Autocoding (or automatic concept indexing) occurs when a software program extracts terms contained within text and maps them to a standard list of concepts contained in a nomenclature. The purpose of autocoding is to provide a way of organizing large documents by the concepts represented in the text. Because textual data accumulates rapidly in biomedical institutions, the computational methods used to autocode text must be very fast. The purpose of this paper is to describe the doublet method, a new algorithm for very fast autocoding.An autocoder was written that transforms plain-text into intercalated word doublets (e.g. "The ciliary body produces aqueous humor" becomes "The ciliary, ciliary body, body produces, produces aqueous, aqueous humor"). Each doublet is checked against an index of doublets extracted from a standard nomenclature. Matching doublets are assigned a numeric code specific for each doublet found in the nomenclature. Text doublets that do not match the index of doublets extracted from the nomenclature are not part of valid nomenclature terms. Runs of matching doublets from text are concatenated and matched against nomenclature terms .The doublet autocoder was compared for speed and performance against a previously published phrase autocoder. Both autocoders are Perl scripts, and both autocoders used an identical text (a 170+ Megabyte collection of abstracts collected through a PubMed search) and the same nomenclature (neocl.xml, containing over 102,271 unique names of neoplasms). In side-by-side comparison on the same computer, the doublet method autocoder was 8.4 times faster than the phrase autocoder (211 seconds versus 1,776 seconds). The doublet method codes 0.8 Megabytes of text per second on a desktop computer with a 1.6 GHz processor. In addition, the doublet autocoder successfully matched terms that were missed by the phrase autocoder, while the phrase autocoder found no terms that were missed by the doublet autocoder.The doublet method of autocoding is a novel algorithm for rapid text autocoding. The method will work with any nomenclature and will parse any ascii plain-text. An implementation of the algorithm in Perl is provided with this article. The algorithm, the Perl implementation, the neoplasm nomenclature, and Perl itself, are all open source materials. Autocoding is a specialized form of machine translation. The general idea behind machine translation is that computers have the patience, stamina and speed to quickly parse through gigabytes of text, matching text terms with equivalent terms from an external vocabulary. Human translators often scoff at the output of machine translators, noting the high rate of comical errors. An often cited, perhaps apocryphal, example of poor machine translation is the English to Russian transformation of "out of sight, out of mind" to the Russian equivalent of "invisible idiot."Despite limitations, machine translation is the only way to transform gigabytes and terabytes of text. As long as clinicians, pathologists, radiologists, nurses, and scientists continue to type messages, reports, manuscripts and notes into electronic documents, we will need computers to parse and organize the resulting text.One of the many problems in the field of machine translation is that expressions (multi-word terms) convey ideas that transcend the meanings of the individual words in the expression. Consider the following sentence:"The ciliary body produces aqueous humor."The example sentence has unambiguous meaning to anatomists, but each word in the sentence can have many different meanings. "Ciliary" is a common medical word, and usually refers to the action of cilia. Cilia are found throughout the respiratory and GI tract and have an important role locomoting particulate matter. The word "body" almost always refers to the human body. The term "ciliary body" should (but does not) refer to the action of cilia that move human bodies from place to place. The word "aqueous" always refers to water. Humor relates to something being funny. The term "aqueous humor" should (but does not) relate to something that is funny by virtue of its use of water (as in squirting someone in the face with a trick flower). Actually, "ciliary body" and "aqueous humor" are each examples of medical doublets whose meanings are specific and contextually constant . Furthermore, the meanings of the doublets cannot be reliably determined from the individual words that constitute the doublet, because the individual words have several different meanings. Basically, you either know the correct meaning of the doublet, or you don't.Any sentence can be examined by parsing it into an array of intercalated doublets:"The ciliary, ciliary body, body produces, produces aqueous, aqueous humor."The important concepts in the sentence are contained in two doublets (ciliary body and aqueous humor). A nomenclature containing these doublets would allow us to extract and index these two medical concepts. A nomenclature consisting of single words might miss the contextual meaning of the doublets.What if the term were larger than a doublet? Consider the tumor "orbital alveolar rhabdomyosarcoma." The individual words can be misleading. This orbital tumor is not from outer space, and the alveolar tumor is not from the lung. The 3-word term describes a sarcoma arising from the orbit of the eye that has a morphology characterized by tiny spaces of a size and shape as may occur in glands . The term "orbital alveolar rhabdomyosarcoma" can be parsed as "orbital alveolar, alveolar rhabdomyosarcoma" Why is this any better than parsing the term into individual words, as in "orbital, alveolar, rhabdomyosarcoma"? The doublets, unlike the single words, are highly specific terms that are unlikely to occur in association with more than a few specific concepts.Very few medical terms are single words. In "The developmental lineage classification and taxonomy of neoplasms" there are 102,271 unique terms for neoplasms. All but 252 of these terms are multi-word terms see  see Add[1]. Of tMedical autocoding can be considered a specialized form of machine translation. Medical autocoders transform text into an index of coded nomenclature terms . Several innovative approaches to autocoding have used the higher information content of multiword terms  to match terms in text with terms in vocabularies or to enhance the content of vocabularies by identifying n-grams occurring in text that qualify as new nomenclature terms -4. UnlikThe purpose of this paper is to describe a novel algorithm for autocoding based on finding runs of word-doublets that match a list of doublets extracted from a medical nomenclature. Using the same hardware and the same nomenclature, the speed and accuracy of the doublet method can be compared with another fast lexical parser.The nomenclature used is neocl.xml, previously described by the author and currently designated as "The developmental lineage classification and taxonomy of neoplasms". In the An example of the variety of synonyms encountered for a single tumor is shown for "adenocarcinoma of the colon." There are 44 synonyms listed in the taxonomy. These are: adenoca arising from colon, adenoca arising in colon, adenoca of colon, adenocarcinoma arising from colon, adenocarcinoma arising from large intestine, adenocarcinoma arising from the colon, adenocarcinoma arising from the large intestine, adenocarcinoma arising in colon, adenocarcinoma arising in large intestine, adenocarcinoma arising in the colon, adenocarcinoma arising in the large intestine, adenocarcinoma of colon, adenocarcinoma of large intestine, ca arising from colon, ca arising in colon, ca of colon, cancer arising from colon, cancer arising in colon, cancer of colon, carcinoma arising from colon, carcinoma arising in colon, carcinoma of colon, colon adenoca, colon adenocarcinoma, colon ca, colon cancer, colon cancers, colon carcinoma, colon carcinomas, colon with adenoca, colon with adenocarcinoma, colon with ca, colon with cancer, colon with carcinoma, colonic adenoca, colonic adenocarcinoma, colonic adenocarcinomas, colonic ca, colonic cancer, colonic cancers, colonic carcinoma, colonic carcinomas, large intestine adenocarcinoma, large intestine with adenocarcinoma.The input file was created by a PubMed query on "pathology [ad] AND neoplasm [all]", at the U.S. government website . The queThe doublet autocoder is supplied as an open source Perl script (doubcode.pl) with this manuscript see . Perl itThe algorithm used by the doublet autocoder is described:1. Each phrase (term) in the nomenclature (neocl.xml) is converted into intercalated doublets, and each doublet is assigned a consecutive number.2. Each nomenclature phrase is assigned the concatenated list of numbers that represent the ordered doublets composing the phrase.3. Every text record (pubmed abstract in this case) is split into an array consisting of the consecutive words in the text record.4. The text array is parsed as intercalated doublets. Intercalated doublets from the text that match doublets found anywhere in the nomenclature are assigned their numeric values (from the doublet index created for the nomenclature). Runs of consecutive doublets from the text that match doublets from the nomenclature are built into concatenated strings of doublet values. The occurrence of a text doublet that does not match any doublet in the nomenclature cannot possibly be part of a nomenclature term. Such text doublets serve as "stop" doublets between candidate runs of text doublets that match nomenclature doublets.5. The runs of matching doublets are tested to see if they match any of the runs of doublets that compose nomenclature terms or if they contain any subsumed terms that match nomenclature terms.6. The array of doublet runs extracted from the text that match nomenclature terms are cached in an external file.The phrase autocoding script, phrase.pl is included as supplementary file with this article see . The scrThe speed of both coders was compared using a 170+ Megabyte text. The doublet method coded the entire text in 211 seconds compared with 1,766 seconds required for the phrase method. Therefore the doublet method coded 8.4 times faster than the phrase method. The text coding rate for the doublet method is 0.8 Megabytes per second on a desktop computer with a modest 1.6 GHz Pentium processor.Both the phrase method and the doublet method coders used the same Programming Language, the same nomenclature, and the same data structure for the nomenclature (a simple associative array). Both coders grab sequentially occurring chunks of text, each chunk delimited by a double-newline