the u.s . food and drug administration ( fda ) has approved around 30 antidepressant medications for the treatment of major depression . among them are selective serotonin reuptake inhibitors ( ssris ) .
these drugs change the balance of serotonin in the brain , such as fluoxetine ( prozac ) , paroxetine ( paxil ) , sertraline ( zoloft ) , escitalopram ( lexapro ) , and citalopram ( celexa ) .
another family of medications , selective serotonin and norepinephrine reuptake inhibitors ( snris ) , help increase serotonin and norepinephrine levels in the brain , such as venlafaxine ( effexor ) , duloxetine ( cymbalta ) , and levomilnacipran ( fetzima ) .
still others in this family include bupropion ( wellbutrin ) , vortioxetine ( brintellix ) , mirtazapine ( remeron ) vilazodone ( viibryd ) , nefazodone ( serzone ) , and trazodone ( desyrel ) .
in addition , tricyclics and monoamine oxidase inhibitors , which are two classes of older antidepressants that work by inhibiting the brain s reuptake of serotonin and norepinephrine , are also approved but tend to cause more side effects than the other classes of antidepressants .
but pharmacotherapy is nt the only option ; two other major classes of treatment are also available  psychotherapy and somatic nonpharmacological treatments . in randomized
, controlled trials , cognitive - behavioral therapy ( cbt ) and interpersonal psychotherapy ( ipt ) repeatedly have been demonstrated to be effective in the treatment of major depressive disorder ( mdd ) .
whether other forms of psychotherapy , such as insight - oriented , psychodynamically based therapy , are effective in major depression remains controversial .
brain stimulation therapies involve activating or touching the brain directly with electricity , magnets , or implants , and the fda has approved three somatic nonpharmacological treatments for depression : electroconvulsive therapy ( ect ) , vagus nerve stimulation ( vns ) , and repetitive transcranial magnetic stimulation ( rtms ) .
ect is generally considered the most effective of all depression treatments , although no head - to - head , randomized , controlled trial has compared it with other interventions .
it generally requires inpatient hospitalization , at least initially , and general anesthesia with nine to twelve treatments over a three- to four - week period .
vns and rtms are both fda - approved for treatment - resistant depression ; the former requires an invasive surgical procedure .
researchers have conducted relatively few controlled studies of these devices compared with the vast number of pharmacotherapy and psychotherapy treatment trials .
with a plethora of drugs and psychotherapy approaches available , let us consider the problem psychiatrists encounter on a daily basis .
a 50-year - old academic physician suffers from a classic major depressive episode associated with severe work stress .
he has difficulty falling asleep , awakens several times during the night , and rises early with severe anxiety .
he has reduced appetite , difficulty concentrating , and trouble enjoying any leisure activities , and he feels pessimistic about the future .
he admits to passive contemplations about suicide , with recurring thoughts that if a car jumped the median and landed on his car , it would be an end to his suffering .
he has no underlying medical disorder that might be contributing to depression , such as hypothyroidism or drug or alcohol abuse
i want to recommend the treatment most likely to be successful in producing a complete remission of his depressive syndrome and relieving him of his considerable misery . what are the known and best - validated predictors of response ?
our group has previously reviewed the scientific findings in this area.36 the most reliable predictor is past response , but in this case the patient has never been treated for depression .
a positive response in first - degree family relatives is also predictive of a beneficial response to antidepressants , but again , this is not applicable to this patient .
some evidence suggests that certain subtypes of depression respond best to certain treatments  monoamine oxidase inhibitors ( the first type of antidepressants developed ) are believed to be the most effective for patients with so - called atypical depression characterized by hypersomnia , overeating , extreme rejection sensitivity , and feeling better in the morning than later in the day .
combinations of antidepressants and antipsychotics or ect are best for patients with major depression with psychotic features .
surely patient choice is an important consideration , but it will likely be guided by my discussion with the patient . in 2013 ,
mayberg , holtzheimer , dunlop , and craighead ) were colleagues of mine for many years , and we continue to collaborate on various projects .
mayberg s study sought to identify a biomarker that could predict which type of treatment would benefit a patient based on the individual s brain activity . using regional brain glucose metabolism as measured by positron emission tomography ( pet ) as a proxy for neural activity , her group sought to determine whether baseline resting state activity predicted remission after twelve weeks of treatment with either the selective serotonin reuptake inhibitor escitalopram ( 10 to 20 mg per day ) or sixteen sessions of cognitive - behavioral therapy .
the study sample initially comprised eighty - two men and women who were randomized between the two treatments .
of these , sixty - five patients completed the study and thirty - eight had clear outcomes and acceptable pet data .
the thirty - eight patients who comprise the analyzable data set were distributed as follows : eleven who went into remission with escitalopram ( six non - responders ) and twelve who did so with cbt ( nine nonresponders ) .
the major finding were that hypometabolism of glucose in the insula , likely reflecting reduced activity of neurons in this brain region , was associated with remission using cbt , and with poor response to escitalopram .
contrariwise , insula hypermetabolism , reflecting increased activity of neurons in this brain region , was associated with remission using escitalopram and with poor response to cbt .
the authors conclude that baseline insula metabolism is the first objective marker to guide initial treatment selection in depression
first they eliminated from their primary analysis the responders to cbt or to escitalopram who did not go into remission . more specifically , partial responders to escitalopram or cbt were excluded from the analysis .
they did so in order to accentuate the differences between the extremes in the depressed population ; the results revealed clear differences in glucose metabolism in six regions : the right
anterior insula , right motor cortex , left premotor cortex , right inferior temporal cortex , left amygdala , and precuneus .
mean regional activity values for remitters and nonresponders segregated by treatment arm are plotted for the six regions showing a significant treatment  outcome analysis of variance interaction effect .
regional metabolic activity values are displayed as region / whole - brain metabolism converted to z scores .
cbt indicates cognitive - behavioral therapy.7 when all six regions were compared , the right insula exhibited the greatest effect as a discriminator of treatment response , followed by the precuneus .
when the whole sample was studied , right insular activity was positively correlated with the depression symptom severity scale , and with the hamilton depression rating scale ( hrsd ) score in the cbt treatment group while right insular activity was negatively correlated with the hrsd in the escitalopram treatment group .
if additional research can replicates these results , it suggests that a simple brain imaging test could reliably predict whether a given patient should be treated with psychotherapy or antidepressant medication .
it also raises a plethora of additional questions :
 a wealth of data , now summarized in a research meta - analysis , indicate that mdd patients with a history of child abuse and neglect exhibit a poorer response to pharmacotherapy and psychotherapy and exhibit unique brain imaging differences.8 mayberg s research does not address this critical clinical characteristic in this population.it is somewhat unclear how the six brain regions of interest were identified and why several regions repeatedly identified to be implicated in the pathophysiology of depression either were not selected or exhibited no significant effect , including the hippocampus , subgenual cingulate , and others.it is hard to know what to make of the findings that only the right anterior insula , right motor cortex , left premotor cortex , left amygdala , left precuneus , and right inferior temporal region show dramatic differences in the cbt versus escitalopram induced remission versus nonresponder groups whereas their counterparts , namely the left anterior insula , left motor cortex , right premotor cortex , right amygdala , right precuneus , and left inferior cortex did not . was a composite of the left and right sides of these structures informative?as the authors themselves point out , the study comprises a relatively small number of patients and our field is replete with pilot study findings that , unfortunately , have not been replicated in larger trials.this study utilized pet instead of the more often used functional magnetic resonance imaging ( fmri ) technology . as mayberg and
her colleagues appropriately point out in their paper , fmri studies have examined regional brain activity and , more recently , resting state connectivity to identify mdd or mdd subtypes , but neither type of imaging has been used to discriminate response either among antidepressants or between antidepressants and psychotherapy.9 a wealth of data , now summarized in a research meta - analysis , indicate that mdd patients with a history of child abuse and neglect exhibit a poorer response to pharmacotherapy and psychotherapy and exhibit unique brain imaging differences.8 mayberg s research does not address this critical clinical characteristic in this population .
it is somewhat unclear how the six brain regions of interest were identified and why several regions repeatedly identified to be implicated in the pathophysiology of depression either were not selected or exhibited no significant effect , including the hippocampus , subgenual cingulate , and others .
it is hard to know what to make of the findings that only the right anterior insula , right motor cortex , left premotor cortex , left amygdala , left precuneus , and right inferior temporal region show dramatic differences in the cbt versus escitalopram induced remission versus nonresponder groups whereas their counterparts , namely the left anterior insula , left motor cortex , right premotor cortex , right amygdala , right precuneus , and left inferior cortex did not . was a composite of the left and right sides of these structures informative ?
as the authors themselves point out , the study comprises a relatively small number of patients and our field is replete with pilot study findings that , unfortunately , have not been replicated in larger trials . this study utilized pet instead of the more often used functional magnetic resonance imaging ( fmri ) technology .
as mayberg and her colleagues appropriately point out in their paper , fmri studies have examined regional brain activity and , more recently , resting state connectivity to identify mdd or mdd subtypes , but neither type of imaging has been used to discriminate response either among antidepressants or between antidepressants and psychotherapy.9 the expanding area of genetics in general , and pharmacogenetics in particular , is also of vital importance .
a burgeoning database documents the role of certain genetic variations in vulnerability to mood disorders , and more recently how variations may affect treatment response to different antidepressants . whether genetic material was collected in mayberg s study is unclear , but this focus is crucial , particularly in view of recent findings in imaging genomics .
the lack of random assignment of the mdd patients as regards , for example , the vulnerability gene variants of the serotonin transporter or others , now shown to be associated with clear alterations in regional brain activity , could have confounded the results .
this research group has always been willing to take great leaps forward , and they should be applauded for it .
subsequent studies will reveal if the insula is truly  the region  that predicts response to cbt versus a selective serotonin reuptake inhibitor such as escitalopram or whether other regions or biomarkers also need to be a component of the ultimate formula .
this is part of the ongoing and exciting scientific process that is emblematic of the marriage of neuroscience and psychiatry .
ultimately , i believe this work will be judged as crucial in eventually attaining the goal all of us seek : a valid predictor of individual treatment response in depression , still the holy grail in psychiatry research .

