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 . Once aligned, the rhythm of CBFV closely tracked the rhythm of CBT as demonstrated by the substantial correlation between these two measures .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 .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  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  to after the subject returned to the bed , t(3) = -5.17, p = .014). Subject 6 showed a decline in CBFV from before  to after the subject returned to the bed , 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  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 . 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 . 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 . 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 . 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  across the 24 hour period. Diastolic blood pressure (DBP) and CBT showed a negative correlation . 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  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  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  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  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 .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 , 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 . Real-time RT-PCR was performed on a Rotor-Gene RG-3000  using the Quantitect SYBR Green real-time RT-PCR kit , 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  (30 ± 2 yr) and was significantly increased after salbutamol inhalation in normal subjects  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  and Δe°T . In asthma patients, Qaw was reduced 30 minutes after the inhalation of budesonide 400 μg  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 ×  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  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) . 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)  compared to normal subjects  compared to normal subjects  compared to steroid treated patients  and normal subjects .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 Placebo had no effect on Qaw and Δe°T were correlated in asthmatic patients . In normal subjects, no significant changes of Δe°T were found at any of the time points after budesonide inhalation .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  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 . Parker medium (Parker 199) was from SBL . 125 I-LPL was prepared as before [Cycloheximide, bovine serum albumin (BSA), the MMP inhibitors GM6001  and Captopril, chloroquine and collagenase were from SIGMA . 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 . 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.


The necessary and sufficient capabilities of cancer cell have been identified. Strikingly, this list does not include one that would seem to be a key property, namely the ability of cancer cells to kill their "host". This is believed to be a self-evident consequence of the other capabilities , although the available evidence suggests a distinct killer function. Taking into account this unlisted property can significantly affect the current paradigm of carcinogenesis.On the assumption that killer function is a key capability of the cancer cell, it is suggested that cancer has evolved as a mechanism of negative selection of mutant alleles of vitally important genes present in population. Similarly to apoptosis, which is an altruistic suicidal act of a damaged cell, cancer is an altruistic suicidal act of an individual who carries dangerous alleles and presents a hazard for genetic stability of the population. From this point of view, apoptosis is not a protection means against cancer as generally believed, but rather they are the first and second lines of defense against genome instability, respectively.The modern DNA array technology is capable of revealing gene expression profiles responsible for killer function of cancer cell as well as those specific targets in the body that are most strongly affected by the tumor growth.This hypothesis suggests new avenues of cancer research as well as principally new therapeutic strategies. We are now witnessing a post-genomic era of cancer research. Thousands of papers are devoted to discovering molecular mechanisms of this extremely complicated program, the latter term being understood as a prescribed sequence of events with an inevitable result. In almost all studies cancer is considered as a given entity with no attempts made, to my knowledge, to search into its evolutionary origin. However, cancer almost certainly fulfills some evolutionary tasks.First, although cancer is usually mentioned as a representative of a large group of age-related diseases, it principally differs from all the others. Cardiovascular diseases, diabetes, Alzheimer's disease, and many other diseases are manifestations of the "loss-of-function" phenotype due to degeneration and/or death of corresponding cells. Cancer, on the contrary, is an active "gain-of-function" process. Cancer cells acquire numerous new functions, including the unique abilities to adapt to a changing environment and to dodge the blows of the body's protective means, as well as a striking capacity to recruit surrounding normal tissues. The tumor transforms its natural enemies  into unnatural allies, being able to grow only having such a paradoxical support -15. The Second, cancer is an inevitable