delimiter  and place the grabbed text chunk into a temporary scalar variables.The difference between the two coders occurs when the chunks of text are parsed. The phrase coder takes the text and creates an array of every ordered combination of 1,2,3,4 and 5-word phrases contained in the record. This number of elements in the phrase array is about 5 times larger than the number of words in the record. The doublet coder parses the record into the set of all ordered doublet terms. This number of elements in the array of doublets is about the same as the number of words in the record. The phrase coder must try to match about 5 times as many terms as the doublet coder. Also, the phrase coder tries to match each phrase in the record array against the entire nomenclature. The doublet coder tries to match each doublet encountered in the input text against the collection of doublet terms extracted from the nomenclature. Because doublets of text, unlike individual words, tend to have unique meanings, only a small subset of the doublets encountered in the input text will match the set of doublets extracted from the nomenclature. Text doublets that do not match any doublets in the nomenclature are "skipped." Text doublets that match doublets from the nomenclature are concatenated to consecutive matching doublets until a non-matching doublet is encountered. The length of matching doublets sets the length of the candidate term. The algorithmic strength of the doublet method is that it eliminates the need to create and match [against a nomenclature] an array of all possible phrases of all possible lengths found in a textual record.The output of the doublet method and phrase method autocoders are provided as supplemental files with this manuscript see  see Add. The anaThe two autocoder methods parsed a text exceeding 170 megabytes in length. The doublet method coded 4807 different [sometimes called unique] terms. The total number of matching term encountered (which includes replicate matches of terms within a record) by the doublet method is 467,391 terms. The phrase method coded 4557 different terms. There were 250 terms that the doublet method matched and the phrase method missed. There were zero terms that the phrase method matched and the doublet method missed.The improved performance of the doublet coder is accounted for by its ability to match terms of any length, while the phrase coder is limited to match terms that are no more than 5 words in length. Because the phrase coder parses chunks of input text into all allowed phrase size combinations, the performance of the phrase coder is slowed dramatically when the allowed phrase length is large. For instance, if the theoretical maximum phrase size were permitted for a text chunk 100 words in length, the phrase parser would need to create an array of a size equal to 100 × 100 or 10,000 phrases, matching each phrase against nomenclature. If the phrase size were restricted to 5 words, then the phrase parser would only need to create a phrase array of size 5 × 100 or 500. It is reasonable to restrict the allowed length of coded term phrases if it is known that the number of nomenclature terms with length exceeding the allowed limit is small. It is also reasonable to restrict the allowed length of coded term phrases is it is believed that terms of long length can be successfully represented by subsumed terms within the longer term.For example, "Refractory anemia with excess blasts in transformation" will be captured as a single term by the doublet method. The phrase method will miss the 7-word term but will code the subsumed terms, "refractory anemia" and "refractory anemia with excess blasts". In this specific instance, the doublet coder outperformed the phrase coder. However, few cancer terms actually exceed 5 words in length, and longer terms almost always contain modifying phrases  that add little to the fundamental meaning of the term.The following terms are examples of neoplasms extracted by the doublet method and missed by the phrase method. All of the listed terms exceed 5 words in length. "Acute myelogenous leukemia with minimal differentiation, acute myeloid leukemia with minimal differentiation, adenocarcinoma arising in the parotid gland, adenoid cystic carcinoma of submandibular gland, aids-related primary central nervous system lymphoma, atypical glandular cells of undetermined significance, atypical squamous cells of uncertain significance, carcinoid tumor arising from meckel diverticulum, extranodal marginal zone b-cell lymphoma of mucosa-associated lymphoid tissue, giant cell tumor of tendon sheath, giant cell tumour of tendon sheath, glandular malignant peripheral nerve sheath tumor, high-grade malignant peripheral nerve sheath tumor, hurthle cell carcinoma arising from thyroid, hyalinizing spindle cell tumor with giant rosettes, interdigitating dendritic cell sarcoma arising from the spleen, intermediate-grade b-cell non-hodgkin lymphoma, intraductal papillary mucinous neoplasm of pancreas, intraductal papillary-mucinous tumor with moderate dysplasia, kaposi sarcoma arising in the heart, large cell calcifying sertoli cell tumor, leiomyosarcoma arising from the pulmonary artery, low grade cervical glandular intraepithelial neoplasia, low-grade malignant peripheral nerve sheath tumor, lymphoma arising in the thyroid gland, malignant epithelioid peripheral nerve sheath tumor, malignant tumor of peripheral nervous system, malt lymphoma arising in the stomach, metanephric adenosarcoma in a young adult, monomorphic clear cell adenocarcinoma of salivary glands, ovarian serous borderline tumor with micropapillary and cribriform patterns, ovarian serous tumor of low malignant potential, paraganglioma arising in the cauda equina, perineurial malignant peripheral nerve sheath tumor, philadelphia chromosome negative chronic myeloid leukemia, phosphaturic mesenchymal tumor mixed connective tissue variant, pineal parenchymal tumor of intermediate differentiation, pleomorphic adenoma of minor salivary glands, polymorphous low-grade adenocarcinoma of minor salivary gland, polymorphous low-grade adenocarcinoma of minor salivary glands, polymorphous low-grade adenocarcinoma of salivary gland, poorly differentiated malignant peripheral nerve sheath tumor, porokeratotic eccrine ostial and dermal duct nevus, primary cutaneous anaplastic large cell lymphoma, primary gastric diffuse large b-cell lymphoma, primary signet-ring cell carcinoma of lung, primitive neuroectodermal tumor arising in the pancreas, pulmonary immunocytoma with massive crystal storing histiocytosis, rhabdoid transformation of tumor cells in meningioma, serous ovarian tumor of low malignant potential, sex cord tumor with annular tubules, signet ring cell adenocarcinoma of prostate, signet-ring cell mucin-producing adenocarcinoma of minor salivary glands, sinonasal desmoplastic small round cell tumor, smooth muscle tumor of uncertain malignant potential, smooth muscle tumor with an uncertain malignant potential, splenic lymphoma with circulating villous lymphocytes, squamous cell carcinoma of fallopian tube, submandibular gland carcinoma ex pleomorphic adenoma, tall cell variant of papillary carcinoma of thyroid, testicular large cell calcifying sertoli cell tumor, uterine tumor resembling ovarian sex cord tumor."The long terms found by the doublet coder and missed by the phrase coder represent a very small percentage of the different terms found by either coder in the long 170+ Megabyte text (250/4807 = 5%). A cursory review of these terms indicates that they represent rare lesions or rare variants of common lesions.Developers of medical autocoders seldom publish manuscripts. It is the author's perception, based on many years of activity in this field, that most autocoders are proprietary products produced for a very specific type of job. The author has never encountered any autocoder vendors who revealed the speed of their autocoders or shared any primary data that measures the performance of their autocoders. There really has never been any publication where one autocoder was compared against another. This is unfortunate because software developers may defer implementing brilliant ideas for autocoders, if they are uncertain whether better autocoders already exist. Even if competing software developers were to share performance data, there are no widely accepted standards for measuring the performance of medical autocoders. One of the purposes of this paper is to provide open access to algorithms, software, performance methodology and performance outcome data.The performance of autocoders is always a contentious issue. The reflexive approach to measuring autocoder performance usually involves selecting a small corpus of text and allowing a human expert in a language domain to carefully read through the lines of the text, creating a so-called gold standard against which the autocoder can be compared. The problems with this seemingly straightforward approach are many and have been explained by myself and others. The many limitations of "precision and recall" as measurements of indexing performance have been reviewed elsewhere ,10.It would be a useful exercise to re-examine what autocoding performance means to different people in contrast to what autocoding performance means within the context of lexical parsers ,12. FirsFinally, some coders seek to create "concept signatures" from text. A concept signature is the list of the concepts contained in a section of text. The relationship of one section of text to another section of text is determined by a quantitative representation of how closely their signatures match. Concept signatures are used to retrieve or organize related documents, not specific concepts .Lexical coders are, in a sense, perfect autocoders because they don't use grammatic rules, they have no "exception" lists, and they never guess or interpret text. The only thing that lexical parsers do is to parse text, examining strings to see if they contain exact matches to terms from a nomenclature. When well-constructed, they do not make mistakes. If they miss a valid term that is present in the text, it is because the term was missing from the nomenclature. Developers of lexical autocoders place enormous demands on the curators of nomenclatures.The most common cause of missed terms arises when the text contains a modifying word that breaks the term into a phrase that no longer matches anything found in the nomenclature. For instance, "adenocarcinoma of the left lung," would be missed by a lexical parser if the nearest term in its terminology list is "adenocarcinoma of the lung." A rule-based parser