Question:
You will be presented with the full text of a biomedical research paper. Please write an abstract for this paper. Your response should include the abstract and no additional text.

Paper text:
we recently demonstrated that diabetes directly induced dental caries in rats and mice , although a direct association between the 2 was less evident and conflicting in humans [ 14 ] .
we demonstrated that dental caries were produced using noncariogenic diets with a low concentration of sugar in diabetic rodent models [ 57 ] , although many studies usually add large amounts of sugar to the diet to induce dental caries [ 8 , 9 ] .
in addition , early studies on the production of experimental dental caries have reported the effects of physical properties of cariogenic diets with high concentrations of sugar on dental caries .
these studies mainly used finely ground cariogenic diet [ 1013 ] because it is more cariogenic compared to a coarsely ground diet in rodents .
moreover , hard and coarse foods have the ability to prevent caries [ 1417 ] .
thus , modifying the dietary formulation may possibly enhance or reduce caries development in diabetic animals .
however , no reports have investigated the effects of the physical properties of non - cariogenic standard diets on dental caries in diabetic animals .
currently , 2 types of non - cariogenic standard diet formulations ( i.e. , powdered and pelletized diets ) are widely used in experimental rodents . in the present study ,
we compared the effects of these diets on the development of dental caries in a diabetic rodent model focusing on the difference in hardness between the powdered and pelletized diets .
six - week - old female f344 rats were supplied by japan slc , inc .
the animals were housed in stainless - steel cages at a temperature of 2026c and a relative humidity of 4070% under a 12/12  h light / dark cycle , that were ventilated with filtered fresh air . to prevent infection ,
the animals were allowed free access to tap water and fed with a widely used standard pelletized or powdered diet ( charles river formula 1 ( crf-1 ) ; oriental yeast co. , ltd . ,
the animals were handled according to the principles for all experimental procedures outlined in the guide for the care and use of laboratory animals prepared by our institution ( setsunan university ) and the japanese association for laboratory animal science .
the glucose levels in the fresh urine were measured semiquantitatively using urine test paper ( wako pure chemical industries , osaka , japan ) daily from day 1 to day 3 after dosing , once every week for 1 month after the first week , and once every month thereafter .
the blood glucose levels in the tail vein samples were also measured semi - quantitatively using the glucose oxidase method ( glutest e ; sanwakagaku , aichi , japan ) once every month from the fourth week after alloxan injection .
blood samples obtained from the tail vein and fresh urine were collected from 1:00 to 4:00  p.m. the severity of hyperglycemia was defined as follows : normal , < 200  mg / dl ; mild , > 200  mg / dl ; moderate , > 300  mg / dl ; or severe , > 400  mg / dl . the severity of glycosuria was defined as follows : normal , < 100  mg / dl ; mild , > 100  mg / dl ; moderate , > 250  mg / dl ; or severe , > 500  mg / dl . the experimental design is shown in figure 1 .
ten rats , aged 7 weeks , were given a single dose ( 35  mg / kg body weight ) of alloxan ( sigma - aldrich japan , tokyo , japan ) via tail vein injection .
the dose of alloxan was determined as the given dose at which a rat survives for a long period after the onset of diabetic symptoms . after the confirmation of hyperglycemia and glucosuria following the dosing of alloxan ,
five rats were given a pelletized crf-1 diet ( diabetes - pelletized diet group ) and the remaining 5 rats were given a powdered crf-1 diet ( diabetes - powdered diet group ) .
as a control , each of the 5 intact rats was fed a pelletized or powdered crf-1 diet ( control - pelletized diet group and control - powdered diet group ) .
the animals were euthanized by exsanguination under deep anesthesia at the end of the observation period .
subsequently , the mandible was removed and fixed in 10% neutral - buffered formalin ( ph 7.4 ) .
after a 24  h fixation , the occlusal , buccolingual , and proximal surfaces of all of the molar teeth were intensively observed under a binocular stereoscope .
soft x - ray images of the mesiodistal plane were taken under conditions of 35  kv and 2  ma for 4  min .
the molar teeth were classified into 5 groups according to caries characteristics by observing and analyzing the radiographs : no radiolucent change ( grade 0 ) , radiolucent area only on the occlusal surface of the crown ( grade 1 ) , radiolucent areas on occlusal surface and either of the mesiodistal surfaces of the crown ( grade 2 ) , radiolucent areas over the entire surface of the crown ( grade 3 ) , and radiolucent areas over most of the surface of the dental root ( grade 4 ) .
the mean score of the caries was used as indicator for comparing the severity of the carious lesions between the groups .
after soft x - ray examination , a histopathological examination was performed on the mandible in all of the rats .
after fixation with 10% neutral - buffered formalin , the sample was decalcified in a 5% solution of ethylenediaminetetraacetic acid 4 na ( edta 4 na ) for 2 weeks at 4c .
after decalcification , the specimens were trimmed , dehydrated in a sequential ethanol series using an automated processor , and embedded in paraffin wax .
serial 7  m thick sections on the mesiodistal plane were made through the centers of all of the molars and then stained with hematoxylin and eosin for examination using light microscopy .
the severity of the caries lesion was graded as follows : slight , dentin caries localized in occlusal surface of dentin ; mild , dentin caries extended into the dental pulp with pulpitis and/or pulp necrosis ; moderate , dental crowns were partially decayed ; and severe , dental crowns were completely decayed ( only the molar roots remained ) .
