text:
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WARNING: This product can expose you to chemicals including one or more listed chemicals which are known to the State of California to cause cancer or birth defects or other reproductive harm. For more information, go to www.P65Warnings.ca.gov
This kind of the Converter is a HDMI to VGA +R/L Audio converter that converts HDMI signal into analog VGA video and R/L audio signal. It allows you to enjoy your high definition image and sound when watching movies or playing games on the tradition VGA monitors or projectors.
It offers solutions for digital entertainment center, HDTV retail and show site, HDTV, STB, DVD and projector factory, noise space and security concerns, data center control, information distribution, conference room presentation, school and corporate training environments.
The HDMI converter has many features that enable it to perform in a superior manner. Among those features you will find:
• Easy to Use: Install in seconds, no need of setting.
• Conversion: It could convert complete HDMI into VGA video and R/L audio output.
• Support HDCP1.2
• Support 165MHz/1.65Gbps per channel (6.75Gbps all channel) bandwidth for HDMI input.
• Support Analogue Video output up to UXGA and 1080p with 10bit DAC
Signal: Inputs/Output:
Input Video Signal: 0.5-1.0 volts p-p
Input DDC Signal: 5 volts p-p (TTL)
Input connection: HDMI a female(19pin)
HDMI video input format: 480i/576i/480p/576p/720p/1080i/1080P
VGA video output format: 480i/576i/480p/576p/720p/1080i/1080P
Audio output format: 2 channel LPCM
Vertical Frequency Range: 50/60HZ
Input Video Amplifier Bandwidth: 1.65Gpbs/165MHz
Size(W-H-D): 88mmW x 25.5mmH x 68mmD
Weight(Net): 0.68kgs
Limited Warranty: 1 Year Parts and Labor
Operating Temperature: 0 ? to +70?
Operating Humidity: 10% to 85 % RH (no condensation)
Storage Temperature: -10? to +80?
Storage Humidity: 5% to 95 % RH (no condensation)
External Power Supply: 5V DC@1A
Converter Unit: FCC,CE,
Power Supply: CE,FCC
AC Power Adapter: US standard, UK standard and EURO standard
User Manual: English Version
Package Includes:
• 1 x HDMI input TO VGA video + R/L RL Audio output converter
• 1 x 5V DV Power Supply
• 1 x user's manual
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text:
Sunny Deol's fitting reply to those who ridicule him for his dancing!
Sunny Deol is in the midst of promoting his next, 'Ghayal Once Again', but wherever he goes, the 'Yaara O Yaara' track from his 1996 flick 'Jeet' is played and the actor's steps are mimicked.
Sunny Deol
He feels this song is stuck in people's memories. Sunny says people ridicule him for his dancing, but he came to the industry to act, not dance. Point noted.

subset: commoncrawl


text:
You might have heard that "*Utils" classes are a code smell.
Lots of people have written about that before, but I tend to find the reasoning a bit vague, and some of us work better with examples.
So here's one I found recently while working on this bug: you can't know what part of the Utils class is used when you require it, unless you do further investigation.
Case in point: if you place a method in VariousUtils.js and then import it later…
var { SomeFunction } = require('VariousUtils');
it'll be very difficult to actually pinpoint when VariousUtils.SomeFunction was used in the code base. Because you could also do this:
var VariousUtils = require('VariousUtils');
var SomeFunction = VariousUtils.SomeFunction;
or this:
var SomeFunction = require('VariousUtils').SomeFunction;
or even something like…
var SomeFunction;
lazyRequire('VariousUtils').then((res) {
SomeFunction = res.SomeFunction;
Good luck trying to write a regular expression to search for all possible variations of non-evident ways to include SomeFunction in your codebase.
You want to be able to search for things easily because you might want to refactor later. Obvious requires make this (and other code manipulation tasks) easier.
My suggestion is: if you are importing just that one function, place it on its own file.
It makes things very evident:
var SomeFunction = require('SomeFunction');
And searching in files becomes very easy as well:
grep -lr "require('SomeFunction');" *
But I have many functions and it doesn't make sense to have one function per file! I don't want to load all of them individually when I need them!!!!111
Then find a common pattern and create a module which doesn't have Utils in its name. Put the individual functions on a directory, and make a module that imports and exposes them.
For example, with an `equations` module and this directory structure:
You would still have to require('equations').linear or some other way of just requiring `linear` if that's what you want (so the search is "complicated" again). But at least the module is cohesive, and it's obvious what's on it: equations. It would not be obvious if it had been called "MathUtils" — what kind of utilities is that? formulas? functions to normalise stuff? matrix kernels? constants? Who knows!
So: steer away from "assorted bag of tricks" modules because they'll make you (or your colleagues) waste time ("what was in that module again?"), and you'll eventually find yourself splitting them at some point, once they grow enough to not make any sense, with lots of mental context switching required to work on them: "ah, here's this function for formatting text… now a function to generate UUIDs… and this one for making this low level system call… and… *brainsplosion*" 😬
An example that takes this decomposition in files to the "extreme" is lodash. Then it can generate a number of different builds thanks to its extreme modularity.
Update: Another take: write code that is easy to delete. I love it!


text:
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text:
Touring the N.Y. show ... with attitudeMarch 28, 2016
Reporters Nick Bunkley and Mike Colias were on the floor of the New York auto show last week, issuing snap judgments on the hardware faster than Dutch Mandel at Detroit Autorama. Here's a bit of their back-and-forth.
Kia's redesigned Cadenza gets a more angular lookNov. 30, 2015
Few exterior images of the redesigned Kia Cadenza for 2017 show a sharper, more angular look than the previous generation, which marked Kia's entry into the U.S. high-end car market.
2017 Kia Cadenza looks sharper, more angularNov. 24, 2015
Kia Motors unveiled today the exterior of the redesigned, sportier 2017 Cadenza luxury car, which will go on sale next year. The new Cadenza, known as the K7 in Korea, has a sharper, more angular look than the 2016 version.
Kia teases a more angular next-gen CadenzaNov. 16, 2015
Kia's decision to redesign the Cadenza sedan signals that the Korean brand will not abandon its high-end cars despite corporate sibling Hyundai's campaign to create a dedicated luxury brand, Genesis.
Kia offers first look at second-gen CadenzaNov. 12, 2015
The decision to redesign the Cadenza, which starts at about $35,000, signals Kia will not abandon its high-end cars despite Hyundai's plan to create a dedicated luxury brand, Genesis.


text:
Less than one week after he resigned over allegations of having sex with minors, former Elmo puppeteer Kevin Clash is facing another charge that he had sex with an underage boy, bringing the total up to three.
As the New York Daily News reports, the third man, known only as John Doe at this time, is accusing Clash of having sex with him in 2000, when he was only 16. As with Clash's second accuser, Cecil Singleton, Doe has alleged that he met Clash on a chat line, and then was invited over to his Manhattan apartment. Doe is said to have detailed the experience in a book he began writing in 2009, pages of which are expected to be released later today.
Clash, who had performed Elmo for more than two decades and won an Emmy for his performance, denies all charges against him. Doe and Singleton are both represented by New York lawyer Jeff Herman. Sheldon Stephens, the original accuser, who recanted his testimony before withdrawing his recantation, is still in the process of returning a settlement he received for dropping his initial charges.


text:
I got a Carbonizzare back when the stimulus came out, and while I like cruising around in it, I hated it for racing. Way too easy to spin out, especially with all the demo derby drivers.
Recently decided to try out the Coquette after hearing good things, and fell in love with it immediately. Handles like a dream around corners, and much much better at holding it's line against impacts from other racers, like it just wants to stay facing in the right direction.
Or you can have both like I do, the carbonizzare has a great engine sound, looks great, great performance, fun handling, the croquette looks good too.


text:
Pollock Pines California in El Dorado county.
Pollock Pines, California is located in El Dorado county within the zipcode 95726. With a total of 0 golf courses in Pollock Pines, it lies at an elevation of 1866 feet above sea level. The current date and time in Pollock Pines California is November 19, 2017, 6:06 pm. Add a course for free by clicking here or contact us here.


text:
|Publication number
|Publication type
|Application number
|Publication date
|May 30, 1995
|Filing date
|Jan 25, 1993
|Priority date
|Jan 30, 1992
|Fee status
|Also published as
|Publication number
|Jacek Kowalski
|Original Assignee
|Gemplus Card International
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (14), Non-Patent Citations (1), Referenced by (78), Classifications (17), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
The present invention relates to PC-cards and in particular those used for obtaining goods or services. The standard example is that of the prepaid telephone card making it possible to make telephone calls from public call boxes without having to use coins or tokens.
To facilitate the description of the present invention reference will solely be made to telephone cards, bat the invention is also usable in much wider applications of very varying types such as bank cards and the like.