attribute of the animal world. It hits every species despite the fact that there are huge differences among them in the intracellular machinery, organization of signal pathways and, accordingly, in anticancer defense . In no case, however, is the anticancer defense of a species high enough to exclude completely this illness, despite presumably high cellular plasticity in this respect. This fact would indicate some evolutionary advantages of possessing such a trait. It seems likely that anticancer defense of a species is counterbalanced by opposing forces acting on the population level. In other words, cancer incidence among a species is presumably maintained at certain favorable level, which is coupled in each particular case with an evolutionary adaptation of the intracellular machinery.In conclusion, evolutionary grounds for cancer seem to be evident; it is the explanation of its expediency which presents difficulties. A clue to this problem would be the fact that cancer cell possesses, apart from the well known necessary and sufficient capabilities , a kille14 cells, can not endure a relatively small fraction of cancer cells , this burden often being incompatible with life. Rarely, the immediate cause of death is evident , but in general it remains obscure. This suggests a deadly influence of cancer upon the body through some unknown mechanisms. Although each of many different forms of cancer has clinical peculiarities, the overall course of the illness and the final result are always the same. So, the notion that the tumor cell, regardless of origin, possesses a special killer function is a statement of an obvious fact. What is surprising in this regard is that no room has been allocated to it in the existing paradigm of carcinogenesis.Carcinogenesis is a multistage process of accumulation of gene defects that determine the characteristic traits of the cancer cell: self-sufficiency in growth signals, insensitivity to anti-growth and pro-apoptosis signals, limitless replicative potential, sustained angiogenesis, tissue invasion, and metastasis . These aParaneoplastic syndromes present evidence that tumors may affect normal tissues remote from the primary site. These syndromes are extremely diverse and affect almost all organs and tissues . The mosparaneoplastic, take place not within the killer function pathway but somewhere aside. Because of this, the term killer function will be used here, without going into its mechanism, as indicating the obvious capability of cancer cells to kill the body.It remains unknown whether the paraneoplastic syndromes are direct manifestations of the killer function itself or they are mere side effects of tumor growth, such as autoimmune diseases. It is noteworthy that cancer is not always accompanied by paraneoplastic syndromes , yet itsde facto only accessory. It seems to be a distinct capability of cancer cells, rather than a derivative of other capabilities, such as uncontrolled proliferation and metastasizing. It is unlikely that active proliferation can by itself exert such a deleterious effect since dozens of billions of cells divide daily in the human body, which is many-fold greater than the proliferation pool of the biggest tumor. Even the metastases, these relatively small foci of ectopic proliferation, can not account by themselves for the inevitable demise. The killer function is also a universal property of the cancer cell since without treatment the lethal outcome is inevitable no matter what is the type of tumor, its ability to develop metastases, recur, induce cachexia, or affect biochemical indices. Finally, this property is specific to the cancer cell, as in normal cell physiology there are no examples of such activity.The killer capability is crucial to the achievement of the final goal, body demise, whereas all the other, promoting proliferation and spread of killer cells are The killer function seems radically different from all other acquired capabilities in that it apparently gives no selective advantage to the cancer cell. On the contrary, for the latter, as a part of the body, acquiring such a function is the same as committing suicide. This changes radically the understanding of the role of cancer cells: they can be regarded not as selfish "cheats" , which pde novo in the same way as the other properties are, namely in the way of numerous step-by-step cycles of mutation-selection. Rather, the cells possess a built-in and ready-for-use program of deadly events, which, just like apoptosis, is launched under certain conditions and then functions automatically. In such a case, mutations of cancer-related genes are not only transformation steps, as generally considered, but also a trigger countdown mechanism that activates the death program directed against the "host". This dual activity leads to appearance of an expanding clone of killer cells progressively strengthening their effect upon the organism . In a certain sense, these are the defects of not the cell  4 to 12 ,26, this14) and large longevity. Indeed, no matter how unlikely is the event by itself it has a real chance to occur under such conditions. If so, one might suggest that animals with a small body weight and short life-span  should not suffer from cancer at all, while big animals  should get cancer in their mothers' wombs. Reality, however, does not follow this theory's predictions . All anThe Peto paradox is explained by various transformation resistance of cells from different species ,30. ThisThe Peto paradox can apparently be applied to body tissues as well . Indeed,Most patients develop only one tumor, which presents carcinogenesis as allegedly a local process. Experiments with exposure of animal skin to chemical carcinogens as well as cases of occupational cancer, which demonstrate clearly the link between site of exposure and tumor localization, support this notion. Without questioning the significance of such observations, they seem to have an exceptional and limited character (see below).