or a semantic parser may have successfully teased out the "left" from the term and found the match. In this instance, the nomenclature failed the lexical parser because it did not list terms that indicated tumor laterality. Lexical parsers do not match terms that are absent from the text . False-positive terms are possible in rule-based and semantic parsers if they create word patterns not present in the original text. Also, since lexical parsers strictly match phrases in text with phrases in nomenclatures, it is possible to achieve accurate results of dubious value. For instance, a lexical parser would parse "adenocarcinoma of the lung is not seen" and find a match against the neoplasm term, "adenocarcinoma of the lung." The concept is present in the text, even if though it is absent from the patient. It is a misleading but "true" positive.The doublet autocoder creates a string of words from a chunk of text. In the case of the example corpus (PubMed abstracts), all of the sentences from the abstract are squeezed into a single string of words, obliterating sentence boundaries. This means that if a sequence of words crossing a sentence boundary happens to match a term in the reference nomenclature, the coder will register a "pseudo-positive" term. Empirical evidence suggests that this theoretical error in the doublet autocoder simply does not occur. A 4 megabyte collection of abstracts chunked as whole abstracts or as chunked sentences (delimited by period-space-space) contained no instances of pseudo-terms created by the obliteration of sentence boundaries in the whole-abstract text chunks. For fastidious developers who wish to ensure that their parsers respect sentence boundaries, it is possible to pre-process text into sentences with a sentence parser ,15.Lexical parsers can be modified to provide an output that preserves the intended sense of the term as used in its context. For example, if the text includes the sentence, "Adenocarcinoma of the lung is not present." The "sensible" lexical parser may be modified to tag the "adenocarcinoma of the lung" with a negation modifier, preserving the intended sense of the term. The author has previously published an "in place" method of inserting codes directly into sentences, preserving modifier terms (including negations) .The two lexical parsers used the same programming language (Perl), the same nomenclature, the same data structure to hold the nomenclature, the same text corpus, and the same method for breaking the complete text into text chunks. Differences between the performance of the two autocoding methods were accounted for entirely by the algorithmic processes by which chunks of text are parsed into phrases that are matched against the common nomenclature. It is easy to compare speeds. The doublet parser required 211 seconds to parse the 170+ Megabyte file, while the phrase parser required 1,776 seconds. Three algorithmic properties contribute to the speed advantage of the doublet coder over the phrase coder: 1) The doublet coder parses text into a number of phrases that is 5-fold smaller than the number of phrases produced by the phrase coder; 2) Doublets that are not found in any of the nomenclature terms can be quickly excluded, 3) Successive text doublets that match doublets from the nomenclature can be quickly concatenated and tested for matches in the nomenclature. These are the algorithmic differences between the phrase coder and the doublet coder and presumably account for the improved speed of the doublet method.Comparing coding accuracy is somewhat trickier. As discussed, lexical parsers have no "false positive" matches. Lexical parsers, unlike natural language processors, do not construct context-based new word phrases from native text. Lexical parsers only match exact phrases found in text with exact nomenclature terms. Therefore, neither the doublet coder nor the phrase coder have false-positive term matches. Coding output was compared by examining the different terms matched by the two coders. The number of different terms found by the doublet method was 4,807, compared with 4,557 terms found by the phrase method. There were no terms found by the phrase method and missed by the doublet method. There were 250 terms missed by the phrase method and found by the doublet method (about 5% of the number of different terms found by the doublet method). For the most part, these terms represented rare neoplasms with pre-coordinated modifiers. The reason that such rare terms were encountered is due to the large size of the text (exceeding 170 Megabytes). When the doublet and phrase methods are judged by their ability to extract nomenclature from text, their output would be similar for small text sizes or when the primary purpose of the coding is to extract and count commonly occurring terms.The doublet method is a novel approach to autocoding. It can autocode 0.8 Megabytes of text per second on desktop computer using a modest 1.6 GHz processor. For this manuscript, the doublet autocoder was tested on a corpus of medical text that exceeded 170 Megabytes in length and used a publicly available nomenclature of neoplasia containing 102,271 unique terms. In a side-by-side comparison with a publicly available fast lexical autocoder, the doublet method was 8.4-fold faster. The doublet algorithm is generalizable to any type or length of plain-text using any nomenclature. The algorithm and the Perl implementation script are available as open source documents.The autocoding scripts are short programs written in Perl. Perl is a freely available open source programming language. Perl interpreters for virtually any operating system are available from several sites on the web ,7. TheseThe Perl scripts (doublet.pl and phrase.pl) will work without modification using the current version of "The developmental lineage classification and taxonomy of neoplasms," which is provided with this manuscript see  see Add.None declared.The work expresses the opinion of the author and does not represent policy of the U.S. government.The pre-publication history for this paper can be accessed here:Neocl.xml is the developmental lineage classification and taxonomy of neoplasms, in XML format. Because neocl.xml exceeds 7 Megabytes when uncompressed, a gzipped version of the file is provided (neoclxml.gz). After downloading from the biomedcentral site, the filename should be provided with a .gz suffix (if absent from the filename as downloaded). After decompressing the file, the file shoud be renamed "neocl.xml". The file can be viewed on current web browsers, but experience has shown that many browsers lack sufficient memory to display the entire file. Otherwise, the file can be viewed on a wordprocessor or an ascii editor.Click here for fileNeoself.gz is the ascii flat-file version of the neocl.xml file. The file exceeds 16 Megabytes when expanded. Each line-record of the file consists of the numbered name of a different term, its code value, and the hierarchical list of its ancestral [class] terms from the developmental lineage classification and taxonomy of neoplasms.Click here for fileDoubcode.pl is a Perl script that implements the doublet method for text autocoding and expects an external file named tumor.txt as the source corpus and a file named neocl.xml as the reference nomenclature.Click here for filePhrase.pl is a Perl script that implements the doublet method for text autocoding and expects an external file named tumor.txt as the source corpus and a file named neocl.xml as the reference nomenclature.Click here for fileDoub.out is the plain-text output of the doublet method for text autocoding (i.e. output of doubcode.pl).Click here for fileQuickcan.txt is the plain-text output file for phrase method for text autocoding (i.e. output of phrase.pl).Click here for fileDoubcomp.pl is the Perl script comparing the output files produced by doubcode.pl and phrase.pl.Click here for fileDoubcomp.txt is the plain-text output of doubcomp.pl.Click here for file


CBFV  is lower in the morning than in the afternoon and evening. Two hypotheses have been proposed to explain the time of day changes in CBFV: 1) CBFV changes are due to sleep-associated processes or 2) time of day changes in CBFV are due to an endogenous circadian rhythm independent of sleep. The aim of this study was to examine CBFV over 30 hours of sustained wakefulness to determine whether CBFV exhibits fluctuations associated with time of day.Eleven subjects underwent a modified constant routine protocol. CBFV from the middle cerebral artery was monitored by chronic recording of Transcranial Doppler (TCD) ultrasonography. Other variables included core body temperature (CBT), end-tidal carbon dioxide (EtCO2), blood pressure, and heart rate. Salivary dim light melatonin onset (DLMO) served as a measure of endogenous circadian phase position.2 = 0.62 and R2 = 0.68, respectively). Circadian phase position of CBT occurred at 6:05 am while CBFV occurred at 12:02 pm, revealing a six hour, or 90 degree difference between these two rhythms (t = 4.9, df = 10, p < 0.01). Once aligned, the rhythm of CBFV closely tracked the rhythm of CBT as demonstrated by the substantial correlation between these two measures (r = 0.77, p < 0.01).A non-linear multiple regression, cosine fit analysis revealed that both the CBT and CBFV rhythm fit a 24 hour rhythm (RIn conclusion, time of day variations in CBFV have an approximately 24 hour rhythm under constant conditions, suggesting regulation by a circadian oscillator. The 90 degree-phase angle difference between the CBT and CBFV rhythms may help explain previous findings of lower CBFV values in the morning. The phase difference occurs at a time period during which cognitive performance decrements have been observed and when both cardiovascular and cerebrovascular events occur more frequently. The mechanisms underlying this phase angle difference require further exploration. It has been well documented that cerebral blood flow velocity (CBFV) is lower in sleep -7 and inAn alternative to these explanations that attribute changes in CBFV to sleep and wake dependent processes is that this pattern of fluctuation reflects an endogenous process with circadian rhythmicity. The decline of CBFV across the sleep period and rise after subjects are awakened in the morning resemble the endogenous circadian changes in core body temperature (CBT), a reliable index of endogenous circadian rhythmicity. Both patterns are low during sleep, start to rise in the morning, reach their peak in the late afternoon, and then drop during the sleep period.The aim of this study was to examine CBFV over ~30 hours of