the wilcoxon rank - sum test was employed to compare the differences in the mean scores of the caries lesions using soft x - ray examination between the groups .
the chi - square test was used to determine the caries incidence using soft x - ray examination and the incidence of histopathological lesions in each group of rats .
severe hyperglycemia ( > 400  mg / dl ) and glucosuria ( > 500  mg / dl ) continued from the day after alloxan injection to the last monitoring day in all of the rats in the alloxan - treated groups .
in addition , the blood glucose levels ranged from 78 to 120  mg / dl ( normoglycemia ) , and the urine glucose levels were less than 100  mg / dl in the control groups .
typical macroscopic appearances of carious molars in diabetic rats are shown in figures 3(a ) , 3(b ) , and 3(c ) .
macroscopically , the dental caries developed mainly in occlusal fissures and were identified as partial coronal defects of the molars in alloxan - treated diabetic rats ( figure 3(b ) ) .
the carious lesions expanded horizontally , until the crown of the carious molar was completely invisible ( figures 3(b ) and 3(c ) ) . in contrast , control nondiabetic rats showed no changes in any of the molars ( figure 3(a ) ) .
soft x - ray images of the carious lesion in diabetic rats are shown in figures 3(d ) , 3(e ) , and 3(f ) . in all of the alloxan - treated diabetic rats ,
radiographically , the dental caries progressed both horizontally and vertically . in diabetic rats fed with a pelletized diet ,
the carious lesion was mainly characterized as grade 2 ( figure 3(e ) ) or 4 type ( table 1 ) .
in addition , the diabetic rats fed a powdered diet demonstrated carious lesions that were nearly of grade 4 type ( figure 3(f ) ) . in the control
non - diabetic rats , there was no change in the molar teeth ( table 1 , figure 3(d ) ) .
the histopathological characteristics of the carious lesion in diabetic rats are shown in figures 3(g ) , 3(h ) , and 3(i ) .
histopathological carious lesions were detected in the crown as eroded dentin with bacterial colonization in alloxan - treated diabetic rats . in diabetic rats fed with the pelletized diet , many of the molars were moderately affected and the dentin caries spread over a wide area of the dental crown ( figure 3(h ) ) . in the diabetic rats fed with the powdered diet , the dental caries were markedly worsened and the crowns were nearly completely decayed ( figure 3(i ) ) .
the incidence of caries teeth on the basis of each scoring in the soft x - ray examination is shown in table 1 .
the incidence of dental caries in the diabetic rats fed on both the pelletized and powdered diets was apparently higher ( p < 0.01 ) compared to the non - diabetic control rats ( table 1 ) . in the diabetic rats fed with the pelletized diet , 96.7% of their molar teeth were affected with dental caries , and the mean caries score was 2.7 .
in addition , in the diabetic rats fed with the powdered diet , all of their molars ( 100% ) were affected with caries , and the mean caries score was 3.7 , which was significantly higher ( p < 0.01 ) than the diabetic rats fed with the pelletized diet ( table 1 , figure 4 ) .
no radiolucent lesions were observed in any of the molars in control non - diabetic rats .
the incidence of histopathological carious lesions on the basis of each grade is summarized in table 2 .
histopathologically , the severity of the carious lesions in the diabetic rats fed with a powdered diet was significantly enhanced compared to the diabetic rats fed with the pelletized diet ( table 2 ) . in control non - diabetic rats , slight carious lesions , which were macroscopically and radiographically normal ,
were detected in a few animals ; however , there were no differences observed between the non - diabetic rats on the pelletized and powdered diets ( table 2 ) .
the physical properties of foods such as hardness , adhesiveness , and cohesiveness are closely related with the caries - producing potential [ 19 , 20 ] . moreover ,
the development of caries is known to be profoundly affected by food hardness , and hard and coarse foods can exert a detergent activity during mastication , which is effective in the prevention of caries [ 17 , 19 ] . furthermore , a lower degree of caries was reported in animals fed with the hardest food diet . in the present study , the severity of caries in diabetic rats was enhanced in the powdered diet group compared to the pelletized diet group .
it was clear that food hardness affected the development of dental caries in diabetic rats fed on a non - cariogenic diet .
hard foods are known to help flush away or neutralize undesirable material within the dental plaque and exert a cleaning effect on smooth surfaces by direct mechanical friction .
therefore , cariogenicity will be higher in diabetic rats fed with a powdered diet than in those fed with a pelletized diet . in this study , a high incidence and severity of dental caries was confirmed in the alloxan - treated diabetic rats fed with 2 types of standard diets with a low concentration of sugar .
furthermore , the mean caries score of the diabetic rats fed on the pelletized diet at 52 weeks after alloxan dosing in this study was almost 2-fold compared to animals at 26 weeks after alloxan dosing described in a previous report , strongly suggesting that the duration of hyperglycemia may affect caries development in diabetic animals . in this study ,
dental caries developed and extended to all of the molars in diabetic rats fed on both the pelletized and powdered diets .
thus , the effect of food hardness on the different diets in caries development in diabetic rats may become clearer when the dental caries mildly develop during the more early stages . in conclusion
, food hardness could have an effect on the development of dental caries in diabetic rats , and appropriate consideration of the food formulation should be made in experimental caries studies using diabetic rodent models .