-4–10-8 per gene per cell division [5 mutations accumulate in the normal stem cell of human colonic crypt [In fact, mutagenesis is intimately related to metabolism and is therefore omnipresent: every day in each cell many thousands of DNA lesions occur due to replication and repair errors, spontaneous depurination, methylcytosine deamination, reactive oxygen species attacks, and telomere shortening. This list should perhaps be extended to include the mutagenic effect of apoptosis resulting from uptake by phagocytosis of DNA from dead cells . Becausedivision ,27; mutadivision ,36. Calcic crypt .TP53, but remaining undetectable by routine diagnostic techniques [When a tumor nodule appears in the body, it seems to be only the tip of an iceberg, maturing in the body for decades and consisting of a multitude of damaged cells at different stages of transformation. This assumption is supported by the clinical experience showing that overt symptoms of the disease are always preceded by precancerous lesions, such as hyperplasia, metaplasia, and dysplasia. This idea finds further development in the concept of "field cancerization", i.e., large (more than 7 cm in diameter) and surrounding the tumor "patches" of damaged cells, recognized on the basis of mutations in chniques .Similar conclusions can be drawn from a notion of mutation as a random and rare event and carcinogenesis as accumulation of genetic defects. Mutagenesis can be described as the process both extensive (measured by number of affected cells) and intensive . Evidently, these parameters are positively correlated with each other: the wider the damaged zone, the deeper the damage of individual cells. The reverse is also true: the deeper the damage of the individual, most "advanced" cells, the wider the lesion area . In other words, appearance of the cell having a complete set of mutations  is accompanied by formation of a large pool of precancerous cells.The simple model of accumulation of mutant cells  in an exponentially growing cell population is shown in Fig. It follows from the model of multistage carcinogenesis  that in  is the mean number of cells with m mutations after k cell generations and u is the mutation rate. If we assume that the body is composed of 3.5 × 1013 cells , and u is 4 × 10-5 , then aion Fig. . In othetal data  and can tal data ). They mtal data .de facto a triple role: (i) a sensor for general mutagenesis (since its "trigger clock" counts down in accordance with accumulation of mutant cells in the body), (ii) an executioner that unleashes a built-in death program after mutagenesis exceeds some threshold level, and (iii) a founder of a clone of killer cells. Any stem cell in an organism can apparently play such a triple role, thus ensuring reliability of the mechanism.As discussed above, the size of the fraction of pre-malignant cells is dependent on trigger tuning – the more mutations needed for a malignant transformation, the bigger the pool of damaged cells. Hence, the first completely transformed cell plays in vitro [Evolutionary conservation of the death program prompts us to look for an explanation for its appearance. It may consist of the counter-selection of genetically defective individuals. As a matter of fact, if an arising tumor manifests significant whole body mutagenesis, then germ cells are most probably not an exception: a similarity of spontaneous germinal and somatic cell mutation rates was shown for human cells in vitro . High lein vitro . Due to in vitro ,43,44. Bin vitro ,46. In cin vitro , at the While in the case of sporadic tumors the notion that cancer is a local manifestation of generalized mutagenesis is only a more or less validated assumption, it is a truism in the case of hereditary tumors . CarcinoIt is evident that the greater the number of pre-cancerous stages, the more strongly the individual is protected against cancer. A computational model of cancer progression was elaborated recently to show that the appearance of an extra stage and the additional buffering, which arises as a result, reduce the impact of any single hereditary mutation and therefore allow the accumulation of more nonlethal mutations in the population . BecauseThere are many germinal mutations with phenotypic expression but just a few of them  are linA favorable trait is retained only if it manifests itself during the reproductive period. So, the fact that cancer is predominantly a disease of the elderly would seem to be at conflict with a supposition of its evolutionary significance. This is perhaps an apparent conflict: the incidence of cancer among the young may be relatively low exactly because of the efficient selection against the adverse alleles that constantly appear in the population and exist as an inevitable background. As regards the high incidence of cancer at the old age, which seems to have no evolutionary significance, this can be explained from the viewpoint of the evolutionary theory of