sustained wakefulness to unmask and quantify contributions of the endogenous circadian system. By not permitting sleep, the evoked changes dependent on this change of state will not contribute to the observed CBFV changes. We hypothesized that time of day changes in CBFV are due to endogenous circadian regulation. Previous studies have been limited by several factors. First, the environmental conditions (light level) and the behavior of the subject  were not controlled ,13,1,16.CO2), three of the main regulators of CBFV, were collected every half hour.We employed the "constant routine" protocol, which was designed specifically to unmask underlying circadian rhythms in constant conditions . CBFV waTwelve subjects  agreed to participate. One subject discontinued her participation because of a headache 15 hours into the study. Subjects were in good health, as assessed by medical history, semi-structured clinical interview, and physical exam. Information regarding menstrual cycle was not obtained from female subjects. Subjects also underwent an independent standard cerebrovascular assessment and were determined to be normal. They reported no symptoms of sleep problems (such as insomnia, obstructive sleep apnea, narcolepsy, or restless legs syndrome).Subjects that were selected to participate kept to a designated sleep-wake schedule  and filled out a sleep diary for the two weeks prior to the time in the laboratory. According to sleep diary reports, bedtimes ranged from 10:30 pm to 1:00 am and waketimes ranged from 6:00 am to 10:00 am. Alcohol and caffeine intake was discontinued for the entire week before the study. During the data collection, subjects were not permitted either alcohol or caffeine. All subjects were non-smokers.® liquid formula plus one-quarter nutritional food bar) every 2 hours. Subject's typical total food and liquid intake for a day and a quarter were divided into 15 relatively equal portions. Only one subject participated in the CR per 30-hour period.The study protocol was approved by the Institutional Review Boards of New York Presbyterian Hospital – Weill Medical College of Cornell University and The City College of New York. Subjects gave written and informed consent before participating. Subjects arrived at the sleep laboratory between 9:30 am and 10:00 am. They were oriented to the study procedures and to their bedroom. Electrodes were placed on the subject's head and face as they sat in a chair next to the bed. Data collection began at 11 am. Subjects remained in bed and awake in a semi recumbent position for 30 hours in an established "constant routine" (CR) protocol. Subjects remained in low (<25 lux) light levels which have been shown to have little or no entraining effect on the circadian pacemaker . They weThis protocol represents a modified CR in two ways. First, subjects were allowed to watch television and were therefore were not in "time isolation." Television content was monitored so that subjects were not exposed to programs with highly emotional themes. Second, subjects needing to defecate were allowed to go to the bathroom, which was located a few steps away from the bedside. We chose this method as an alternative to using the bedpan to ensure subject's comfort and study compliance. Three subjects (subjects 05, 06, and 10) got out of bed once at 3:30, 21:30, and 15:30, respectively, to defecate. One subject, subject 12, got out of bed twice, at 22:30 and 6:35. Subject 10 used the bathroom only during the adaptation period. A paired-samples t-test was conducted to evaluate the impact of getting out of bed to defecate on subject's CBT and CBFV values. The CBT and CBFV values in the two hours before getting up were compared to the two hours after the subject got up. Subjects 5 showed a slight decrease in CBT from before (M = 98.12, SD = 0.14) to after the subject returned to the bed (M = 97.91, SD = 0.08), t(3) = -5.17, p = .014). Subject 6 showed a decline in CBFV from before (M = 56.14, SD = 2.3) to after the subject returned to the bed (M = 45.67, SD = 3.7), t(3) = 5.49, p = 0.012). There were no other significant differences detected between these two time periods for subject 5's CBFV, subject 6's CBT, or for both times subject 12 got out of the bed. By visual inspection, the overall shape of the curves in these subjects was not affected and therefore these subject's data were included in subsequent analyses.The current study utilized TCD ultrasonography to measure cerebral blood flow velocity. TCD is a non-invasive instrument  that is used predominantly as a diagnostic tool to assess cerebral hemodynamics in normal and pathological conditions. TCD ultrasonography is predicated on a theory that involves the measurement of moving objects when combined with radar. When the instrument emits the sound wave, it is reflected by the blood cells that are moving in the vector of the sound wave .CBFV was measured using either the right or left middle cerebral artery (MCA) using TCD sonography (TCD: DWL Multidop X-2, DWL Elektronische Systeme GmbH, D-78354 Sipplingen/Germany) through the temporal window. An observer who was present continuously during the recordings evaluated the quality of the signal. This enabled long-term recording of CBFV throughout the study. Fast Fourier Transformation (FFT) of the signal was used to analyze the velocity spectra. The mean velocity of the MCA was obtained from the integral of the maximal TCD frequency shifts over one beat divided by the corresponding beat interval and expressed in cm/sec. Analysis was conducted off line.Core body temperature was recorded at 1-minute intervals with an indwelling rectal probe (MiniMitter, Co. Bend, OR). A wire lead connected the sensor out of the rectum to a data collection system worn on the belt. Temperature readings were collected and saved into the device and monitored at hourly intervals by the investigator. After the study, the recordings were visually inspected and artifacts resulting from removal or malfunction of the probe were excluded from further analysis.Salivary samples of 3 ml were collected every hour from 11:00 a.m. on Day 1 to 4:00 p.m. on Day 2. Ten of these samples were used only for the determination of the timing of the salivary dim light melatonin onset (DLMO). For nine subjects, salivary DLMO was assessed across a ten-hour time window that included the ten hours before the CBT minimum. Immediately after collection, each saliva sample was frozen and stored at -20°C. Saliva samples were assayed using Bühlmann Melatonin Radio Immunoassay (RIA) test kit for direct melatonin in human saliva (American Laboratory Products Co., Windham, NH). Analysis was conducted at New York State Institute for Basic Research. Salivary DLMO time was selected based on two criteria. The saliva sample needed to have melatonin concentration 3 pg/ml or above and later samples needed to show higher levels (Bühlmann laboratories). Second, the 3 pg/ml threshold needed to occur within 6–10 hours before core body temperature minimum [Electroencephalography (EEG) was continually assessed across the 30 hours to ensure that subjects maintained wakefulness. The following montage was used according to the international 10–20 system: C3-A2, C4-A1, O1-A2, O2-A1, ROC-A1, LOC-A2, and submentalis electromyogram (EMG). One channel of electrocardiogram was continuously recorded by monitoring from two electrodes (one on each side of the body at the shoulder chest junction). The EEG software (Rembrant Sleep Collection Software Version 7.0) was used for data acquisition and display of the signals on a personal computer. Throughout the CR, the investigator (DAC) monitored the quality of the recordings. The recordings were scored by RQS and DAC.CO2 was continuously obtained. A nasal cannula for monitoring expired gases was placed under the nose. Relative changes in carbon dioxide content were measured by an Ohmeda 4700 Oxicap . Mean EtCO2 levels were analyzed off-line. EtCO2 recordings were not measured in subjects 11 and 12.An automated blood pressure cuff was placed on the bicep of the subject and inflated two times each hour in order to determine changes in blood pressure and heart rate over time. Blood pressure and heart rate in one subject (02) was recorded via a finger blood pressure monitor . Blood pressure and heart rate in subjects 03, 04, 05, 06, and 07 were recorded with Omron Healthcare, Inc, Vernon Hills, Illinois 60061 Model # HEM-705CP Rating: DC 6V 4W Serial No: 2301182L. Blood pressure and heart rate for subjects 08, 09 and 10 was recorded with a similar blood pressure monitor (CVS Pharmacy Inc, Woonsocket, RI 02895 Model # 1086CVS). Blood pressure and heart rate recordings were not measured in subjects 11 and 12. EtCBT and CBFV values were first subjected to data rejection. All CBT values less than 96 degrees were determined to be artifact and were rejected. All CBFV values less than 20 cm/sec were determined to be artifact according to the clinical criteria set by the staff neurologist. Data reduction was accomplished by averaging into one minute, 30 minute or hourly bins. Correlations presented here were performed on mean values in 30 minute bins. To ensure that circadian measurements were made under basal conditions, the first five hours of the constant routine were excluded from all analyses to eliminate effects of study adaptation. The last hour was excluded to eliminate confounding effects such as expectation effects.The data are presented in this article in three ways. First, CBT and CBFV values were plotted according to time of day Figures  and 2. STo align the CBFV nadir to the CBT nadir, first, the lowest value of CBT and the lowest value of CBFV were identified and set to circadian time 0, or 0°. Each half hour data point after the CBT nadir and CBFV nadir were then set to a circadian degree. There were a total of 48 data points across the 24 hour period. Therefore, each data point was equal to 7.5 degrees so that each data point would accumulate to 360°. Lastly, mean values were obtained for CBT and CBFV at each circadian degree.A 24-hour non-linear multiple regression -cosine curve fit analysis was performed on the CBT and CBFV data (SAS Institute, Cary, NC). This technique constrains the circadian period of CBT and CBFV to be within 24 hours. This technique used the following equations: model cbt = &avg_cbt + r * cos((2 * 3.1415) * (hours-&max_cbt)/24; model cbfv = &avg_cbt + r * cos((2 * 3.1415) * (hours-&max_cbfv)/24, where & = constants that center the curve at the actual average for each series  and the predicted maximum at the actual maximum ; r = the amplitude of the cosine wave. An additional analysis