A problem which is ever more frequently encountered is the compatibility between the PC-cards issued by a first service provider and those issued by another service provider, even when the services supplied are very similar.
The case of the telephone card is a perfect example of this problem. A card issued by the telecommunications bodies of one country are physically very similar, i.e. with the same standard dimensions, to those issued by another country. The position of the electrical contacts on the card and the number of such contacts are also fixed by the same international standard. This means that a card issued in one country can be physically inserted into a reader corresponding to the system of another country. However, it will still not operate, because the communication protocols between the card and the reader do not correspond, so that the reader emits signals in accordance with a protocol which the card does not understand.
The term "protocol" is intended to cover beth the time organization of electrical signals passing between the card and the reader and their spatial organization, i.e. their presence on a particular physical contact of the card or reader.
In numerous cases, it would e.g. be preferable for a French card holder to be able to use his card in Germany should the need arise, despite the protocol difference between the readers of the two countries.
To provide a more complete illustration of this problem, reference can be made to the standards which define the protocols for different systems using conventional PC-cards with eight contacts and a synchronous and sequential communication mode. In France the standard used is called TS1001, in Germany SLE4403, whilst other countries use standard TS1001 or SLE4403 or TS1301. These are standards corresponding to synchronous operations of the card, i.e. controlled by a clock based on the card reader. The operations to be carried out are extremely simple: reinitialization; reading a data item at the current address; incrementation by one address position; programming or erasing at the current address, etc.
If the eight contacts of the card are called VCC, VSS, VPP, CLK, I/O, RST, FUSE and PROG, it can be seen that the first five symbols are the same for all these standards and respectively correspond to:
VCC: positive supply voltage 5 V
VSS: earth/ground
VPP: programming voltage (approx. 15 V)
CLK: clock
I/O: data input/output.
However, the three other contacts have different meanings according to the different standards.
In standard TS1001, the FUSE contact is a general activation contact. If it is at zero, a resetting operation is performed and it must be at 1 for the operations of reading, address incrementing or programing in the card. The contact RST defines whether it is an incrementation operation (RST=0) or programming operation (RST=1) on the rising front of the clock CLK. Reading takes place in all cases on the falling front of the clock CLK.
In standard TS1301, it is the signal RST which defines the activation (RST=1) or the reinitialization (RST=0), the contact PROG defining whether or not there is programming. Reading takes place on the falling front of the clock CLK and address incrementation on the rising front of the clock CLK.
Finally, in standard SLE4403, the contact RST fulfills a double function, namely reinitialization if it lasts sufficiently long and programming on the rising front of the clock CLK if there has previously been a short positive pulse on RST and address incrementation in the absence of such a short prior pulse on RST. In all cases reading takes place on the falling front of the clock CLK.
This makes it clear that it is impossible to make a card operate in a reader, which applies to it signals which are different from what it can understand.
It could be imagined that the reader would be able to communicate with several types of cards and its operation is adapted to the card which is inserted. However, in practice, the physical processing of the signals produced by the reader and intended for the card takes place in a fixed manner, either in hardware (i.e. cables logic) or in firmware, i.e. under the control of a ROM. It would therefore be necessary to change or modify the readers and not merely their operating software if the readers would have to be made adaptable to several card types. Moreover, even if the card reader has several different protocol circuits, the user would have to indicate which type of card he was going to insert.
According to the invention, in order to be able to insert cards in readers operating according to different communication protocols, it is proposed that in the card there are:
several conversion circuits, each of which is able to convert into instructions performable by the card the electrical signals received from the reader according to a given protocol, each of the different conversion circuits corresponding to a different communication protocol,
and a protocol selection circuit, incorporating an auxiliary conversion circuit, the latter being able to produce specific instructions performable by the card, said specific instructions being used for the selection of one of the conversion circuits and being produced from electrical signals which can be produced in all the protocols.
In practice, the invention consists of essentially using those card contacts which have identical or very similar meanings in all the protocols in order to make a prior selection of the protocol which will be subsequently used in practice. Then use is made of the appropriate conversion circuit according to which the communication takes place with the chosen protocol.
The reader communicates with the card during the initial protocol selection phase temporarily using a simplified protocol performable by all reader types, no matter what their normal exchange protocol.
Preferably it is ensured that said simplified protocol is mainly constituted by a part common to all possible protocols, so that the reader can communicate simply using its own protocol, no matter what this is. This makes it unnecessary for the reader to have to use two successive protocols, i.e. firstly a simplified protocol and then a complete protocol and this in particular makes it possible to use the readers of different types which have already been installed.
During the initial phase, the card then interprets the protocol of the reader as a special protocol, which makes it possible to perform all the operations necessary for the selection of a future exchange protocol. It is the auxiliary conversion circuit which carries out this interpretation of the signals received from the reader during the initial phase.
Specifically, in the example of the cards having eight contacts referred to hereinbefore with five common contacts (VCC, VSS, VPP, CLK, I/O), use will essentially be made of the clock contact (CLK) for producing the selection instructions. The contact RST will be used for confirming the selection made. The data input/output contact (I/O) will be used for reading operations. The supply terminals are also used because they are common to the different protocols. However, in principle, the terminals PROG and FUSE will not be used.
Preferably, in a simple PC-card application in which the instructions performable by the card relate to the memory and are five in number (initialization, reading, address incrementation, programming, erasing), use is made of part of the card memory for storing a bit representing the protocol which is to be used. Thus, use is made of a simplified protocol for entering in said memory the bit identifying the protocol to be used.
Preferably, if a clock contact is one of the common contacts to the different protocols, according to the invention for the protocol selection phase specific instructions will be performed, whose meaning will be modified as a function of the duration of a clock square wave pulse appearing on said contact. In this case, it is particularly desirable to ensure that programming or erasing instructions in a memory correspond to a long square wave pulse time and reading instructions correspond to a short square wave pulse time.
The auxiliary conversion circuit is then able to receive the clock signals from the reader and is also connected to the reader by a data input-output. It preferably at least supplies the following instructions: erasing the state of a bit in the presence of a long clock signal; programming said bit in the presence of a second long clock signal immediately following the first without interposing a short clock signal between them; and incrementing by one address bit the memory activated by the rising front of a short clock signal.
The following specific supplementary instruction can then be performed: reading of the state of a bit and transmission on the data input-output contact activated by the falling front of a short clock signal.
The invention is described in greater detail hereinafter relative to non-limitative embodiments and with reference to the attached drawings, wherein show:
FIG. 1 The general circuit diagram illustrating the performance of the invention.
FIGS. 2 & 3 In exemplified manner portions of the conversion circuit of the diagram of FIG. 1 with the corresponding timing charts.
FIG. 4 An embodiment of the auxiliary conversion circuit according to the invention.
FIG. 5 The timing charts corresponding to the diagram of FIG. 4.
FIG. 1 is the circuit diagram making it possible to perform the present invention in the example of a PC-card (such as a prepaid telephone card). FIG. 1 more specifically shows the circuits incorporated into the PC-card and enabling the latter to be inserted in readers operating according to different protocols.
In a conventional PC-card chip the following elements would be present:
external access contacts connecting the chip to the reader, there being eight such contacts here which are designated VCC, VPP, VSS, CLK, I/O, RST, FUSE, PROG;
a memory MEM, e.g. an EEPROM (electrically erasable programmable read only memory). This memory is sequentially addressable in the most widely used case, i.e. a unit-incremented address counter CPT controls an address decoder DEC in order to successively designate the different memory words;
logic control circuits for the memory, generally designated CMD, which make it possible to establish on the rows and columns of the memory cells the voltages suitable for performing the various operations necessary for the function of a memory; in this case there are e.g. five such operations: reading the content of a cell at a given address, programming a cell at said address, erasing said cell; incrementation of the address to pass to a following cell; reinitialization of the address counter CPT, which defines the current address--said circuits CMD will not be described here because they are of a conventional nature and are dependent on the memory type used;
a conversion circuit able to receive the signals emitted by the PC-card reader on the card contacts and able to transform then into instructions intended for the control circuit CMD in order to make the latter perform the operations indicated hereinbefore as a function of the instruction received; the conversion circuit directly receiving the signals from the reader and establishes the orders necessary as a function of the spatial and time organization of said signals and these orders are transmitted to the control circuit CMD.
According to the invention, on the chip there are the sane access contacts, the memory MEM, the same control circuit CMD, but there are several different conversion circuits CNV1, CNV2 and CNV3, each fulfilling the function of the prior art conversion circuit, but each for a different protocol. In addition, on the chip there is a protocol selection circuit able to select a particular conversion circuit and connect it to the control circuit CMD.
The protocol selection circuit is intended to carry out this selection on the basis of an order received from the reader. Thus, the reader will start a dialogue with the PC-card by indicating to it the protocol with which it functions, so that the card adapts to said protocol. The difficulty is that the card must be able to understand the instructions from the reader, whilst still not knowing which is the protocol.
Thus, according to a feature of the invention, the protocol selection circuit mainly has an auxiliary conversion circuit CNVA able to temporarily communicate with the reader according to a concise dialogue mode making it possible to determine the protocol to be used subsequently in order to continue the data exchange. The auxiliary conversion circuit has the special feature of essentially reacting as a function of signals, which can be very easily produced in all protocols liable to be used.
In the present case, the auxiliary conversion circuit CNVA receives the supply voltages present on the terminals VPP, VCC, VSS and also receives the clock signals from the reader appearing on the terminal CLK. It receives the signal present on the terminal RSF. Finally, it is connected to the input/output terminal I/O, mainly for transmitting information to the reader. In the example described here, it is considered that these five contacts or terminals receive signals of the same nature in all the protocols and make it possible to temporarily establish a simplified protocol which is only used during the protocol selection phase.
Thus, the auxiliary conversion circuit will establish a small number of checking or control signals intended for the control circuit CMD, but only during an initial phase during the introduction of the card into the reader. Several practical possibilities exist. In a first possibility, the protocol selection circuit can designate the protocol to be used during the operation taking place and without permanently recording in the card a protocol identification to be used in the future. This would be the simplest solution.
However, preference is given to a solution in which the protocol identification to be used is recorded in non-volatile manner in the card and remains for as long as the card is not introduced into a reader having a different protocol. When the card is introduced into a reader of a different type, the information relative to the protocol is modified in a non-volatile manner. This is the case described in greater detail hereinafter.
Preferably, according to the invention and in the case of a PC-card, the first addresses of the memory are used for recording a word representing the protocol to be used.
In a simple illustrative example, it will be considered that each of the memory words comprises a bit and that the successive addresses of the memory designate successive binary cells. The first three cells of the memory are used for designating the protocol to be used. For example, if protocol 1 is to be used, a bit 1 has to be stored in the first address, the other two being at zero. If it is the second protocol which is to be used, a bit 1 will be entered in the second cell and the two others are at zero. Finally, if it is the third protocol, the bit of the third address must become 1 and the first and second are at zero.
The state of the three first memory cells is used for defining the state of three bistable flip-flops L1, L2, L3, each of which controls the putting into or out of operation of a respective conversion circuit CNV1, CNV2, CNV3. In normal operation, only one of these three flip-flops is in a state putting a conversion circuit into operation and only one protocol can operate at once. Moreover, a supplementary bistable flip-flop LO makes it possible to put out of service the three conversion circuits CNV1, CNV2, CNV3, whilst putting into service the auxiliary conversion circuit CNVA.
The control of the conversion circuits by the flip-flops is represented symbolically in the drawing by transistors, which are controlled by a flip-flop and which connect the outputs of a respective conversion circuit to the control circuit CMD. The prevention of putting into service of the conversion circuits CNV1, CNV2, CNV3 by the flip-flop LO is symbolized by the logic AND gates G1 to G3, each placed between a flip-flop L1 to L3 and a respective conversion circuit CNV1 to CNV3. These AND gates all have an input controlled by the flip-flop LO and an input connected to the output of a flip-flop L1 to L3.
The positioning of the flip-flops L1 to L3 as a function of the content of the memory MEM takes place by means of the gates G4 to G6 making it possible to transmit to each flip-flop the state of a respective bit from among the first three bits of the memory. These gates are opened by output lines of the decoder DEC (corresponding to the first three memory positions). They allow the passage to the flip-flops of the state read on an output line of the memory.
The auxiliary conversion circuit CNVA, the flip-flops L0 to L3 and the logic gates associated therewith together form the protocol selection circuit. The procedure for putting into service a particular conversion circuit takes place in the following way. On making the chip live, there is a zero initialization of the address counter CPT, which then makes it possible to designate the first memory cell by means of the decoder DEC. In parallel, at the time of activation or rendering live, the bistable flip-flop L0 is placed in a state where it puts into service the auxiliary conversion circuit CNVA or places out of service all the other conversion circuits. The following operations are then performed under the control of the auxiliary conversion circuit CNVA.
The first three memory bits are read by a succession of reading operations followed on each occasion by an incrementation of the address counter. During the reading stages, the flip-flops L1, L2, L3 assume the state corresponding to the bit stored in the respective memory cell corresponding thereto (the flip-flop L1 assuming the state of the first cell, flip-flop L2 that of the second cell, etc.).
Only one of the flip-flops will consequently be placed in a state authorizing the putting into service of one of the conversion circuits, e.g. flip-flop L2 authorizes the putting into service of the circuit CNV2.
If this corresponds to the desired protocol, i.e. that of the reader, the reader supplies a signal which switches the flip-flop L0 inhibiting the operation of the auxiliary conversion circuit CNVA and putting into service the conversion circuit CNV2 authorized by the flip-flop L2. The signal which makes the flip-flop L0 switch is preferably the combination of a clock signal CLK at logic level 1 and the signal RST at level 1. An AND gate receiving these two signals makes it possible to establish this switching control of L0.
It should be noted that the software controlling the operation of the readers must be designed so as to permit this operation. Thus, in the case of a reader according to standard SLE4403 or TS1301, it is sufficient to supply an instruction "RESET", because said instruction supplies the combination RST=1, CLK=1. However, in the case of a reader according to standard TS1001, the reader must supply a programing instruction in order to obtain the combination RST=1 and CLK=1 which will switch the flip-flop L0. The software used in the readers for said protocol selection phase must therefore take account of this requirement.
However, if the protocol information stored in the first three bits of the memory is not satisfactory, it must be changed and then the following procedure will be used. It should be noted that the reading stages which have preceded are not absolutely necessary, because it would be possible to write a protocol information in the memory without previously reading that already entered there.
It will be necessary to erase the first three bits from the memory, so as to only program that corresponding to the selected protocol. For this purpose the erased instruction and then the programming instruction will be constituted by the presence of a long clock signal, e.g. exceeding approximately 1 millisecond on the terminal CLK, the terminal RST remaining at 0. The presence of a long clock signal will be interpreted as an erase instruction. The presence of two successive long clock signals, i.e. not separated by a reading operation (short clock signal) will be interpreted as an erase instruction, followed by a programming instruction.
Thus, it would be possible to perform the following protocol by e.g. accepting that the third bit has to be programmed: the reader is positioned on a first bit, erases it, then on the second bit, erases it, then on the third bit, erases it and programs it by means of two long clock signals not separated by a short clock signal. During this time, the signal RST remains at 0.
Following the programming of the cell corresponding to the desired protocol, e.g. the third memory cell, the flip-flop corresponding to said cell (L3) will be placed in a state authorizing the putting into service of a given conversion circuit (CNV3 in this case).
Finally, simultaneously a reinitialization signal is supplied to the terminal RST and a short clock signal to the terminal CLK, which will switch the flip-flop L0. The latter stops inhibiting the selected conversion circuit and also prevents the operation of the auxiliary circuit CNVA.
It is then possible for the reader to exchange in normal manner data with the card in accordance with its own protocol, which it bas imposed on the card. The protocol number will remain recorded in the RCM for subsequent uses of the card with the same reader type and until said card is inserted in a different reader type. Thus, temporary use has been made for a protocol selection of the signals present on the terminals VCC, VPP, VSS (supply terminals), the clock terminal CLK and the terminal RST, as well as the terminal I/O for the reading operations.
In order to illustrate the significance of the conversion circuits such as CNV1, CNV2, CNV3, FIG. 2 shows the circuit portion which establishes a writing instruction WR intended for the control circuit CMD in a protocol corresponding to standard TS1301. The corresponding timing charts are also shown. The writing instruction appears during the duration of a clock signal on the terminal CLK, provided that the signal present on the terminal PROG is at state 1 at the time of the rising clock front. The instruction WR makes it possible to write or erase, as a function of the state of the input I/O.
Once again in exemplified manner, FIG. 3 shows the corresponding conversion circuit portion for the protocol SLE4403 with the corresponding timing charts. The writing instruction WR (programming or erasing, as a function of the state of I/O) appears during the time of a clock square wave pulse on the terminal CLK, provided that said pulse has been preceded by a short pulse on the terminal RST.
FIG. 4 shows the main portions of the auxiliary conversion circuit according to the invention making it possible to establish the read, write and erase signals on the basis of signals appearing on the terminal CLK and which can be produced from readers operating in accordance with any random one of the three protocols TS1001, TS1301 or SLE4403.
The simplified protocol which is used is as follows: if the clock signal is a short square wave pulse, it determines an incrementation operation on the rising front of said pulse and a reading operation on the falling front. If the clock signal is a long square wave pulse it determines a writing operation and consists of an erase operation if there is only one long square wave pulse and a programming operation if there are two successive pulses of this type.
For this purpose, the auxiliary conversion circuit can be constituted in the way shown in FIG. 4. A monostable flip-flop DT1 is connected to the terminal CLK and establishes a square wave pulse of duration t1 from the rising front of the clock CLK. An AND gate G7 receives the output of the flip-flop DT1 and the inverted output of the clock CLK. The AND gate G7 establishes a reading instruction RD only if the duration of the clock signal is shorter than t1 (reading instruction only for square wave pulses of short duration). An AND gate G8 receives the signal on CLK and the inverted output of the flip-flop DT1. It establishes a signal S1, which is a writing instruction if the clock is a shorter duration than t1.
The writing instruction S1 is transformed into an erase instruction ER or a programming instruction WR in the following way. A flip-flop RS B1 receives the logic compliment S1* of the signal S1 on a first input and the read signal RD on a second input. Its output S2 passes to 1 during a rising front of S1*, i.e. during a falling front of S1, i.e. at the end of a long square wave clock pulse and remains there for as long as it is not brought to 0 by a rising front of the read signal RD.
Said output S2 authorizes the opening of an AND gate G9, which can allow the passage of the signal S1. The logic compliment S2* of S2 authorizes the opening of an AND gate G10, which can also permit the passage of the signal S1. The output of the gate G9 can be used for forming a programming signal WR, whereas the output of the gate G10 can be used for forming an erase signal ER. The durations of these signals are substantially equal to the duration of the long square wave pulse on CLK, from which must be deducted the duration t1 of the monostable flip-flop DT1.
Operation is as follows. When the clock square wave pulses are short, the output S2 of the flip-flop RS B2 remains at zero, the gate G10 being open, but there is no signal S1 to pass through. When a long square wave pulse appears, a signal S1 is produced and passes through the still open gate G10. An erase operation is performed. At the end of the long square wave pulse on CLK, the flip-flop RS changes state and its output S2 opens the gate G9.
Two possibilities will then occur. Either a further long square wave pulse is emitted and a signal S1 is again produced, but on this occasion the signal will pass through the open gate G9, thus producing a programming operation WR. Alternatively a short square wave clock pulse is emitted (reading operation), which restores the flip-flop RS to the initial state (output S1 at zero), so that the next writing operation can only be an erase operation.
Finally, another monostable flip-flop DT2 receives the signal present on the clock terminal CLK and supplies a square wave pulse from the rising front of the clock square wave pulse. The output of said flip-flop constitutes a signal INC, which controls the incrementing of the address counter CPT. However, this signal must not appear between two successive long square wave pulses, because the erase and programming operation relate to the same bit. Not shown, supplementary logic means are used for eliminating the incrementing instructions INC for long clock signals representing a programming following an erase.
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|U.S. Classification
|235/492, 710/11
|International Classification
|Cooperative Classification
|European Classification
|Jan 25, 1993
Owner name: GEMPLUS CARD INTERNATIONAL, FRANCE
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:KOWALSKI, JACEK;REEL/FRAME:006417/0425
Effective date: 19930108
|Oct 26, 1998
|Fee payment
Year of fee payment: 4
|Nov 4, 2002
|Fee payment
Year of fee payment: 8
|Oct 30, 2006
|Fee payment
Year of fee payment: 12
|Mar 17, 2009
Owner name: GEMALTO SA, FRANCE
Free format text: MERGER;ASSIGNOR:GEMPLUS;REEL/FRAME:022399/0850
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Free format text: CHANGE OF NAME;ASSIGNOR:GEMPLUS CARD INTERNATIONAL;REEL/FRAME:022399/0835
Effective date: 19950113


text:
MOVIE SYNOPSIS [ +16 years-old ] With direction of Joe Ferraro, this film in English (original language) was presented in 2015. This film, a creation of Tim Biasi and Joe Ferraro, was produced in USA. You can watch The Getaway Car online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2015.
Rating: 5 by 10 users./
Runtime: 90 minutes
Gregg Prosser, Marc I. Daniels, Bryan Fitzgerald, James Sampson, Connor Galvin, Joe Ferraro, Jean Bonavita, Chris Byrne, Alex Recine, Chris Marth, James Castro, Joe Ferraro Sr., Joe Monteleone, Danielle Guidi, Evan Califates and Anthony Pallino.
Drama, Criminal.
90-minute film (full movie) directed by Joe Ferraro.
The original audio of The Getaway Car has dialogues in Dolby Digital, it was produced in USA.
It premiered in cinemas or television in 2015.
Screenplay written by Tim Biasi and Joe Ferraro.
Joe Ferraro.
Edward Blom and Marc I. Daniels.
This film was produced and / or financed by Jack Deuce Productions.
Jack Deuce Productions.
Without commercial breaks, the full movie The Getaway Car has a duration of 90 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, The Getaway Car in FULLTV Movies.


text:
Traveling Luck for Golo Brdo, Serbia (general), Serbia
Where is Golo Brdo?What's around Golo Brdo?
Wikipedia near Golo Brdo
Where to stay near Golo Brdo
The timezone in Golo Brdo is Europe/Belgrade
Sunrise at 06:43 and Sunset at 16:10. It's Dark
Latitude. 43.4064°, Longitude. 19.7900° , Elevation. 1627m
Satellite map around Golo Brdo
Loading map of Golo Brdo and it's surroudings
Geographic features & Photographs around Golo Brdo, in Serbia (general), Serbia
- populated place;
- a city, town, village, or other agglomeration of buildings where people live and work.
- Gornje Pravoševo (2km)
- Podkonik (2.3km)
- Bakalovina (3km)
- Zagaj (3.4km)
- Rupčine (4.2km)
- Turska Mala (4.2km)
- Gornje Pravoševo (4.4km)
- Grablje (4.5km)
- Gornje Selo (4.6km)
- Svijetnjak (4.7km)
- Pravoševo (5.1km)
- Vukovina (5.1km)
- Biskupići (5.3km)
- Donja Kosatica (5.5km)
- Vranjak (5.8km)
- Dobroila (6km)
- Ljubići (6.2km)
- Liješća (6.5km)
- Brdo (6.6km)
- mountain;
- an elevation standing high above the surrounding area with small summit area, steep slopes and local relief of 300m or more.
- Golo Brdo (0km)
- Mala Merica (1.5km)
- Velika Merica (2.4km)
- Glavice (3.5km)
- Borovniče (3.7km)
- Alin Brdo (3.8km)
- Velika Orlovača (3.9km)
- Veternik (4.4km)
- Ravna Gora (4.7km)
- Promaha (6.3km)
- Klik (6.3km)
- Pliješ (6.4km)
- peak;
- a pointed elevation atop a mountain, ridge, or other hypsographic feature.
- Velika Krševa (0.4km)
- Runjeva Glava (0.8km)
- Česte (3.7km)
- Crni Vrh (3.9km)
- Crna Stena (4km)
- Bigalj (5km)
- Gradac (5km)
- Borja (6km)
- populated locality;
- an area similar to a locality but with a small group of dwellings or other buildings.
- Drmanovići (5km)
- Bulatovići (5.5km)
- Gornja Kosatica (5.9km)
- Smreka (6km)
- Draževići (6.2km)
- locality;
- a minor area or place of unspecified or mixed character and indefinite boundaries.
- Točilo (2.4km)
- Crkvina (3.9km)
- Bakarovina (5.6km)
- Gradina (5.6km)
- Gradina (6.1km)
Accommodation around Golo Brdo
- spring(s);
- a place where ground water flows naturally out of the ground.
- Točurnica (3.1km)
- Tubića Voda (4.6km)
- Crno Vrelo (5.7km)
- hill;
- a rounded elevation of limited extent rising above the surrounding land with local relief of less than 300m.
- Zmijnjak (5.6km)
- Markovo Brdo (6.1km)
- ridge(s);
- a long narrow elevation with steep sides, and a more or less continuous crest.
- Strane (6km)
- well;
- a cylindrical hole, pit, or tunnel drilled or dug down to a depth from which water, oil, or gas can be pumped or brought to the surface.
- Brajski Bunar (2.7km)
- spur(s);
- a subordinate ridge projecting outward from a hill, mountain or other elevation.
- Ljeljenak (3.6km)
- mountains;
- a mountain range or a group of mountains or high ridges.
- Zlatar (5.8km)
- valley;
- an elongated depression usually traversed by a stream.
- Duboki Dol (5.8km)
- stream;
- a body of running water moving to a lower level in a channel on land.