antagonistic pleiotropy . A genetGerminal mutations in functionally important genes are a strong stimulus for development of a mechanism to prevent their spread in the population. Apart from these highly penetrant rare alleles with a strong hereditary predisposition to cancer, which are merely eradicated from the population, there are many alleles that determine a weak predisposition  .In carcinogenesis, the hypothetical sensor/executor functions under the conditions of continuous interference and noise, which are generated by numerous external and internal locally acting mutagens. From this, many false actions ensue. If one takes the hypothesis that cancer is a means of protection, one must admit that in humans this mechanism is hyperactive and operates beyond the originally set objectives. Both the high cancer incidence in old age and the multitude of cancer cases resulting from local exposure to carcinogens are examples of this hyperactivity that obscure the true evolutionary nature of the phenomenon.Evolution hypotheses that attempt at explaining the appearance of cooperation and altruistic behavior are based on the ideas of kin or group selection ,57. TheiMaintaining DNA integrity is one of the main priorities of living organisms. Depending on the extent of DNA damage, three outcomes are possible: (i) small damage induces repair which restores the initial state; (ii) strong damage launches apoptosis thus preventing cell-to-cell transfer of damaged DNA; (iii) accumulating lesions, when apoptosis is impossible, trigger cancer thus preventing individual-to-individual transfer of damaged alleles of vitally important genes. From this point of view, apoptosis is not a protection means against cancer as generally believed, but rather they are the first and second lines of defense against genome instability, respectively.A striking discovery was made recently that a tumor can survive, propagate, and spread in the body only through the unnatural help coming from normal tissues ,13,59. OBesides, identification of genes responsible for killer function is to be supplemented, using the same technology, with serial analyses of expression profiles of various organs and tissues of tumor-bearing animals at various stages of tumor progression. This can help to identify those specific targets within the body that are most strongly affected by the tumor growth.Recognition of killer function as a crucial capability of cancer cells suggests not only new avenues of cancer research (see above), but also principally new therapeutic strategy. Achievement of better understanding of mechanisms of body death may help to pinpoint new targets for therapy, such as some factors presumably emitted by cancer cells , that exert the deadly effect. These factors are likely to be cancer-specific, so their elimination would not entail severe side effects. The present day cancer therapy is based, without much success, on the imperative to "exterminate the evil" . The essence of an alternative strategy which may turn out to be more effective is not to kill the cancer cells, but to neutralize them.The author(s) declare that they have no competing interests.


During the 1990s, health care restructuring in Nova Scotia resulted in downsized hospitals, reduced inpatient length of stay, capped physician incomes and restricted practice locations. Concurrently, the provincial homecare program was redeveloped and out-of-hospital cancer deaths increased from 20% (1992) to 30% (1998). These factors all pointed to a transfer of end-of-life inpatient hospital care to more community-based care. The purpose of this study was to describe the trends in the provision of Family Physician (FP) visits to advanced cancer patients in Nova Scotia (NS) during the years of health care restructuring.Design Secondary multivariate analysis of linked population-based datafiles including the Queen Elizabeth II Health Sciences Centre Oncology Patient Information System , the NS Hospital Admissions/Separations file and the Medical Services Insurance Physician Services database. Setting Nova Scotia, an eastern Canadian province . Subjects: All patients who died of lung, colorectal, breast or prostate cancer between April 1992 and March 1998 . Outcome Measures Inpatient and ambulatory FP visits, ambulatory visits by location , time of day , total length of inpatient hospital stay and number of hospital admissions during the last six months of life.In total, 139,641 visits were provided by family physicians: 15% of visits in the office, 10% in the home, 5% in the emergency department (ED), 5% in a long-term-care centre and 64% to hospital inpatients. There was no change in the rate of FP visits received for office, home and long-term care despite the fact that there were 13% fewer hospital admissions, and length of hospital stay declined by 21%. Age-sex adjusted estimates using negative binomial regression indicate a decline in hospital inpatient FP visits over time compared to 1992–93 levels  and an increase in FP ED visits .Despite hospital downsizing and fewer deaths occurring in hospitals, FP ambulatory visits (except for ED visits) did not rise correspondingly. Although such restructuring resulted in more people dying out of hospital, it does not appear FPs responded by providing more medical care to them in the community. Canadians have become increasingly vocal about the need for improved care for the dying. In response, the Special Senate Committee on Euthanasia and