was performed which also yielded the estimated clock time for the CBT nadir and CBFV nadir . Third, the minimum of the circadian rhythm of CBT and salivary DLMO were also used as markers of the endogenous circadian phase. A paired t-test was used to determine the overall phase difference between CBT and CBFV.Eleven subjects completed the protocol. The TCD probe was placed on either the right or left temple, whichever gave the better signal. Mean isonation depth of the TCD signal was 56.5 mm for the right MCA and 55.6 mm for the left MCA (range 53–60 mm). The constant routine ranged from 28 to 30 hours in duration. Polygraphic recordings confirmed sustained wakefulness across essentially the entire protocol in all but one subject. Subjects that had difficulty remaining awake were monitored closely and aroused when needed by engagement in conversation. Results from the polygraphic recordings are not presented here. We do not present the results of the polygraphic recordings because, for the purposes of this study, these recordings were used solely to monitor whether subjects were awake or asleep. The first five hours and the final hour of data from the constant routine were excluded from analysis.2 = 0.62, p < 0.01), Figure 2 = 0.67, p < 0.01), Figure A 24 hour non-linear multiple regression, cosine fit analysis revealed that the overall mean CBT rhythm n = 11) fit a 24 hour cosine rhythm . Blood pressure, heart rate, and EtCO2 served only as regulators of CBFV and were not analyzed according to circadian phase.A Pearson correlation revealed a positive relationship between CBT and heart rate (r = 0.40, p < 0.01) across the 24 hour period. Diastolic blood pressure (DBP) and CBT showed a negative correlation (r = -0.30, p < 0.05). EtThis study is the first to use the constant routine (CR) protocol to determine whether the endogenous circadian pacemaker contributes to the previously reported diurnal changes in CBFV. The current work demonstrates that, with limited periodic external stimuli and a constant posture, there is 24-hour rhythmicity in CBFV. Subjects showed a cycle of approximately 24 hours in CBT, which has been previously demonstrated with the CR .Figure Figure Earlier studies using simultaneous EEG and TCD to continuously measure CBFV across the sleep period have concluded that, except for periods of REM sleep, ,27, therMoreover, our interaction with the subjects and the monitoring of EEG for signs of sleep resulted in no sleep in all but one subject. The one exception was in a subject who lapsed into brief periods of sleep. Therefore, the fall in CBFV in 10 out of 11 subjects cannot be explained by the occurrence of non-REM sleep. It is possible, however, that the decline of CBFV across the night and early morning may be secondary to the sleep deprivation that is part of the constant routine. Brain imaging studies across sustained periods of wakefulness have shown significant decreases in absolute regional cerebral glucose metabolic rate in several areas of the brain -34.CO2, is unlikely present during waking while lying in bed at night. Therefore, CBT declines remain a plausible explanation for the portion of the 24 hours when CBFV declined.The drop in CBT which preceded the parallel fall in CBFV needs to be considered as a possible explanation for the CBFV changes. The fall in CBT during sleeping hours is attributed in part to sleep-associated changes and in part to strong regular circadian forces independent of the sleep period. CBT is, in fact, one of the key and most extensively studied indices of the circadian phase. It is also known that CBT is highly correlated with brain temperature and brain metabolic rate . ImagingCO2 in the absence of sleep, in subjects with constant posture, and highly restricted movements. While blood pressure clearly falls during sleep in normal individuals, the absence of sleep in the current study obviates the explanation that CBFV declines are secondary to lowered blood pressure. Furthermore, we sampled blood pressure throughout the day and night and found a weak inverse relationship between DBP and CBT. This finding is in contrast to a careful study of circadian influence on blood pressure in the absence of sleep which showed no change in blood pressure during the descending portion of the body temperature curve [CO2 and CBT is similar to that found by Spengler et al. [CO2 on a CR protocol. EtCO2 showed a trend towards a direct relationship with CBFV, which is consistent with previous studies showing that changes in EtCO2 are associated with changes in CBFV [This protocol allowed the unique opportunity to evaluate blood pressure, heart rate, and Etre curve . Neverthr et al. , who sho in CBFV ,42. Hear in CBFV .The approximate 6 hour (90 degree) phase angle difference between the CBFV and CBT suggests that CBFV continues to decline into the early to mid-morning hours. This finding is consistent with a time window in the morning during which several physiological changes have been observed. For example, cerebral vasomotor reactivity to hypocapnia, hypercapnia, and normoventilation has been found to be most reduced in the morning ,16. It iOverall, the results demonstrate that CBFV, in the absence of sleep, exhibits properties of a circadian rhythm, as it rises and falls across a 24 hour period. The 6 hour (90 degree) phase angle difference in the CBFV rhythm with respect to the CBT rhythm may help explain previous findings of lower CBFV values in the morning. The phase difference occurs at a time period during which cognitive performance decrements have been observed and when both cardiovascular and cerebrovascular events occur more frequently. The mechanisms underlying this phase angle difference require further exploration.CBFV Cerebral Blood Flow VelocityCBT Core Body TemperatureTCD Transcranial DopplerEtCO2 End tidal Carbon DioxideDLMO Dim Light Melatonin OnsetEEG ElectroencephalogramMCA Middle Cerebral ArteryFFT Fast Fourier TransformationCR Constant routineEMG ElectromyogramSBP Systolic Blood PressureDBP Diastolic Blood PressureCVA Cerebrovascular accidentMI Myocardial infarctionThe author(s) declare that they have no competing interests.DAC coordinated, carried out, analyzed, and interpreted the study. AJS participated in the analysis and interpretation of the findings. DAC drafted the manuscript and AJS provided final approval of this version. RQS participated in data collection and data analysis. DAC and AJS co-designed the study. All authors read and approved the final manuscript.


In the present review, we concisely explore the conceptual framework of stem cell biology and recent advances pertinent to the lungs. We illustrate lung diseases in which manipulation of stem cells may be physiologically significant and highlight the challenges facing stem cell-related therapy in the lung.Fueled by the promise of regenerative medicine, currently there is unprecedented interest in stem cells. Furthermore, there have been revolutionary, but somewhat controversial, advances in our understanding of stem cell biology. Stem cells likely play key roles in the repair of diverse lung injuries. However, due to very low rates of cellular proliferation  According to Greek mythology, the immortal Prometheus stole fire from the Gods as a gift for humankind. As punishment, he was shackled to a rock, whereupon each day for 30,000 years an eagle consumed as much of his liver as would regenerate. There is some debate whether the eagle ate his liver or heart, but what if the bird had a taste for lung? And what if Prometheus was a mere mortal?Analogous to Prometheus and the eagle, the ambient air-exposed lung is subject to an array of potentially damaging agents, including chemical oxidants and proteolytic enzymes. Presumably, daily oxidant and protease wear and tear on structural components such as elastin and collagen contributes to inevitable age-related declines in pulmonary function in normal individuals ,2. Acute. During embryonic development, the inner cell mass of the blastocyst forms three primary germ layers, which generate all fetal tissue lineages , or preparations enriched for hematopoietic stem cells (HSC). Whole body irradiation, which may injure lung tissue, is typically used to deplete the host bone marrow. Importantly, lung injury apparently enhances engraftment into lung -29. Wholculation .Sex-mismatched lung and bone marrow transplantation in humans provides a natural model for analysis of donor and recipient cell behavior. Bronchial epithelial and gland cells and type II pneumocytes of host origin were reported in one study of lung allografts , but notin vitro and in vivo, which likely explains why some of the cells contain both donor and lung cell markers [see [Many questions remain unanswered. The mechanism whereby cells assume lung cell phenotypes remains uncertain. Several studies have demonstrated that cell fusion occurs both ers [see  for a sters [see ,41 for rers [see . New, moers [see  and the ers [see , while aers [see . MitigatMajor lung diseases likely involving stem cells and the cellular targets for stem cell therapy are summarized in Table in vivo [What are the realistic prospects for beneficial stem cell therapy of the lung? First, we must conclusively identify lung diseases/cases/timing in which cell and tissue damage occurs in excess of the capacity for timely endogenous repair. Second, we must establish standardized sources of relevant stem/progenitor cells and methods for their delivery to the appropriate lung sub-compartment. Once delivered, therapeutic cells must home to microscopic sites of need and integrate to serve a beneficial function. There is clearly potential for adverse effects, as exemplified by the propensity of embryonic stem cells to form teratomas when implanted in vivo . Major lProvocative, but controversial, recent evidence suggests that circulating stem cells may home to the lung. There is great excitement and hope that exogenous and/or mobilized endogenous stem cells may be harnessed to prevent or treat acute and chronic lung diseases and even regenerate abnormally developed or lost tissue. Our understanding of lung stem cells and the regulation of lung morphogenesis is still rudimentary, and the complex, integrated function of multiple cell types underlying normal lung structure and function poses unique challenges. Thus, the therapeutic prospects for stem cell therapy in lungs appear more distant than in some other organs. This realization should stimulate meaningful new studies from the lung research community. Unlike the mythical hero Prometheus, patients with lung disease cannot wait 30,000 years!None declared.


Similar genes are found in monocots; however, they do not accumulate in leaves as storage proteins, and the ability of monocot leaves to support accumulation of an ectopically expressed soybean VSP is in question. To test this, transgenic maize (Zea Mays L. Hi-II hybrid) lines were created expressing soybean vspB from a maize ubiquitin Ubi-1 promoter.Developing monocots that accumulate more vegetative tissue protein is one strategy for improving nitrogen-sequestration and nutritive value of forage and silage crops. In soybeans (a dicotyledonous legume), the vspB transcripts and VSPβ polypeptides. In leaves from seven-week-old plants (prior to flowering), VSPβ accumulated to 0.5% of the soluble leaf protein in primary transgenic plants (R0), but to only 0.03% in R1 plants. During seed-filling (silage-stage) in R1 plants, the VSPβ protein was no longer detected in leaves and stems despite continued presence of the vspB RNA. The RNA transcripts for this peptide either became less efficiently translated, or the VSPβ protein became unstable during seed-fill.From 81 bombardments, 101 plants were regenerated, and plants from five independent lines produced vspB transcript level, VSPβ protein that is readily detected in leaves of preflowering plants, becomes undetectable as seeds begin to develop.Developmental differences in the accumulation of soybean VSPβ when transgenically expressed in maize show that despite no changes in the  Although genetic variation for protein content has been found in forage plants, this variability is narrower than that observed for other traits such as digestibility . Since tGenes encoding seed storage proteins of various plant species have been transgenically expressed to test for improvement of nutritional quality. Most experiments were conducted with tobacco and legume species including alfalfa, soybean, canola, clover and lupins. For nuclear-targeted genes, accumulation of these seed storage proteins in vegetative tissue of transgenic plants was either undetectable or very low. These included pea vicilin ,3, soybeThe greatest accumulation of a seed storage protein from a nuclear-targeted gene was achieved using zeins, a maize seed storage protein that is targeted to "protein bodies" directly from the endoplasmic reticulum (ER), thus avoiding the secretory route to the cellular vacuole. Transgenic expression from the CaMV 35S promoter in tobacco resulted in the formation of these protein bodies containing the zein within vegetative tissues ,9. AltervspA and/or vspB genes fused to the 35S promoter were expressed in transgenic tobacco to study their accumulation in a heterologous dicot plant. Nuclear targeted genes produced VSP ranging between 2 and 6% of the soluble protein in leaves of the transgenic plants [In many legumes, accumulation of specific vegetative storage proteins (VSPs) in leaves and stems is the main source of increased nitrogen content -14. Use c plants ; whereasc plants ,18. Soybc plants . Howeverbar gene  were shown by Southern blot analysis to contain a 1.5 kb hybridizing band corresponding to the intact and vspB .0) transgenic maize plants at vegetative stage (7 weeks old). A distinct VSPβ band was not visible in silver-stained SDS-PAGE separated maize extract proteins due to complexity of the total protein pattern and the relatively low level of VSPβ expression  were easily visible on total protein stained gels Fig. , relativ1 plants were produced by back-crossing the R0 plants with Hi II control non-transformed pollen. Back-crossing was performed because R0 plants directly regenerated from tissue culture did not have synchronized production of pollen and receptive female flowers. The R1 families segregating for bar expression were analyzed for the presence of the vspB gene by Southern blot analysis. From 57 R1 plants analyzed, Southern blot analysis showed that 35 (61%) contained the vspB gene integrated into the genome. Figure 1 from five R0 parental lines. The ~1.9 kb vspB band can be seen in nine of the 16 R1 lines. There are also fainter bands one slightly larger than the 1.9 kb band and one or two migrating between 4 and 5 kb. These are often observed as incomplete restriction of all Eco RI sites internal to plasmid DNA that is integrated into the maize genome. Similar bands are observed even with the plasmid control.R1 plants at the vegetative stage (7 weeks after planting). Eighteen transgenic R1 plants, including the ones originating from R0 plants representing different levels of VSPβ accumulation , were analyzed for vspB transcripts. Because of high sequence similarity (85%) between vspA and vspB cDNAs, the same probe hybridized in soybean with both mRNAs as demonstrated by Staswick [Ubi-1 promoter for both the vspB and bar gene probably led to a high number of transgenics with the vspB silenced, and a higher level of VSPβ accumulation will likely result in future work using a combination of different promoters.Total RNA was isolated from leaves of young RStaswick . The tra0) and their R1 progeny, with the greatest variation observed with the highest VSPβ expressing R0 plants  and silage stage plants (plants with developing seeds at the 18 DAP-days after pollination stage), as well as, stems from the silage stage plants, . The relative level of RNA among the different plant samples was not consistent across the different lines with some having more transcripts in the young leaves while others had more in the older leaves and stems.Despite the overall low level of VSPβ expression, for the purposes of this work, VSPβ accumulation in several of the plants was high enough to study the relationship of plant developmental stage and , Figure . The vspvspB RNA abundance was compared to VSPβ protein accumulation there was no correlation between the two  were altered in the silage stage leaves and stems as compared to the leaves of immature plants.When two Fig. . AntiservspB gene was successfully introduced into R0 regenerated maize and transferred to the R1 progeny, of which vspB transcript and VSPβ protein were detected and studied. This is the first report on introduction and expression of a legume vegetative storage protein in a monocot plant. The inability to detect VSPβ from the maize vegetative tissue at the time of seed development, even when the vspB transcript was still expressed, must have arisen from either reduced translational efficiency of the vspB transcript or a decrease in the stability of the VSPβ protein. The reduction of seed storage protein level in leaves of transgenic eudicots was also observed with expression of vicillin in alfalfa [The  alfalfa  and toba alfalfa ; however alfalfa  or ovalb alfalfa . These dPlants of hybrid "Hi-II" maize were established in a greenhouse and immature tassels were used for embryogenic type II callus production, as described by Armstrong .vspB cDNA clone (998 bp) from pKSH3 [EcoRI, blunt ends of this fragment were produced using S1 nuclease and the fragment was cloned into similarly blunt ended BamHI restricted pAHC17 [vspB coding region was inserted downstream of the Ubi-1 promoter and a 5' untranslated region (exon) and intron; and upstream of the nos terminator sequence. The plasmid pAHC25 carried the bar gene and the uidA reporter gene, both under the control of the Ubi-1 promoter. Bombardments were performed with a Biolistic® PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, Hercules, CA) and an osmotic treatment was applied to reduce the cell damage caused by the gene transfer method [Microprojectile bombardment of callus was performed using the procedure of Somers et al. . Calli wom pKSH3  with Ecod pAHC17 . As a rer method . Putativr method , and groEcoRI, which released a 1.9 Kb fragment containing the vspB gene, the nos terminator and part of the Ubi-1 promoter. DNA was separated on a 0.8% agarose gel, blotted onto Hybond N+ membrane (Amersham Pharmacia Biotech, Inc. Piscataway, NJ) by capillary blotting [vspB gene segment amplified from pRSVP-1 and gel purified. The forward and reverse primers 5'-GTTCTTCGGAG GTAAAAT-3' and 5'-TTCGCCTCTGTGGT-3' were used, respectively, to amplify a 611 bp segment, and the primer pair 5'-GCAGGCTACCAAAGGT-3' and 5'-TAGGTGACTTACCCACAT-3' was used to amplified the product of 843 bp.One gram of frozen young leaf tissue was ground in liquid nitrogen and genomic DNA was extracted using the Dellaporta procedure . Twenty blotting , and UV bar transgenic plants, the DNA was digested with EcoRI, which released a fragment of ~1.5 Kb that contained part of the Ubi-1 promoter, the bar gene, and the nos terminator, and was identified with a 419 bp digoxigenin labeled probe produced by PCR amplification of pAHC25 using the forward and reverse primers: 5'-GGCGGTCTGCACCATCGT-3' and 5'-GCCAAGTTCCCGTGCTTGA-3', respectively.For identification of 10E1 and treated with 2 μl RNasin® (4U/μl) RNAse inhibitor (Promega, Madison, WI) and stored at -70°C until use. Thirty micrograms of total RNA were separated on 1.2% agarose formaldehyde gels and transferred to Hybond N+ membrane by capillary blotting overnight or by pressure blotting for one hour using a PosiBlot® 30-30 pressure blotter (Stratagene, La Jolla, CA) according to the manufacture's instructions. Membranes were UV-crosslinked, and probed with the same probes as described for Southern blot analysis. Chemilluminescence was captured using a Kodak Digital Science Image Station 440 CF (Eastman Kodak Company, Rochester, NY).Total RNA was isolated from 2 g of tissue using acid guanidine isothiocyanate-phenol-chlorophorm extraction , resuspevspB using the following primers: 5'-TGGTTCAACGCACTCTTC-3' and 5'-GGCTATGGTGAGCGTTCTTC-3'. Reverse transcription was performed for 30 min at 50°C followed by a 15 min denaturing at 95°C, and 40 cycles of 40 s at 95°C, 40 s at 58°C and 40 s at 72°C. Quantification was based on relative abundance to maize 18S RNA by amplifying a 174 bp fragment with primers: 5'-CCTGCGGCTTAATTGACTC-3' and 5'-GTTAGCAGGCTGAGGTCTCG-3', and using the comparative quantification function of the Rotor-Gene RG-3000 software. All real-time RT-PCR experiments were conducted in triplicate and on triplicate RNA preparations for each sample. Melting curve analysis and agarose gel electrophoresis were performed to verify single product formation.Total RNA extractions for real-time RT-PCR were performed using 500 mg of tissue ground to fine powder using mortar and pestle in the presence of liquid nitrogen, then processed with the RNeasy midiprep Kit (Qiagen, Germany), following the manufacturer's protocol. Trace DNA contamination was removed from total RNA by a combination of acid phenol: chloroform 5:1 pH= 4.7 extraction and Dnase I treatment (Ambion, Texas). Real-time RT-PCR was performed on a Rotor-Gene RG-3000  using the Quantitect SYBR Green real-time RT-PCR kit (Qiagen, Germany), and the manufacturers protocols with 300 ng of Dnase I treated total RNA. Primers were designed to amplify a 108 bp fragment of the soybean 2HPO4, 2 mM KH2PO4) supplemented with 1 tablet/10 ml buffer of the Complete-Mini protease inhibitor cocktail  by homogenizing in the presence of zirconia/silica 0.1 mm dia. beads. Centrifuged extract supernatants were removed and used for protein concentration determination [Protein was extracted from 100 mg of leaves and stems with 0.5 ml of phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Namination . BecauseThirty micrograms of protein were separated on 12% SDS-polyacrylamide gels. Proteins in the gel were either stained with silver nitrate  or transRT-PCR, reverse transcriptase-polymerase chain reaction; VSP, vegetative storage proteinMFG participated in experimental design, carried out the transgenic plant development, plant crosses, and molecular blotting/detection methods, and participated in manuscript draft preparation. RLS participated in experimental design, and provided guidance and training in development of transgenic maize. CM performed RT-PCR experiments. BTS participated in experimental design and provided expertise and training in plant crosses. RGS conceived of the study, participated in experimental design, coordinated the experimental plan, and wrote the draft manuscript. All authors read and approved the final manuscript.


Science published draft genome sequences for the two major subspecies of cultivated rice, Oryza sativa. The release of the rice genome—the first plant crop to be sequenced—was big news. Rice is a staple crop for more than half of the world's population, and it was hoped that the availability of the genome sequence might enable scientists to develop more productive rice strains or strains that are more environmentally friendly. Furthermore, the rice genome might provide the key to understanding the genetics of other major cereal crops, all of which have much larger genomes.In April 2002, indica and japonica subspecies of O. sativa, which are eaten in India and China, and Japan, respectively. Their analysis team, led by Gane Ka-Shu Wong, provides important insights into the evolution of rice.But the sequences published in 2002 were only draft genomes, containing many gaps and errors—works-in-progress rather than finished products. Now, a large group of scientists led by the Beijing Institute of Genomics is publishing a much improved, near-complete genome analysis of the indica by generating significantly more sequence data, and then they used better methods to assemble these data. In whole-genome shotgun sequencing, the entire genome is chopped into random fragments, each fragment is sequenced, and then powerful computer programs search for overlaps and put all these data in order. It's like putting a fiendishly difficult jigsaw puzzle together by looking for patches of matching color.First of all, the team improved their original whole-genome shotgun sequencing of indica or the japonica dataset, on one piece of DNA whose position along the chromosomes is well defined.The key to the improvement in the genome sequence analysis is that the researchers have used the combined DNA sequence data from the two subspecies to facilitate the sequence assembly. The result is a nearly 1,000-fold increase in contiguity for the two genome sequences. In other words, while the original draft was very fragmented, in the new version, 97.7% of the genes can be found, in either the The researchers have also used their improved genome sequence to investigate the evolutionary history of rice. Central to evolution is the development of new functions through mutation of existing genes. But when mutations occur in functional genes, the result is rarely beneficial, so it is thought that evolution is more likely to proceed first by duplicating existing genes and then experimenting on the “backup” copy of the gene. Wong and colleagues report that there is evidence in the rice DNA sequences for a whole-genome duplication event just before the grasses diverged from other flowering plants, about 55–70 million years ago. This genome duplication may have played a role in the origin of the grasses, which then spread rapidly across the world to provide important sources of food that, among other things, possibly influenced human evolution.Analysis of the rice genomes also indicates that a small chromosomal segment was duplicated about 21 million years ago and that there is massive ongoing duplication of individual genes. These individual gene duplications provide a continuous source of raw material for gene genesis and very likely contribute to the differences between members of the grass family. Now the challenge is to use the rice sequences as a basis for detailed genetic analyses of additional cereal crops and for the development of improved strains of not only rice, but wheat, maize, and other important food crops.


We also studied the anti-inflammatory and vasoactive effects of inhaled corticosteroid and β2-agonist.We investigated the relationship of Δe°T with Q1 74 ± 6 % predicted) compared to 16 normal volunteers (4.23 ± 0.41 Δ°C/s, p < 0.01) (30 ± 2 yr) and was significantly increased after salbutamol inhalation in normal subjects (7.8 ± 0.6 Δ°C/ s, p < 0.05) but not in asthmatic patients. Qaw, measured using an acetylene dilution method was also elevated in patients with asthma compared to normal subjects (49.47 ± 2.06 and 31.56 ± 1.6 μl/ml/min p < 0.01) and correlated with exhaled NO (r = 0.57, p < 0.05) and Δe°T (r = 0.525, p < 0.05). In asthma patients, Qaw was reduced 30 minutes after the inhalation of budesonide 400 μg (21.0 ± 2.3 μl/ml/min, p < 0.05) but was not affected by salbutamol.Δe°T was confirmed to be elevated (7.27 ± 0.6 Δ°C/s) in 19 asthmatic subjects (mean age ± SEM, 40 ± 6 yr; 6 male, FEVaw and exhaled NO in asthmatic patients and therefore may reflect airway inflammation, as confirmed by the rapid response to steroids.Δe°T correlates with Q To test this, we investigate the relationship between airway inflammation as assessed by exhaled NO, with Qaw and Δe°T measured non-invasively. Qaw is an expression of bronchial blood flow, whereas Δe°T reflects the rate of temperature increase in the exhaled breath. We hypothesised that Qaw changes may contribute to the levels of Δe°T and we studied their relationship considering that, potentially, minor changes of bronchial blood flow may not affect exhaled breath temperature.Asthma is an inflammatory disease of the airways. Vasodilatation is a critical feature of inflammation, and angiogenesis and vascular remodelling are features of chronic inflammatory diseases, such as asthma . The incaw and Δe°T.Elevated levels of exhaled NO in asthma ,7 are liaw is non-invasive and was standardised and adapted from a previously validated technique [aw) were also measured non-invasively allowing us to make repeated measurements and to study the interactions of these two markers and exhaled NO.The measurement of Qechnique ,13. Δe°Taw has been advocated to assess airway steroid sensitivity. In order to evaluate further the correlation of Qaw and Δe°T with inflammation, we studied the acute effect of inhaled budesonide on these parameters and their reciprocal changes. Furthermore, to validate our methods, we also evaluated the vasodilating effect of the short-acting β2-agonist salbutamol.The inhalation of corticosteroids has been shown to have an acute vasocostrictive effect on the bronchial circulation  and the 1 74 ± 6 %, 9 patients were on inhaled steroid treatment and 10 patients had mild persistent asthma and were on β2-agonist inhalers only). These two groups of patients were chosen because they are representative of the larger majority of asthmatic patients; in addition this allowed us to verify cross sectionally the effect of inhaled steroids on bronchial blood flow and exhaled NO.Nineteen asthmatic patients were studied  recruited from our outpatient clinic and from volunteers . In one time constant the response reaches 63% of its final change.The exhaled breath temperature changes exponentially with time. The shape of the curve depends upon the The response is of the formV2 is the final value, V1 the initial value, and t is the time after exhalation was started. As t > infinity, the exponential term > 0. Hence the response rises asymptotically to its final value. When performing experiments to determine the time response of a system, it is difficult to tell at which time the final value is obtained, since there will be a small change in the response for a relatively long time as the asymptote is approached. Jitter and noise in the signal will also add to this problem. It is easier to estimate the asymptotic value and find the time at which a certain percentage of this is reached. Various percentages are possible, the most commonly used in physics and biology is the time constant, although in some applications (particularly electronic engineering) the rise time to 95% of the final value is used.Where The 63% arises because when t = T, the expression above becomesV2-V1)(1-1/e) = (V2-V1)(1-0.37) = 0.63(V2-V1)(n)In n time constants the percentage reached is 100 × (1-1/ewhich, approaches 98% after about 5 time constants.a" is 63% of Δ°T and "b" the time to reach "a") proved to be the more reproducible parameter to characterize the curves.The rate of temperature increase (Δe°T) calculated between the beginning of exhalation and 63 % of the total temperature increase  compared to higher (5–6 L/min) exhalation flow rates (6.25 ± 0.4°C/s and 4.45 ± 0.8°C/s, p < 0.01) in 8 normal subjects. When the thermocouple was inserted close to the edge of the mouthpiece (1 cm) the Δe°T was significantly higher (7.15 ± 0.2°C/s) compared to when it was located farther (2 cm) (4.45 ± 0.8°C/s, p < 0.01). There was a tendency for faster Δe°T when the subjects were starting exhaling from higher baseline ambient temperatures but this was not significant for temperature changes within ± 3°C (5.05 ± 0.8°C/s at 28°C and 4.45 ± 0.8°C/s at 22°C p > 0.05). The volume ventilation did not influence the Δe°T value. The difference in exhaled breath Δe°T and plateau temperatures measured during two successive collections at five minutes intervals (single session variability) was 4.4%, while between sessions variability  was 6.8%. The reproducibility of the test was confirmed by the Bland and Altman test .Exhaled NO was measured using a modified chemiluminescence analyzer  as previously described .aw, using acetylene rather than the potentially explosive dimethylether [aw was calculated from the Fick principle (dilution of acetylene concentration) .. Data were expressed as means ± standard error of mean. The relationship between the exhaled breath temperature, Qaw was elevated to a similar extent in patients with mild persistent and moderate asthma  (46.0 ± 51 μl/ml/min) and patients with mild intermittent asthma (on β2-agonists as needed only) (52.64 ± 3.0 μl/ml/min, p > 0.05) compared to normal subjects  compared to normal subjects  compared to steroid treated patients (7.5.6 ± 2.3 ppb, p < 0.05) and normal subjects (4.7 ± 0.3 ppb, p < 0.05).aw was significantly reduced 30 minutes after the inhalation of budesonide compared to baseline , while this effect was not present in asthmatic patients  nor in normal subjects (34.0 ± 3.0,38.1 ± 4.5 and 35.1 ± 8.1 μl/min/ ml/ p > 0.05)Placebo had no effect on Qaw and Δe°T were correlated in asthmatic patients (r = 0.78, p < 0.05). In normal subjects, no significant changes of Δe°T were found at any of the time points after budesonide inhalation (3.5 ± 2 Δ°C/sc at baseline, 3.5 ± 1 Δ°C/sc at 30 minutes, 3.53 ± 2 Δ°C/sc at 60 minutes, p > 0.05).The inhalation of budesonide was associated with a tendency for a decrease of Δe°T in asthmatic patients , while this effect was not present in asthmatic patients  and in normal subjects (3.50 ± 0.29 Δ°C/ sec and 4.20 ± 0.32 Δ°C/ sec p > 0.05) after the inhalation of placebo.aw and Δe°T are correlated with one another and with airway inflammation as assessed by exhaled NO. We propose that high levels of NO generated in asthmatic patients as a result of airway inflammation may cause vasodilatation of the bronchial circulation contributing to increased heat exchange. This is supported by the demonstration that lower Δe°T levels, after the inhalation of corticosteroids, are correlated with reduced levels of bronchial blood flow. We propose that these non-invasive measurements may be useful to evaluate airway inflammation and may provide a tool to assess steroid sensitivity.This is the first study to show that elevated levels of QAngiogenesis and microvascular remodelling are features of chronic inflammatory diseases, such as asthma . As inflaw and Δe°T and airway inflammation as assessed by exhaled NO. Even though the patients enrolled in this study were significantly older than the control group, our previous studies [aw which has also been suggested as a marker of inflammation. We also studied the interaction of these two parameters after inhaled corticosteroids β2 agonists.In a previous study  we have  studies ,24 indicaw in asthmatic subjects compared to normal volunteers, using a modified method developed by Onorato et al [aw in this study presented the same response pattern and timing to the inahaltion of steroids [aw.We confirmed previous data showing elevated levels of Qto et al . In the steroids  and betasteroids  as previaw produces results which include the contribution of the dead space and trachea blood flow to the total bronchial blood flow. We acknowledge that the trachea may not be the main site of inflammation in asthma, but inflammation in asthma extends from the larynx to the terminal bronchioles and the tracheal mucosa certainly appears inflamed in many asthmatic patients. When tracheal inflammation occurs there will be a good separation between normal subjects and patients with asthma because the measurements of bronchial blood flow and exhaled breath temperature will particularly reflect the contribution of this part of the respiratory tractThe method used in this study for the measurement of Qaw and Δe°T but we have also shown that these measurements respond similarly to steroids and beta 2 agonists. Therefore, patients with asthma have a significantly faster rise of breath temperature and Qaw and these are correlated. We presume that this is due to the increased vascularity of the bronchial vessels [aw and Δe°T may appear to be weak, it is noteworthy that the acute changes of these variable was significantly correlated, reinforcing the hypothesis that elevated breath temperature gradients in asthmatic patients may reflect increased bronchial blood flows.For the first time we have shown, using non-invasive methods, that changes in bronchial blood flow can alter exhaled breath temperature indicating that the bronchial circulation may control airstream temperature. Furthermore, in this study not only we have shown a correlation between Q vessels  and elevWe were able to show a positive correlation between exhaled NO and Δe°T. NO is a gas produced by several types of pulmonary cells, including inflammatory, endothelial and airway epithelial cells. Elevated levels of exhaled NO in asthma , and intaw in corticosteroid-treated compared to untreated asthmatic patients, despite the efficacy of steroids in reducing bronchial blood flow [aw [2 induced vasodilatation resulting in minimal changes in bronchial artery diameter and blood flow and therefore no changes of Qaw and Δe°T. However, these results must be confirmed by placebo controlled studies.In this cross-sectional study we could not show any differences of Δe°T or Qood flow . This isood flow  and Qaw flow [aw  comparedaw which returned to baseline one hour after inhalation. This is also consistent with a previous publication [aw and Δe°T and their interaction were studied simultaneously in the same group of patients for the first time. Corticosteroids may cause vasoconstriction by numerous mechanisms. They can potentiate the vasoconstrictor actions of noradrenalin and angiotensin II by upregulating their vascular receptors [1-adrenergic vasoconstrictor response [1-adrenergic steroid interplay in the regulation of vascular tone.In contrast to the effect of chronic treatment with corticosteroids, the acute inhalation of budesonide caused a significant temporary reduction of Qlication , notablyeceptors . Furthereceptors , thus caeceptors . The gluresponse . This adFurther studies are required, investigating the dose response relationship for inhaled steroids would provide valuable information.aw and exhaled NO which was shown for the first time in this study. Measurement of exhaled breath temperature and bronchial blood flow may provide means of detecting airway inflammation and vascular remodelling in a non-invasive way.The cardinal signs of inflammation are rubor (redness), calor (heat), tumor (swelling), dolor (pain), and impaired function (functio laesa). Exhaled breath temperature and bronchial blood flow may reflect rubor and calor in the airways and therefore may be markers of tissue inflammation and remodelling as confirmed by the positive correlation between Δe°T, Q


It has not been possible to reproduce this process with isolated adipocytes suggesting that other cells are needed, and perhaps mediate the regulation. The objective of the present study was, therefore, to explore if explants of adipose tissue could be used for studies of the regulatory process.Recent studies 125I-labeled LPL added to the medium was degraded to acid soluble products, indicating that the degradation occurred extracellularly. Fragmentation of the labelled lipase occurred in conditioned medium and this process was virtually abolished by two MMP inhibitors.When explants of rat epididymal adipose tissue were incubated, LPL mass and activity decreased rapidly. Mass and activity within adipocytes remained constant for at least six hours, demonstrating that it was the extracellular portion of the enzyme that decreased. Adipocytes isolated from the explants after three or six hours of incubation retained their ability to secrete LPL to the medium. Addition of a cocktail of protease inhibitors to the incubation medium slowed down the decrease of LPL mass. Chloroquine was without effect, indicating that the degradation was not lysosomal. The decrease of LPL mass and activity that occurs when explants of rat adipose tissue are incubated is due to proteolysis of extracellular LPL. The adipocytes continue to produce and secrete the enzyme. The effect of inhibitors indicates, but does not prove, that the degradation is mediated by MMPs. It appears that this process is accelerated in the tissue fragments compared to intact tissue. To study the underlying mechanism an in vitro model is urgently needed. Experiments with isolated adipocytes do not seem to bring out the mechanism and the in vivo experiments indicate that it is the extracellular LPL that is the target for the regulation [Lipoprotein lipase (LPL) hydrolyzes triglycerides in very low-density lipoproteins and chylomicrons . Tissue-gulation . We haveIn the first set of experiments we incubated explants of rat adipose tissue and followed LPL mass and activity Figure . LPL masWe tried variations in technique and several different incubation media in experiments such as those in Figure One possible explanation could be that LPL was released from the tissue into the medium. The amounts of LPL mass or activity that appeared in the medium were, however, small Table . To testAnother possibility was that the cells lost their ability to produce LPL. Isolated adipocytes, incubated under the same conditions as the tissue explants, released LPL activity Figure  and massHeparin release is often used to assess the LPL activity of tissues and releases mainly extracellular LPL . Figure These results indicated that the decline of LPL mass occurred through proteolytic cleavage of the extracellular enzyme. To test this hypothesis, we included a cocktail of protease inhibitors in the medium used for incubation of tissue explants. This slowed down the decrease of LPL mass Figure . Chloroq125I-LPL was added to the incubations. TCA soluble material appeared in the medium demonstrating that proteolytic degradation took place . Unless otherwise stated, the rats were 23 days old and weighed around 60 g. After transport to Umeå they were allowed to acclimatize for seven to ten days by which time they had reached a weight of approximately 120 g. The rats were kept in a well ventilated, temperature (21°C) and humidity (40–45%) controlled room with free access to a standard laboratory chow (Laktamin AB, Stockholm) and tap water. The light in the room was on between 6 a.m. and 6 p.m. In experiments where the rats were to be fasted, food was withdrawn from the cages at 6 a.m. and a grid was placed at the bottom of the cages to prevent coprophagia. The adipose depot used in all experiments was the periepididymal one. The rats were killed by decapitation. Animal experiments were approved by the animal ethics committee in Umeå.3H-labelled triolein in Intralipid (10%) kindly prepared by Pharmacia-UpJohn (Stockholm, Sweden). Parker medium (Parker 199) was from SBL (Stockholm, Sweden). 125 I-LPL was prepared as before [Cycloheximide, bovine serum albumin (BSA), the MMP inhibitors GM6001  and Captopril, chloroquine and collagenase were from SIGMA (St. Louis, MO, USA). Protease inhibitor cocktail tablets "Complete Mini" were from Roche Diagnostics, Mannheim, Germany. Heparin was from Lövens . Substrate for the LPL activity assay was m Parker 9 was froLPL was extracted from tissues by homogenization in a Tris-HCL buffer (pH 8.2) containing detergents and protease inhibitors as described . The homLPL activity was measured as described previously . BrieflyLPL mass was measured with an ELISA as described . The chiDNA content was assayed using Labarca's method .2: 95 % O2 with continuous gentle shaking motion in a Cellstar Incubator (Queue Systems, Asheville, Canada). After incubation, the tissues were prepared for measurement of LPL activity and mass as described above. In some experiments samples of the medium were taken for assay of LPL activity and/or mass.Epididymal adipose tissue was dissected out from fed or 24 h fasted rats. The tissue was cut into small pieces (5 mg or less). A total of about maximal 100 mg tissue pieces were immediately put into culture plates. Each well contained 15 ml of Parker Medium 199 supplemented with 2% BSA, 0.5% casein hydrolysate, 10 mM glucose and adjusted to pH 7.4. Incubations were at 37°C and 5 % COIn some experiments adipocytes were prepared after collagenase treatment of the tissue pieces as described . To measStudent's t-test was used for analysis of the data.GW carried out the experiments and participated in their design and in writing of the manuscript, TO participated in the design of the study and drafted the manuscript. GO conceived of the study and coordinated the work. All authors read and approved the final manuscript.