Answer:
we have previously shown that dental caries may be produced in diabetic rodent models fed with noncariogenic standard diets ; however , many studies usually add large amounts of sugar to the diet to induce dental caries .
 moreover , the physical properties of cariogenic diets have been reported as an important factor in the formation of caries .
 the aim of this study was to clarify the effect of the hardness of non - cariogenic diets on the development of dental caries in diabetic rodents .
 seven - week - old female f344 rats were divided into 4 groups : intact rats fed with a standard pelletized or powdered diet and alloxan - induced diabetic rats fed with a standard pelletized or powdered diet .
 all of the rats were sacrificed at 52 weeks of age for morphological examinations on their dental tissue .
 dental caries had developed and extended to all the molars in the diabetic rats that were fed with both the pelletized and powdered diets .
 moreover , the lesion was significantly enhanced in the powdered diet group compared to that in the pelletized diet group . in conclusion , food hardness is an important factor influencing the development of dental caries in diabetic rats .


Question:
You will be presented with the full text of a biomedical research paper. Please write an abstract for this paper. Your response should include the abstract and no additional text.

Paper text:
vestibular schwannoma ( vs ) usually arises within the internal auditory canal ( iac ) from either the superior or inferior vestibular nerve .
these tumors usually grow very slowly with the growth rate 0.66 to 2 mm per year6,7 ) . with time , they cause erosion of iac .
the location of tumor in iac was closely related in erosion of iac , tumor that arise laterally along the course of eighth nerve could tend to cause varying degree of erosion before they extend into cerebellopontine angel ( cpa ) cistern4 ) .
almost all vs usually widens iac only and could not invade the temporal bone beyond the confines of the bone of iac .
in this report , we describe a patient without previously known bone disease who presented with aggressive temporal bone invasion of unilateral vs.
a 51-year - old male presented with progressive hearing loss of last 4 years and dizziness of few months duration .
he did not have a history of a head trauma or of any brain surgery . on admission , neurological and physical examination revealed left side deafness , no facial weakness , or no other low cranial nerve deficit like loss of gag reflex , uvular deviation , tongue deviation .
the patient did not have any known systemic disease of bone and family history or physical characteristics of neurofibromatosis .
his blood profile , including his serum calcium , phosphorus , and alkaline phosphates , was normal .
ct scan showed that the tumor mass had destructed iac completely and temporal bone to petrous apex , grown around the cochlea , reached the carotid artery in its petrous portion , extended to jugular foramen ( fig .
we noticed pneumatized area of opposite temporal bone corresponded to eroded area by tumor ( fig .
the tumor was a 43.83 cm sized , heterogeneous lobulated , well - defined mass with destruction of iac and extended to cpa cistern .
the tumor was confined to posterior fossa and not extended to meckel 's cave ( fig .
the combined translabyrinthine with retrosigmoid suboccipital approach was used for the resection of the tumor . at the first time of surgery , we removed the tumor piece by piece within temporal area with translabyrinthine approach , but we could not find facial nerve with this approach .
so we performed additional retrosigmoid suboccipiral approach , and it allowed the exposure the lateral recess area easily and origin site of facial nerve from brain stem .
after detection of facial nerve with intraoperative facial nerve monitoring , we dissected facial nerve from proximal to distal area .
the dissection of the facial nerve within the temporal bone was very difficult , because the facial nerve was severely adhered to the tumor mass ( fig .
the tumor was extended to jugular foramen by destruction of superior wall of jugular foramen .
the consistency of temporal bone was felt to be normal and the colors and the consistency of the tumor were typical of vs. we successfully achieved total removal of tumor and preserved facial nerve in continuity .
the postoperative course was uneventful with normal facial nerve function and the vertigo was resolved .
the patient was good condition after 18 months of follow - up without cranial nerve dysfunction .
he also did not show any evidence of the recurrence or residual tumor in postoperative mri performed at 15 months of follow up .
vs typically originates lateral to the glial - schwann junction , and this point is usually located within the iac , and thus can erode the iac . but the mechanism of widening of iac by a vs still unknown .
takada et al.5 ) hypothesized that the mechanism involved in the destruction of iac is as follows : 1 ) the repeated strokes of the tumor caused by brain pulsation gradually eroded the bony wall of iac ( especially , that of the posterior wall ) .
the apparently less destruction of iac in cases with mostly cystic large tumors might be due to the restriction of this pulsatile movement .
2 ) increased cerebrospinal fluid pressure on the site had some influence on the mechanism of bone erosion as also does solidity of the tumor .
this case presented with aggressive pattern of invasion of temporal bone , which is more extensive than expected in case of unilateral vs. temporal bone destruction over iac in vs is very rare . to our knowledge
feghali and kantrowitz2 ) reported 4 cases of temporal invasion of unilateral vs , in which one cases had experienced previous schwannoma surgery .
they suggested that the invasion to temporal bone occurred through two mechanism in growth of vs. one of them was that the destruction of bone in the petrous apex can actually follow the pneumatized tracts when the cortex of the iac was eroded .
the second mechanism was that a thick membranous scar had " trapped " and confined the tumor within the temporal bone and separated it from the recurrent tumor in the cpa .
we examined various factors related in the excessive erosion of temporal bone in this patient . in this case , the patient showed no signs of osteoporosis and no abnormal laboratory findings including serum level of calcium , phosphorus and alkaline phosphatase .
histologic findings demonstrated typical nature of benign schwannoma , and there were not excessive unusual vascularity nor any malignant features .
if cortex of iac was eroded , the tumor could grow into the temporal bone through the pneumatized area .
but we can not determine why this unilateral tumor first eroded cortex of iac and destructed temporal bone to the petrous apex before growing along the area of least resistance toward the cpa cistern .
trigeminal schwannoma usually widen meckel 's cave and extend into middle fossa and posterior fossa , may destruct petrous apex and grow into temporal bone .
mri showed that the tumor was confined to posterior fossa and not extended to meckel 's cave . and
tympanic segment is the most frequently involved3 ) , so temporal bone might be destructed .
the patient presented no facial palsy , no facial sensory change , and no facial pain .
the most common surgical approaches for vs is the translabyrinthine or suboccipital approaches1 ) . because of hearing loss and mass in temporal bone , we performed combined translabyrinthine with retrosigmoid suboccipital approach to detect the facial nerve earlier and remove the tumor in temporal bone . the facial nerve was severely compressed and irregularly distorted by tumor , bony edges , and crests along the invaded petrous apex .
temporal bone destruction beyond the cortex of iac in vs is very rare , which cause facial nerve compressed and distorted severely . during operation
, it is very difficult to dissect and preserve the facial nerve because of uneven erosion of temporal bone .

Answer:
vestibular schwannoma ( vs ) usually present the widening of internal auditory canal ( iac ) , and these bony changes are typically limited to iac , not extend to temporal bone .
 temporal bone invasion by vs is extremely rare .
 we report 51-year - old man who revealed temporal bone destruction beyond iac by unilateral vs. the bony destruction extended anteriorly to the carotid canal and inferiorly to the jugular foramen . on histopathologic examination ,
 the tumor showed typical benign schwannoma and did not show any unusual vascularity or malignant feature .
 facial nerve was severely compressed and distorted by tumor , which unevenly eroded temporal bone in surgical field .
 vestibular schwannoma with atypical invasion of temporal bone can be successfully treated with combined translabyrinthine and lateral suboccipiral approach without facial nerve dysfunction .
 early detection and careful dissection of facial nerve with intraoperative monitoring should be considered during operation due to severe adhesion and distortion of facial nerve by tumor and eroded temporal bone .


Question:
You will be presented with the full text of a biomedical research paper. Please write an abstract for this paper. Your response should include the abstract and no additional text.

Paper text:
lignocaine ( n - diethylaminoacetyl-2,6-xylidide ) was first synthesized by lofgen a swedish chemist in 1943 and was first introduced into clinical use in 1948 .
lignocaine hydrochloride ( c14h22n2o.hcl ) is most soluble in water and so this is most commonly used injectable solution as local anesthetic agents .
the efficacy profile of lidocaine as a local anesthetic is characterized by a rapid onset of action and intermediate duration of efficacy .
lidocaine or lignocaine along with adrenaline has the advantage of a rapid onset of action .
epinephrine ( adrenaline ) vasoconstricts arteries , reducing bleeding and also delays the resorption of lidocaine , almost doubling the duration of anesthesia . for surface anesthesia ,
several available formulations can be used , e.g. for endoscopies , before intubations , etc .
topical lidocaine has been shown in some patients to relieve the pain of postherpetic neuralgia ( a complication of shingles ) , though not enough study evidence exists to recommend it as a first - line treatment .
although not completely curing the disorder , it has been shown to reduce the effects by around two - thirds .
lidocaine is also the most important class-1b antiarrhythmic drug ; it is used intravenously for the treatment of ventricular arrhythmias ( for acute myocardial infarction , digoxin poisoning , cardioversion , or cardiac catheterization ) if amiodarone is not available or contraindicated .
lidocaine should be given for this indication after defibrillation , cardiopulmonary resuscitation , and vasopressors have been initiated .
a routine prophylactic administration is no longer recommended for acute cardiac infarction ; the overall benefit of this measure is not convincing .
the onset of action of lidocaine is about 4590 s and its duration is 1020 min .
it is about 95% metabolized ( dealkylated ) in the liver mainly by cyp3a4 to the pharmacologically active metabolites monoethylglycinexylidide ( megx ) and then subsequently to the inactive glycine xylidide .
megx has a longer half - life than lidocaine but also is a less potent sodium channel blocker .
the elimination half - life of lidocaine is biphasic and around 90120 min in most patients .
this may be prolonged in patients with hepatic impairment ( average 343 min ) or congestive heart failure ( average 136 min ) .
lidocaine is excreted in the urine ( 90% as metabolites and 10% as unchanged drug ) .
bupivacaine ( 1-butyl-2 , 6- pipecoloxylidide ) was first synthesized in 1957 by ekenstam , a scandinavian chemist , and was first introduced into clinical use in 1963 .
it is the longer side chain with four methylene groups on the piperidine ring that is responsible for the different properties of bupivacaine when compared to lignocaine .
it has a molecular weight of 288.4 and an empirical formula of c18h28n2o and exists as white crystalline base with a melting point of 251258c .
the base is not soluble in water , but the acid salt , bupivacaine hydrochloride ( c18h28n2o.hcl ) is slightly soluble .
the rate of systemic absorption of bupivacaine and other local anesthetics is dependent upon the dose and concentration of drug administered , the route of administration , the vascularity of the administration site , and the presence or absence of epinephrine in the preparation .
duration of action ( route and dose - dependent ) : 29 h. half - life in neonates is 8.1 h and in adults is 2.7 h. time to peak plasma concentration ( for peripheral , epidural or caudal block ) is 3045 min .
the rate of systemic absorption of local anesthetics is dependent upon the total dose and concentration of drug administered , the route of administration , the vascularity of the administration site , and the presence or absence of epinephrine in the anesthetic solution .
a dilute concentration of epinephrine ( 1:200,000 or 5 mcg / ml ) usually reduces the rate of absorption and peak plasma concentration of bupivacaine , permitting the use of moderately larger total doses and sometimes prolonging the duration of action .
the onset of action with bupivacaine is rapid and anesthesia is long lasting . the duration of anesthesia is significantly longer with bupivacaine than with any other commonly used local anesthetic .
it has also been noted that there is a period of analgesia that persists after the return of sensation , during which time the need for strong analgesics is reduced .
local anesthetics are bound to plasma proteins in varying degrees . in general , the lower the plasma concentration of drug the higher the percentage of drug bound to plasma proteins .
the rate and degree of diffusion are governed by ( 1 ) the degree of plasma protein binding , ( 2 ) the degree of ionization , and ( 3 ) the degree of lipid solubility
. fetal / maternal ratios of local anesthetics appear to be inversely related to the degree of plasma protein binding because only the free , unbound drug is available for placental transfer .
bupivacaine with a high protein binding capacity ( 95% ) has a low fetal / maternal ratio ( 0.20.4 ) . depending upon the route of administration ,
local anesthetics are distributed to some extent to all body tissues , with high concentrations found in highly perfused organs such as the liver , lungs , heart , and brain .
pharmacokinetic studies on the plasma profile of bupivacaine after direct intravenous injection suggest a three - compartment open model .
the second compartment represents the equilibration of the drug throughout the highly perfused organs such as the brain , myocardium , lungs , kidneys , and liver .
the third compartment represents an equilibration of the drug with poorly perfused tissues , such as muscle and fat .
the elimination of drug from tissue distribution depends largely upon the ability of binding sites in the circulation to carry it to the liver where it is metabolized .
after injection of bupivacaine hydrochloride for caudal , epidural , or peripheral nerve block in man , peak levels of bupivacaine in the blood are reached in 3045 min , followed by a decline to insignificant levels during the next 36 h. various pharmacokinetic parameters of the local anesthetics can be significantly altered by the presence of hepatic or renal disease , addition of epinephrine , factors affecting urinary ph , renal blood flow , the route of drug administration , and the age of the patient .
the half - life of bupivacaine in adults is 2.7 h and in neonates 8.1 h. in clinical studies , elderly patients reached the maximal spread of analgesia and maximal motor blockade more rapidly than younger patients .
amide - type local anesthetics such as bupivacaine are metabolized primarily in the liver via conjugation with glucuronic acid .
patients with hepatic disease , especially those with severe hepatic disease , may be more susceptible to the potential toxicities of the amide - type local anesthetics .
when administered in recommended doses and concentrations , bupivacaine hydrochloride does not ordinarily produce irritation or tissue damage and does not cause methemoglobinemia .
local anesthetics block the generation and the conduction of nerve impulses , presumably by increasing the threshold for electrical excitation in the nerve , by slowing the propagation of the nerve impulse , and by reducing the rate of rise of the action potential . in general ,
the progression of anesthesia is related to the diameter , myelination , and conduction velocity of affected nerve fibers .
clinically , the order of loss of nerve function is as follows : ( 1 ) pain , ( 2 ) temperature , ( 3 ) touch , ( 4 ) proprioception , and ( 5 ) skeletal muscle tone .
systemic absorption of local anesthetics produces effects on the cardiovascular and central nervous systems ( cns ) . at blood concentrations achieved with normal therapeutic doses , changes in cardiac conduction ,
however , toxic blood concentrations depress cardiac conduction and excitability , which may lead to atrioventricular block , ventricular arrhythmias , and cardiac arrest , sometimes resulting in fatalities . in addition
, myocardial contractility is depressed , and peripheral vasodilation occurs , leading to decreased cardiac output and arterial blood pressure .
recent clinical reports and animal research suggest that these cardiovascular changes are more likely to occur after unintended intravascular injection of bupivacaine
apparent central stimulation is manifested as restlessness , tremors and shivering progressing to convulsions , followed by depression and coma progressing ultimately to respiratory arrest . however , the local anesthetics have a primary depressant effect on the medulla and on higher centers
in most of the studies 0.5% bupivacaine with adrenaline 1:200,000 and 2% lignocaine with adrenaline 1:200,000 were used as local anesthetic agents for third molar surgery as they are roughly equipotent .
studies have proved that bupivacaine is superior to lidocaine plus diflunisal in controlling postoperative pain after lower third molar surgery .
surprisingly there have been records of patients with no or mild pain at 8 h after the bupivacaine procedure was almost twice as many as after the lidocaine procedure , but is explained by the reason for preference being shorter duration of the anesthetic agent . in an australian study , most of the patients preferred the long - acting anesthetic agent bupivacaine .
many authors suggest that it seems reasonable that the combination of a long - acting local anesthetic with a weak analgesic is more efficient in relieving pain than the combination of a rather short - acting local anesthetic and a weak analgesic .
bupivacaine is considered to have a therapeutic ratio of 2:0 while lignocaine in combination with adrenaline has a therapeutic ratio of 2:3 .
however , it has shown that the injection route alters the relative toxicity of local anesthetics . in a study by de jong and bonin in 1980 they found out that the bupivacaine has a greater therapeutic ratio than lignocaine when used for surgical removal of impacted third molars .
studies have proved that long - acting bupivacaine can be safely administered for surgical removal of lower third molar and it does have a long period of nalgesia postoperatively compared to lidocaine , but the cardio depressant property of bupivacaine should be kept in mind and should be administered judicially .
even studies have also shown that bupivacaine combined with methylprednisolone reduced the postoperative pain and swelling compared with the use of lidocaine and placebo , lidocaine and methylprednisolone , or bupivacaine and placebo .
right now clinical trials are going on bupivacaine versus lidocaine anesthesia under university of british columbia 2014 with the idea that the longer duration of anesthesia offered by bupivacaine when administered preoperatively in elective outpatient hand surgeries , will offer more effective postoperative pain control compared to using lidocaine only .
it has been found that both bupivacaine and lignocaine have their merits and demerits but beyond any doubt it has been proven by the clinical trials that bupivacaine provides better and prolonged analgesia and anesthesia post operatively during minor surgical procedures done at chair side along with surgical removal of impacted third molars .
hence , bupivacaine can be regularly used as the anaesthetic solution along with adrenaline 1:200,000 for surgical removal of impacted third molars provided care being taken regarding the dosage and the cardiodepressant property of bupivacaine . right now , further studies are going on .

Answer:
one of the most important goal in minor surgical procedures is to achieve proper and sufficient anesthesia and analgesia preoperatively , intraoperatively and in the immediate postoperative period .
 several local anesthetic agents have been cited in the literature and studied .
 bupivacaine is one of the most common long - acting anesthetic agents being used for surgical removal of impacted third molars .
 lignocaine is one of the commonest short - acting anesthetic agents being used for the same procedure . in this review article ,
 the analgesic and anesthetic abilities of the bupivacaine versus lignocaine have been reviewed while surgical removal of impacted third molars .


Question:
You will be shown the title of a chemistry paper, together with sections excerpted from the body of the paper. The paper sections may include HTML tags like `<p>` and `</p>`; please ignore these. Your task is to write an abstract for the paper. Your response should include the abstract and no other text.

Title:
Comparison of Mineral Element Content in a Functional Food Maca (Lepidium meyenii Walp.) from Asia and South America

Paper sections:
1. Introduction

Maca (Lepidium meyenii Walp.) is an endemic highland crop of the Central Andes which is grown from central Peru to Bolivia and northwestern Argentina [1, 2]. This plant has great potential as an adaptogen and appears to be promising as a nutraceutical in the prevention of several diseases [3]. Maca roots have been traditionally used to increase the rate of fertility in both humans and livestock [4]. Over the past 20 years, interest in maca has increased in many parts of the world [5]. Commercial maca products have gained popularity as dietary supplements for aphrodisiac purposes and for increasing fertility and stamina [6]. Recently, maca industry develops fast in Yunnan Province, China. In Yunnan, until the end of 2010, maca growing and promotion areas reached 175 hm2 and maca yield achieved 780 t, taking up to 90% and 93%, nationwide [7].

Mineral elements in food are very important because the quality of many functional foods and medicines depends on the content and type of minerals [8]. In the US, national surveys show that micronutrient inadequacies are widespread and mineral supplement helps fulfill micronutrient requirements in adults and children [9]. Up to now, only limited studies on selected elements in maca from different origins have been carried out [10, 11]. Moreover, in some studies, no reference material had been certified for elemental analysis, so there has been doubt about accuracy of the determination.

