutureless bioprosthesis, combined with mitral valve replacement. The present case confirms the feasibility and advantages of using sutureless valve implantation in complex and high-risk redo procedures. Sutureless bioprostheses represent a valid alternative to stented valves in isolated aortic valve replacement (AVR) in patients with calcific aortic stenosis.A 79-year-old man had undergone in 2006 a modified Bentall procedure using a 27-mm Prima Plus porcine aortic root . Eleven years later, he presented with recent onset of exertional dyspnea. He denied any previous episode of fever. He was in atrial fibrillation. The chest X-ray was negative, and routine blood tests were unremarkable.A two-dimensional (2D) echo demonstrated preserved left ventricular function, moderate aortic regurgitation, and severe mitral incompetence. Coronary angiography revealed diffuse coronary artery disease without significant stenoses of the major branches. Computed tomography demonstrated marked calcification of the aortic root, with multiple calcific spots in the aortic arch and coronary arteries.At reoperation, the heart was approached through a repeat median sternotomy and completely isolated by dissection of pericardial adhesions. After cannulation of the distal aorta and both venae cavae, moderately hypothermic cardiopulmonary bypass (CPB) was instituted, and the aorta was cross clamped and opened approximately 5 mm above the previous distal suture line. The heart was arrested with antegrade cold blood cardioplegia into the coronary buttons repeated every 30 minutes.Through a left atriotomy, the mitral valve was replaced by inserting a 27-mm stented porcine bioprosthesis, preserving the entire posterior mitral leaflet.At inspection, the aortic valve showed only pinpoint calcifications, dehiscence of one commissure, and no evidence of infection. Based on the pathologic intraoperative findings also replacement of the porcine aortic valve was considered necessary.After excision of the cusps, the presence of a grossly calcified annulus prevented implantation of any standard bioprosthesis. Therefore, it was decided to implant an M size PSP . The toPrior to discharge to a rehabilitation center, on postoperative day 8, a transthoracic 2D echo confirmed normal function of both prostheses, with a mean gradient of 12 mm Hg for the PSP. At 1-year follow-up, transthoracic 2D echo showed normal function of both mitral and aortic prostheses.The use of sutureless bioprostheses for AVR, particularly the PSP, has been associated with gratifying overall results in the medium term.The patient herein reported presented with a degenerated Prima Plus porcine aortic root 11 years after prior AVR, also with concomitant mitral valve disease. Although symptoms were mostly related to the severe mitral regurgitation, the finding of mild cusp calcifications with dehiscence of one commissure in the porcine aortic valve suggested the need for concomitant AVR. The presence of a porcelain aortic root, with a severely calcified aortic annulus prevented AVR with any other stented or stentless bioprosthesis. The alternative of excising the entire root and performing a repeat Bentall operation was considered too challenging and at significantly higher risk, as previously highlighted.A recent review of the literature reported a total of 25 patients with a degenerated porcine aortic root or aortic homograft in whom the use of either a PSP or a rapid deployment bioprosthesis has proved effective in simplifying a challenging reoperation.We believe that the present case confirms the efficacy of implanting a stentless bioprosthesis in complex and high-risk reoperations. This approach appears to be indicated in recipients of any type of biological conduit when tissue degeneration occurs, particularly widespread calcification of the aortic root.