Assisted Suicide declaredAs these calls for better care for the dying go out, hospital roles in Canadian communities have been redefined. Hospital restructuring has transferred many aspects of inpatient care to community-based care, including the end-of-life or palliative care of those with cancer. End-of-life care is defined broadly as all care provided to dying persons. Multiple providers are involved in such care and include generalist providers such as family physicians, community nurses and other primary care providers, hospital-based providers, specialists, specialist palliative care providers, volunteers and family. Unless otherwise stated "end-of-life care" reflects any or all of these kinds of care as received by dying patients. When warranted, we will refer to "palliative care program" (PCP) as the comprehensive, organized and specialized program of care for the dying. Such PCPs are often hospital based and include an inpatient palliative care unit, consultant nurses and physicians and may provide consultations and / or care in the community. End-of-life care has been provided in the community in the past, however, this health system restructuring has forced an even greater emphasis on this location for care.Nova Scotia is a small, east coast Canadian province of just under one million people where the percentage of cancer deaths occurring outside of hospitals rose from 19.8% in 1992–93 to 30.2% in 1997–98, an increase of over 50%).Study subjects included all Nova Scotians who died due to lung, colorectal, breast or prostate cancer from April 1, 1992 to March 31, 1998 as indicated on the Vital Statistics death certificate  the Queen Elizabeth II Health Sciences Centre Oncology Patient Information System (OPIS) which encompasses the provincial Nova Scotia Cancer Registry (NSCR) and includes provincial Vital Statistics information, (2) the Nova Scotia Medical Services Insurance Physician Services (MSIPS), and (3) the provincial Hospital Admissions and Separates file (HAS). The MSIPS includes data pertaining to all visits provided by physicians in the province who are remunerated via fee-for-service schedules and for physicians who are paid alternatively that provide 'shadow' billing information. More than 96% of FPs in Nova Scotia were fee-for-service at the time of this study.Health services were limited to the 'end-of-life (EOL)', defined in this study as the six months (180 days) prior to the date of death, or from the date of initial cancer diagnosis as recorded in the NSCR to death for persons living less than six months after diagnosis. Service fee codes  and health service identification numbers  were used to identify FP visits.All visits provided by a family physician were counted for each patient during their end-of-life including those in the FP office, patient's home, long-term-care facility (LTC), emergency department (ED), and hospital inpatient settings. Ambulatory visits were defined as all visits, including those to the emergency department, but excluding those made to a patient during a hospital inpatient stay or outpatient procedures. Two 'time of care' categories were created: regular hours (8:01 am to 5 pm), and after hours care . Because a common code was not available to identify the time of visit to hospitalized patients, all hospital inpatient visits were considered as occurring during regular hours.In addition to the total length of inpatient hospital stay and the number of hospital admissions, we also examined the total number of specialty visits received by these patients. Demographic and clinical variables included sex, age, fiscal year of death, geographic region, time from diagnosis to death, and tumour site .Initial analyses focused on frequency counts and descriptive measures  of all FP visits, hospital admissions, length of inpatient stay and specialty visits, overall and by fiscal year of death. Each count was expressed as the number of visits provided per 100 "end-of-life (EOL) person-days". Linear temporal trends were assessed using negative binomial regression with a logarithmic link function linking the dependent variable  to fiscal year. Fiscal year was included as a linear predictor adjusting for age and sex, and with log (EOL person-days) as an offset variable. Assessment of a nonlinear trend was made by including fiscal year as both a linear predictor and a qualitative predictor. The Type 3 analysis of year as a qualitative predictor in this model relates to the nonlinear component of trend. Adjusted regression coefficients were exponentiated and reported as rate ratios (RR) with associated 95% confidence intervals (CI). All analyses were conducted using SAS software[In total, 7212 Nova Scotians were identified as having died due to lung, colorectal, breast or prostate cancer over the six-year study period. Males comprised a larger proportion of deaths, as did adults aged 65 years and older, those who died due to lung cancer and survivors of at least 150 days from date of initial cancer diagnosis , were provided by FPs to patients during their end-of-life with 94% of patients receiving at least one FP visit. Variability across fiscal years was minimal, ranging from 92.9% receiving at least one FP visit in 1993–94 to 95.3% in 1997–98.The majority of FP visits were provided to hospital inpatients (64%), followed by the office (15%), home (10%), the emergency department (5%) and long-term care (5%). Of visits provided to hospital inpatients, almost 71% were