 editor s note:holy grail  is a well - known metaphor for the eternal spiritual pursuit for truth and wisdom .
 it suggests that in order for us to find what no one has found , we must search where few have looked . in 2013 ,
 a group led by helen mayberg published a groundbreaking paper that sought an answer to one of the most discussed conundrums in psychiatry and neuroscience : can specific patterns of brain activity indicate how a depressed person will respond to treatment with medication or psychotherapy ? our author examines the findings and discusses their potential impact on treatment for a public health problem that affects millions of people worldwide .


primary mitochondrial disorders due to impaired oxidative phosphorylation ( oxphos ) are a well - established cause of severe disability and precocious death in both children and adults ( koopman et  al . , 2012 ) .
no effective therapies are currently available for these conditions , but encouraging results have recently been obtained by stimulating mitochondrial biogenesis acting on either the ppar system ( wenz et  al . , 2008 ) or the amp - kinase ( ampk)/pgc1 axis ( viscomi et  al . , 2011 ) .
importantly , these approaches can in principle be extended to several mitochondrial diseases with different genetic causes , as they do not point to the correction of a specific defect but are based on a more general strategy aimed at increasing the overall residual activity of the respiratory chain .
additional targets able to activate the mitochondriogenic program and boost mitochondrial function are sirtuins 17 ( houtkooper et  al . , 2012 ) , the mammalian orthologs of the yeast silent information regulator ( sir ) 2 gene ( imai et  al . , 2000 ;
sirtuins have different subcellular localization , sirt1 and sirt6 being mainly found in the nucleus , sirt2 in the cytosol , sirt3sirt5 in mitochondria , and sirt7 in the nucleolus .
sirtuins are important regulators of several proteins , including key metabolic players , acting as either deacetylases or adp - ribosylases ( houtkooper et  al . , 2012 ) .
the most - investigated member of the family is sirtuin1 ( sirt1 ) , a nad - dependent type iii nuclear deacetylase that utilizes nad as a cosubstrate to remove acetyl groups from lysine residues of a target protein .
known targets of sirt1 are the tumor suppressor p53 , the myocyte - specific enhancer factor 2 ( mef2 ) , the forkhead box o ( foxo ) , and pgc1 , all of which regulate transcriptional programs related to increased mitochondrial function ( andreux et  al . , 2013 ) .
sirt1 activity is directly regulated by nad availability , by substrate - dependent activation , raising the hypothesis that nad acts as a metabolic sensor .
for instance , both nad levels and sirt1 activity increase in mammalian tissues in response to energy / nutrient stresses such as exercise ( cant and auwerx , 2009 , 2010 ) and fasting ( cant and auwerx , 2010 ; chen et  al .
recent studies have shown that sirt1 activation can prevent diet - induced obesity in mice .
this effect was achieved by increasing the content of nad in cells and tissues essentially through ( 1 ) dietary supplementation of suitable nad precursors , such as nicotinamide riboside , nr ( cant et  al . , 2012 ) , or ( 2 ) inhibition of nad - consuming enzymes , such as the poly(adp - ribose ) polymerase 1 , parp1 ( bai et  al . , 2011 ) .
here , we have tested the therapeutic efficacy of these strategies on a genetic mitochondrial disease model , the sco2 knockout / knockin ( sco2 ) mouse ( yang et  al . , 2010 ) .
sco2 encodes a metallochaperone involved in the formation of the copper redox centers into nascent complex iv ( cytochrome c oxidase , cox ) ( leary et  al . , 2009 ) .
mutations in this gene lead to infantile fatal encephalocardiomyopathy ( papadopoulou et  al . , 1999 ) .
most of the patients  carry at least one allele encoding the common mutation p.e140k , corresponding to the p.e129k mutation in the knockin murine sco2 allele .
the second knockout allele in this animal model is functionally null ( yang et  al . , 2010 ) .
while homozygous knockout individuals are embryonic lethal , the sco2 mice show a predominantly myopathic phenotype characterized by  exercise intolerance and associated with ubiquitous cox deficiency .
to test the effects of persistent increase of the nad pool on  the sco2 mouse , we first crossed it with a constitutive  parp1 mouse ( de murcia et  al . ,
1997 ) , which shows increased levels of nad in skeletal muscle ( bai et  al . , 2011 ) .
the sco2-parp1 double mutants showed reduced fasting blood glucose levels , body weight and epididymal white adipose tissue ( wat ) compared to sco2 littermates , whereas no differences were observed between wt and parp1 littermates ( see figure  s1 available online ) .
the endurance motor performance of sco2-parp1 double mutants , sco2 , parp1 , and wt littermates ( four animals per group ) was monitored weekly by a standard treadmill test for 4  weeks starting at 2  months of age . while sco2 mice showed markedly reduced motor performance compared to wt littermates throughout the observation time , the double mutant individuals performed as well as the wt and parp1 littermates ( figure  1a ) .
biochemically , significant reduction of complex iv activity and , to a lesser extent , complex iii as well ( yang et  al . , 2010 ) , was measured in skeletal muscle of sco2 mice . in the muscle homogenate of sco2-parp1 double mutants ,
these activities were comparable to that of parp1 and wt littermates , whereas complex i and ii activities were even higher ( figure  1b ; table s1 ) .
accordingly , the intensity of the histochemical staining specific to cox was increased in skeletal muscle of the double mutants , compared to the sco2 mutants ( figure  1c ) .
the activities of complex iii and iv ( figure  1d ; table s2 ) were also increased in the brains of double mutants relative to sco2 animals , but remained significantly lower than those measured in parp1 and wt littermates ; the histochemical reaction to cox was concordant with the biochemical data ( figure  1e ) . to test the effects of pharmacological intervention , we first administered nr to sco2 mice and wt littermates ( n  = 4/group ) , as a food admix ( 400  mg / kg ) ( cant et  al . , 2012 ) for four weeks .
metabolic parameters in the treated groups , including reduced blood glucose , plasma fatty acids , and epididymal wat , mirrored those of parp1 mice ( figure  s2 ) , confirming that nr , or its derivative nmn , was pharmacologically active in  vivo .
nr - treated sco2 mice significantly improved their motor performance compared to the vehicle - treated sco2 group , rapidly achieving the levels of motor endurance displayed by treated and untreated wt mice ( figure  2a ) , which showed no difference to each other .
these results suggest that nr treatment increases mitochondrial function in the sco2 mice through nad activation of sirt1 ( cant et  al . , 2012 ) .
accordingly , the nad / nadh ratio was significantly increased ( figure  2b ) , and the ratio between acetylated and total foxo1 , a direct target of sirt1 , was clearly reduced ( figure  2c ) in skeletal muscle of nr - treated versus vehicle - treated sco2 and wt animals .
we did not observe differences in mtdna content ( data not shown ) and citrate synthase ( cs ) activity ( figure  2f ) , but mrna levels of several genes related to either fatty acids oxidation ( fao ) , including acox and  cd36 , or oxidative phosphorylation ( coxi , coxii , coxiv , coxva ) were significantly increased in nr - treated versus vehicle - treated sco2 but not in wt animals ( figure  2d ) .
tfam , a key factor of mtdna transcription , was also increased , and ucp3 and pdk4 , which were downregulated in sco2 mice , returned to control levels upon nr treatment .
as expected , we found no change in transcripts specific to pgc1 , which is activated posttranscriptionally by sirt1 , and to two pgc1 partners , nrf1 and nrf2 .
accordingly , western blot immunovisualization demonstrated increased levels of several nuclear- and mitochondrial - dna - encoded oxphos - related proteins in nr - treated versus vehicle - treated skeletal muscle samples ( figure  2e ) . in the same specimens , mitochondrial respiratory chain activities were significantly increased ( figure  2f ; table s3 ) in nr - treated versus vehicle - treated sco2 mice , but not in wt animals .
the histochemical staining for cox reflected the biochemical results ( figure  2 g ) . in both c.  elegans and mammalian cells
, nr - dependent sirt1 activation can induce the mitochondrial unfolded protein response ( mtupr ) , a stress - response protective mechanism which can improve mitochondrial function ( durieux et  al .
we found that the mtupr - specific transcripts clpp , hsp60 , and sod2 were significantly increased in muscle samples of nr - treated sco2 mice , whereas sod3 , which is unrelated to mtupr , was unchanged ( figure  2h ) .
no effect of the nr treatment was observed on the mitochondrial respiratory chain activities in the brain of our animals ( table s4 ) .
next , we administered a pan - parp inhibitor ( mrlb-45696 , ic50 for parp  < 1  nm ; pirinen et  al . , 2014 , in this issue of cell metabolism ) at 50  mg /
kg as a food admix for 4  weeks . in mrlb-45696-treated sco2 mice , we observed metabolic effects similar to those of nr treatment ( figure  s3 ) .
weekly treadmill tests showed progressive increase , up to normal , of the motor endurance in mrlb-45696-treated sco2 , whereas no change was seen in wt mice ( figure  3a ) .
the nad / nadh ratio was significantly increased in treated versus untreated wt , but not in the sco2 , animals ( figure  3b ) , whereas the acetylated / total foxo1 ratio was reduced in both treated groups ( figure  3c ) , indicating activation of sirt1 by mrlb-45696 .
similar to nr , mrlb-45696 increased the mrna expression levels of oxphos- and fao - related genes in both sco2 and wt mice , whereas mtdna content ( data not shown ) and cs activity ( figure  3f ) remained unchanged . in treated sco2 but not in wt animals , western blot analysis showed increased content of mitochondrial respiratory chain subunits ( figures 3d and 3e ) , which was paralleled by significantly increased mitochondrial respiratory chain activities ( figure  3f ; table s3 ) .
histochemistry for cox showed increased staining in treated versus vehicle - treated sco2 mice ( figure  3 g ) .
again , we found that expression of the mtupr genes hsp60 , clpp , and sod2 was significantly increased , unlike the mtupr - unrelated gene sod3 ( figures 3h and 3i ) .
in contrast to the nr treatment , the mrlb-45696 treatment determined significant increase of cox transcripts ( figure  4a ) ,  and respiratory chain activities in the brain ( figure  4b ; table  s4 ) . the intensity of cox staining was increased as well ( figure  4c ) .
similar results were obtained in both skeletal muscle and brain by using pj34 , a commercially available pan - parp inhibitor ( figures s3 and s4 ; table s4 ) .
the nad pool is set by the balance between de novo and  salvage biosynthetic pathways and utilization by nad - consuming enzymes .
nad is synthesized de novo from tryptophan , but the main source of nad is from salvage pathways ( houtkooper et  al . , 2010 ) .
these require the uptake of other nad precursors from the diet , including nr . upon its entry in the cell , nr is phosphorylated by nr kinases into nicotinamide mononucleoside ( nmn ) , which is then converted to nad by nmn adenylyltransferase ( bieganowski and brenner , 2004 ) .
nad biosynthesis and cellular levels are also controlled by a circadian clock related to the feeding / fasting cycle .
for example , in the mitochondrial compartment nad levels regulate sirtuin 3 , a deacetylase targeting respiratory chain subunits ( peek et  al . , 2013 ) , while in the nucleus nad is a substrate of both parp1 and sirt .
parp1 , the highest consumer of nad in mammalian tissues , is activated upon binding to damaged or abnormal dna ( durkacz et  al . , 1980 ) and catalyzes the formation of poly(adp - ribose ) polymers ( par ) using nad as a substrate , onto different acceptor proteins , including parp1 itself ( adamietz , 1987 ) .
ablation of the parp1 gene , supplementation of nr or administration of parp inhibitors ( parpi ) can expand the nad pool and activate sirtuins , particularly sirt1 , a master regulator of mitochondrial homeostasis .
these effects can protect mice from high - fat ( hf)-induced metabolic disease ( cant et  al . ,
we have shown here that these treatments can correct the biochemical and clinical phenotype of the sco2 mouse , a model of genetically determined mitochondrial disease . increased transcription of genes related to both oxphos and mtupr
was associated with activation of oxidative metabolism , increase of mitochondrial respiratory chain activities , and normalization of the endurance motor deficit , displayed by naive sco2 animals .
notably , these effects were hardly seen in wt littermates , suggesting that mitochondrial dysfunction sensitizes muscle and possibly other tissues to activators of mitochondriogenic programs .
increased mitochondrial function can be achieved in wt animals only by much longer - term treatments ( > 6  months ) ( pirinen et  al . , 2014 ) .
in contrast with previous results , we found no change in mtdna copy number and cs activity in nr- or parpi - treated versus untreated animals , possibly because of the shorter timeframe of our experimental protocol ( 4  weeks ) compared to that of  other studies ( 12  weeks ) .
this observation suggests time - dependent activation of different mitochondriogenic programs , with induction of oxphos- and fao - related genes occurring much  earlier than stimulation of mitochondrial proliferation and increase in mtdna content .
likewise , prolonged nr supplementation up to 6  months induced mitochondrial biogenesis in the brain and improvement of cognitive dysfunction of an alzheimer disease mouse model ( gong et  al . , 2013 ) .
while we observed hardly any effect of nr in our 4  week trial , two pan - parp inhibitors did correct the respiratory chain defect in the brains of our sco2 mice .
this observation is particularly relevant , as the brain is an exquisite target of mitochondrial dysfunction , and progressive encephalopathy is the most frequent clinical presentation of mitochondrial disease in infancy and childhood .
our work supports the idea that the increase of nad levels in critical tissues is an effective therapeutic option for mitochondrial disease .
nr is a natural vitamin with no known adverse effects , which could be administered as a dietary supplement , particularly in case of isolated mitochondrial myopathy .
our results are concordant with very recent works reporting beneficial effects of nad precursors in mouse models characterized by reduced nad / nadh ratio , such as aging ( gomes et  al . , 2013 ) or complex i deficiency ( karamanlidis et  al . , 2013 )
initially shown to boost oxidative metabolism in diet - induced models of obesity , parp1 ablation or inhibition has recently been reported to remarkably rescue pharmacological models of liver cirrhosis , partly by correcting the associated mitochondrial impairment ( mukhopadhyay et  al . , 2013 ) .
several parp inhibitors are currently under clinical trial as anticancer molecules , and seem to be associated with relatively mild side effects ( bundred et  al . , 2013 ; tutt et  al . ,
however , more work is needed to evaluate their use in chronic conditions such as primary mitochondrial disorders in view of their potential genotoxic effects .
mouse tissues were homogenized in 15 volumes of 10  mm potassium phosphate buffer ( ph 7.5 ) .
mitochondrial - enriched fractions were collected after centrifugation at 800  g for 10  min in the  presence of protease inhibitors , and frozen and thawed three times in liquid nitrogen .
aliquots , 70  g each , were run through a 12% sds - page and electroblotted onto a  nitrocellulose membrane , which was then matched with different antibodies .
carlo besta  neurological institute , in accordance with guidelines of the italian ministry of health .
mice were maintained in a temperature- and humidity - controlled animal - care facility , with a 12  hr light / dark cycle and free access to water and food .
a standard treadmill apparatus ( columbus instruments , columbus , oh ) was used to measure motor exercise endurance , as described in viscomi et  al .
series of 8  m thick sections were stained for cox and sdh , as described ( sciacco and bonilla , 1996 ) .
muscle quadriceps samples stored in liquid nitrogen were homogenized in 10  mm phosphate buffer ( ph 7.4 ) , and the spectrophotometric activity of ci , cii , ciii , and civ , as well as cs , was measured as described ( bugiani et  al . , 2004 ) .
note that in all panels the activity of cii has been multiplied by 10 for  visualization clarity .
nad was extracted using acidic and alkaline extraction methods , respectively ( yang and sauve , 2006 ) .
tissue nad was analyzed with mass spectrometry as previously described ( yang and sauve , 2006 ) .
mtdna content and transcripts analysis was carried out by sybr green real - time pcr , as described ( viscomi et  al . , 2011 ) .

 summarymitochondrial disorders
 are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain . pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling , often fatal , disorders .
 pathways related to mitochondrial biogenesis are targets of sirtuin1 , a nad+-dependent protein deacetylase . as nad+ boosts the activity of sirtuin1 and other sirtuins , intracellular levels of nad+ play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell .
 we show here that supplementation with nicotinamide riboside , a natural nad+ precursor , or reduction of nad+ consumption by inhibiting the poly(adp - ribose ) polymerases , leads to marked improvement of the respiratory chain defect and exercise intolerance of the sco2 knockout / knockin mouse , a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis .
 this strategy is potentially translatable into therapy of mitochondrial disorders in humans .


hypertension is one of the most common if not the commonest medical complication in pregnancy.19 hyper - tensive disorders in pregnancy are found to be the greatest single cause of maternal mortality.911 it also causes a lot of prenatal mortality.12 most of the complications caused by this problem could be reduced by early detection and proper management.1314 studies on the incidence of hypertensive disease in pregnancy in most developing countries , including saudi arabia , are scarce.15 few of those studies , if any , have looked at the extent of knowledge , attitude or practice of doctors or nurses dealing with hypertensive pregnant mothers .
hence , the objective of this study was to determine the status of knowledge , attitude and practice ( kap ) of doctors and nurses in phc centers with regard to hypertension in pregnancy and to analyze factors affecting kap in the al - khobar area of the eastern province of the kingdom of saudi arabia .
terms used to describe hypertension and its complications in pregnancy differ according to its presentation , gestational age of discovery and the presence of previous history of hypertension before pregnancy .
the one defined by the 1972 committee on terminology of the american obstetricians and gynecologists ( acog)131519 was adopted for the purpose of this kap study .
doctors and female nurses working at all 8 phc centers in al - khobar area were enrolled for the study .
two self - administered questionnaires structured to examine the essential knowledge , attitude , and practice of doctors and nurses in dealing with hypertensive disorders during pregnancy .
the first was intended to gather demographic characteristics and information on the respondent that might affect the kap .
these variables included age , sex , nationality , year of graduation , certification , training and experience in obstetrics , duration of work in phc in the country and any in - service training received .
the second part of the questionnaire consisted of 49 questions divided into three sections dealing with practice , attitude and knowledge .
the practice sections included questions designed to test competence of doctors and nurses in the skills of measuring blood pressure , management of the hypertensive pregnant mother and practices of health education .
the knowledge section was composed of 28 questions to test basic medical knowledge regarding blood pressure reading , management of the problem and its complications .
both questionnaires were given to three consultant obstetricians and two nurse educators for consent validity and elimination of non - essential questions .
both knowledge and practice areas were scored on a zero and one additive scoring system in which each correct answer or practice was given one score and no mark given for a wrong response .
the attitude part included questions that measured attitudes of participants towards the seriousness and commonness of hypertension in pregnancy and their motivation to improve their knowledge on the subject .
attitude questions were scored using the five - point likert scaling system . in this scale ,
a high attitude was assigned to the answer if the respondent 's answer was to  agree  or
strongly agree  to the question in the scale and a low attitude level for  not sure  or
final scoring of the knowledge and practice section was satisfactory if the participant scored at least 60% of the total marks in these two sections .
this was an arbitrary cut - off point based on the pilot study and judgment of the experts . on attitude
, the questionnaire was handed over to five postgraduate doctors in general practice and their responses to attitude questions were used as a reference standard . the questionnaire for nurses
both versions of the questionnaire were pilot tested on a pilot group of 10 doctors and 22 nurses , selected randomly from phc centers in a nearby city , al - dammam to assess level of difficulty , clarity , suitability and time required for their completion .
data were fed into a personal computer , cleaned and analyzed using epi info and spss statistical packages .
frequency distribution tables were constructed and appropriate tests including the multiple regression analysis to identify significant independent factors were applied .
the total number of phc doctors in al - khobar area was 44 . among those 36 ( 81.4% ) who were present at time of data collection
their ages ranged between 25 - 47 with mean of 37.1 years ( sd  6.4 ) and 56% were in the 35 - 44 years group .
arab nationals other than saudis including egyptians , palestinian , and jordanian formed the majority of doctors ( 41.7% ) , while saudis constituted 22.2% of the group and the rest were from the indian subcontinent . the mean number of years in practice after graduation was 12.6 years ( sd  7.38 ) .
distribution of phc doctors according to years of practice after graduation the mean number of years working in phc was 4.7 years ( sd  3.4 ) and 17 doctors ( 47.2% ) had worked for over 5 years in phc centers . only , 4 doctors ( 11.1% ) had postgraduate qualifications ; out of these only one held a diploma in obstetrics .
five doctors ( 13.9% ) had had 3 - 6 months post - internship training in obstetrics but no certification .
of the entire group , only 2 doctors ( 5.6% ) had had courses in hypertensive disorders in pregnancy in the course of their work . in describing their general attitude towards managing hypertension in pregnancy ,
a majority of doctors ( 80% ) stated clearly that they did not feel confident in managing hypertensive pregnant mothers and would prefer to see children or adult male patients in their clinic . nevertheless , the attitude of 34 doctors ( 94.4% ) toward learning more about hypertension in pregnancy was positive .
out of the maximum attitude score of 45 marks , the mean score for the attitude questions for the group reached a reasonably high figure of 30.69 marks .
doctors attitudes towards hypertension in pregnancy doctors management of hypertension in pregnancy was quite deficient as 16 doctors ( 44.4% ) reported that they had not actually seen any hypertensive pregnant mother during last year .
around 53% of the doctors depended on bp measurement taken by a competent staff and did not check it .
the doctors scores for practice was generally low , as only 60% of them got the correct answers to the skill of blood pressure measurement , a basic skill necessary for any medical graduate .
in addition , almost all doctors wrongly reported that they had advised their pregnant hypertensive patients to lose weight and reduce their salt intake .
another major mistake noted among 25% of the doctors was that they said they would start treatment of hypertensive cases immediately after diagnosis in their clinic without considering referral .
the scores on practice of 75% of the doctors were below the satisfactory cut - of point .
again the doctors score of 58.3% on all areas of knowledge was unsatisfactory since this directly affected the diagnosis and quality of care for hypertensive mothers .
some of the responses dealt with normal and abnormal readings of blood pressure and the presenting symptoms of preeclampsia .
doctors mean score 12.1 marks out of a total score of 21 on knowledge was moderate .
the total number of phc nurses in al - khobar area was 120 . among these ,
the 91 ( 75.8% ) who were present at the time of data collection answered the questionnaire .
their mean age was 29.8 years ( sd  6.3 ) ranging between 20 - 44 years , 74% of them fell within the 20 - 34 age group .
indians formed the second largest group ( 37.4% ) followed by filipinos ( 11% ) . according to their qualification , 14 nurses ( 15.4% ) held bachelor degrees and 77 ( 84.6% ) had diplomas .
mean years of practice after registration was 8.7 ( sd  5.4 ) and their distribution is shown in table 3 .
mean duration of work in phc was 4.1 years ( sd  2.8 ) and most nurses ( 69.2% ) had spent < 5 years working in phc in the country .
distribution of phc nurses according to years of practice after registration thirty - seven nurses ( 40.7% ) had spent < 6 months in obstetrics training and 10 nurses ( 11% ) had spent > 1 year .
most of the group ( 48.3% ) , mainly the saudis , had minimal training of 3 weeks in obstetrics ward during their internship period as required by the saudi female health institution .
a majority of the nurses  73 ( 80.2% ) had not worked in a phc setting before their current positions . out of those who had had previous phc experience ,
nine of them had had courses in hypertensive disorders in pregnancy during their work in phc . in describing their general attitude towards hypertension in pregnancy
, 86% of them reported that it was a common health problem and they were all positive about learning more .
they were all keen to talk to pregnant women about their problem and advice them to take bed rest .
out of the maximum attitude score of 35 marks , the highest mean score was 30.02 marks . table 4 shows some of the attitude questions and their responses . nurses attitude towards hypertension in pregnancy
the nurses were quite good on practice in the area of hypertension in pregnancy , as 95.6% of them scored  satisfactory  .
the mean score of this section on practice was quite high for nurses being 8.3 marks out of a total score of 12 marks .
the nurses level of knowledge was very low since only five of them ( 5.5% ) scored  satisfactory  on the questions in this section .
the deficiencies were in areas dealing with diagnosis and quality of care for hypertensive mothers .
most of them ( 96.7% ) wrongly considered salt restriction and weight reduction as important in the health education of hypertensive pregnant women .
mean scores on knowledge was 9.02 marks out of a total score of 20 marks which was quite low . in the regression analysis of independent factors associated with kap of doctors and nurses , all variables in the first part of the questionnaire such as age , nationality and certification
were included in a series of multiple regression equations against each of the dependent variables , namely , scores on knowledge , attitude , and practice for doctors and nurses .
there was a positive significant association of the doctors attitude with their sex i.e. , male , nationality , being older and previous enrollment in a course on hypertension ( r square = 0.26 p=0.02 ) .
their knowledge was only associated with previous training in obstetrics ( r square = 0.14 , p=0.02 ) , while practices of both doctors and nurses were not associated with any of the factors under study .
the attitude of nurses , on the other hand , showed different associations as non - saudi nationals and training in obstetrics had a positive significant effect on their attitude ( r square = 0.08 , p=0.02 ) .
their knowledge was only associated significantly with being non - saudi ( r square = 0.19 , p=0.001 ) .
hypertension in pregnancy is one of the major causes of prenatal morality and morbidity.20 it is responsible for about 18% of maternal mortality,102122 maternal mortality in hypertensive disorders of pregnancy is primarily due to low standard of care and delay in referral.14 antenatal care ( anc ) is a major part of maternal and child services in phc .
one of the most important functions of anc is to detect high - risk pregnancies and to give them the necessary care .
findings from this study will help to identify the status kap of workers in phc on hypertensive disorders in pregnancy .
it was discovered in this study that the definition of raised blood pressure and its management was not standardized among doctors and nurses in phc .
this is not surprising as previous study by bisson in bristol23 in which a large group of general practitioners , hospital doctors , hospital midwives , community midwives and student midwives who were questioned , gave variety of action plans according to their understanding of diagnostic criteria .
they considered the reading of 90 mmhg the model value of diastolic bp at which further action would be taken , whether proteinuria was present or absent .
edema was considered a useful indicator by 93% of the respondents and 49% would use ankle edema in their assessment .
another questionnaire - based survey by hutton24 on the management of hypertension in pregnancy completed by 65 new zealand obstetricians found that 40 ( 61.5% ) doctors considered the diastolic of 80 - 85 mmhg , the lowest abnormal reading , at 28 weeks , and by 18 ( 27.7% ) doctors at 36-week gestation . however , 20 ( 30.8% ) doctors considered the diastolic of 90 mmhg , at 28 weeks and 42 ( 64.6% ) doctors at 36 weeks gestation , the lowest abnormal reading . around 47% of phc doctors
had spent more than 5 years in phc service in ksa and only 14% of them had had post - internship training in obstetrics but being males had not been involved in anc activity in phc . only 2 had had courses in hypertensive disorders of pregnancy .
about 44% of the doctors had not seen any cases of hypertensive pregnant mothers during the last year .
questions asked were concerned with four aspects namely , techniques of blood pressure measurements , history and physical examination , health education practice and action management to be taken by the phc doctors on discovered cases .
it is obvious that having such little contact with hypertensive cases and lacking the basic training or refresher courses , their management skills will be inadequate .
since hypertension is one of the commonest medical complications in pregnancy , it is important to educate the pregnant mother about it .
almost all doctors report that they advise hypertensive mothers on the need for bed rest .
this is an important non - pharmacological measure in the management of the problem.25 on the other hand , almost all doctors report that they advise their patients to restrict their salt intake and to go on a weight - reducing diet .
salt restriction in hypertensive non - pregnant women can be effective but in pregnancy may aggravate the condition .
moreover , diet restriction in pregnancy can lead to delivery of small - for - date fetus.25 similar findings of wrong advice were reported in other studies by trudinger26 and bisson.23 around 85% of phc doctors reported that they would not refer hypertensive cases after diagnosis and 25% of them would start medication immediately after diagnosis .
in fact , the cornerstone of management of hypertensive cases starts in the phc center with accurate diagnosis and undelayed referral of these cases from the phc to the specialist or hospital.1621 phc doctors should be aware of their limitation in the management of these cases and not to jeopardize the health of the mother and her fetus .
around 31% did not recognize this is a common health problem in pregnancy , while 97% of them stressed the importance of taking blood pressure reading at each anc visit .
that 53% of phc doctors depended on blood pressure measurement taken by clinical staff without confirming it , the reading themselves is an alarming negative attitude as well as wrong practice .
the diagnosis of hypertension in general or in pregnancy in any patient is not an easy task and proper management and modifications depend on it .
it is therefore , important that the person responsible for the management , namely , the doctor , should confirm the reading again . in looking for factors affecting the attitude of doctors
, it was found that gender i.e. , male , arab , older and previous training in a course on hypertension were factors associated with higher scores . longer experience in phc work , being male and older and the absence of a language barrier for arabic speakers , including saudis , might explain to some extent their better attitude scores as compared to those of the females and younger doctors .
the knowledge of about 58% of doctors on questions that bare on the diagnosis and quality of care of hypertensive mothers as well as
nurses provide most of the vital anc services in the phc . around 53% of the phc nurses
were non - saudis and non - arabic speakers and the language barrier adversely affects the health education in hypertensive cases .
it is obvious from the results that there is a shortage of specialized courses for nurses .
none of the saudi nurses had more than a diploma in nursing and there did nt seem to be any arrangements for their further movement at universities .
a period of 3 weeks in an obstetrics ward for new graduate nurses as part of an internship period is not enough to give clear , pragmatic information on anc services in a phc setting .
about 31% of the nurses have spent > 5 years in the phc centers in the ksa , but only 10% had taken courses in hypertension .
this clearly , demonstrates the necessity of a better arrangement for on - job training classes for them .
their responses to the question of history and physical examinations were 87% correct and about 90% of them responded correctly to the questions on health education .
their erroneous responses were on the items on restriction on salt intake and weight reduction .
these good scores indicate that except for their wrong ideas on salt and diet restriction , which should be corrected at refresher courses , the correct procedure have been learnt . although the phc nurses were very good with practice in general , around 95% of them had poor knowledge .
knowledge and facts , on the other hand , need to be updated by continuous education .
around 86% of them felt that it was a common health problem and reported that they needed to know more about it .
the importance of a health education as a vital task for nurses can not be ignored .
the presence of these cases in the clinic , therefore , provides a good opportunity for the performance of this task .
there was a strong positive association of attitude of nurses to non - saudi nationality and training in obstetrics .
while all saudi nurses hold diplomas , about 30% of the non - saudi nurses have bachelors degrees .
there is a masked difference in the type , duration and content of curriculum at the pre - graduate level for both groups and non - saudi nurses had higher knowledge scores than saudis .
the implication of these findings clearly points out the need to improve the knowledge and attitude of phc nurses through refresher courses .
good  in attitude questions but had low scores on the practice and knowledge component of the questionnaire .
the nurses scored high on practice and attitude but had low scores on the knowledge component .
it is recommended that appropriate regular refresher courses on common and serious problems like hypertension be organized for doctors and nurses in the phcs .
there should be opportunities for effective training of reasonable duration with clearly defined objectives under proper supervision in good hospitals to improve their knowledge and practice
. it would be also appropriate to offer saudi doctors extra incentives for postgraduate study in family medicine to deal with these common problems , and to institute a suitable program of continuing medical education within the health centers for both doctors and nurses .
it is also vital to review the curriculum of the female nursing institutions to update both its theoretical and practical content , and extend the duration of training in such common problems as hypertension in obstetrics .

 objective : to assess the status of knowledge , attitude and practice ( kap ) of doctors and nurses in primary health care ( phc ) centers with regard to hypertension in pregnancy and to identify factors associated with kap in al - khobar , saudi arabia.methodology:using a self - administered comprehensive questionnaire , all available doctors and nurses in phc centers of the al - khobar area were approached to determine their knowledge , attitude and practice in hypertension during pregnancy .
 questionnaires were validated and pilot tested .
 each section of the questionnaire was scored and the mean scores calculated .
 factors affecting each section were identified by means of multiple regression analysis.results:a total of 36 doctors and 91 nurses were enrolled in the study .
 saudis formed 22.2% of the doctors and 47.3% of nurses .
 mean years of practice after graduation were 12.6 and 8.7 years for doctors and nurses , respectively .
 saudi nurses spend only 3 weeks in the obstetrics ward during the whole period of their internship .
 all saudi nurses hold only diplomas and not many courses on the hypertensive disorder are offered to both doctors and nurses after graduation .
 the practice of doctors particularly in the management of patients after reaching a diagnosis and educating them on diet and salt intake was poor .
 furthermore , their knowledge was also poor . though their level of knowledge was poor , the nurses practice was satisfactory , particularly in taking history and physical examination
 .
 the attitude of both doctors and nurses towards hypertensive disorders was in general , positive and satisfactory towards health education .
 nurses nationality and duration of post - internship training were the factors that influenced their attitude and scores on knowledge.conclusion and recommendation : the study revealed that both doctors and nurses working in the phc lacked training and knowledge in this area of their work .
 it is therefore necessary to give phc doctors and nurses refresher courses on common and serious problems like hypertension . a longer period of training in action management is needed to improve the knowledge and practice of doctors and nurses working in antenatal clinics in this area .


renal cell carcinoma ( rcc ) is the most common primary malignant neoplasm of the kidney and accounts for about 3% of all adult neoplasms .
the incidence of rcc has been increasing during the past two decades in all age groups .
the greatest increase has been in patients with localized tumors as a result of incidental detection , which is thought to be the result of more widespread use of abdominal ultrasound and computed tomography . however , about 30% of patients with rcc already have metastatic disease at the time of diagnosis , and 10% to 14% of patients experience local recurrence or distant metastasis after nephrectomy despite a finding of pathologically confined disease at the time of nephrectomy [ 3 - 5 ] .
well - known pathological and clinical variables have been identified and sometimes integrated into nomograms to better predict disease recurrence , but with only partial success . with a better understanding of the biology of rcc
, laboratory values and molecular markers are increasingly being investigated as potential predictive factors for rcc [ 6 - 8 ] .
disease progression appears to depend on a complex interaction between the tumor and host inflammatory responses .
it is estimated that underlying infection and inflammatory responses are linked to 15% to 20% of all deaths from cancer worldwide .
the serum level of c - reactive protein ( crp ) , a nonspecific inflammatory acute - phase protein , is frequently increased in patients with metastatic rcc and is a predictor of prognosis .
however , secondary thrombocytosis has been reported with many malignant diseases ; the survival of such patients has been reported to be significantly shorter . the prognostic value of the platelet count has been studied in patients with localized and metastatic rcc ; patients with thrombocytosis had a poorer survival than did patients with a normal platelet count . the purpose of this study was to investigate the association of preoperative crp elevation and thrombocytosis with the prognosis of patients with non - metastatic rcc .
the medical records of 177 patients with non - metastatic rcc who underwent a radical nephrectomy between march 2000 and may 2008 and for whom preoperative crp and platelet levels were available for analysis were retrospectively reviewed .
there were 130 men and 43 women , with a mean age of 53.5 years ( range , 28 - 83 years ) .
preoperatively , all patients were evaluated with a physical examination , routine hematology and biochemical analysis , and radiology studies , including abdominal computed tomography and chest x - ray .
the tumors were staged by using the 2002 tnm classification of the american joint committee on cancer ( ajcc )   and were graded according to fuhrman 's nuclear grading system .
all patients were evaluated postoperatively every 3 months for the first 2 years , every 6 months for the next 2 years , and yearly thereafter .
crp elevation was defined as a crp level 0.8 mg / dl , and thrombocytosis was defined as a platelet count 380,000/l in men and 370,000/l in women according to the normal reference range in our hospital .
disease recurrence was defined as local failure in the tumor bed or regional lymph nodes or distant metastasis .
the chi - square test or fisher 's exact test was used to analyze the correlation between preoperative crp elevation or thrombocytosis and the clinical and pathological variables , including age , gender , anemia , histology subtype , tumor size , t stage , nuclear grade , and metastasis during follow - up .
univariate and multivariate analyses were performed by using the log - rank test or the cox proportional hazards regression model . for all tests ,
the clinical and pathological characteristics of the 177 patients with non - metastatic rcc are summarized in table 1 .
the mean tumor size was 5.12 cm ( median , 4.50 ; range , 1 - 22 cm ) .
regional lymph node dissection was performed in 24 patients who showed suspicious lymph node metastasis on computed tomography . however , none had a pathological diagnosis of lymph node metastasis .
there were 38 patients ( 21.5% ) with crp elevations and 11 patients ( 6.2% ) with thrombocytosis . among patients with crp elevation ,
the mean follow - up period was 48.3 months ( median , 48.0 ; range , 13 - 111 months ) .
twenty - three patients ( 13.0% ) developed metastases and six patients died during the follow - up period .
preoperative crp elevation was significantly correlated with anemia ( p=0.001 ) , t stage ( p=0.004 ) , grade ( p=0.025 ) , and metastasis ( p<0.001 ) , but not with age , gender , tumor histology subtype , or tumor size ( table 2 ) .
preoperative thrombocytosis was significantly correlated with anemia ( p=0.003 ) , t stage ( p=0.002 ) , and metastasis ( p=0.001 ) , but not with age , gender , tumor histology subtype , tumor size , or grade ( table 3 ) .
no significant correlation was found between preoperative crp levels and platelet counts when analyzed by using pearson 's correlation coefficient ( r=0.132 , p=0.079 ) .
kaplan - meier recurrence - free survival curves according to crp level showed that the survival rate of patients with crp elevation ( crp0.8 mg / dl ) was significantly lower than that of patients with normal crp levels ( p<0.001 ) ( fig .
the univariate analysis identified anemia , crp elevation , thrombocytosis , tumor histology subtype , tumor size , t stage , and grade as significant prognostic factors for recurrence - free survival , whereas the multivariate analysis showed that crp elevation ( p=0.033 ) and tumor size ( p=0.007 ) were independent prognostic factors ( table 4 ) .
crp is an acute phase reactant that is produced exclusively by hepatocytes in response to cytokines such as interleukin ( il)-6 , which are known mediators of ongoing inflammatory processes .
an elevation of the serum concentration of this protein indicates the presence of acute inflammation , which is observed 8 to 12 hours after the onset of infection . the relationship between cancer and crp elevation
hepatic production of crp is induced by cytokines such as il-1 , tumor necrosis factor ( tnf ) , and primarily il-6 , which is frequently overproduced by the tumor cells themselves .
experimental studies on rcc cell lines and expression studies of renal surgical specimens have shown that at least some renal tumors produce il-6 , which functions as an autocrine growth factor of the rcc .
therefore , the presence of a systemic inflammatory response might be associated with aggressive rcc behavior .
the prognostic value of crp levels has been reported in many recent studies of cancers , including those of the esophagus , ovaries , and colon [ 21 - 23 ] .
however , the mechanism by which a systemic inflammatory response affects cancer - specific survival in patients with rcc is unclear .
hwang et al studied the relationship between crp and survival in patients with rcc who underwent a radical nephrectomy .
they reported a statistically insignificant difference in the survival rates of patients with and without crp elevation and concluded that crp was not a significant prognostic factor for rcc when compared with tumor stage , grade , tumor size , and cell type .
however , komai et al demonstrated that the 5-year and 10-year disease - specific survival rates ( 75% and 30% , respectively ) in patients with high crp levels were significantly worse than the rates in patients with normal crp levels ( both 93% , p<0.001 ) and suggested that the preoperative crp level is associated with poor survival in patients with localized rcc .
in addition , they reported that the crp level is a useful preoperative screening tool in patients with localized rcc .
blay et al reported that pretreatment il-6 and crp were significantly associated with the effects of cytokine therapy and high il-6 and that the crp level indicated a poor response to cytokine therapy . in the present study ,
preoperative crp elevation was associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc .
this result confirms the importance of the preoperative crp level as an independent prognostic factor for tumor recurrence and is consistent with the findings of earlier reports showing that a high crp level is associated with poor survival in localized rcc .
secondary or reactive thrombocytosis has been associated with tumor metastasis and poor survival in cancer patients .
the pathophysiologic mechanism of reactive thrombocytosis may be tumor - associated elevation of circulating platelets by tumor - derived humoral factors such as il-6 and macrophage colony - stimulating factor ( m - csf ) that may be responsible for the development of cancer - related thrombocytosis .
il-6 is a potent stimulator of megakaryocytopoiesis , and tumor cells have been shown to release il-6 both in vitro and in vivo .
these observations suggest that il-6 plays a role in the development of tumor - associated thrombocytosis .
karakiewicz et al analyzed 1828 patients with rcc by univariable , multivariable , and predictive accuracy analyses with regard to rcc - specific mortality .
in that study , they showed that the addition of thrombocytosis to the base model ( age , tumor size , tnm stage , ecog - performance status , fuhrman grade , and histology subtype ) increased the predictive accuracy by only 0.3% ( from 85.3% to 85.6% ) ; these changes in predictive accuracy were not statistically significant .
consequently , they concluded that patients who presented with thrombocytosis did not have poorer prognosis than did their counterparts who did not exhibit these apparently unfavorable characteristics , as long as the effects of the tnm stage , histology , tumor grade , and ecog - performance status were considered .
however , many investigators have reported that thrombocytosis is related to a poor prognosis in patients with rcc .
, they showed that patients with thrombocytosis had a mean survival of 45.2 months compared with 76.6 months in patients without thrombocytosis ( p=0.0002 ) and concluded that preoperative thrombocytosis is associated with a poorer survival than is a normal platelet count . in the present study
, we showed that preoperative thrombocytosis was significantly correlated with anemia , t stage , and metastasis .
in addition , thrombocytosis was a significant prognostic factor associated with recurrence - free survival in the univariate analysis .
therefore , thrombocytosis was associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc ; these findings are consistent with those of previous studies .
ito et al showed that preoperative crp levels and platelet counts had a significant correlation and suggested that this was because the reactive thrombocytosis and crp elevation in rcc were both caused by the production of inflammatory cytokines such as il-6 .
as in the study by ito et al , we analyzed the relationship between preoperative crp and platelet count .
however , we did not find a statistically significant association between preoperative crp and platelet count ( r=0.132 , p=0.079 ) .
therefore , the exact relationship between preoperative crp and platelet count remains to be determined .
a limitation of this study is that several variables could not be considered in the current analysis .
nonetheless , all available variables were included , and our findings indicate that several known prognostic factors were valuable as prognostic factors
the reason for this result was , to some extent , too many patients in stage t1 rather than other t stages and a small number of patients with grade 4 compared with other grades according to the early detection of rcc nowadays .
preoperative crp elevation and thrombocytosis were associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc . moreover ,
however , preoperative thrombocytosis was not an independent prognostic factor for tumor recurrence and prognosis .