- Šaša (4.6km)
- pass;
- a break in a mountain range or other high obstruction, used for transportation from one side to the other [See also gap].
- Kose (4.4km)
Wikipedia entries close to
Golo Brdo
- Prijepolje city city h
- Sjenica Airport airport airport
- Pljevlja city city i
- Golija mountain k
- Rudo city city o
- Bijelo Polje city city i
- Mokra Gora city city m
- Küstendorf city city o
- Užice city city g
- Mojkovac city city l
- Užice-Ponikve Airport airport airport
- Žabljak Airport airport airport
- Drinabrücke (Višegrad) landmark landmark
- Višegrad city city o
- Čajniče city city o
- Žabljak (Montenegro) city city m
Airports close to Golo Brdo
Podgorica(TGD), Podgorica, Yugoslavia (147.1km)
Sarajevo(SJJ), Sarajevo, Bosnia-hercegovina (148.9km)
Pristina(PRN), Pristina, Yugoslavia (162.4km)
Tivat(TIV), Tivat, Yugoslavia (167.1km)
Dubrovnik(DBV), Dubrovnik, Croatia (183.9km)


text:
Influenza causes an acute infection characterized by virus replication in respiratory epithelial cells. The severity of influenza and other respiratory diseases changes over the life course and during pregnancy in women, suggesting that sex steroid hormones, such as estrogens, may be involved. Using primary, differentiated human nasal epithelial cell (hNEC) cultures from adult male and female donors, we exposed cultures to the endogenous 17β-estradiol (E2) or select estrogen receptor modulators (SERMs) and then infected cultures with a seasonal influenza A virus (IAV) to determine whether estrogenic signaling could affect the outcome of IAV infection and whether these effects were sex dependent. Estradiol, raloxifene, and bisphenol A decreased IAV titers in hNECs from female, but not male, donors. The estrogenic decrease in viral titer was dependent on the genomic estrogen receptor-2 (ESR2) as neither genomic ESR1 nor nongenomic GPR30 was expressed in hNEC cultures and addition of the genomic ER antagonist ICI 182,780 reversed the antiviral effects of E2. Treatment of hNECs with E2 had no effect on interferon or chemokine secretion but significantly downregulated cell metabolic processes, including genes that encode for zinc finger proteins, many of which contain estrogen response elements in their promoters. These data provide novel insights into the cellular and molecular mechanisms of how natural and synthetic estrogens impact IAV infection in respiratory epithelial cells derived from humans.
- antiviral defenses
- estradiol
- respiratory disease
- SERMs
- zinc finger proteins
biological differences exist between males and females, yet these differences are often overlooked in experimental studies in immunology and infectious diseases (2). The recent policy announcement by the National Institutes of Health (NIH) that applicants consider balancing the sexes in preclinical, cell culture, and animal studies speaks to the importance of sex as a biological variable that can help predict the outcome of diseases (4, 19). Clinical observations reveal that respiratory diseases, such as asthma, chronic obstructive pulmonary disorder, and influenza are often more severe in women than men, being highly dependent on both age and hormonal status (3, 18). Influenza, in particular, is a major recurring cause of morbidity and mortality worldwide, affecting 10% of the population annually through epidemics, pandemics, and seasonal outbreaks. When age and sex are included in analyses of influenza pathogenesis, young adult women experience more severe outcomes compared with age-matched men (42). Young adult women were reportedly two to six times more likely to die from H5N1 or H7N9 avian influenza infection and during the initial wave of the 2009 H1N1 influenza pandemic (14, 51, 64). Influenza is considered an immune-mediated disease, in which the disease severity is largely determined by the strength of the host inflammatory response to infection (56). Murine models have been instrumental in characterizing sex differences in the outcome of influenza A virus (IAV) infection (26, 46, 47). These studies collectively illustrate that young adult female mice suffer a worse outcome from infection with IAV than their male counterparts. Sex differences in the outcome of IAV in mice are not caused by differences in virus replication in the lower respiratory tract (i.e., lungs) but rather are associated with an exacerbated proinflammatory cytokine and chemokine response in the lungs (26, 47). Regulating the balance between immune responses causing protection or pathology is integral for repair of pulmonary tissue and recovery from infection.
Murine models further reveal that sex differences in the outcome of respiratory diseases, including IAV, are mitigated in the absence of sex steroid hormones (46, 47), suggesting that the hormonal milieu plays a fundamental role in determining sex-specific differences in the outcome of infection. Endogenous sex steroid hormones, such as 17β-estradiol (E2), are immunomodulatory and regulate cellular immune responses to infection (54, 62). In mouse models of asthma, allergy, and IAV, females suffer a worse outcome from pulmonary diseases compared with males (30, 47). Among female rodents, endogenous changes in circulating E2 concentrations alter lung function and exogenous E2 treatment reduces pulmonary inflammation and disease symptoms (22, 30, 45, 65). Continuous E2 exposure, but not cyclical or ablated E2, in adult female mice prolongs survival following IAV infection (47). In women, sustained concentrations of E2 are associated with improved outcome of respiratory diseases, such as asthma. For example, administration of oral contraceptives containing E2 reduces allergic inflammation in the respiratory tract and asthma exacerbations associated with premenstrual asthma (22, 59).
Estrogens, primarily E2, regulate cellular function in diverse cell types, via binding to intracellular, genomic estrogen receptors (ERs), such ERα (ESR1) and ERβ (ESR2), or to membrane-associated, nongenomic ERs, such as the G protein-coupled estrogen receptor (GPER). These receptors are cell type specific and can activate gene expression directly by serving as ligand-dependent transcriptional factors (ESR1 and ESR2) or indirectly by altering signaling pathways, including ERK/MAPK, NF-κB, and STAT activity (10, 54). Both immune cells and nonimmune cells (e.g., respiratory epithelial cells) have intracellular ERs that regulate cellular functions, including inflammatory responses (10). Targeting ER activity might be therapeutic for diseases that are caused by excessive inflammation and that disproportionately affect females. Selective estrogen receptor modulators (SERMs) bind to ERs to cause conformational changes that alter engagement with coactivator and corepressor proteins to initiate either induction or suppression of target gene transcription (21, 38). A recent screen revealed antiviral properties of two SERMs against Ebola virus infection: clomiphene, which is used to treat infertility, and raloxifene, approved for treatment of osteoporosis and to decrease the risk of breast cancer in postmenopausal women (17). Environmental estrogens, including xenoestrogens, such as bisphenol A (BPA), also can interact with ERs to alter transcriptional activity (57), but the effects on cellular responses to viruses have not been evaluated. In this study, we used primary differentiated human nasal epithelial cell (hNEC) cultures isolated from healthy tissue of male and female donors (8, 20, 44) to study the effects of estrogenic compounds on the human cellular response to IAV infection. Nasal epithelial cells are the primary cell type infected with IAV, and these cultures allowed us to investigate IAV infection and pathogenesis based on the sex and hormonal milieu of the donor cultures.
Madin-Darby canine kidney cells.
Madin-Darby canine kidney (MDCK) cells were propagated in Dulbecco's modified Eagle's low-phenol, high-glucose medium (DMEM; Invitrogen) containing 10% fetal bovine serum (FBS; Life Technologies), 100 U/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), and 2 mM l-glutamine (Life Technologies). The cells were maintained at 37°C in a humidified environment with 5% CO2.
The recombinant influenza A/Udorn/307/72 H3N2 virus (IAV) used in this study was described previously (41). The hemagglutinin (HA) protein contains amino acid changes (L226Q/S228G in the H3 numbering system) that allows for preferential binding to α2,3-linked sialic acid. Viral working stocks of were generated by infecting MDCK cells at a multiplicity of infection (MOI) of ∼0.01 infectious units per cell in low-phenol, high-glucose DMEM containing 0.25% bovine serum albumin (BSA; Life Technologies), acetylated trypsin from bovine pancreas (5 μg/ml; N-acetyl trypsin; Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin, 6 mM l-glutamine, and 1 mM sodium pyruvate (Life Technologies), which we refer to as infection media. The infected cells were incubated at 37°C for 72 h, and infected cell supernatant was harvested, clarified by centrifugation, aliquoted, and stored at −80°C. Infectious virus titers were determined by 50% tissue culture infectious dose (TCID50) as described previously (33).
Stock solutions of 17β-estradiol (E2; 1 mM; Sigma) were made in ethanol, while raloxifene (100 mM; Sigma), ospemifene (100 mM; Sigma), clomiphene citrate (100 mM; Sigma), ICI 182,780 (ICI; 2 mM; Tocris-Cookson), and BPA (44 mM; Sigma; provided by Dr. Jodi A. Flaws, University of Illinois at Urbana-Champaign) were made in dimethyl sulfoxide (DMSO; Sigma). All stock solutions were stored at −20°C.
hNEC cultures.