categorized as a 'subsequent hospital visit', which represents continuing in-hospital care. Other inpatient visits included initial hospital visits (4.7%), visits after four weeks (17.5%), supportive care visits (4.6%) and urgent or emergency care visits (2.5%).Temporal trends associated with ambulatory and inpatient FP visits per 100 EOL person-days are shown in Figure Examination of ambulatory FP visits provided during regular hours (8:01 am-5 pm) showed no significant change over time. However, ambulatory visits provided during 'after' hours  were found to have increased significantly over the study period (p < 0.03). Compared to patients in 1992–93, patients in 1997–98 received 12% more ambulatory visits after hours . Although this increase represented a significant change over time, after hour visits were relatively few compared to the large number of other ambulatory visits and therefore exerted no impact on the overall temporal effect among all ambulatory visits.Significant temporal trends were evident with respect to both the total number of hospital admissions experienced by the patient and the total number of days they spent as a hospital inpatient. A decline in hospital admissions was seen over time, from 1.2 admissions in 1992–93 to 1.1 admissions per 100 EOL person-days in 1997–98. Compared to 1992–93, patients in all subsequent years experienced fewer hospital admissions after accounting for sex and age. By 1997–98 patients experienced 13% fewer hospital admissions than patients in 1992–93 .Over 85% of patients spent at least one day as a hospital inpatient. Patients spent on average a total of 22.7 days in hospital  or 15.6 days per 100 EOL person-days. Total length of hospital inpatient stays declined from 18.6 days per 100 EOL person-days in 1992–93 to 14.8 in 1997–98. Results from the age and sex adjusted regression analysis indicate total length of hospital inpatient stays in 1997–98 were 21% shorter than experienced in 1992–93 .In total, 82,575 visits were made to a medical specialty during the end of life. The number of visits ranged from one to 169, with a median of 11 visits per patient . Age and sex adjusted regression analysis indicate visits to a medical specialty did not change significantly over time.Despite the move to a greater percentage of cancer patients dying out of hospital and despall types of patients in the province from 1992–1999, the total number of office-based, home and long-term-care visits has declined slightly[When we compare these trends to concurrent trends in the province of Nova Scotia for all types of patients, a number of interesting points emerge. For  slightly. This isWe expected to see an increase in home visits for those dying of cancer given the longer period people are spending out of hospital and the greater numbers dying at home. This was not the case. Home and long-term care visits among patients remained stable over time. This trend may have been facilitated by a revamped home care system in 1995 providing more nursing and assisted care visits in the home in the latter part of the decade perhaps reducing the need for family physician visiting.Hospital visits declined quite substantially in the early years of our study and then increased in the final year. Provincial information suggests this trend was true for non-cancer patients as well. Since bThe increase in emergency department visits by family physicians parallels a rise during the study period among the general Nova Scotian population. . This greater emergency department utilization may be a reflection of reduced access to inpatient beds experienced by all patients, including those with advanced cancer.The lack of increased visits made by family physicians in end-of-life care in the community is concerning when one considers the evidence that their participation is associated with a greater likelihood of home death -12 and lIt is important to note that the advantage of using provincial administrative health databases is that we have information for the entire population regarding cancer mortality and physician and hospital utilization. However, there is no clinical information on severity of disease, which would provide a much better understanding of factors influencing health service utilization. In addition, we do not have concurrent home care utilization data or private long-term care facility data to factor into our modeling.Despite health care restructuring of the 1990s which resulted in fewer days in-hospital for those dying with cancer in Nova Scotia, there was no concurrent increase in the family physician visits provided to the dying. This may represent a growing unmet need for community-based medical care. However, further research is needed to examine whether end-of-life-care needs are being met by other health providers such as specialized home palliative care nurses, or general home care nursing services.The author(s) declare that they have no competing interests.FB and BL participated in the conceptualisation and design of the project, the analysis and interpretation of the data, created the first draft of the article, and incorporated co-authors' comments into the final draft. GJ participated in the design of the project, the interpretation of data, and revising the manuscript. GF participated in the analysis and interpretation of data and the revising of drafts. All authors gave approval to the final version.The pre-publication history for this paper can be accessed here: