text:
Q: Error (InvalidChangeBatch) in adding multiple DNS Records from aws command line I am trying to add multiple DNS records using this script add_multipleDNSrecord.sh and i am getting this error
A client error (InvalidChangeBatch) occurred when calling the ChangeResourceRecordSets operation: FATAL problem: UnsupportedCharacter (Value contains unsupported characters) encountered with ' '

But i am able to add single record without any issue from aws cli. can anyone please tell me what went wrong in this script?
#!/bin/bash
# declare STRING variable
STRING="Hello World"
#print variable on a screen
echo $STRING

# Hosted Zone ID
ZONEID="Z24*************"

#Comment
COMMENT="Add new entry to the zone"

# The Time-To-Live of this recordset
TTL=300

# Type
TYPE="A"

# Input File Name
FILENAME=/home/ec2-user/awscli/route53/scripts/test.json

cat >> $FILENAME << EOF
    {
      "Comment":"$COMMENT",
      "Changes":[
        {
          "Action":"CREATE",
          "ResourceRecordSet":{
            "ResourceRecords":[
              {
                "Value":"$IP"
              }
            ],
            "Name":"$RECORDSET",
            "Type":"$TYPE",
            "TTL":$TTL
          }
        }
      ]
    }
EOF

echo $FILENAME

A: After Replacing the space and using dot instead of space solves the problem.
Now,The script works fine and its able to add multiple records to the hosted zone.

redpajama_set_name: RedPajamaStackExchange


This is from a Columbia news, views & reviews post from last year | TODAY IS THE ANNIVERSARY OF THE FIRST DAY OF THE BATTLE OF GETTYSBURG. John Rentz was wonded on the July 1, 1863. John Rentz was the son of an immigrant. Great-great grandfather John Rentz's name is on Pennsylvania's 151st Infantry monument at Gettysburg.
What is the name of the person who mentions John Rentz? He is my great great grandfather too! I grew up in Pennsylvania but now live in Massachusetts.


text:
Кі́рстовщина — одна з течій профспілкового руху в Україні 1919 р. Названа за прізвищем інженера Костянтина Кірсти, який з початку денікінської окупації Київщини у серпні — вересні 1919 р. разом зі своїми однодумцями сформував «Організаційний комітет по об'єднанню всіх професійних робітничих профспілок м. Києва».

Комітет мав тісні зв'язки з денікінською контррозвідкою. К.Кірсту підтримало чимало робітників, які
повірили в легенду про його героїчне минуле. На одержані від влади гроші (500 тис. рублів) К.Кірста почав видавати газету «Путь рабочего», на її сторінках проповідувалися ідеї «гармонії класових інтересів» та підтримки Добровольчої армії. В одному з номерів були опубліковані дані реєстру «підприємств, що приєдналися до Організаційного комітету». У реєстрі зазначалося, що комітет підтримали 32 тис. осіб, переважно члени профспілок (однак, як було вказано в цій же газеті, реєстр складався на основі даних про кількість членів усіх професійних спілок Києва на 1 січня 1919 р.). У жовтні 1919 р. з ініціативи комітету було створено військову організацію — «робітничо-офіцерську роту», вона взяла участь у боях під Києвом проти військ Червоної армії. Наступ більшовиків змусив денікінців залишити Київ, разом з ними вийшли з міста й провідники комітету. Свою організацію вони почали відроджувати в Одесі. При цьому акцент робився на ті підпрємства, де робітники виявили незадоволення більшовицькою владою. Таким, зокрема, був завод «РОПІТ». Однак уже восени 1919 р. комітет почав втрачати підтримку робітників, а взимку 1920 р. в одеських листівках, прокламаціях і відозвах почали звучати мотиви до згуртування робітників в ім'я перемоги над зрадниками робітництва. Цими настроями одразу ж скористалася більшовицька влада й посилила свої пропагандистські акції.

Див. також
 Денікінці

Джерела та література
 О. П. Реєнт. Кірстовщина  //
 Реєнт О. П. Крах кірстовщини (Провал контрреволюції у профспілковому русі України в період денікінської окупації). В кн.: До питання про розстановку політичних сил в Україні в 1917—1920 рр.
 Реєнт О. П. Крах кірстовщини. «УІЖ», 1989, № 11.

Історичні громадські організації України

redpajama_set_name: RedPajamaWikipedia


So, No Peace Process Then?
Ehud Olmert is leaving the building. Slowly. Which nevertheless means that the Middle East process is frozen. It's amazing to think that someone of the stature of Morris Talansky could bring down an Israeli prime minister. But he's a Long Islander, and us Long Islanders have an infinite capacity for mischief.
In any case, please enjoy my story from earlier this year about, among other things, the tortured life of Ehud Olmert.


text:
Location: 相模原市立博物館
XW-2B - Visible Passes Home | Info. | Orbit | Close encounters
Search period end: 01 February 2022 00:00
22 Jan ? 05:23:57 10° NNE 05:27:31 45° E 05:31:03 10° S visible
22 Jan ? 17:34:36 10° SSW 17:37:56 30° W 17:41:18 10° NW visible
23 Jan ? 05:08:25 10° NE 05:11:36 27° E 05:14:47 10° SSE visible
24 Jan ? 04:53:10 10° NE 04:55:40 17° E 04:58:09 10° SE visible
25 Jan ? 04:38:51 10° E 04:39:41 11° E 04:40:30 10° ESE visible
26 Jan ? 18:06:54 10° WSW 18:08:34 13° W 18:10:15 10° WNW visible
27 Jan ? 17:49:47 10° SW 17:52:35 20° W 17:55:23 10° NW visible
28 Jan ? 17:33:15 10° SSW 17:36:37 32° W 17:39:59 10° NNW visible
31 Jan ? 04:37:52 10° E 04:38:13 10° E 04:38:34 10° E visible

redpajama_set_name: RedPajamaCommonCrawl


text:
Biografia
Figlio di Lodovico Mortara, ministro della giustizia con il primo governo Nitti, seppure nipote del rabbino di Mantova non venne educato religiosamente. La madre era Clelia Vivanti, sorella del matematico di fama internazionale Giulio Vivanti.

Mortara conseguì molto precocemente ottimi risultati negli studi tanto da laurearsi in Giurisprudenza a Napoli già nel 1905. Successivamente, interessato in particolar modo di statistica e demografia, trascorse un anno accademico a Berlino con lo statistico ed economista russo di origine polacca, Ladislaus von Bortkiewicz studiando la variabile casuale poissoniana che venne applicata da Mortara ai suoi studi statistici su omicidi e alcolismo.

Rientrato in Italia non ancora venticinquenne, iniziò la libera docenza universitaria, prima a Messina (1909-14), poi a Roma (1915-24) e infine a Milano (1924-38) dove ottenne la cattedra di statistica all'Università Statale e poi la direzione dell'Istituto di statistica della Università Bocconi.

Mortara nel 1910 acquistò con Alberto Beneduce la comproprietà del Giornale degli economisti. Della rivista fu ininterrotto direttore o condirettore fino al 1938, quando entrarono in vigore le leggi razziali fasciste. A causa di ciò Mortara, seppure nel 1933 avesse sottoscritto la tessera del Partito Nazionale Fascista, dovette lasciare la Bocconi e la direzione del Giornale degli economisti. Alla scopo di sottrarre alle mire del regime fascista il più importante giornale italiano di materie economiche e statistiche, Mortara acconsentì che il Giornale degli economisti fosse ceduto a titolo gratuito all'Università Bocconi.

Negli anni immediatamente precedenti l'applicazione delle leggi razziali, fu chiamato dal Governatore Vincenzo Azzolini a collaborare all'istituzione e organizzazione del Servizio studi della Banca d'Italia. Talmente fu apprezzato il suo apporto, che l'istituto d'emissione creò borse di studio "Giorgio Mortara", ancor oggi distribuite, che sono assegnate per il perfezionamento degli studi all'estero sulle metodologie  matematiche, statistiche ed econometriche.

Nel 1939 emigrò in Brasile dove collaborò con il "Servizio nazionale di censimento" realizzando il primo censimento demografico. Nel Paese che lo accolse in fuga dal regime fascista e gli concesse la cittadinanza, Mortara fondò anche una scuola di demografia. Nel 1956 tornò a insegnare a Roma.

Pubblicazioni
 Le popolazioni delle grandi città italiane, Unione Tipografico-Editrice Torinese, 1908;
 Le popolazioni di Basilicata e di Calabria all'inizio del secolo ventesimo (1910);
 Tavola di mortalità, secondo le cause di morte, per la popolazione italiana (1901-10), Cecchini, 1914;
 Statistica economica e demografica (1920);
 Prospettive economiche(1921–37), Università commerciale Luigi Bocconi;
 Lezioni di statistica metodologica (1922);
 La salute pubblica in Italia durante e dopo la guerra, G. Laterza & figli, 1925;
 Sui metodi per lo studio della fecondità dei matrimoni, Giornale degli economisti, 1933;
 La realtà economica (1934);
 L'Economia della popolazione, Unione Tipografico-Editrice Torinese, 1960;
 Previsioni sull'incremento della popolazione nel mondo, L'industria (1958);
 Raccolta di Saggi di metodologia demografica (1963).

Note

Bibliografia
 Alberto Baffigi e Marco Magnani, Giorgio Mortara dal sito della Società Italiana Statistica, 27 novembre 2008 (bozza provvisoria). consultato in data 23 giugno 2012.
 Libero Lenti, Il «Giornale degli economisti» compie cento anni, "Nuova Antologia", ott.-dic. 1986, fasc. 2160,  97-111.
 Marco Magnani, «MORTARA, Giorgio», in Dizionario Biografico degli Italiani, Volume 77, Roma, Istituto dell'Enciclopedia Italiana, 2012.

Altri progetti

Collegamenti esterni
 Mortara, Giorgio la voce nella Treccani.it L'Enciclopedia Italiana. URL visitato il 21 gennaio 2013.
 Mortara, Giorgio la voce di Alberto Baffigi in Il Contributo italiano alla storia del Pensiero: Economia (2012) Treccani.it. URL visitato il 14 marzo 2023.

Ebrei italiani
Italiani emigrati in Brasile
Professori della Sapienza - Università di Roma
Professori dell'Università commerciale Luigi Bocconi
Professori dell'Università degli Studi di Messina
Professori dell'Università degli Studi di Milano
Studenti dell'Università degli Studi di Napoli Federico II


Although nearly six years have passed since Hurricane Katrina struck Louisiana in 2005, 88 families in Plaquemines Parish are still homeless or displaced, many of them still living in temporary Federal Emergency Management Agency (FEMA) trailers.
Malcolm Savage, a retired pipefitter in his 70s, is one Plaquemines Parish resident for whom temporary housing has become a long-term solution. Malcolm, who was hospitalized because of heart problems during the storm, remained safe amid the destructive gales – but his uninsured home was completely flooded, and he has been living in a FEMA trailer ever since.
However, FEMA is now mandating that residents either leave the trailers or pay a steep penalty – if Malcolm doesn't vacate his trailer by April 30, he will have to pay an $800 monthly fine. The Fuller Center Disaster ReBuilders (FCDR) team plans to help Malcolm renovate his gutted home by this deadline, as well as the home of another family, the Demolles, who face the same looming penalty.
Bart Tucker, director of the FCDR, said the new penalty has added "insult to injury" for a community that has already faced a multitude of recent hardships.
The FCDR also just received a grant from the United Way to build four new homes for families who had their homes destroyed by Katrina. Like Malcolm and the DeMolle family, all four families have been living in temporary housing for almost six years.
Because the grant stipulates that all construction must be completed by June 30, the FCDR has already started the foundations on one of those four new houses.
But in order to complete these projects by the deadlines, FCDR needs as many volunteers as possible.


text:
nubbin/POU domain protein 1
EVOLUTIONARY HOMOLOGS (part 1/2)
pdm-1 and pdm-2 belong to the class II type of POU genes.
As a POU domain transcription factor, VVL is related to an evolutionary complex group of genes consisting of at least 5 classes. Pit-1 is a class one POU domain transcription factor. Pit-1 is involved in the development of the anterior pituitary gland in mammals. Class II POU domain transcription factors include mammalian Oct1, Oct2, Oct11 and Drosophila PDM-1 and PDM-2. Mammalian Brn1, Brn2, Brn4, SCIP/Oct6 and Xenopus XLPOU1 and XLPOU2 and well as Drosophila Ventral veins lacking (Drifter/Cf1a) and C. elegans ceh6 are Class III proteins. Acj-6 is in POU domain group IV, along with C. elegans unc86 and vertebrate Brn3. Oct-3/4 is a class V POU domain protein. There is no known Drosophila class 1 or class V homolog (Verrijzer, 1993).
The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for 'POU' domain signatures.
Analysis of nubbin expression patterns in insects
nubbin expression patterns were studied in three hemimetabolous insect groups: zygentomans (Thermobia domestica, firebrat), dyctiopterans (Periplaneta americana, cockroach), and hemipterans (Oncopeltus fasciatus, milkweed bug). Three major findings are reported: (1) nub patterns were observed in the ventral central nervous system ectoderm represent a synapomorphy (shared derived feature) that is not present in other arthropods. Furthermore, each of the analyzed insects exhibits a species-specific nub expression in the central nervous system. (2) Recruitment of nub for a role in leg segmentation occurred early during insect evolution. Subsequently, in some insect lineages (cockroaches and flies), this original role was expanded to include joints between all the leg segments. (3) The nub expression in the head region shows a coordinated change in association with particular mouthpart morphology. This suggests that nub has also gained an important role in the morphological diversification of insect mouthparts. Overall, the obtained data reveal an extraordinary dynamic and diverse pattern of nub evolution that has not been observed previously for other developmental genes (Li, 2004).
Overall, the cloned Nub/Pdm sequences showed a high degree of conservation with other arthropod nub/pdm orthologs and were quite distinct from sequences of a related vvl gene. Among insects, nub sequences showed perfect conservation in the POU domain except for a single amino acid replacement. In addition, insect Nub/Pdm sequences showed a high degree of sequence similarity in the homeodomain. Even a variable 'linker' region was conserved between firebrats, cockroaches, and milkweed bugs. Conservation was especially noticeable at the beginning of the 'linker' region, where all examined insect species (except Drosophila) contained an NSLN motif, compared with a KTIT/S motif in crustaceans (Pc nub and Ps nub) or to an ASMN motif in spiders (St nub). Intriguingly, the Drosophila nub sequence in this region differed from those of other insects and other arthropods. Although suggestive, the functional significance of these changes is not known (Li, 2004).
nub is one of the few developmental genes for which extensive comparative data are available. Among arthropods, nub expression has been examined in chelicerates, including spiders and horseshoe crabs, and in several crustacean species. Within insects, this gene was studied only in Drosophila. Because higher flies have a highly derived mode of development and a relatively recent phylogenetic origin, it is unclear whether the pattern observed in Drosophila is representative of all insects. With this analysis of an apterygote and two hemimetabolous insects, it is now possible to consider the evolution of nub expression in arthropods in general. nub expression is class specific and sometimes even species specific. Nonetheless, all examined arthropods share a common expression pattern in appendages, indicating that nub was originally an 'appendage' gene. In chelicerates, generally thought to be basal arthropods, nub is localized exclusively to the walking legs and other leg-derived structures. In the spider Steatoda triangulosa, a single band of nub expression was detected in the tarsus of all prosomal legs. This basic chelicerate pattern was substantially altered in the crustacean Porcelio scaber. (1) nub expression spread anteriorly into the head region. Note that this head expression is incomplete, encompassing some but not all the segments. (2) Although nub is still restricted to the distal leg segments, it is expressed in a set of rings (instead of in a single band as in spider embryos). This refinement of the nub expression continues even further in insect embryos. In firebrat and cockroach embryos, there was no expression in the antennal and mandibular appendages. However, milkweed bug embryos exhibit a strong antennal and mandibular staining Thus, the spread of nub expression in the anterior direction is complete in insects and now includes all the head segments. In addition, insect embryos also exhibit a further proximal expansion of nub expression in the legs. Whereas nub is localized only in the distal leg segments in spiders and crustaceans, its expression in insects encompasses proximal leg regions as well. This is particularly pronounced in cockroaches, in which nub is expressed in all leg segments (Li, 2004).
The other key trend in the evolution of nub expression in arthropods is in its expansion from the periphery of the embryo (appendages) to the mid-ventral region. In both chelicerates and crustaceans, there is complete absence of nub in the center of the embryo. In insects, however, mid-ventral expression in neuroectoderm is one of the most noticeable aspects of nub expression. Intriguingly, each insect species exhibits a unique species-specific CNS pattern. Functional studies in Drosophila show that nub is part of a gene regulation cascade specifying the identity of developing neuroblasts in the CNS. These findings, combined with the current observations, suggest that nub expression in the CNS is unique to the insects and has continued to evolve toward more elaborate spatial and temporal patterns that coincide with diversification of insect morphology (Li, 2004).
nub expression patterns exhibit substantial differences between and within the major arthropod classes. However, in groups such as spiders and crustaceans, these differences are rather static. More specifically, once the pattern of expression is established, it does not change during development. In contrast, the data clearly indicate that variation is a key feature of nub expression within insect embryos. The best illustration of this variation is provided by dynamic temporal and spatial changes of nub expression in the CNS and antennae (Li, 2004).
If one compares the timing of its expression in the CNS versus appendages, it is evident that the onset of nub expression within the CNS is highly variable. In firebrat embryos, nub is first expressed in the CNS and is subsequently detected in the appendages. In cockroach embryos, the earliest expression of nub is detected in the appendages followed by the CNS. And in milkweed bug embryos, expression of nub in appendages and CNS occurs simultaneously at a very early stage. In addition to this temporal variation, nub also exhibits spatial variation in its pattern. nub expression in the CNS was found to have a unique species-specific pattern for each insect species studied. In firebrats, nub is expressed in groups of cells aligned along the midline of each segment in a uniform pattern that persists throughout development. In the cockroach, nub expression appears in an irregular triangular pattern encompassing a wider region in the thoracic than in the abdominal segments. Once established, this pattern does not change from early to late development. In contrast, CNS expression of nub in milkweed bugs is very dynamic. In early developmental stages, nub is expressed in cells aligned along either side of the midline, with expression excluded from any of those cells within the midline. As development proceeds, cells within the midline form an 'x' pattern of expression in each segment, beginning in T1 and ending at the posterior abdominal segment (Li, 2004).
Antennal expression represents another example of a highly variable nub pattern. In embryos of basal insect groups such as firebrats and cockroaches, nub is absent in antennae. This observation is also consistent with those on spiders and crustaceans. No nub expression is found in the first two prosomal segments (cheliceral and pedipalpal) in spiders. Similarly, nub is absent from the antennal 1 segment of crustaceans (Li, 2004).
These observations indicate that lack of nub expression in the anterior-most appendages is an ancestral arthropod feature. However, in more derived hemimetabolous insects such as milkweed bugs, nub is observed in this new domain. Moreover, its expression undergoes dramatic temporal and spatial change during development. Early on, nub is strongly expressed throughout the antennae. Then, as the appendages start to elongate, its expression is lost. As development of appendages continues, nub accumulation reappears again in a cluster of cells at the posterior-distal portion of antennae and then shifts to their most distal part and continues to be strongly expressed during dorsal closure. Toward the end of development, its expression disappears again. nub accumulation has also been detected in the eye/antennal disc of Drosophila larvae, further suggesting this novel pattern to be of relatively recent origin and to be shared among phylogenetically more derived insect groups (Li, 2004).
This study revealed that nub function may be evolving more rapidly than that of homeotic or other leg patterning genes such as Dll and dac. Also, the most dynamic temporal and spatial changes in expression occurred in milkweed bugs, a relatively derived hemimetabolous insect. Additional dynamic changes in nub pattern were observed in firebrat and cockroach embryos, especially in legs and mouthparts. These observations pose an intriguing question as to what level of developmental variation actually exists in nature. Whereas traditional views support relatively high levels of conservation in developmental pathways, the present analysis of nub suggests that variation may be more prevalent than previously thought (Li, 2004).
Mainly based on insights from leg development in Drosophila, it is now known that the Notch (N) signaling pathway plays a fundamental role in the process of segmental boundary formation in legs. In flies, the actual joint formation is mediated by establishing distinct rings of N expression in these regions of the leg. nubbin is also expressed in a series of concentric rings in third instar Drosophila leg discs, and its mutant expression results in shortened and gnarled legs. Mutant clones of Notch also cause loss of nub expression in Drosophila legs. Conversely, ectopic expression of nub is induced within clones of cells expressing activated Notch. Thus, in Drosophila legs, nub is positively regulated downstream of Notch (Li, 2004).
Whereas this information indicates that nub is also involved in leg patterning in Drosophila, in addition to its previously described roles in CNS and wing development, the question remains as to when this acquisition of leg function occurred. In spider embryos nub is localized to a single distal band on all walking legs. In crustacean embryos with joint-less trunk appendages such as Artemia franciscana, no expression of nub has been detected. However, in crustaceans with segmented endopods (main leg branches), nub is localized to a series of rings that correspond to joints in the distal leg region. These findings indicate that nub may have a partial role in leg segmentation in chelicerates and crustaceans, mainly during the establishment of distal leg segments. However, formation of most leg joints in those groups has to be under the control of other genes (Li, 2004).
There are two dominant bands of nub expression in firebrat legs, a proximal band and a mid-distal band. The locations of these bands roughly correspond to the positions of the future coxa-trochanter and femur-tibia boundaries, respectively. But leg segmentation in firebrats occurs very late in development when it is technically very difficult to perform the in situ experiments (due to the deposition of embryonic cuticle). This prevents inference of the complete pattern of nub in this species and relation of this expression to the formation of joints. Leg development in Periplaneta embryos is quite different, and clearly identifiable leg segments can be recognized by mid-stages of embryogenesis. In this species, the appearance of five nub stripes precedes any visible demarcation of distinct leg regions. Only afterward does continuing nub expression begin to coincide with the formation of segmental boundaries in the legs. This coordination between the precise patterning of nub expression and leg segmentation in the cockroach embryos is highly indicative of a role in formation of joints. The observed pattern in Periplaneta is also reminiscent of Notch signaling-regulated nub expression in Drosophila leg development. These similarities suggest that regulation of leg segmentation in two widely divergent insect species may be homologous (Li, 2004).
The timing of leg segmentation in milkweed bug embryos is intermediate compared with firebrats and cockroaches. Proximal leg segments in Oncopeltus are established relatively early during germ-band extension, but segmentation of distal leg regions does not occur until late development. As in Periplaneta, the leg patterns of nub expression in Oncopeltus roughly correspond to the position of future joints and its appearance precedes formation of segments. There are also two main differences between observed nub patterns between milkweed bugs and cockroaches: (1) nub expression in Oncopeltus legs is composed of only three bands versus five bands in Periplaneta; (2) two of these bands in Oncopeltus begin to fade much earlier (compared with cockroach). The possible explanation for the first discrepancy is that milkweed bug nub may be involved in formation of some, but not all, leg segments. The likely explanation for the second discrepancy is that nub may play a role in initiating the formation of some leg joints in Oncopeltus but is not required for its maintenance (Li, 2004).
Overall, this analysis suggests that recruitment of nub for a role in leg segmentation may have occurred early in insect evolution. In both insects and crustaceans, nub expression is associated with establishment of some but not all leg segments. In insects, the primary association is with proximal and mid-leg segments. However, only the two most distal segments may be involved in crustaceans. These findings indicate that nub cannot be a necessary element in the regulation of overall leg segmentation in arthropods. Rather, the observed expression patterns suggest that this gene was independently recruited in insects and crustaceans for a possible role in development of specific leg joints. Subsequently, in some insect lineages (cockroaches and flies) this original role was expanded to include joints between all leg segments (Li, 2004).
The insect head has a modular organization and is composed of the three pregnathal (ocular, antennal, and intercalary) and three gnathal segments (mandibular, maxillary, and labial). A pair of appendages grows from each gnathal segment and forms the future mouthparts. Diversity in organization of the insect head is best represented by the variation in structure and morphology of the mouthparts. There are two basic types of mouthparts: mandibulate (specialized for chewing and biting), and haustellate (specialized for piercing and sucking). The mandibular type represents the ancestral form and is characteristic of members of most basal hexapod lineages (Zygentoma, Orthoptera, Blattoidea, etc.). Its main feature is that the mandibles function as 'jaws,' whereas the maxillary and labial limbs form branched appendages that are structurally identical until the midline fusion of the latter. In the haustellate type, it is the mandibular and maxillary appendages that are similar, whereas the labial appendages exhibit a completely different structure (Li, 2004).
Studies in firebrats, crickets, milkweed bugs, and flies have shown that the basic programming and identity of the gnathal segments are determined by three Hox genes: proboscipedia (pb), Deformed (Dfd), and Sex combs reduced (Scr). Changes in pb expression have been directly associated with changes in mouthpart structure. In insects with biting and chewing mouthparts, pb is expressed in the labium and maxillae but not in the mandibles. However, in insects with piercing and sucking mouthparts, such as milkweed bugs, pb expression is mainly detected in the labial appendages Thus, it has been proposed that loss of pb expression in the maxillae was responsible for the transformation of the mandibulate to the haustellate mouth type. More specifically, the loss of pb expression in milkweed bug embryos would 'free' the maxillary appendages to diverge from their original labium-like phenotype. However, such change in pb regulation cannot account for the development of stylets in both maxillary and mandibular segments. This suggests that other nonhomeotic genes are involved in the latter process (Li, 2004).
Consistent with the morphological diversification of head appendages, the sharpest difference of nub expression is observed in this region. Patterns of expression show a coordinated change in association with particular mouthpart morphology. In firebrat and cockroach embryos, which have typical mandibulate mouthparts, nub is localized in the maxillary and labial appendages in a similar pattern. This is particularly pronounced in Periplaneta embryos, which exhibit highly coordinated patterns of expression in the labium and maxillae from early to late development. Such an observation is consistent with the fact that these segments are structurally very similar and differ from the mandibular segment. nub is generally not expressed in the mandibles except locally and transiently in cockroaches. This suggests that in insects with biting and chewing mouthparts, mandibles were originally devoid of nub expression (Li, 2004).
In milkweed bug embryos, which have the stylate-haustellate (sucking) mouthparts, nub is expressed in a very different pattern: (1) there is novel expression along the mid-ventral region of the mandibular appendages; (2) this mandibular expression is similar to the maxillary pattern. Expression in the labium, however, is localized only in the distal portion of the appendage and exhibits a completely different timing and pattern. This differential expression correlates with a change in the morphology of the maxillary and labial segments that is characteristic for piercing and sucking mouthparts: development and morphology of the maxillary segment parallels that of the mandibular segment. These observations provide the first nonhomeotic gene example of a linkage between a change in expression pattern and a corresponding change in the morphology of insect mouthparts. They also provide a strong indication that nub may function in the development of haustellate mouthparts (Li, 2004).
The emerging insight from this and other recent studies suggests that the evolution of the maxillae and mandibles in Oncopeltus and other hemipterans was governed by regulatory changes at both higher levels (Hox genes such as pb and Dfd) and lower levels (genes such as nub) in the developmental hierarchy. This underlines the critical importance of understanding the functional roles and relationships between these two classes of genes. For example, Dfd is expressed in the mandibular segments in both Tribolium (which has mandibulate mouthparts) and in Oncopeltus (which has haustellate mouthparts). Functional studies have shown that in the absence of Dfd, mandibles are transformed into antenna-like appendages in these two insects. These findings show that Dfd orthologs control mandibular identity in both species. Yet the actual morphology of mandibles in Tribolium and Oncopeltus is very different, a result of distinct mandibulate and haustellate modifications. Additional factors, either acting in parallel with Dfd, or downstream targets, or independently of Dfd, must be involved in the establishment of these distinct mandibular morphologies. Based on this analysis of its expression patterns, nub is a good candidate for being such a target gene in the mandibular segment. Further studies of other genes at lower levels in the developmental hierarchy will be necessary to fully understand the mechanisms underlying the morphological diversification of insect mouthparts (Li, 2004).
Patterning of the adult mandibulate mouthparts in the red flour beetle, Tribolium castaneum
Specialized insect mouthparts, such as those of Drosophila, are derived from an ancestral mandibulate state, but little is known about the developmental genetics of mandibulate mouthparts. The metamorphic patterning of mandibulate mouthparts of the beetle Tribolium castaneum was studied RNA interference to deplete the expression of 13 genes involved in mouthpart patterning. These data were used to test three hypotheses related to mouthpart development and evolution. First, the prediction was tested that maxillary and labial palps are patterned using conserved components of the leg-patterning network. This hypothesis was strongly supported: depletion of Distal-less and dachshund led to distal and intermediate deletions of these structures while depletion of homothorax led to homeotic transformation of the proximal maxilla and labium, joint formation required the action of Notch signaling components and odd-skipped paralogs, and distal growth and patterning required epidermal growth factor (EGF) signaling. Additionally, depletion of abrupt or pdm/nubbin caused fusions of palp segments. Second, the hypotheses was tested for how adult endites, the inner branches of the maxillary and labial appendages, are formed at metamorphosis. The data reveal that Distal-less, Notch signaling components, and odd-skipped paralogs, but not dachshund, are required for metamorphosis of the maxillary endites. Endite development thus requires components of the limb proximal-distal axis patterning and joint segmentation networks. Finally, adult mandible development is considered in light of the gnathobasic hypothesis. Interestingly, while EGF activity is required for distal, but not proximal, patterning of other appendages, it is required for normal metamorphic growth of the mandibles (Angelini, 2012).
In D. melanogaster, Dll mutants lack maxillary structures and portions of the proboscis (i.e., labium), although Dll expression in the maxillary anlagen is weaker than in the leg or antennal discs. Paralleling the results for T. castaneum, in the horned beetle Onthophagus taurus distal regions of the adult mouthparts were deleted with larval Dll RNAi (Simonnet 2011). The embryonic and metamorphic functions of Dll in T. castaneum are also similar: the gene is required for the development of distal structures at both stages, and during embryogenesis Dll is expressed throughout the developing palps. Interestingly, removal of T. castaneum Dll expression earlier during larval life led to delayed metamorphosis, as well as changes in appendage morphology (Suzuki, 2009). Many insects delay molting after appendage loss to allow time for regeneration, and this dual role of Dll suggests a mechanism linking these processes (Angelini, 2012).
The data from T. castaneum provide evidence for a conserved gap gene role of dac during patterning of mouthparts and legs of this species. dachshund is not expressed in or required for development of the labial and maxillary anlagen of D. melanogaster. In T. castaneum embryos dac is expressed strongly in the proximal maxilla and part of the developing endite. Embryonic dac expression is weaker in the distal maxillary palp and the labium. The current data show a clear metamorphic requirement for dac in the intermediate regions of the maxillary and labial palps, as does a recent study of O. taurus (Simonnet, 2011). A function for dac in the development of an intermediate portion of the maxillary and labial appendages has so far only been observed in these two beetles, while data from two species with specialized mouthparts (the milkweed bug O. fasciatus and D. melanogaster) found that dac is not required for PD patterning of the mouthparts. Thus, comparative data from other species do not support the hypothesis that this mouthpart patterning role is ancestral. However, if mandibulate mouthparts evolved from leg-like structures similarities in the expression and function of genes patterning both legs and mouthparts are expected to be plesiomorphic. This hypothesis can be further tested by examining the role of dac in mouthpart development in additional insect orders, particularly those that retain mandibulate mouthparts, and in other arthropods (Angelini, 2012).
The effects of hth depletion are distinct in different species, but typically involve some degree of homeotic transformation. In D. melanogaster, hth is expressed in the labial discs, but without nuclear expression of its cofactor Extradenticle. Maxillary palps are retained in hth loss-of-function flies, but they may possess bristles typical of legs, indicating a partial proboscis-to-leg transformation. In the cricket Gryllus bimaculatus, which has mandibulate mouthparts, hth depletion causes transformation of proximal mouthpart structures towards antennal identity, with a loss of endites, while distal structures are transformed towards leg identity (Ronco, 2008). hth RNAi in T. castaneum transformed intermediate regions of the maxilla and labium towards distal mouthpart identity. Proximal regions also appeared transformed, but their identity could not be established, while distal regions appeared wild type. In the beetle O. taurus, proximal regions of the labium are transformed towards maxillary endite identity, but distal regions of the labium and the entire maxilla remain relatively unaffected (Angelini, 2012).
These results highlight the similarity between patterning of the maxilla, labium and legs in T. castaneum. Functional data from two species with highly derived mouthpart morphologies, D. melanogaster and the milkweed bug Oncopeltus fasciatus, suggest only limited similarity between mouthpart and leg patterning. One explanation for this low degree of conservation is that evolution of the ancestral patterning mechanism has occurred in concert with the functional and morphological diversification of these mouthparts. A correlation between generative mechanisms and structural morphology has been used as a common null hypothesis, although exceptions in which similar morphologies result from different developmental pathways are documented. Nevertheless, this hypothesis predicts that developmental patterning should be more highly conserved across appendage types in species that retain the ancestral mandibulate mouthpart morphology (Angelini, 2012).
The maxillary and labial palps are an interesting case of serial homology. Despite a difference in overall size, their shape and arrangement of sensillae are similar. The intermediate segments of each palp type are also similar, but differ in number, which suggests that segment number is regulated independently from other morphological traits. The RNAi depletion of pdm in T. castaneum caused the reduction and deletion of the third maxillary palp segment, producing a phenotype closely resembling the wildtype morphology of the labial palps. While a role for pdm in the labium cannot be excluded, the absence of observed labial phenotypes was significant compared to maxillary results. Therefore, it is hypothesized that the difference in the number of palp segments results from specific activation of pdm in the maxillary palp. Loss of function in the Hox gene Deformed during T. castaneum embryogenesis causes a transformation of the larval maxillae towards labial identity. Since Hox genes are the primary determinants of body segment identity, it is proposed that pdm is activated by Deformed, and repressed by the labial Hox gene Sex combs reduced. RNAi targeting pdm in another mandibulate insect, the cricket Acheta domesticus, generated defects in the antenna and legs, but no defects in the mouthparts, despite similar pdm expression in these appendages (Turchyn; 2011; Angelini, 2012).
Endites are a primitive component of arthropod appendages, and they are retained in insect mouthparts, as well as in the mouthparts and thoracic appendages of many crustaceans (Boxshall 2004). At least three hypotheses have been put forward for how endites are patterned, and these hypotheses are not mutually exclusive. The first hypothesis states that multiple PD axes result from redeployment of a PD axis patterning mechanism shared by palps and endites. A second hypothesis posits that endites and appendage segments form by the same mechanism, Notch-mediated in-folding of the cuticle. A third hypothesis states that dac expression initiates endite branching from the main appendage axis. The axis redeployment hypothesis predicts that depletion of genes involved in PD axis patterning will have similar effects on the development of palps and endites. Some support for this hypothesis comes from studies of endite morphogenesis and the expression and function of leg gap genes in the embryos of T. castaneum and the orthopteran Schistocerca americana, but not all data are consistent with it. The segmentation hypothesis predicts that endites will fail to differentiate if genes required for joints are depleted. This hypothesis was posed based on a comparative developmental study of segmented and phyllopodous crustacean limbs. Finally, the dac-mediated hypothesis predicts that depletion of dac will lead to reduced endites. This hypothesis emerged from the observation that dac expression is reiterated along the medial edges of larval endites in the crustacean Triops longicaudatus. Comparative expression data from the isopod Porcellio scaber are also consistent with the dac-mediated hypothesis (Angelini, 2012).
The current data are consistent with predictions of the axis redeployment and segmentation hypotheses but do not support a role for dac in endite metamorphosis. Adult endites were disrupted by depletion of Dll, Krn, the odd-related genes, and Notch signaling, and to a lesser degree hth. In the maxilla depletion of most of these genes led to the failure of the single larval endite to divide into two distinct branches, while in the labium, their depletion caused reduction of the ligula. Their requirement in the endites is consistent with the hypothesis that these structures are generated by redeploying appendage PD axis determinants. Depletion of Notch signaling components and the odd paralogs produced reductions and fusions between palp segments, between the palps and endites, and between the lacinia and galea. Thus, these data are compatible with both the hypothesis that a reiterated PD axis is used to pattern the endites and the hypothesis that endite formation is linked to joint formation. Normal endite development in dac-depleted specimens is inconsistent with the dac-mediated hypothesis (Angelini, 2012).
It is noteworthy that endite specification and the division of the single larval endite into the adult galea and lacinia appear to be separable functions. For example, Ser RNAi resulted in a single endite lobe with lacinia identity medially and galea identity laterally. In contrast, severe Dll RNAi individuals had a single endite that lacked also obvious lacinia identity (Angelini, 2012).
The mandibulate structure of Tribolium mouthparts is the pleisomorphic state for insects and is shared by a majority of insect orders. These mouthparts are characterized by robust mandibles, lacking segmentation. A classic debate in arthropod morphology concerns whether the mandibles of insects and myriapods are derived from a whole appendage or only from proximal appendage regions; the latter are called gnathobasic mandibles. Palps are retained on the mandibles of many crustaceans, making it clear that the biting regions of their mandibles are gnathobasic. Phylogenetic support for the gnathobasic hypothesis comes from phlyogenetic studies that place insects nested within crustaceans (Regier, 2010). The first developmental genetic support for the gnathobasic hypothesis came from the discovery that insect mandibles lack Dll expression. Furthermore, neither mutations in Dll nor its depletion through RNAi have been observed to alter mandible development in insects, including T. castaneum. This evidence has led to widespread acceptance of the gnathobasic hypothesis. Of the 13 genes depleted in this study, two (Krn and hth) produced results that would not be predicted by the most straightforward form of the gnathobasic hypothesis for mandible origins (Angelini, 2012).
Loss of EGF function in insects leads to distal appendage defects, including pretarsal or tarsal deletions. The role of EGF signaling in distal appendage regions is conserved in T. castaneum metamorphosis, since depletion of the EGF ligand Krn leads to reduction of the antennal flagellum, and maxillary and labial palps, as well as to deletion of the pretarsus and malformation of the tarsus. In light of the restriction of Krn's role to distal appendage regions and regulation of distal EGF ligand expression by Dll in D. melanogaster, the gnathobasic hypothesis predicts that Krn should not be required for normal development of the mandible in T. castaneum. In contrast to this prediction, depletion of Krn produced a significant reduction in mandible length (Angelini, 2012).
The hypothesis of a gnathobasic mandible also predicts that hth depletion should produce effects in the mandible similar to those in the proximal regions of other appendage types. In T. castaneum, hth RNAi during metamorphosis caused homeotic transformation of proximal regions of the maxilla, labium and legs. However, the mandibles were not affected by hth depletion. In the beetle O. taurus, hth depletion slightly altered mandible shape, but also without apparent homeosis. In contrast, hth RNAi in embryos of the cricket G. bimaculatus transformed the mandible towards a leg-like structure distally and an antenna-like structure proximally, paralleling the transformation observed in other appendages. Because these results come from only two lineages and from different life stages, additional data are needed to determine whether a homeotic role for hth was present ancestrally in insect mandibles (Angelini, 2012).
These data must be weighed alongside other evidence bearing on the gnathobasic hypothesis. In T. castaneum, the lack of phenotypic effects on mandible metamorphosis of other genes in this study is consistent with the gnathobasic hypothesis. In particular, it was observed that mandible metamorphosis was normal following depletion of genes involved in distal growth and patterning or joint formation. Moreover, homology at one biological level, such as anatomy, does not preclude divergence at other levels, such as development. Nevertheless, since developmental genetic studies of Dll and other appendage-patterning genes have been used as strong support for the gnathobasic homology of the insect mandible, the findings of Krn function highlight the difficulties in establishing serial homology based solely on developmental data (Angelini, 2012).
This study provides a genetic model of adult mouthpart development in Tribolium castaneum based on 13 genes. While previous studies have examined patterning in species with derived mouthpart morphologies, T. castaneum retains the pleisomorphic, mandibulate state of insect mouthparts. These results demonstrate the conservation of many gene functions in the maxilla and labium, relative to the legs, thus supporting the interpretation of novel gene functions in groups with derived mouthpart morphology as indicative of their specialized morphogenetic roles in those species. Mandibulate mouthparts such as those of T. castaneum include medial maxillary and labial endites, and the current data are consistent with hypotheses of reiteration in the PD axis and specification by Notch signaling, but rule out a direct role for dac in branch generation or patterning at metamorphosis. Additionally these results demonstrate that a regulator of distal leg development, Krn, which encodes an EGF ligand, is required for normal mandible elongation. This finding underscores the complex relationship between homology at the levels of anatomy and developmental patterning (Angelini, 2012).
POU homeodomain protein interaction with DNA
The homeodomain defines a family of transcription factors broadly involved in the regulation of gene expression. DNA recognition, as observed in three representative complexes (Engrailed, Antennapedia, and MAT alpha 2), is mediated in the major groove by a helix-turn-helix (HTH) element and in the minor groove by an N-terminal arm. The three complexes share similar overall features, but they also exhibit significant differences in DNA interactions. Because these differences may distinguish the biological activities of different classes of homeodomains, the contribution of the Oct-2 POU-specific homeodomain (POUHD) to the specificity of the bipartite POU motif has been investigated. Comparative studies of variant protein-DNA complexes demonstrate the following:
Mutations in an invariant residue in the POUHD HTH (N347; residue 10 of the putative recognition alpha-helix) reduce octamer binding with the relaxation of specificity at one position (5'-ATGCAAAT). The inferred HTH side chain-base interaction, although not observed in the solution structure of the Antennapedia complex, is in accord with homologous contacts in the Engrailed and MAT alpha 2 cocrystal structures.
Comparison of the DNA-binding properties of POU and POUHD demonstrates that POU side chain and POUHD independently regulate specificity at opposite ends of the DNA site (5'-TATGCAAAT). Both domains contact the two central bases (5'-TATGCAAAT) where coordinate binding of POU side chain in the major groove overrides the intrinsic specificity of POUHD in the minor groove.
The differential sensitivity of POU side chain and POUHD to 2'-deoxyinosine substitutions (a minor-groove modification) suggests that POU side chain binding repositions the POUHD N-terminal "arm" (Botfield, 1994).
The bipartite DNA binding domain of the POU family of transcription factors contains a 'POU-specific' domain unique to this class of factors and a 'POU homeodomain' homologous to other homeodomains. DNA binding of the Oct-2 factor POU domain and the Antennapedia (ANTP) homeodomain were compared with a chimeric Oct-2/ANTP protein in which the distantly related Antp homeodomain was substituted for the Oct-2 POU homeodomain. The Oct-2/Antp chimeric protein binds both the octamer and the Antp sites efficiently, indicating that DNA binding specificity is contributed by both components of the POU domain (Brugnera, 1992).
Two crystal structures of Oct-1 POU domain bound to DNA provide a rationale for differential, conformation-dependent recruitment of transcription cofactors. The POU-homeo and POU-specific subdomains of Oct-1 contain two different nonoverlapping pairs of surface patches that are capable of forming unrelated protein-protein interfaces. Members of the POU factor family contain one or two conserved sequence motifs in the interface that are known to be phosphorylated, as noted for Oct-1 and Pit-1. Modeling of Oct-4 reveals the unique case where the same conserved sequence is located in both interfaces. These studies provide the basis for two distinct dimeric POU factor arrangements that are dictated by the architecture of each DNA response element. It is suggested that interface swapping in dimers could be a general mechanism of modulating the activity of transcription factors (Remenyi, 2001).
Members of the POU transcription factor family are involved in a broad range of biological processes ranging from housekeeping gene functions (Oct-1) to programming of embryonic stem cells (Oct-4) and the development of immune responses (Oct-1, Oct-2). However, according to the latest global sequencing reports, human, fly, and worm genomes encode only fifteen, five, and four POU factors, respectively. Therefore, members of this transcription factor family need to rely on multilevel control mechanisms such as posttranslational modification, interaction with heterologous transcription regulators, and flexible DNA binding to perform these multiple tasks. A linker joining the POU-specific (POUS) and the POU-homeo domain (POUH) confers the flexibility inherent to members of the POU factor family. This linker is variable both in sequence and length (15-56 residues). Since both domains are structurally and functionally autonomous in DNA binding, various arrangements on DNA are possible (Remenyi, 2001 and references therein).
POU factors were originally identified to function as monomeric transcription regulators, for instance, when they bind to the DNA octamer motif. However, more recently, their capability to homo- and hetero-dimerize on specific DNA response motifs has received substantial attention. The Palindromic Oct factor Recognition Element (PORE), ATTTGAAATGCAAAT, within the first intron of the osteopontin (OPN) gene, was initially identified as an Oct-4 DNA responsive element. It mediates strong transcriptional activation in preimplantation mouse embryos and cell lines derived thereof. Oct-4 binds to the PORE in a monomer/dimer equilibrium, in which single nucleotide replacements are sufficient to enhance or diminish dimerization. In vitro, the PORE behaves as a general Oct factor recognition element. To further investigate the general dimerization potential of Oct factors, sequences related to the Prl Pit-1 response element were characterized. These so-called MORE sequences (More palindromic Oct factor Recognition Elements: ATGCATATGCAT) are found in various promoters. All members of the Oct family tested bind cooperatively as homo and heterodimers to the consensus MORE (Remenyi, 2001 and references therein).
The transcriptional activity of Oct-1 on the octamer motif in B cells is regulated by the lymphoid-specific coactivator OBF-1 (OCA-B, BOB-1) that clamps the POUH and POUS subdomains together and thus enhances their DNA binding affinity. However, the Oct-1 dimer formed on MOREs within immunoglobulin heavy chain promoters (VH) fails to interact with OBF-1. In contrast, the Oct-1/PORE dimeric complex can interact and synergize in transcriptional activation with this cofactor. These findings established the paradigm of differential transcriptional regulation mediated by two distinct POU dimer configurations (Remenyi, 2001).
To elucidate the structural basis of this phenomenon, two crystal structures of the Oct-1 POU domain bound to the MORE and PORE have been solved. By direct comparison, these structures demonstrate how the same polypeptide chain can form two different dimer arrangements with two distinct protein-protein interfaces. These data introduce the concept of distinct transcription factor dimerization that depends on the sequence and the spacing of the protein domain binding motifs of the DNA response element. Thus, it extends previous models of protein-DNA complex formation mediated by ligand-induced allosteric effects. The members of the nonsteroid nuclear receptor family, for example, provide another mode of transcription factor dimerization dictated by the binding site, the hormone response element (HRE). HREs consist of two core hexad sequences, AGGTCA. These could form direct, inverted, or everted repeats. On inverted and everted repeats, the receptors dimerize only via a specific surface patch within the carboxy-terminal ligand binding domain. On direct repeats, however, the receptors form an additional interface between the conserved zinc finger DNA binding domains. The DNA sequence-mediated dimerization of POU proteins is different since the distinct surface patches involved in dimer interface formation are both located on the DNA binding POU domain. The results of this study show how swapping protein-protein interfaces between different quaternary arrangements of POU factor/DNA complexes can regulate transcriptional activity (Remenyi, 2001).
The Oct-1 POU factor binds to DNA in a bipartite fashion. The binding topography is the same in both complexes in the sense that the POUS domains are situated proximal to the center of each DNA element, while the POUH domains bind at distal positions. In the MORE complex, the DNA binding sites for POUH and POUS overlap by 2 base pairs within each MORE half-site, whereas in the PORE complex, the two POUS domains share 1 base pair in the center of the PORE motif. Due to the nonpalindromic nature of the PORE, the two POUS domains bind to nonidentical sites, AGGC and TTTC (Remenyi, 2001).
Each POUH domain is bound to a distal AT-motif. On the MORE, this motif is located at positions 1-2 and 11-12, while on the PORE it is located at positions 1-2 and 14-15. In both complexes, the C-terminal helix 3 of each POUH domain is situated in the major groove, with base-specific interactions of the side chains of Asn151 and Gln154. In the PORE complex, the arginine residues of the N-terminal part of POUH (Arg102 and Arg105) form DNA sequence-specific hydrogen bonds (H bonds) with the base pairs in positions 3-4 and 12-13. These interactions are formed via the minor groove within each half-site of the PORE motif. They are not visible in the Oct-1/MORE complex because this part is disordered. The POUS domains interact with bases in the major groove via their third alpha helix. In the two complexes, the side chains of residues Gln44, Thr45, and Arg49 almost identically contribute with base-specific interactions to each POUS binding site (Remenyi, 2001).
In the Oct-1/MORE structure, there are two identical protein-protein contacts forming between a POUS and a POUH domain within each half-site of the palindromic MORE motif. The buried surface per domain is about 550 Å2. A central feature of this interface is the docking of the C-terminal residues 157-160 of the POUH domain onto the loop region between helix 3 and 4 of the POUS domain. The side chain of Ile159 fits into a hydrophobic cavity of the POUS subdomain, which forms a 'knob-in-the-hole' structure. Furthermore, additional interactions are mediated by several H bonds, mostly from main chain atoms except for one side chain contribution by Asn160. This interface is reminiscent of those found in the Pit-1/DNA complexes (Remenyi, 2001).
The two main interfaces formed in the Oct-1/PORE complex are located across the center of the PORE motif, as opposed to the interface observed in the Oct-1/MORE structure. These interfaces are not identical, reflected in the different amounts of buried surface areas of 220 Å2 and 500 Å2, hence termed IF1 and IF2, respectively. Central to this interface is the N terminus of the POUH domain, which interacts with the minor groove of the PORE-DNA as well as with residues of a POUS domain. In the larger interface (IF2), one phosphate group within the minor groove (position 10 of the PORE) forms H bonds with Arg20 from the POUS and Ser107 from the POUH domain. This DNA-mediated POUS-POUH contact is surrounded by two POUS-POUH salt bridges, Asp29-Lys104 and Lys22-Glu109. Like the knob-in-the-hole interaction by Ile159 in the Oct-1/MORE complex, the exposed Ile21 from the POUS domain penetrates into a hydrophobic surface patch of the POUH domain (Remenyi, 2001).
Within the smaller interface (IF1), the only specific interaction is the POUS-POUH salt bridge between Asp29 and Lys104. Ser107 is more exposed to the solvent than in the IF2 interface. Thus, the phosphate group from the minor groove forms only one H bond with Arg20. The observed asymmetry in the PORE interface (IF1 and IF2) can be explained by the different minor groove parameters at the two half-sites of the semipalindromic PORE. Only the minor groove at the nonoctamer half-site, which is about 10% narrower and deeper than that of the octamer half-site, allows a tight fit of the two interacting POU domains within the IF2 interface (Remenyi, 2001).
It is concluded that the two structures of the Oct-1 POU factor in complex with the MORE and PORE DNA motifs reveal two alternative and unrelated dimer arrangements of the same polypeptide chain. Direct evidence is provided for the unique property of POU transcription factors to regulate their functional properties by forming different dimer assemblies. The members of the POU transcription factor family could serve as a paradigm to regulate protein function by protein-protein interface swapping (Remenyi, 2001).
At high DNA concentration, as found in the nucleus, DNA-binding proteins search for specific binding sites by hopping between separate DNA strands. This study used NMR spectroscopy to characterize the mechanistic details of intermolecular hopping for the multidomain transcription factor, human Oct-1. Oct-1 is a member of the POU family of transcription factors and contains two helix-turn-helix DNA-binding domains, POU(HD) and POU(S), connected by a relatively short flexible linker. The two domains were found to exchange between specific sites at significantly different rates. The cotranscription factor, Sox2, decreases the exchange rate and equilibrium dissociation constant for Oct-1 > or = 5-fold and approximately 20-fold, respectively, by slowing the exchange rate for the POU(S) domain. DNA-dependent exchange rates measured at physiological ionic strength indicate that the two domains use both an intersegmental transfer mechanism, which does not involve the intermediary of free protein, and a fully dissociative or jumping mechanism to translocate between cognate sites. These data represent an example of dissecting domain-specific kinetics for protein-DNA association involving a multidomain protein and provide evidence that intersegmental transfer involves a ternary intermediate, or transition state in which the DNA-binding domains bridge two different DNA fragments simultaneously (Doucleff, 2008).
Lamin B1 controls oxidative stress responses via Oct-1
Interaction of lamins with chromatin and transcription factors regulate transcription. Oct-1 has previously been shown to colocalize partly with B-type lamins and is essential for transcriptional regulation of oxidative stress response genes. Using sequential extraction, co-immunoprecipitation (IP), fluorescence loss in photobleaching, and fluorescence resonance energy transfer, it was confirm Oct-1-lamin B1 association at the nuclear periphery, and this association was shown to be lost in Lmnb1(Delta/Delta) cells. Several Oct-1-dependent genes, including a subset involved in oxidative stress response, are dysregulated in Lmnb1 cells. Electrophoretic mobility shift assay and chromatin IP reveal that Oct-1 binds to the putative octamer-binding sequences of the dysregulated genes and that this activity is increased in cells lacking functional lamin B1. Like Oct1-/- cells, Lmnb1δ/δ cells have elevated levels of reactive oxygen species and are more susceptible to oxidative stress. Sequestration of Oct-1 at the nuclear periphery by lamin B1 may be a mechanism by which the nuclear envelope can regulate gene expression and contribute to the cellular response to stress, development, and aging (Malhas, 2009).
Transcriptional regulation of Pdm homologs
Nerve growth factor (NGF) has been found to have differential effects on the levels of three POU protein transcription factors that are expressed in adult rat sensory neurons. A sensory neuron octamer-binding protein with the properties of the transcription factor Oct-2 is up-regulated 3-4-fold by NGF, as measured using nuclear extracts from adult rat dorsal root ganglion neurons grown in the presence or absence of NGF. There is a parallel increase in Oct-2 mRNA levels. In contrast, the levels of mRNA encoding the ubiquitous POU protein Oct-1 or the neuron-specific POU protein Brn-3, also present in sensory neurons, are unaffected by NGF. These observations suggest a role for Oct-2 in mediating transcriptional effects induced by NGF. In particular, as Oct-2 is known to inhibit herpes simplex virus immediate-early gene expression in neuronal cells, these findings provide a mechanism for the known action of NGF in the maintenance of latent herpes virus infections in sensory neurons (Wood, 1992).
Transcriptional targets of Pdm homologs
During S phase, transcription of mammalian histone H2B genes requires a specific promoter element and its cognate transcription factor Oct1 (OTF1). A possible mechanism for regulating histone H2B transcription during the cell cycle is direct modulation of Oct1 activity by phase-specific posttranslational modifications. Analysis of Oct1 during progression through the cell cycle reveal a complex temporal program of phosphorylation. A p34cdc2-related protein kinase that is active during mitosis may be responsible for one mitotic phosphorylation of Oct1. However, the temporally controlled appearance of Oct1 phosphopeptides suggests the involvement of multiple kinases and phosphatases. These results support the idea that cell cycle-regulated transcription factors may be direct substrates for phase-specific regulatory enzymes (Roberts, 1991).
Oct-1 and Oct-2 represent the prototypical example of related transcription factors with identical DNA recognition properties. They bind functionally critical octamer elements found in diverse regulatory sequences. It has not been possible to determine directly if these factors are functionally redundant or selective when interacting with different regulatory sequences in the appropriate cell type. An equivalent pair of altered DNA-binding specificity mutants of Oct-1 and Oct-2 were used to examine their function from varied regulatory contexts in B cells. These factors function as redundant activators of immunoglobulin (Ig) gene promoters (Vkappa and VH) and a histone H2B promoter. The structural basis of redundancy resides in the highly conserved DNA-binding POU domain, because this domain of either protein can activate transcription from both Ig and H2B promoters. The nature of a distal enhancer dictates the relative potency of Oct-1 versus Oct-2 bound to a promoter. Oct-1 preferentially stimulates transcription from a VH or Vkappa promoter in combination with enhancers from the IgH or Igkappa locus, respectively. In this context, the more potent action of Oct-1 is dependent on a region external to the POU domain. These results suggest that Oct-1 may be the critical regulator of Ig gene transcription during B cell development and provide an explanation for selective Ig isotype expression defects in Oct-2 and OCA-B null mice (Shah, 1997).
The cellular aging-associated transcriptional repressor that has been previous termed Orpheus is identical to Oct-1, a member of the POU domain family. Oct-1 represses the collagenase gene, one of the cellular aging-associated genes, by interacting with an AT-rich cis-element upstream of the gene. This repression occurs in preimmortalized cells at earlier population-doubling levels and in immortalized cells. In cells at these stages, considerable fractions of the Oct-1 protein are prominently localized in the nuclear periphery and colocalize with lamin B. During the cellular aging process, however, this subspecies of Oct-1 disappears from the nuclear periphery. The cells lacking the nuclear peripheral Oct-1 protein exhibit strong collagenase expression and carry typical senescent morphologies. Concomitantly, the binding activity and the amount of nuclear Oct-1 protein is reduced during the aging process and resumes after immortalization. However, the amounts of Oct-1 protein in the cells are not significantly changed during either process. Thus, the cellular aging-associated genes, including the collagenase gene, seem to be derepressed by the dissociation of Oct-1 protein from the nuclear peripheral structure. Oct-1 may form a transcriptional repressive apparatus by anchoring nuclear matrix attachment regions onto the nuclear lamina in the nuclear periphery (Si, 1997).
An enhancer sequence found in the Protease Nexin-1 (PN-1) gene has been shown to drive lacZ expression specifically at the met-/mesencephalic junction in transgenic mouse embryos. A functional study of this enhancer has been performed to better understand the mechanisms regulating isthmic gene expression. An octamer-binding site for POU domain factors is crucial for the activity of the enhancer in vivo. Comparative expression studies of POU domain factors, electrophoretic mobility shift assays and transient transfection experiments, strongly suggest that Brn-1/-2 regulates the enhancer activity in vivo. In addition, in vitro experiments indicate that FGF-8 is required for the maintenance of the enhancer activity, but not for the synthesis of Brn-1/-2. These data represent the first functional evidence for a role of POU factors in the regulation of met-/mesencephalic gene expression. It also implies that at least two regulatory pathways, namely the FGF-8 signaling and the octamer-binding site pathway, synergistically interact to control the PN-1 enhancer activity in vivo (Mihailescu, 1999).
Genetic variation in cytokine promoter regions is postulated to influence susceptibility to infection, but the molecular mechanisms by which such polymorphisms might affect gene regulation are unknown. Through systematic DNA footprinting of the TNF promoter region, a single nucleotide polymorphism (SNP) has been identified that causes the helix-turn-helix transcription factor OCT-1 to bind to a novel region of complex protein-DNA interactions and alters gene expression in human monocytes. The OCT-1-binding genotype, found in approximately 5% of Africans, is associated with fourfold increased susceptibility to cerebral malaria in large case-control studies of West African and East African populations, after correction for other known TNF polymorphisms and linked HLA alleles (Knight, 1999).
Invertebrate Pdm homologs
Two hypotheses have been proposed to explain the origin of insect wings. One holds that wings evolved by modification of limb branches already present in multibranched ancestral appendages that probably first functioned as gills. The second hypothesis proposes that wings arose as novel outgrowths of the body wall, not directly related to any pre-existing limbs. If wings derive from dorsal structures of multibranched appendages, it is expected that some of their distinctive features have been built on genetic functions that were already present in the structural progenitors of insect wings, and in homologous structures of other arthropod limbs. Crustacean homologs have been isolated for two genes that have wing-specific functions in insects: pdm and apterous. Their expression patterns support the hypothesis that insect wings evolved from gill-like appendages that were already present in the aquatic ancestors of both crustaceans and insects. Artemia franciscana PDM and Apterous are specifically expressed in cells of the distal epipodite before these acquire their characteristic differentiated morphology (large nuclei, large intercellular spaces). These expression patterns contrast markedly with that of Distal-less which is expressed in all outgrowing appendages (including insect legs and wings, and all crustacean limb branches). Artemia pdm and apterous are associated specifically with a distal epipodite of crustacean limbs. Crustacean epipodites are dorsally located limb branches with respiratory and osmoregulatory functions, precisely the type of structures that would have given rise to insect wings, according to some hypotheses. An alternative interpretation might be that wings did not derive from epipodites but have nevertheless independently coopted a number of gene functions that already existed in epipodites (Averof, 1997).
Developmental roles of Pdm genes
The roles of the POU domain genes Skin-1a/i (Skn-1a/i/Epoc/Oct-11) and Testes-1 (Tst-1/Oct-6/SCIP), respectively related to Drosophila pdm-1 and drifter) have been examined in epidermis where proliferating basal keratinocytes withdraw from the cell cycle, migrate suprabasally, and terminally differentiate to form a multilayered, stratified epithelium. The expression of the Skn-1a/i and Tst-1 genes is linked to keratinocyte differentiation in vivo and in vitro, whereas the ubiquitous POU domain factor Oct-1 is expressed highly in both proliferating and post-mitotic keratinocytes. Analysis of Skn-1a/i gene-deleted mice reveals that the Skn-1a/i gene modulates the pattern of expression of the terminal differentiation marker loricrin and inhibits expression of genes encoding markers of the epidermal keratinocyte wounding response. Although epidermis from Tst-1 gene-deleted mice develops normally, epidermis from mice deleted for both Skn-1a/i and Tst-1 is hyperplastic and fails to suppress expression of K14 and Spr-1 in suprabasal cells when transplanted onto athymic mice. This suggests that Skn-1a/i and Tst-1 serve redundant functions in epidermis. Therefore, at least two POU domain genes, Skn-1a/i and Tst-1, serve both distinct and overlapping functions to regulate differentiation of epidermal keratinocytes during normal development and wound healing (Andersen, 1997).
To examine the role of the Oct-6 gene in Schwann cell differentiation, the chicken and zebrafish homologues of the mouse Oct-6 gene have been cloned and characterized. Oct-6 has no known close Drosophila homolog. While highly homologous in the Pit1-Oct1/2-Unc86 (POU) domain, sequence similarities are limited outside this domain. Both genes are intronless and both proteins lack the amino acid repeats that are a characteristic feature of the mammalian Oct-6 proteins. However as in mammals, the aminoterminal parts of the chicken and zebrafish Oct-6 proteins are essential for transactivation of octamer containing promoters. By immunohistochemistry it is found that the chicken Oct-6 protein is expressed in late embryonic ensheathing Schwann cells of the sciatic nerve and is rapidly downregulated when myelination proceeds. This expression profile in glial cells is identical to that in the mouse and rat. The zebrafish Oct-6 homolog is expressed in the posterior lateral nerve at a time when it contains actively myelinating Schwann cells. Thus, despite extensive primary sequence divergence among the vertebrate Oct-6 proteins, the expression of the chicken and zebrafish Oct-6 proteins is consistent with the notion that Oct-6 functions as a 'competence factor' in promyelin cells to execute the myelination program (Levavasseur, 1998).
The vertebrate midbrain-hindbrain boundary (MHB) organizes patterning and neuronal differentiation in the midbrain and anterior hindbrain. Formation of this organizing center involves multiple steps, including positioning of the MHB within the neural plate, establishment of the organizer and maintenance of its regional identity and signaling activities. Juxtaposition of the Otx2 and Gbx2 expression domains positions the MHB. How the positional information is translated into activation of Pax2, Wnt1 and Fgf8 expression during MHB establishment remains unclear. In zebrafish spiel ohne grenzen (spg) mutants, the MHB is not established, neither isthmus nor cerebellum form, the midbrain is reduced in size and patterning abnormalities develop within the hindbrain. In spg mutants, despite apparently normal expression of otx2, gbx1 and fgf8 during late gastrula stages, the initial expression of pax2.1, wnt1 and eng2, as well as later expression of fgf8 in the MHB primordium are reduced. spg mutants have lesions in pou2, which encodes a POU-domain transcription factor. Maternal pou2 transcripts are distributed evenly in the blastula, and zygotic expression domains include the midbrain and hindbrain primordia during late gastrulation. Microinjection of pou2 mRNA can rescue pax2.1 and wnt1 expression in the MHB of spg/pou2 mutants without inducing ectopic expression. This indicates an essential but permissive role for pou2 during MHB establishment. pou2 is expressed normally in noi/pax2.1 and ace/fgf8 zebrafish mutants, which also form no MHB. Thus, expression of pou2 does not depend on fgf8 and pax2.1. These data suggest that pou2 is required for the establishment of the normal expression domains of wnt1 and pax2.1 in the MHB primordium (Belting, 2001).
Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities. In mouse, Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes. spiel ohne grenzen (spg) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape. Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed. Segment-specific expression of hoxa2, hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 (Krox20 ortholog) and valentino (val; kreisler ortholog) expression is impaired. spg mutants carry loss-of-function mutations in the pou2 gene (most closely related to Drosophila vvl). pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val. Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants, but does not induce ectopic expression of these genes. This suggests that spg/pou2 acts in a permissive manner and is essential for normal expression of krx20 and val. It is proposed that spg/pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories (Hauptmann, 2002).
The POU homeobox protein Oct-1 regulates radial glia formation downstream of Notch signaling
Radial glia cells function as guide cells for neuronal migration and a source of neural progenitor cells, and play a crucial role for the development of the central nervous system. To date, several signals have been demonstrated to promote the formation of radial glia cells and Notch signaling is one such signal. However, the mechanism of the signaling hierarchy of radial glia developmental cascade promoted by Notch signaling still remains incomplete. This study shows that Notch signaling promotes Xenopus radial glia formation and that the Notch activation is sufficient for radial glia formation prior to neural tube closure. Moreover, Oct-1 (POU2f1), a POU transcription factor, has been identified as a downstream target of Notch signaling by microarray based screen. The expression of Oct-1 in the brain is regulated by Notch signaling and Oct-1 is sufficient and necessary for radial glia formation. Together, Oct-1 is a downstream effector of Notch signaling during radial glia formation (Kiyota, 2008).
Protein interactions of Pdm homologs
Continued: Evolutionary Homologs part 2/2
nubbin/POU domain protein 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.


NVIDIA is discounting the SHIELD TV ahead of Prime Day, bringing the higher-end model, which comes with the remote and controller, down to just $179. That's good for $20 off of its regular price and also brings it back down to its all-time low. The SHIELD TV doesn't typically drop in price, so it's good to see it drop just ahead of Prime Day. This deal is going to run all week long as well, starting today, through Saturday July 21.
The NVIDIA SHIELD TV is really the only option for an Android TV set-top box, but it is also a gaming console. With Android TV, you're going to get all of the great apps that you'd expect on Android. That includes YouTube, Google Play Movies & TV, Netflix, Hulu, Sling TV and many other apps. It's a great set-top box for those that are looking to cut the cord and get rid of cable, because there are so many options for watching content without the cable box. Now on the gaming side, the SHIELD TV is one of the best gaming consoles out there. Since it does support 4K and HDR, the SHIELD TV doesn't just provide impressive performance for games, but also rather impressive graphics - as long as your TV supports it. There are a slew of games available as well as NVIDIA's GeForce system.


The Muramatsu EX-RE has a solid silver head joint, silver plated body and silver plated open hole keywork mechanism.
Since the first Muramatsu Flute was created in 1923 by the late Koichi Muramatsu, thousands of artists throughout the world have chosen Muramatsu over all other makes. The inspiration given to Osamu Muramatsu by his father is felt by the staff, who are instrumental in making the worlds finest Flute.
The research department constantly seeks to improve the instrument and their endless efforts are creating the unique Muramatsu sound prized by artists in every part of the world. Extensive technical research in the Muramatsu laboratory has led to the development of the amazingly accurate Muramatsu scale, acclaimed as the finest available. All Muramatsu Flutes are supplied complete with a deluxe case.


text:
Glossary of Grammar Terms
Glossary of Literary Terms
Allusions and Symbols in Dickens
Grammardog Teacher's guide contains 6 quizzes that identify allusions and symbols in 6 novels by Charles Dickens. 135 multiple choice questions. All sentences are from these 6 novels: A Christmas Carol, A Tale of Two Cities, David Copperfield, Great Expectations, Hard Times, and Oliver Twist.
Allusions and Symbols in Twain
Grammardog Teacher's Guide contains 6 quizzes that identify allusions and symbols in 6 novels by Mark Twain. 150 multiple choice questions. All sentences are from these 6 novels: A Connecticut Yankee in King Arthur's Court, Huckleberry Finn, The Innocents Abroad, Life on the Mississippi, The Prince and the Pauper, and Tom Sawyer.
Figurative Language in Dickens
Grammardog Teacher's guide contains 6 quizzes that identify figurative language (metaphors, similes, personification, hyperbole, onomatopoeia) in 6 novels by Charles Dickens. 150 multiple choice questions. All sentences are from these 6 novels: A Christmas Carol, A Tale of Two Cities, David Copperfield, Great Expectations, Hard Times, and Oliver Twist.
Figurative Language in Twain
Grammardog Teacher's Guide contains 6 quizzes that identify figurative language (metaphors, similes, personification, hyperbole, onomatopoeia) in 6 novels by Mark Twain. 150 multiple choice questions. All sentences are from these 6 novels: A Connecticut Yankee in King Arthur's Court, Huckleberry Finn, The Innocents Abroad, Life on the Mississippi, The Prince and the Pauper, and Tom Sawyer.
Poetic Devices in Dickens
Grammardog Teacher's guide contains 6 quizzes that identify poetic devices (alliteration, assonance, consonance, repetition, rhyme) in 6 novels by Charles Dickens. 150 multiple choice questions. All sentences are from these 6 novels: A Christmas Carol, A Tale of Two Cities, David Copperfield, Great Expectations, Hard Times, and Oliver Twist.
Poetic Devices in Twain
Grammardog Teacher's Guide contains 6 quizzes that identify poetic devices (alliteration, assonance, consonance, repetition, rhyme) in 6 novels by Mark Twain. 150 multiple choice questions. All sentences are from these 6 novels: A Connecticut Yankee in King Arthur's Court, Huckleberry Finn, The Innocents Abroad, Life on the Mississippi, The Prince and the Pauper, and Tom Sawyer.
Sensory Imagery in Dickens
Grammardog Teacher's guide contains 6 quizzes that identify sensory imagery (sight, sound, taste, touch, smell) in 6 novels by Charles Dickens. 145 multiple choice questions. All sentences are from these 6 novels: A Christmas Carol, A Tale of Two Cities, David Copperfield, Great Expectations, Hard Times, and Oliver Twist.
Sensory Imagery in Twain
Grammardog Teacher's Guide contains 6 quizzes that identify sensory imagery (sight, sound, taste, touch, smell) in 6 novels by Mark Twain. 150 multiple choice questions. All sentences are from these 6 novels: A Connecticut Yankee in King Arthur's Court, Huckleberry Finn, The Innocents Abroad, Life on the Mississippi, The Prince and the Pauper, and Tom Sawyer.
Alice's Adventures in Wonderland by Lewis Carroll
Grammardog Teacher's Guide contains 16 quizzes for this classic. All sentences are from the novel. The language is full of fun and familiar characters like the White Rabbit, Mad Hatter and Cheshire Cat. Figurative language includes lots of hyperbole (All persons more than a mile high to leave the court!) and simile combined with rhyme (Up above the world you fly, like a tea tray in the sky). Sophisticated allusions pertain to mathematics, time, law and order and toys and games.
All's Well That Ends Well by William Shakespeare
Grammardog Teacher's Guide contains 16 quizzes for this Shakespearean comedy. All sentences are from the play. Some sentences sound like proverbs (Love all, trust a few, do wrong to none). Figurative language centers on love, marriage and courtship. Alliteration creates a light-hearted tone (A young man married is a man that's marred). Allusions to mythology include Cupid, Juno, Mars, Hercules and Jove.


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Pu Lisi understood that even though this potion was only considered as an intermediate level potion, its effects was absolutely comparable to high level potions.
After Pu Lisi break away from that memory, he looked extremely shocked at the bottle of purple potion in his hands; his old eyes were full of astonishment.
How could this brat concoct a Fake Death Potion?!
It must be known that although this potion was an intermediate level potion, the concoction process of it was extremely harsh. The person must first concoct two different potions at the same time, and after completing them, that person must then use a very strong crepis gra.s.s as a medium to mix these two potions together.
Pu Lisi gasped and stared at the side where Shen Yanxiao was. He was practically wis.h.i.+ng that he could use his eyes to burn two holes into her body.
Before, he had ridiculed her when he thought that Shen Yanxiao mistook crepis gra.s.s for another medicinal herb.
But now, it seemed that she didn't make a mistake!
Her goal, since from the very beginning, was to pick a crepis gra.s.s!
After that, she concocted two different potions at the same time, which he had even witnessed right before his eyes. Even if Pu Lisi didn't want to believe it, he could only accept the facts laid before him.
Shen Yanxiao had truly concocted a Fake Death Potion!
This child who had just been in school for less than six month, and a mere thirteen-year-old stinking brat at that, had actually managed to concoct such a potion!
Actually, it wasn't too difficult to concoct two different potions, but the dose of each potion should undergo some changes, and determining the parts that needed a change was the most difficult part in concocting a Fake Death Potion.
Just a little bit of bad control and it would fail.
But the color of this bottle of Fake Death Potion in his hands was pure and no different from Ye Qing's own potion.
Pu Lisi took a deep breath and stared at Shen Yanxiao.
How could this little demon have such a great skill! Not only did she managed to create Ye Qing's Fake Death Potion that still wasn't announced in public, she even managed to do it perfectly!
And as far as he remembered, she just entered the library last week ago!
Yet in just a week, she actually managed to learn a potion that could be said to be the pinnacle of intermediate level potions. This was just crazy!
"Master Pu Lisi?" Pu Lisi's breathing became so frightening that the mentors at the side needed to carefully ask him.
Pu Lisi coldy humphed before he handed the Fake Death Potion to the mentor. He then looked at the direction where Shen Yanxiao was.
Everyone was dumbfounded by Pu Lisi's remark.
After inspecting the potion that Shen Yanxiao concocted, how could Pu Lisi suddenly announce that Shen Yanxiao won the compet.i.tion?
What exactly was going on here? What did Shen Yanxiao concoct in the end that even Shangguan Xiao's Deep Magic Potion couldn't be compared to it?
"Master Pu Lisi, what's going on here?" At the side where the mentors were, two people who were unable to make sense of anything asked.
Although unwilling to admit it, Pu Lisi had to recognize Shangguan Xiao's failure this time.
Even though the Deep Magic Potion was powerful, compared to Fake Death Potion, the distance between them was completely far away!
"Master Ye Qing's... new... potion?" At this moment, the tone of the mentors had changed.
Who was Ye Qing? Shen Yanxiao could actually concoct Ye Qing's new potion that he had researched?
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You're reading The Good for Nothing Seventh Young Lady Chapter 319. This novel has been translated by Updating. Author: North Night,夜北 already has 10340 views.
Current Time is Apr-22-2019 00:13:03 AM.


text:
Q: Newsstand application I have one application in iTunes which download books and magazines from server. I have integrated Non-consumable IAP for every books and magazines. Now, I want to integrate Newsstand to application. For that I need to integrate at least one subscription to application. I am confusing how to manage it? How can application get idea that any user has subscripted for that IAP or not?
How server will allow any particular device to download new subscription ?

A: A user buys a new subscription the same way he buys a standard IAP.
After the purchase you will get a receipt (in iOS < 7) or an entry in the App Receipt (iOS >= 7) that tells you when the subscription was bought and when it expires.
When a new issue is available, (or when a user restores his purchases etc) you will need to check that information and determine if his subscription is valid and provide him with the issues he has subscribed for.
Your server can store device tokens and the base64-encoded receipt to know which users are valid subscribers.
That's the really really really basic info on newsstand and subscriptions. There is much to read and learn in the documentation pages and in Stackoverflow.
I would suggest reading through the tutorials 1 and 2 listed below FIRST. They are a bit outdated now but they will give you a more clear view of what is ahead of you.
Subscription Guide
Newsstand notes
Tutorial 1
Tutorial 2


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Nashville Man Booted From Kenny Chesney Concert for Looking Like Singer
By Jennifer Abbey June 25, 2012
(Image Credit: WKRN-TV Nashville)
The Nashville, Tenn., man who was kicked out of Kenny Chesney's Saturday night concert in the Music City for looking too much like the country-music singer says the resemblance is out of his control.
"I can't help the way I look," Nathan Blankenship told ABC affiliate WKRN-TV in Nashville.
Blankenship was dressed similar to Chesney, wearing a sleeveless shirt and straw cowboy hat, and he says people sometimes say he looks like the star.
Security at the concert venue, LP Field, claims Blankenship was causing a disturbance among fans who thought they were seeing Chesney in the audience. Security personnel escorted Blankenship out of the stadium as a result.
"I think the way they handled it made it seem worse, like they were really escorting Kenny Chesney through the crowd," he said.
It seems Blankenship has been through this kind of thing before. An official at LP Field says Metro Nashville police knew about Blankenship's looking like Chesney, 44, before the concert began. Country stars at previous Nashville concerts have apparently spotted Blankenship in the crowd and thought he was causing a similar disturbance.
Blankenship is also upset about having to give up his $200 concert ticket to the show. Chesney's camp released a statement Monday saying it didn't know about Blankenship's getting kicked out of the concert until it was reported in the media
Concert promoter Kate McMahon of the Messina Group says they are reaching out to Blankenship to remedy the situation.


text:
package com.github.ambry.router;

/**
 * All the error codes that accompany a {@link RouterException}.
 */
public enum RouterErrorCode {
  // General errors. May occur for any operation.
  /**
   * Insufficient Ambry DataNodes could be contacted to successfully complete an operation. The caller should retry the
   * operation. An operation that changes the state of an existing blob (delete) may have partially completed and so may
   * eventually complete in the future.
   */
  AmbryUnavailable, /**
   * Caller passed in an invalid blob id and so operation could not be attempted. May occur for
   * {@link Router#getBlob(String, GetBlobOptions)}, {@link Router#deleteBlob(String)} (and their variants) operations.
   */
  InvalidBlobId, /**
   * Caller passed in an illegal argument for
   * {@link Router#putBlob(com.github.ambry.messageformat.BlobProperties, byte[], ReadableStreamChannel)}
   * operation (and its variant).
   */
  InvalidPutArgument, /**
   * Operation did not complete within specified time out. The caller should retry the operation. An operation that
   * changes the state of an existing blob (delete) may have partially completed and so may eventually complete in the
   * future.
   */
  OperationTimedOut, /**
   * Thrown when an operation is attempted after the {@link Router} is closed.
   */
  RouterClosed, /**
   * Router experienced an unexpected internal error. The caller should retry the operation. An operation that
   * changes the state of an existing blob (delete) may have partially completed and so may eventually complete in the
   * future.
   */
  UnexpectedInternalError,

  // Errors on write path. May occur for put operations.
  /**
   * Blob is too large. Cannot store blob of such size.
   */
  BlobTooLarge, /**
   * Unexpected error reading from the input channel for puts.
   */
  BadInputChannel, /**
   * Insufficient capacity available in Ambry for object to be stored.
   */
  InsufficientCapacity,

  // Errors on read path. May occur for getBlobInfo, getBlob or deleteBlob operations.
  /**
   * Blob has been deleted and so cannot be retrieved.
   */
  BlobDeleted, /**
   * No Blob could be found for specified blob id.
   */
  BlobDoesNotExist, /**
   * TTL of Blob has expired and so Blob cannot be retrieved.
   */
  BlobExpired, /**
   * The range offsets provided for a getBlob operation are invalid for the specified blob.
   */
  RangeNotSatisfiable, /**
   * The channel returned to the user in a getBlob operation has been closed before operation completion.
   */
  ChannelClosed
}

redpajama_set_name: RedPajamaGithub


Home Tutors Directory helps you to find a tutoring agency or tutor in Gravesend. View free contact details by clicking on the tutor profile link to contact the Gravesend teachers on this page. Find Maths tutors in Gravesend or search for English tutors in Gravesend who can provide individual classes. We also list 11+, Physics, French, Biology, Chemistry and Science tutors in Gravesend who can offer lessons. The teachers listed with us offer coaching for primary (KS1 and KS2), SATS, entrance exams, secondary (KS3 and GCSE), A-Level, degree and postgraduate courses for many subjects.
Can't find a tutor close to you, why not try contacting some of our tutoring agencies and tuition centres in Gravesend listed with us.
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Decorative vintage lace doily DECORATIVE The decorative arts are traditionally defined as ornamental and functional works in ceramic, wood, glass, metal, or textile. The field includes ceramics, glass, furniture, furnishings, interior design, and architecture. Decorative items are used to make ones home more beautiful and to bring joy to daily life. Add this Belgian handmade doily to your home today!
Shows normal wear to fabric and minor stains due to age and use.