Primary hNECs were collected from nondiseased nasal mucosa of patients undergoing endoscopic sinonasal surgery for nonsinusitis indications, including dacryocystorhinostomy, approach to anterior skull base pathology, or removal of benign nasal masses. The cells were collected from male (n = 10) and female (n = 42) donors (age range 18–45 yr). The research protocol was approved through the Johns Hopkins Institutional Review Board, and all subjects gave signed informed consent. The cells were differentiated at an air-liquid interface in 24-well Falcon filter inserts (0.4-μM pore; 0.33 cm2; Becton Dickinson) coated with human type IV placental collagen (Sigma-Aldrich) as described previously (8, 20, 44).
Infection and treatment of hNEC cultures.
Before infection, the apical surface of hNEC cultures was washed with Dulbecco's PBS with calcium and magnesium (DPBS; Life Technologies). The cultures were infected via the apical chamber with a MOI of 0.1 TCID50/cell or mock infected at 32°C in 200 μl of infection media. After 2 h, the inoculums were aspirated, the apical surfaces washed twice with DPBS, and cells were incubated at 32°C. At the indicated hours postinfection (hpi), 200 μl of infection media were added to the apical surface, incubated at 32°C for five min, and then collected. All samples were stored at −80°C. At the indicated times pre- or postinfection, the basolateral media were replaced by media containing doses of vehicle control (EtOH or DMSO as appropriate), E2 (0.1, 1.0, or 10 nM), BPA (44 μM), raloxifene (50 μM), ospemifene (50 μM), clomiphene citrate (50 μM), or ICI (100 nM). Basolateral media were collected and replaced with media containing fresh vehicle or compound at 48 and 96 hpi.
TCID50 assay.
MDCK cells were plated in a 96-well plate and cultured for 72 h in growth DMEM until 90–100% confluent. The cells were washed twice with DPBS and then covered with 180 μl of infection media. Serial dilutions (10−1 to 10−8) of hNEC apical samples at each time point were made in separate, 96-well plates by adding 20 μl of each apical sample to 180 μl of infection DMEM and then serially diluted 10-fold. Twenty microliters of each dilution were then added to the MDCK plates in replicates of six, resulting in final dilutions of each sample ranging from 10−2 to 10−9. The infection proceeded for 7 days at 32°C, and then the cells were fixed with 4% formaldehyde and stained with napthol blue-black solution. The cytopathic effect was scored visually and the Reed and Muench calculation was used to determine the titer of infectious virus at each time point.
Multiplex chemokine assay analysis.
The Meso Scale Discovery (MSD) multiplex assay system was used to measure chemokines collected from apical samples of hNECs after infection. In each well, the Chemokine Panel 1 kit quantitatively determined the concentration of eight C-C ligand motif (CCL2, CCL3, CCL4, CCL11, CCL13, CCL17, CCL22, and CCL26) and two C-X-C ligand motif (CXCL8 and CXCL10) chemokines, according to the manufacturer's protocol. All samples were run in duplicate. MSD plates were read on the MSD SECTOR Imager 2400 at the Becton Dickinson Core Facility at the Johns Hopkins Bloomberg School of Public Health and analyzed on the accompanying software (MSD Discovery Workbench version 4).
Enzyme-linked immunosorbent assay.
The PBL Assay Science DIY Human IFN Lambda 1/2/3 (IL-29/28A/28B) enzyme-linked immunosorbent assay (ELISA) was used to measure levels of interferon-λ (IFNλ) collected from apical samples of hNECs after infection. All samples were run in duplicate and read on the FilterMax F3 Multi-Mode Microplate Reader (Molecular Devices). The data were analyzed with the accompanying software (SoftMax Pro Data Acquisition and Analysis Software).
Real-time RT-PCR.
At the indicated times postinfection, hNEC cultures were treated with TRIzol Reagent (Life Technologies) and total RNA was extracted using the PureLink RNA Mini Kit (Ambion by Life Technologies) according to the manufacturer's protocol. Reverse transcriptase generation of complementary DNA (cDNA) was performed with 0.5 μg of total RNA, primed with a blend of random hexamer and oligo (dT)s against the RNA library, using an iScript RT Kit (Bio-Rad Laboratories). Real-time PCR (qPCR) using Ssofast qPCR Supermix with EvaGreen (Bio-Rad Laboratories) was conducted using the StepOnePlus Real-Time PCR System (Life Technologies) and accompanying software (StepOne Software) according to the manufacturer's instructions. A dilution curve was generated from a compilation of the samples and qPCR analysis was performed using forward and reverse primers for estrogen receptor-α (ESR1), estrogen receptor-β (ESR2), G protein-coupled estrogen receptor 1 (GPER), and the zinc finger protein genes ZNF91, ZMYM6, and ZFAND4. An initial incubation of 95°C for 10 min was followed by 94°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 10 s, for 40 cycles, followed by a final extension at 72°C for 10 min. A melting curve was generated at 55–90°C to monitor the generation of a single product. 18S RNA (RNA18S) was used as a reference gene for each sample. Final values of relative gene expression were calculated and expressed as the ratio normalized to RNA18S. All analyses were performed in duplicate or triplicate.
Western blot analysis.
At the indicated times postinfection cells were lysed with 1% sodium dodecyl sulfate (SDS; Fisher Scientific) in PBS and total protein was measured using the Pierce BCA Protein Assay Kit (Life Technologies) according to the manufacturer's instructions. BSA dilutions (Pierce BCA Protein Assay Kit) were used as the standards. For Western blot analysis, 7–30 μg of total protein from each sample were separated on 4–15% Mini-PROTEAN TGX Precast Gels (Bio-Rad) and transferred to a polyvinylidene fluoride Immunobilon-FL membrane (PVDF; Millipore). The membranes were blocked with PBS containing 5% dry milk powder and 0.05% Tween-20 (Sigma). Wash buffer contained PBS with 0.05% Tween-20. Membranes were blocked for 30 min at room temperature (RT), incubated for 1.5 h at RT with primary antibody, washed three times each for 5 min, incubated for 1 h at RT with secondary antibody, and then washed three times each for 5 min. Primary and secondary antibodies were diluted in blocking buffer. The primary antibodies used were mouse anti-β-actin (AC-15 MAb; 1:5000 dilution; Abcam-ab6276), rabbit anti-ESR2 (PAb; 1:100 dilution; Thermo- PA1-310B), and rabbit anti-ESR1 (1:500 dilution; Genetex-GTX62423). The Alexa Fluor 647-conjugated secondary antibodies used were a goat anti-mouse immunoglobulin G (IgG) and a donkey anti-rabbit IgG (both at a 1:1,000 dilution; Invitrogen). For visualization, membranes were imaged using a FluorChemQ phosphorimager (ProteinSimple) and signal intensities quantified by ImageJ analyses (NIH).
Identification of putative estrogen response elements.
The Dragon ERE Finder version 3.0 (http://datam.i2r.a-star.edu.sg/ereV3/) was used to identify putative estrogen response elements (EREs) in the promoters of select genes (1). The NCBI BLAST program was used search for the annotated start sites of genes of interest against ∼11,000 human promoters. These sequences were then used to pinpoint the location of the putative ERE pattern by expanding them to cover an extended region of [−5,000, +3,000] relative to genes of interest start site. We used the recommended search sensitivity of 83%.
Microarray analysis.
Trizol reagent and the PureLink RNA Mini kit (Ambion/Life Technologies) were used for extraction and purification of RNA. Cells were processed according to the manufacturer's protocol. Following elution of purified RNA from the PureLink columns with Nuclease-free water, quantitation was performed using a NanoDrop spectrophotometer and quality assessment was determined by RNA Nano LabChip analysis on an Agilent BioAnalyzer 2100 or RNA Screen Tape on an Agilent TapeStation 2200. One hundred nanograms of total RNA were processed for hybridization to Affymetrix Human Gene ST 2.0 microarrays using the Affymetrix GeneChip WT PLUS Reagent Kit according to the manufacturer's recommended protocol. The signal amplification protocol for washing and staining of eukaryotic targets was performed in an automated fluidics station (Affymetrix FS450) using Affymetrix protocol FS450_0002. The arrays were scanned in the GCS3000 laser scanner with autoloader and 3G upgrade (Affymetrix). Quality assessment of hybridizations and scans was performed with Expression Console software (Affymetrix).
Statistical analyses.
For analysis of virus titers, cytokine/chemokine levels, and real-time PCR data, comparisons among treatment groups were performed using multivariate analysis of variance (MANOVA; for repeat measures) followed by planned comparisons or one-way ANOVA with appropriate post hoc tests using SigmaPlot 12 (Systat Software). For microarray analyses, Partek Genomics Suite version 6.6 was used, with the robust multichip average (RMA) algorithm used for background correction, normalization, and summarization of probes. ANOVAs with linear contrasts were performed to generate relative fold change, P values, and gene lists. Statistical significance was assigned at P ≤ 0.05.