 purposethe aim of this study was to investigate the association of preoperative c - reactive protein ( crp ) elevation and thrombocytosis with the prognosis of patients with non - metastatic renal cell carcinoma ( rcc).materials and methodsthis was a retrospective review of the medical records of 177 patients ( 130 men and 47 women ) with non - metastatic rcc who underwent a radical nephrectomy between march 2000 and may 2008 and for whom preoperative crp and platelet data were available for analysis .
 preoperative crp elevation and thrombocytosis were compared with clinical and pathological variables.resultsthere were 38 patients with crp elevation and 11 patients with thrombocytosis .
 the mean follow - up time was 48.3 months ( median , 48.0 ; range , 13 - 111 months ) .
 twenty - three patients ( 13.0% ) developed metastases and six patients died during the follow - up period .
 crp elevation was significantly correlated with anemia ( p=0.001 ) , t stage ( p=0.004 ) , grade ( p=0.025 ) , and metastasis ( p<0.001 ) .
 thrombocytosis was significantly correlated with anemia ( p=0.003 ) , t stage ( p=0.002 ) , and metastasis ( p=0.001 ) .
 the univariate analysis identified anemia , crp elevation , thrombocytosis , tumor histology subtype , tumor size , t stage , and grade as significant prognostic factors associated with recurrence - free survival , whereas the multivariate analyses showed that crp elevation ( p=0.033 ) and tumor size ( p=0.007 ) were independent prognostic factors.conclusionspreoperative crp elevation and thrombocytosis were associated with a poorer prognosis and a higher recurrence rate in patients with non - metastatic rcc .
 moreover , preoperative crp elevation appeared to be an independent predictor of tumor recurrence and prognosis .
 preoperative thrombocytosis , however , was not an independent prognostic factor for tumor recurrence and prognosis .


angiogenesis is a constant hallmark of multiple myeloma ( mm ) progression and has prognostic potential .
we have previously isolated endothelial cells ( ec ) from bone marrow of patients with mm ( mmec ) .
they show intrinsic angiogenic ability , because they rapidly form a capillary network in vitro , and extrinsic ability , because they generate numerous new vessels in vivo in the chick embryo chorioallantoic membrane ( cam ) .
moreover , mmec secrete angiogenic cytokines , such as fibroblast growth factor-2 ( fgf-2 ) and vascular endothelial growth factor ( vegf ) , as well as matrix metalloproteinase-2 and 9 ( mmp-2 and mmp-9 ) and express adhesion molecules facilitating dissemination of these cells .
studies by rajkumar et al . , dominici et al . , and laroche et al .
the bone marrow microenviromnent plays a crucial role in inducing the angiogenic response in mm [ 6 , 7 ] .
reciprocal positive and negative interactions between plasma cells and bone marrow stromal cells , namely haematopoietic stem cells , fibroblasts , ostoblasts / osteoclasts , ec , ec progenitor cell , t lymphocytes , macrophages and mast cells are mediated by an array of cytokines , receptors and adhesion molecules .
these cells secrete angiogenic factors , such as interleukin-6 ( il-6 ) , vegf , fgf-2 , hepato - cyte growth factor / scatter factor ( hgf / sf ) , transforming growth factor- ( tgf- ) and tumour necrosis factor  ( tnf- ) [ 6 , 7 ] . here , we have attempted a fine characterization of the angiogenic response induced by mmec , as compared with monoclonal gammopathy of undetermined significance ec ( mgusec ) in the cam assay .
we investigated the angiogenic response induced by gelatin sponges soaked with the cell suspensions and implantedonthecamsurfacefrom8today12ofincu - bation and we studied the effects of mmec and mgusec on the expression of endogenous levels in the cam of vegf , fgf-2 , angiopoietin-1(ang-1 ) , hypoxia - inducible factor-1 ( hif-1 ) and endostatin by reverse transcriptase - polymerase chain reaction ( rt - pcr ) .
mmec were obtained from 10 patients with mm and mgsuec were obtained from 10 patients with mgus , as previously described .
fertilized white leghorn chicken eggs were incubated at 37c at constant humidity . on day 3 of incubation a square window was opened in the egg shell after removal of 23 ml of albumen so as to detach the developing cam from the shell .
the window was sealed with a glass and the eggs were returned to the incubator . gelatin sponges ( gelfoam , upjohn company , kalamazoo , mi , usa ) were cut to a size of 1 mm3 and placed on top of a growing cam at day 8 incubation under sterile conditions .
the sponges were then adsorbed with 2 l of cell suspension ( 18,000 cells per sponge ) of mmec or mgusec or with rpmi-1640 medium alone or supplemented with fgf-2 ( 200 g / ml ) ( r & d systems , abington , uk ) , used as negative and positive control , respectively .
the angiogenic response was evaluated 96 hrs after the implants by means of a stereomicroscope connected to an image analyser system ( olympus italia , italy ) .
blood vessels entering the sponges within the focal plane of the cam were counted by two observers in a double blind fashion at a magnification of 50x .
means  1 standard deviation ( sd ) were evaluated for all the parameters and the statistical significance of the differences between the counts was determined by student 's t - test for unpaired data .
total rna was extracted from 12 cam treated with ec obtained from six patients with mgus and six patients with mm and from six control cam using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufactures 's protocol . the concentration and the purity of the isolated rna
were determined using a spettrofotometric analysis and the integrity of the rna was verified by means of electroforesis in a formaldehyde agarose gel followed by ethidium bromide staining .
the cdna was then synthetized with 5 g of total rna using m - mlv and oligo ( dt)1820 as primer ( invitrogen , carlsbad , ca , usa ) .
level of mrna expression of pro - angiogenic molecules vegf , fgf-2 , ang-1 , hif-1 and mrna expression of anti - angiogenic factor endostatin were evaluated by pcr using specific primers and the relative level of the each amplified transcripts were normalized to those of the housekeeping gene -actin .
pcr reactions were performed in a final volume of 25 l containing 2 l of rt products , 10 picomoles of each primer and 1x of red - taq polymerase mix ( sigma - aldrich , milano , italy ) .
preliminary experiments were performed to determine the appropriate conditions ( ta and specific number of cycles ) for semi - quantitative rt - pcr and transcript were amplified using different cycle numbers:25 cycles for -actin , 30 cycles for vegf , fgf-2 , ang-1 and for endostatin and 35 cycles for hif-1. pcr products were subjected to electrophoresis in a 1.5% agarose gel and ethidium bromide stained , and then photographed under an ultraviolet transilluminator ( amersham biosciences ) .
intensity of bands was quantified as arbitrary optical density units ( od ) using the scion image software ( based on nih image ) .
six cam treated with ec obtained from three patients with mgus and three patients with mm and three control cam were homogenized in ice cold buffer containing 250 mm sucrose , 10 mm tris - hcl , ph 7.4 and protease inhibitors [ 1g / ml leupeptin , 1g / ml pepstatin and 1 mm phenyl - methylsulfonyl fluoride ( pmsf ) ] .
after homogenization in a potter apparatus and centrifugation at 1000 g for 10 min , the protein content of the supernatant was measured using a bradford assay ( bio - rad laboratories , hercules , ca , usa ) . for sds - page , samples ( 60 g / lane ) were solubilized in laemmli buffer , boiled at 90c for 10 min and runned on a 12% polyacrylamide .
the proteins were blotted to a pvdf membrane ( millipore corporate , billeira , ma , usa ) and immunoblotting was performed using a monoclonal mouse primary antibody against endostatin ( novus biologicals , littleton , co , usa ) diluted 1:500 in tbst .
peroxidase activity was revealed by chemiluminescence ( ecl kit , santa cruz biotechnology , santa cruz , ca , usa ) and blot was immediately exposed to x - ray film .
control experiments were performed using a mouse - anti--actin ( abcam , cambridge , ca , usa ) to detect housekeeping protein -actin to normalize the protein load .
stained bands were scanned and intensity was quantified using the scion image system ( based on nih image ) .
mmec were obtained from 10 patients with mm and mgsuec were obtained from 10 patients with mgus , as previously described .
fertilized white leghorn chicken eggs were incubated at 37c at constant humidity . on day 3 of incubation a square window was opened in the egg shell after removal of 23 ml of albumen so as to detach the developing cam from the shell .
the window was sealed with a glass and the eggs were returned to the incubator .
gelatin sponges ( gelfoam , upjohn company , kalamazoo , mi , usa ) were cut to a size of 1 mm3 and placed on top of a growing cam at day 8 incubation under sterile conditions .
the sponges were then adsorbed with 2 l of cell suspension ( 18,000 cells per sponge ) of mmec or mgusec or with rpmi-1640 medium alone or supplemented with fgf-2 ( 200 g / ml ) ( r & d systems , abington , uk ) , used as negative and positive control , respectively .
the angiogenic response was evaluated 96 hrs after the implants by means of a stereomicroscope connected to an image analyser system ( olympus italia , italy ) .
blood vessels entering the sponges within the focal plane of the cam were counted by two observers in a double blind fashion at a magnification of 50x .
means  1 standard deviation ( sd ) were evaluated for all the parameters and the statistical significance of the differences between the counts was determined by student 's t - test for unpaired data .
total rna was extracted from 12 cam treated with ec obtained from six patients with mgus and six patients with mm and from six control cam using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufactures 's protocol . the concentration and the purity of the isolated rna
were determined using a spettrofotometric analysis and the integrity of the rna was verified by means of electroforesis in a formaldehyde agarose gel followed by ethidium bromide staining .
the cdna was then synthetized with 5 g of total rna using m - mlv and oligo ( dt)1820 as primer ( invitrogen , carlsbad , ca , usa ) .
level of mrna expression of pro - angiogenic molecules vegf , fgf-2 , ang-1 , hif-1 and mrna expression of anti - angiogenic factor endostatin were evaluated by pcr using specific primers and the relative level of the each amplified transcripts were normalized to those of the housekeeping gene -actin .
pcr reactions were performed in a final volume of 25 l containing 2 l of rt products , 10 picomoles of each primer and 1x of red - taq polymerase mix ( sigma - aldrich , milano , italy ) .
preliminary experiments were performed to determine the appropriate conditions ( ta and specific number of cycles ) for semi - quantitative rt - pcr and transcript were amplified using different cycle numbers:25 cycles for -actin , 30 cycles for vegf , fgf-2 , ang-1 and for endostatin and 35 cycles for hif-1. pcr products were subjected to electrophoresis in a 1.5% agarose gel and ethidium bromide stained , and then photographed under an ultraviolet transilluminator ( amersham biosciences ) .
intensity of bands was quantified as arbitrary optical density units ( od ) using the scion image software ( based on nih image ) .
six cam treated with ec obtained from three patients with mgus and three patients with mm and three control cam were homogenized in ice cold buffer containing 250 mm sucrose , 10 mm tris - hcl , ph 7.4 and protease inhibitors [ 1g / ml leupeptin , 1g / ml pepstatin and 1 mm phenyl - methylsulfonyl fluoride ( pmsf ) ] .
after homogenization in a potter apparatus and centrifugation at 1000 g for 10 min , the protein content of the supernatant was measured using a bradford assay ( bio - rad laboratories , hercules , ca , usa ) . for sds - page , samples ( 60 g / lane ) were solubilized in laemmli buffer , boiled at 90c for 10 min and runned on a 12% polyacrylamide .
the proteins were blotted to a pvdf membrane ( millipore corporate , billeira , ma , usa ) and immunoblotting was performed using a monoclonal mouse primary antibody against endostatin ( novus biologicals , littleton , co , usa ) diluted 1:500 in tbst .
peroxidase activity was revealed by chemiluminescence ( ecl kit , santa cruz biotechnology , santa cruz , ca , usa ) and blot was immediately exposed to x - ray film .
control experiments were performed using a mouse - anti--actin ( abcam , cambridge , ca , usa ) to detect housekeeping protein -actin to normalize the protein load .
stained bands were scanned and intensity was quantified using the scion image system ( based on nih image ) .
the ability of mmec and mgusec to induce an angiogenic response in vivo was assessed with the cam - gelatin sponge assay .
1 ) . cam implanted with the medium containing fgf-2 or mmec gave significantly higher vessel counts and numerous allantoic vessels converging like spokes toward the sponges were recognizable ( table 1 and fig .
when the sponges were loaded with the medium alone and with mgusec , physiologic angiogenesis was observed in the form of few allantoic vessels partly around and partly converging toward the sponge ( table 1 and fig .
angiogenic response assessed by the chick embryo chorioallantoic membrane ( cam)-gelatin sponge assay p<0.001 versus rpmi-1640 and mgusec angiogenic activity in the chorioallantoic membrane ( cam ) assay . gelatin sponges loaded with fgf-2 ( a ) , mmec ( b ) , rpmi-1640 ( c ) and mgusec ( d ) were implanted on top of the cam on day 8 .
macroscopic view of the cam on day 12 shows numerous allantoic vessels converging like spokes toward the sponge in a and b , while few allantoic vessels are recognizable in c and d. original magnification : a  d , x 50 . in order to evaluate the angiogenic effects of mmec and mgusec , mrna expression of pro - angiogenic factors including vegf , fgf2 , ang-1 , hif-1 and mrna expression of anti - angiogenic molecule endostatin
the expression level of each transcript was compared to those detected in control cam . as shown in fig .
2 , treatment of cam with mmec and mgusec did not produce significant difference in the expression of pro - angiogenic factors mrna in comparison with control cam treated with medium alone .
moreover , no statistical difference was found in vegf , fgf-2 , ang-1 , hif-1 and endostatin mrna content between mm and mgus treated cam ( p<0.05 ) .
in contrast , the expression of endostatin mrna detected in both mgus and mm treated cam was significantly lower respect to control cam ( mgus od:120.113 and mm od:48.319.6 versus ctrl od:205.84  19.6 ; p<0.05 ) .
expression levels of mrna coding for vegf , fgf-2 , ang-1 , hif1 and endostatin evaluated by semi - quantitative rt - pcr .
transcript levels from the cam assay are referred to ec obtained from 6 mm and 6 mmgus patients and the error band represents the standard deviation of six experiments .
western blot analyses were also performed to study whether the expression of endostatin changes in mmec and mgusec treated cam respect to control . as shown in fig .
3 , according to rt - pcr quantification , endostatin was significantly lower also at the protein level in both mgusec and mmec treated cam respect to control cam ( mgus od : 97.1410 and mm od : 52.313.6 versus ctrl od : 158.715.52 ;
p<0.05 ) . immunoblotting analysis and quantification after western blot analysis of endostatin expression in cam treated with ec obtained from 3 mm and 3 mgus patients .
mm plasma cells secrete vegf , whereas both vegfr-1 and vegfr-2 are markedly elevated in the bone marrow stromal cells , suggesting that a paracrine growth pathway mediated by vegf - activated stromal cells , secretion of il-6 on their part , and subsequent activation of plasma cells may occur . in active mm , we found secretion of the vegf - a isoform by plasma cells , overexpression of vegfr-2 by bone marrow microvessels and isolated ec and of vegfr-1 by the other stromal cells , secretion of vegf - c and vegf - d by stromal cells , and expression of their cognate receptor vegfr-3 by plasma cells , which suggests another paracrine loop of angiogenesis and tumour growth in mm .
parallel studies by kumar et al . found secretion of vegf by several mm cell lines and fresh patients ' bone marrow plasma cells , as well as expression of vegfr-1 and vegfr-2 , which suggests multiple vegf - mediated autocrine pathways of tumour growth and paracrine stimulation of angiogenesis .
when we studied the angiogenic potential of plasma cell suspension obtained from patients with active mm and implanted them onto the cam , we demonstrated a vasoproliferative response , significantly higher than that induced by cell suspension obtained from patients either with non - active mm or mgus .
hgf / sf is yet another angiogenic factor identified in the culture medium of both human mm cell lines   and freshly isolated plasma cells .
still other factors may be responsible for bone marrow angiogenesis in mm : the plasma cell secretion products , such as tgf- , il-1 , il-6 and il-8 ; granulocyte - colony stimulating factor ( g - csf ) , granulocyte macrophage - colony stimulating factor ( gm - csf ) and tnf- , secreted by bone marrow microenvironment cells recruited and activated by plasma cells .
since human mm cell lines produce ang-1 that up - regulates the tie-2 cognate receptor in bone marrow ec , involvement of the ang-1/tie-2 loop in mm neovascularization has been demonstrated .
conversely , mmec secrete angiogenic cytokines , such as fgf-2 , vegf and ang-1 as well as matrix metalloproteinase-2 and -9 ( mmp-2 and mmp-9 ) and express adhesion molecules facilitating dissemination of these cells .
other studies , however , showed that the expression levels of vegf , fgf-2 , and their receptors were similar among plasma cells from mgus and mm , suggesting that increasing angiogenesis from mgus to mm is , at least in part , explained by increased tumour burden rather than increased expression of vegf / vegfr-2 by plasma cells .
another possibility is that the inhibition of angio - genesis in mgus is lost with progression , hence that the switch from mgus to mm may involve a loss of an anti - angiogenic activity .
our data are in accord with this statement , because the expression of endostatin mrna detected in both mgus and mm treated cam was significantly lower respect to control cam .
the phenotypic switch to angiogenesis in tumour growth involves more than simple up - regulation of angiogenic activity and is thought to be the result of a net balance of positive and negative regulators , and our study suggests that angiogenic switch in mm , investigated by using an in vivo experimental model such as the chick cam , may involve loss of an endoge - nous angiogenesis inhibitor , namely endostatin .

 abstractwe have attempted a fine characterization of the angiogenic response induced by multiple myeloma endothelial cells ( mmec ) by using the chick embryo chorioallantoic membrane ( cam ) assay and by reverse transcriptase - polymerase chain reaction ( rt - pcr ) .
 results showed that in the cam assay mmec induced an angiogenic response comparable to that of a well - known angiogenic cytokine , namely fibroblast growth factor-2 ( fgf-2 ) , while rt - pcr demonstrated that the expression of endostatin mrna detected in mm treated cam was significantly lower respect to control cam .
 these data suggest that angiogenic switch in mm may involve loss of an endogenous angiogenesis inhibitor , such as endostatin .


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You know, I saw a movie - "Crocodile Dundee."


We're consuming 50 percent of the world's cocaine.


That answer was about as clear as Boston harbor.


Let me help the governor.


We've run up more debt in the last eight years than under all the presidents from George Washington to Jimmy Carter combined.


I was in Houston on Tuesday meeting with a group of good citizens, working citizens.


The last time the Vice President used that phrase, I suspect he remembers it, don't you?


I think we have to demonstrate some flexibility and I think the FDA is attempting to do so now in trying to make it possible for new and experimental drugs to be available to people who are at risk at AIDS and I would hope that we could bring that kind of a policy to bear beginning in January.


This isn't a question of a little charity for the homeless.


Go out and take a look at St. Louis at some of that effort.


I shouldve stayed at home
Cause right now I see all these people that love me
But I still feel alone
Cant help but check my phone
I couldve made you mine
But no, it wasnt meant to be and see I wasnt made for you
And you werent made for me
Though it seemed so easy
And thats because I wanna be your favorite boy
I wanna be the one that makes your day
The one you think about as you lie awake
I cant wait to be your number one
Ill be your biggest fan and youll be mine
But I still wanna break your heart and make you cry
But wont you wait?
You know its too late
Im on my own shit now
Let me tell you how it feels to be fucking great
I feel great
Whoa, oh, oh, oh, oh
You need to be yourself
Love someone for loving you instead of someone really cool
That makes your heart melt
Who knows what you truly felt
Youre still my favorite girl
You better trust me when I tell you there aint no one else
More beautiful in this damn world
In this damn world
Youre gonna wanna be my best friend baby
Youre gonna wanna be my best friend
I said that
Youre gonna wanna be my best friend baby
Youre gonna wanna be my best friend
Youre gonna wanna be my best friend baby
Youre gonna wanna be my best friend
Best friend
Youre gonna wanna be my best friend baby
Youre gonna wanna be my best friend
I say that Im happy
I say that Im happy
But no, no, no, no
No, no, no, oh
I still wanna be your favorite boy
I wanna be the one that makes your day
The one you think about as you lie awake
And I cant wait to be your number, your number one
Ill be your biggest fan and youll be mine
But I still wanna break your heart and make you cry
I still wanna be your favorite boy
I wanna be the one
I might just be the one


Oh, I want to know
Where I can go
When youre not around
And Im feeling down
So wont you stay for a moment
So I can say
I, I need you so?
Cause right now, you know
That nothing heres new
And Im obsessed with you
Then I fell to the ground
And you smiled at me and said
I dont wanna see you cry
You dont have to feel this emptiness
She said I love you til the day that I die
Well, maybe shes right
Cause I dont wanna feel like Im not me
And to be honest, I dont even know why
I let myself get down in the first place
Tryna keep my mind at bay
Sunflower still grows at night
Waiting for a minute til the suns seen through my eyes
Make it down, down, do-down-down
Diggy, dig down, doo-doo-doo-doo
Waiting for a minute til the suns seen through my eyes
You know you need to get yourself to sleep and dream
A dream of you and I
Theres no need to keep an open eye
I promise Im the one for you
Just let me hold you in these arms tonight
Im lucky to be me and you can see it in my face
Back when I fucked my shit up too many times
Why would I let myself get down in the first place?
Tryna keep my mind at bay
Sunflower still grows at night
Waiting for a minute til the suns seen through my eyes
Make it down, down, do-down-down
Diggy, dig down, doo-doo-doo-doo
Waiting for a minute til the suns seen through my eyes
And so she sat me down and told me that I didnt have to cry
Said I didnt need to get down or feel empty inside
And told me that shell love me for as long as shes alive
And well, maybe shes right
Cause I hate it when I feel like Im not me
See, I honestly dont even know why
I-I honestly dont even know why
Tryna keep my mind at bay
Sunflower still grows at night
Waiting for a minute til the suns seen through my eyes
Make it down, down, do-down-down
Diggy, dig down, doo-doo-doo-doo
Waiting for a minute til the suns seen through my eyes


Always felt like I needed to please her, or impress her
Though only in the hope that one day I would undress her
Dont be offended
You think Im bout to tell you that I love you
But I really, really, really fuckin dont
See the months of obsession
And crying for hours
I even started sittin down in the shower, girl
Ill take my time on my own
And Ill be fine, now Im alone
Constantly told stories with a stretch of the truth
Trying hard to make memories to remember from youth
Desperately wanting you to be interested too
Its not clear what I need yet
But its clear its not you
Heres what Id say to any young man thats still interested in you
Do you prioritize the things in your life?
The things that you hope to do?
Cause if not, then
Mate, youd better trust me when I tell you that
Its not worth forgettin about yourself
Because of one fucking girl
She may seem perfect, and gorgeous, and lovely
Youll think she likes you
I mean I thought she loved me, no
But no
Neither of us will get down from the shelf
The only one she loves is herself


Bitch, fuck!
You have a question?
How many cars can I buy til I run outta drive?
How much drive can I have until I run outta road?
How much road can they pave until I run outta land?
How much land can it be until I run in the ocean?
Niggas go with the motions and all the plans
See, I was never into the beaches and all the sands
See, I was in the woods with flowers, rainbows, and posies
Fallin outta my pocket, but yall want to know if I swam to cool down
How much cooler can I get until I run out of fans?
How many fans can I have until they turn on the AC?
If the AC blow up, then Im TNT, Im gone
Im gone and Im finished
And I aint seen my friends in a minute
Guessing nothing lasts forever
Yeah, nothing lasts forever
Nothing sticks together
Sick of sitting in doubt
Please let me figure this out, out
Shout out to the girls that I led on
For occasional head and always keepin my bed warm
And tryin their hardest to keep my head on straight
And keepin me up enough til I had thought I was airborne
How many raps can I write til I get me a chain?
How many chains can I wear til Im considered a slave?
How many slaves can it be til Nat Turner arise?
How many riots can it be until them Black lives matter?
Niggas click-clack splatter, pew that nigga
Life a game of basketball, you better shoot that nigga
Cause if that cop got trigger, he better pull
Cause when I get pulled over, I usually play it cool
Cause I know what Im drivin is usually paid in full
And my ego and possessions, will not let me be one
Because I got a mansion, my mansion got some rooms
Them rooms got some windows and my windows got some views
Them views get some stares and my backyard does too
And if you walk to the bottom youll prolly see a pool
You better not drown, keep them ten toes up
Cause if them ten toes down, that mean that you fucked up
And thats what I swim in
And if I drown and dont come back
Whos gonna know?
And if I crash and dont come back
Whos gonna know?
And if I fall and dont come back
Whos gonna know?
Im wondering if I dont come back
Maybe then Ill know
Dont come back
Dont come back
Ayy, dont come back
Dont come back
No, no


Loving is easy
You had me fucked up
It used to be so hard to see
Yeah, loving is easy
When everythings perfect
Please dont change a single little thing for me
Listen, girl
When you cant even hide it
And it didnt take forever to find it
I was all on my own
Almost glad to be alone
Until love came in on time, on time
So loving is easy
You had me fucked up
It used to be so hard to see
Yeah, loving is easy
When everythings perfect
Please dont change a single little thing for me
Oh, oh
Ah, ah
Oh, oh
Ah, ah
So listen, girl
When you cant even hide it
And it didnt take forever to find it
I was all on my own
Almost glad to be alone
Until love came in on time, oh, on time
Loving is easy
You had me fucked up
It used to be so hard to see
Yeah, loving is easy
When everythings perfect
Please dont change a single little thing for me
Oh, oh
Ah, ah
Oh, oh
Ah, ah


Ill be the one that stays till the end
And Ill be the one that needs you again
And Ill be the one that proposes in a garden of roses
And truly loves you long after our curtain closes
But will you still love me when nobody wants me around?
When I turn 81 and forget things, will you still be proud?
Cause I am the one thats waited this long
And I am the one that might get it wrong
And Ill be the one that will love you the way Im supposed to, girl, oh
But will you still love me when nobody wants me around, around?
When I turn 81 and forget things, will you still be proud?
Proud of me and my short list of accomplishments, say
And me and my lack of new news
Me and my selfishness, oh, me and myself
Wish you nothing but a happy new version of you
Because I, I mmm, mmm, yeah
I want you to tell me you find it hard to be yourself so I can say
Its gonna be alright, yeah
And I want you to love me the way you love your family
The way you love to show me what its like
To be happy


Context: At the same time the Mongols imported Central Asian Muslims to serve as administrators in China, the Mongols also sent Han Chinese and Khitans from China to serve as administrators over the Muslim population in Bukhara in Central Asia, using foreigners to curtail the power of the local peoples of both lands. Han Chinese were moved to Central Asian areas like Besh Baliq, Almaliq, and Samarqand by the Mongols where they worked as artisans and farmers. Alans were recruited into the Mongol forces with one unit called "Right Alan Guard" which was combined with "recently surrendered" soldiers, Mongols, and Chinese soldiers stationed in the area of the former Kingdom of Qocho and in Besh Balikh the Mongols established a Chinese military colony led by Chinese general Qi Kongzhi (Ch'i Kung-chih). After the Mongol conquest of Central Asia by Genghis Khan, foreigners were chosen as administrators and co-management with Chinese and Qara-Khitays (Khitans) of gardens and fields in Samarqand was put upon the Muslims as a requirement since Muslims were not allowed to manage without them. The Mongol appointed Governor of Samarqand was a Qara-Khitay (Khitan), held the title Taishi, familiar with Chinese culture his name was Ahai

Question: Where in Central Asia did the Han Chinese move?

Besh Baliq, Almaliq, and Samarqand
Besh Baliq, Almaliq, and Samarqand
Bukhara


Context: The Social Chapter is a chapter of the 1997 Treaty of Amsterdam covering social policy issues in European Union law. The basis for the Social Chapter was developed in 1989 by the "social partners" representatives, namely UNICE, the employers' confederation, the European Trade Union Confederation (ETUC) and CEEP, the European Centre of Public Enterprises. A toned down version was adopted as the Social Charter at the 1989 Strasbourg European Council. The Social Charter declares 30 general principles, including on fair remuneration of employment, health and safety at work, rights of disabled and elderly, the rights of workers, on vocational training and improvements of living conditions. The Social Charter became the basis for European Community legislation on these issues in 40 pieces of legislation.

Question: How many general principles does the Social Charter declare?