Pretreatment with E2 reduces viral titers in hNECs from female, but not male, donors.
To test the hypothesis that E2 has antiviral effects on IAV infection, hNEC cultures from female and male donors were pretreated in the basolateral media with E2 (0.1, 1, or 10 nM) or vehicle for 24 h before infection with IAV. During peak virus replication (i.e., 72–96 hpi), E2 pretreatment of hNEC cultures from female (Fig. 1A) but not male (Fig. 1B) donors significantly reduced virus titers compared with vehicle-treated hNEC cultures (P < 0.05). We interpret these data to suggest that E2 has a sex-specific antiviral activity in hNEC cultures.
To determine whether the duration of E2 exposure could shift the timing of the antiviral effects of E2, hNEC cultures from female donors were exposed to E2 72 h before or 24 h after infection with IAV. Pretreatment of hNEC cultures with E2 for 72 h resulted in a significant reduction in virus titers 48 and 72 hpi compared with vehicle-treated cultures (P < 0.05; Fig. 2A), while treatment of hNEC cultures 24 hpi significantly reduced virus titers at 72 hpi (P < 0.05; Fig. 2B). Taken together, these data suggest that the antiviral activity of E2 in hNEC cultures from female donors can be induced with treatments over an extended timeframe.
E2 does not affect production of cytokines in response to IAV infection in female donors.
Respiratory epithelial cells are significant producers of cytokines, chemokines, and type III interferons in response to IAV infection (36, 49). To test the hypothesis that E2 reduces IAV titers by altering host immune responses to infection, hNEC cultures from female donors were pretreated with E2 (0.1, 1, and 10 nM) or vehicle for 24 h before infection, and the apical supernatants were analyzed at 72 hpi (i.e., during peak virus replication) for production of a panel of chemokines and type III interferons. Concentrations of IFNλ and all chemokines measured, with the exception of CCL13 and CCL26, were significantly increased in IAV-infected hNEC cultures compared with mock infected cultures derived from females (P < 0.05 in each case; Table 1). In contrast to the immunomodulatory effects of E2 reported in reproductive epithelial cells (11, 50, 52), E2 did not significantly alter secretion of any chemokine or IFNλ in either the mock- or IAV-infected hNECs compared with the vehicle-treated hNECs. Therefore, the antiviral effects of E2 on IAV infection do not involve altered apical production of IFNλ or chemokines.
The antiviral effects of E2 require intracellular ESR2 signaling in hNECs from female donors.
The ER family is comprised of many ER and ER-like proteins, including the intracellular genomic E2 receptors ESR1 and ESR2 and the nongenomic G protein-coupled receptors (e.g., GPER). To test the hypothesis that the antiviral effects of E2 were mediated by signaling through genomic rather than nongenomic receptors, we pretreated hNEC cultures from females with E2 (10 nM), the genomic ER blocker ICI 182,780 (100 nM), E2 + ICI, or vehicle for 24 h before inoculation with IAV. Pretreatment of hNEC cultures with E2 significantly reduced virus titers at 72 and 96 hpi compared with vehicle-treated cells (P < 0.05; Fig. 3), but the addition of ICI with E2 reversed the antiviral effects of E2 resulting in virus titers that were indistinguishable from vehicle-treated cells (Fig. 3). These data suggest that the antiviral effects of E2 are mediated by genomic signaling through intracellular ERs.
To determine whether hNEC cultures expressed genomic and nongenomic ERs, we measured transcripts of ESR1, ESR2, and GPER in hNECs derived from male or female donors treated with either vehicle or E2 before mock or IAV infection. At 24 hpi, transcripts of GPER were not detected in hNEC cultures from male or female donors (data not shown). Although ESR1 was expressed in hNECs derived from both male and female donors, the expression level was low and was not altered in the presence of E2 (Fig. 4A). In contrast, ESR2 was expressed to higher levels in hNECs derived from female than male donors following exposure to E2 (P < 0.05; Fig. 4B). To evaluate the kinetics of genomic ER expression, transcripts of ESR1 and ESR2 were measured 24 and 48 hpi in hNEC cultures from female donors. Detection of ESR1 mRNA remained low at both 24 and 48 hpi and was not altered by treatment with E2 (Fig. 4C). In contrast, treatment of hNECs from females with E2 significantly increased the expression of ESR2 at 24 hpi but not 48 hpi (P < 0.05; Fig. 4D).
To confirm that hNEC cultures derived from female donors expressed genomic ER proteins, cell lysates at 24 hpi were subjected to Western Blot analysis. At 24 hpi, ESR2, but not ESR1, was detected in hNECs derived from females (Fig. 5, A and B). Both proteins were detected in MCF-7 cells, indicating the lack of ESR1 expression in hNEC cultures was not due to an inability of the antibodies to detect the protein. Taken together, these data suggest that the activation of ESR2 by E2 in female, but not male, hNECs mediate the sex-specific antiviral effects of E2.
E2 reduces metabolic processes in hNEC cultures from females following IAV infection.
To uncover potential pathways affected by E2-ESR2 signaling in hNEC cultures derived from females, we conducted microarray analyses on RNA isolated at 24 and 48 hpi. To determine the selective effects of E2 on gene expression during IAV infection, samples from IAV-infected cultures were normalized to mock-infected cultures from the same hormone treatment condition. Overall, significantly more genes were altered by E2 treatment at 24 compared with 48 hpi. Using a fold change of 1.3 and P < 0.05, we determined that 666 transcripts were differentially expressed in hNECs treated with E2 compared with vehicle at 24 hpi and only 234 transcripts were differentially expressed at 48 hpi (Fig. 6A). There were only seven overlapping genes that were differentially expressed in hNECs treated with E2 compared with vehicle at both 24 and 48 hpi (Fig. 6A). Gene Ontology Enrichment revealed that the biological functions with the greatest number of differentially expressed genes in hNECs treated with E2 compared with vehicle at 24 hpi included organelle organization and cellular metabolic processes, both of which received significant enrichment scores (i.e., > a score of 3; Fig. 6B). Further analyses revealed that a family of zinc finger protein (ZFP) genes was the largest cluster of genes differentially expressed at 24 hpi in response to E2 treatment of hNEC cultures (Fig. 6C).
To determine whether E2 could transcriptionally regulate the activity of these zinc finger genes, we used Dragon ERE Finder version 3.0 (http://datam.i2r.a-star.edu.sg/ereV3/) to identify putative EREs in the promoter regions of ZFP genes. Of the 38 ZFP genes that were downregulated during IAV infection by E2, 21 had at least one putative or known EREs present in their promoters (Fig. 7A). Of the 21 ZFP genes that had putative EREs in their promoters, 20 of these genes had two or more putative EREs, 11 of which have been experimentally confirmed to bind to ERs (1). ZFPs play diverse roles in regulating cellular function and responses to damage; their role in IAV infection, however, is largely unknown. Of the 38 ZFP genes that were downregulated in the presence of E2 during IAV infection, ZNF91, ZMYM6, and ZFAND4 were of particular interest because of the large number of EREs present in their promoters (i.e., ZNF91 and ZMYM6) or because they belong to a family of ZFPs that can have antiviral activity (i.e., ZFAND4) (28). The effects of E2 on the expression of ZNF91, ZMYM6, and ZFAND4 transcripts were validated using real time RT-PCR, which confirmed that E2 significantly downregulated ZNF91 and ZMYM6 at 24 hpi but not 48 hpi (P < 0.05; Fig. 7B). While ZFAND4 expression was reduced at 24 hpi, this did not achieve statistical significance (P = 0.057; Fig. 7B). Taken together, these data suggest that E2 has the potential to directly regulate transcription of ZFP genes, which may provide a novel mechanism for reducing late stage virus replication.
Estrogenic chemicals that selectively bind to ESR2 reduce IAV titers in hNECs from females.
Selective estrogen receptor modulators are a class of FDA-approved therapeutic estrogenic chemicals, while BPA is a xenoestrogen forming the backbone of various plastic and consumer products. These estrogenic chemicals have differential affinity for ERs in a variety of tissues (21). To determine if compounds that bind genomic ERs could decrease IAV titers, similar to E2, hNEC cultures from females were pretreated with raloxifene, clomiphene citrate, ospemifene, BPA, or vehicle 24 h before infection with IAV. Raloxifene, but not clomiphene citrate or ospemifene, significantly reduced viral titers at 72 and 96 hpi compared with vehicle-treated cultures (P < 0.05; Fig. 8A). Treatment of hNECs with BPA also significantly reduced viral titer at 48 and 72 hpi compared with vehicle-treated hNECs (P < 0.05; Fig. 8B). Both raloxifene and BPA have stronger binding affinities for ESR2 than ESR1 (24), suggesting that chemicals that selectively bind ESR2 have antiviral activity against IAV infection of respiratory epithelial cells from females.
In this study, E2 and select estrogenic chemicals had antiviral effects against IAV infection. These estrogenic compounds reduced peak viral titer, which did not involve changes in the secretion of chemokines or IFNλ, but rather was associated with reduced metabolic processes. The effects of estrogens were specific for hNEC cultures from female, but not male, donors. The antiviral effects were most likely mediated by signaling through ESR2 as ESR2, but not ESR1 or GPER, was expressed in hNEC cultures and the protective effects of E2 were eliminated in the presence of the genomic ER antagonist ICI 182,780.
Antiviral effects of E2 and estrogenic chemicals have been reported previously against viruses including HIV, hepatitis C virus (HCV), Ebola virus, and human cytomegalovirus (HCMV). Estradiol can prevent HIV transcription by blocking HIV promoter activity in peripheral blood mononuclear cells and preventing HIV entry in CD4+ T cell and macrophages (48, 55). Estradiol also prevents the production of infectious HCV particles (12). Certain FDA-approved SERMs inhibit Ebola and other Marburg viruses in vivo and in vitro (17). Furthermore, SERMs, including clomiphene and raloxifene, inhibit HCV infection at multiple stages of the virus life cycle and, raloxifene, in particular, acts as an adjuvant to improve antiviral HCV therapies in postmenopausal women (9, 37). While E2 has antiviral activities against a wide range of viruses, it is still not clear whether the molecular mechanisms involved are conserved across virus families or result from changes in different cellular pathways resulting from the broad effects of E2 on cellular gene expression. Implications of these findings are far reaching as a significantly greater proportion of the world population is exposed to IAV and suffer from influenza annually than Ebola, HCV, HCMV, or HIV (63).
The inhibition of IAV infectious virus production in E2 or SERMs treated hNECs from female donors only occurred at late times after low MOI infection, with the magnitude of the antiviral effects being greater when cells were treated before rather than after the initiation of infection. Because the inhibition of virus titer occurs at a time when the infection in the hNECs is no longer synchronized among the cells in the cultures, the E2-induced reduction in titer could be resulting from E2 effects at multiple stages of the virus life cycle (i.e., entry, replication, reinfection). Therefore, we hypothesized that there may be some combination of host response and virus infection occurring synergistically to impair virus replication at later time points of infection.
Our microarray data indicate that E2 significantly alters the expression of genes involved in metabolic processes. Within the differentially regulated genes comprising metabolic processes, the largest family of E2-downregulated genes in IAV-infected hNECs from females was the one that encodes for ZFPs. Very little is known about the function of many of these ZFPs, let alone their potential roles in the IAV life cycle. In general, ZFPs are transcriptional regulators that can induce or repress host gene transcription (25). Because these ZFPs are all similarly downregulated in response to E2 treatment and IAV infection and expressed EREs in their promoters, this may point to a common transcription factor or activation pathway responsible for the antiviral effects of E2. There is a growing appreciation of the importance of various metabolic, cell cycle, and lipid metabolism pathways for productive IAV infection (60), and downregulation of these ZFPs may be exerting an antiviral activity by altering those pathways.
Both ESR1 and ESR2 are present in the upper and lower respiratory tract and are required for the development and function of the lung but exhibit distinct cell-type-specific expression patterns (5, 15, 29). ESR2 has been found in bronchial epithelial cells and alveoli, whereas ESR1 is mostly limited to alveolar macrophages and endothelial blood vessel cells, with modest expression in bronchial epithelial cells (15, 29, 40, 58). In primary human bronchial epithelial cells (HBECs) and HBEC cell lines from males and females, the amount of ESR2 is almost doubled compared with ESR1 (15). In the nasal mucosa, ESR2 is also the dominant ER present and, in some studies, the only ER detectable (34, 43, 53), which is consistent with our data. Similar to our results, in lung adenocarcinoma cells, only the ERs in cells from females respond to E2 and other ER ligands (7). Although no studies have investigated the ESR1:ESR2 relationship in the respiratory tract, in mammary, uterine, and prostate tissue, where both subtypes of ER are expressed, ESR2 is antagonistic to ESR1, inhibiting ESR1-mediated gene expression (13, 31). The expression of ESR2 has also been shown to alter the recruitment of ESR1 transcriptional cofactors and increase the degradation of ESR1, suggesting that ESR2 can mediate or dominate ESR1 signaling in the same cell (31, 32), which may explain why low levels of ESR1 mRNA, but not ESR1 protein, is detectable in hNECs.
ESR1 and ESR2 have similar DNA binding homology but only share 57% homology in their ligand binding domains, contributing to differences in transcriptional activities and tissue-specific cellular responses triggered through ligand-dependent ER activation (27). For example, E2, through ESR1, the dominant receptor in the female reproductive tract, decreases the production of human β-defensin 2 (HBD2) and elafin in the vaginal epithelium but increases HBD2 and elafin in the uterine epithelium (61). ESR2 is required for overall lung function, whereas ESR1 is required to control inflammation in the lung (35, 58). In mice, signaling through ESR1, but not ESR2, reduces pulmonary concentrations of proinflammatory cytokines and chemokines in the lungs and improves the outcome of influenza in female mice (47). In addition to the tissue type, the biological effects of E2 are contingent on the concentration of E2. Physiological levels of E2, signaling through ESR1, promote proinflammatory type 1 IFN production via Toll-like receptor stimulation and induction type 1 IFN genes (23). Conversely, high E2 levels consistent with those found before ovulation and during pregnancy are protective against excessive inflammatory responses (54). In our study, physiologically relevant levels of E2 had no effect on the induction of chemokines or IFNλ in cells from the upper respiratory tract. This may reflect a fundamental difference between signaling through ESR2 in upper respiratory tract epithelial cells and signaling through ESR1 in immune cells in the lower respiratory tract and reproductive tract. Future studies will need to compare ER signaling in mouse and human epithelial and immune cells in the upper and lower respiratory tract to definitively resolve the differences in how E2 protects against IAV in both hNECs and mice. Collectively our data illustrate that cell type-specific differences in ER expression result in differential modes of protection.
In the current study, of the SERMs tested, only raloxifene and BPA significantly decreased IAV titer in a manner similar to E2. Raloxifene can bind to both ESR1 and ESR2 but has a higher affinity for ESR2 (16). Furthermore, raloxifene binding to ESR2 results in an increase in ESR2 reporter gene expression but minimal expression after binding to ESR1 (39). BPA can have varying affinity for ERs depending on the cell or tissue in which it is measured (24). Ospemifene can bind both ERs with similar affinity, and, while the affinities of clomiphene citrate for ESR1 and ESR2 are unknown, clomiphene citrate is a well-documented ESR2 antagonist, inhibiting ESR2-mediated cellular events. We hypothesize that ospemifene and clomiphene citrate did not inhibit virus replication because they do not signal primarily through ESR2 (21). The data from the present study implicate signaling through ESR2 as being critical for the antiviral effects of E2 in hNEC cultures from female donors.
After the Women's Health Initiative raised concerns about estrogen replacement therapy, the use of SERMs as safer alternatives has greatly increased in the United States (21). Repurposing SERMs as antivirals could be a beneficial and a novel therapeutic for the many women currently prescribed SERMs for other diseases. The expression of ERs in the respiratory tract implies that E2 and SERMs can affect the function of tissues and organs outside of the reproductive tract. Understanding these effects will help shed light on additional novel functions of E2 in pulmonary cellular physiology.
This work was funded by the Center for Alternatives to Animal Testing Grant 2013-09 (to S. L. Klein) and National Institutes of Health Grants R01-AI-097417 and HHSN272201400007C (to A. Pekosz), R01-AI-72502 (to A. P. Lane), and T32-AI-007417 (to J. Peretz).
No conflicts of interest, financial or otherwise, are declared by the author(s).
J.P., A.P., and S.L.K. conception and design of research; J.P. performed experiments; J.P. analyzed data; J.P., A.P., A.P.L., and S.L.K. interpreted results of experiments; J.P. prepared figures; J.P. and S.L.K. drafted manuscript; J.P., A.P., A.P.L., and S.L.K. edited and revised manuscript; J.P., A.P., A.P.L., and S.L.K. approved final version of manuscript.
We thank the Klein and Pekosz laboratory members for insightful discussions about these data. We also thank Anne Jedlicka and Amanda Dziedzic of the Johns Hopkins Bloomberg School of Public Health Genomic Analysis and Sequencing Core for assistance with microarray analyses.
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