In the present study, inductively coupled plasma optical emission spectroscopy (ICP-OES) was used to determine the contents of eight elements (B, Co, Cr, Cu, Li, Na, Ni, and Zn) in maca samples, and a comparison was made between the samples from China and Peru.

2. Materials and Methods

All reagents used in the present work were of analytical reagent grade. Maca sample digests were prepared using HNO3 and H2O2 of ultrapure grade. Standard sample solutions of B, Co, Cr, Cu, Li, Na, Ni, and Zn obtained from Standard Material Center of China were used to make a mixed calibration curve in the range of 0–400 μg mL−1.

Ten maca samples were collected from four places of Yunnan, China, and four samples were from Peru. These samples were washed with deionized water thoroughly, dried to a constant weight, grounded into powder using an agate mortar, passed through a 60-mesh sieve, and stored in the plastic bags. 500 mg of each maca sample was weighed into an acid washed Teflon digestion tube. 8 mL of HNO3, 2 mL of H2O2, and 1 mL ultrapure water were added to the vessel.

Samples were digested in a microwave dissolver (Ethos One, Milestone, Italy) equipped with PTFE vessels. A three-step program was applied to the samples (Table 1). The extract was transferred into a cuvette and made up to 25 mL with ultrapure water.

Simultaneous multielement detection of B, Co, Cr, Cu, Li, Na, Ni, and Zn was performed with ICP-OES (ICPE-9000, Shimazu, Japan). The optimal instrumental conditions for ICP-OES are shown in Table 2. The emission wavelengths and detection limits for each element are shown in Table 3. Every sample was measured with three replicates. Blank experiments were prepared in the same way.

To ensure the precision of the experiment, the certified reference material GBW10028 (dried herb powder Astragalus membranaceus) was used for validation of the method (Table 4). The relative standard deviation (RSD) was found to be below 8%, and the recoveries range from 92% to 109%, which proved that this method was accurate and precise.

3. Results and Discussion

Contents of B, Co, Cr, Cu, Li, Na, Ni, and Zn in maca samples are shown in Table 5. All element contents were determined on a dry weight basis.

Boron is essential for humans. Boron affects fat and lipid metabolism, minerals and mineral metabolism, vitamin D, and bone development [12]. Our B values in maca from China and Peru were 8.1–21 mg kg−1 dw and 6.6–12 mg kg−1 dw, respectively, which were in agreement with that reported in the literature (8.8 mg kg−1 dw) [13].

Cobalt is an essential micronutrient in the form of vitamin B12, but cobalt is toxic in larger doses or long-term exposure at a low level. The adverse effects of cobalt relate to various organs and tissues and may include a possible carcinogenic potential [14]. Chromium can induce the oxidative stress and genotoxicity in chromium exposed population [15]. Lithium appears to play an especially important role during the early fetal development [16]. The contents (mg kg−1 dw) of Co, Cr, and Li were, respectively, <0.023, <1.1–3.5, and 0.020–0.17 for the samples from China and <0.023, <1.1–2.3, and 0.035–0.063 from Peru. Up to now, there was no other report on the contents of Co, Cr, and Li in maca.

Copper plays role as a cofactor for numerous enzymes in humans. Its compounds show vast array of biological actions, such as anti-inflammatory, antiproliferative, and biocidal activities [17]. Cu contents in the samples from China (2.5–31 mg kg−1 dw) were higher than the samples from Peru (<2.1 mg kg−1 dw). Published data available on Cu in maca from China were 4.0–32 mg kg−1 dw [10, 11, 13, 18], while those from Peru were 1.5–62 mg kg−1 dw [10, 11, 19–22].

Sodium is an essential element. In mammals, sodium concentrations in blood are held high by strict homeostatic mechanisms [23]. Na in the samples from China was in the range <30–2600 mg kg−1 dw which is similar to the value (67–2400 mg kg−1 dw) in maca from China in literatures [11, 13, 18]. However, our data on Na in the samples from Peru (<30–41 mg kg−1 dw) are lower than the values reported (110–190 mg kg−1 dw) in the literatures [11, 19–22].

Nickel compounds have showed an increased risk of lung and nasal cancer in humans [24]. The contents of Ni in the samples were 0.085–4.5 mg kg−1 dw from China and 0.68–1.7 mg kg−1 dw from Peru. However, higher value (11.3 mg kg−1 dw) was found in the literature on the maca from China [13].

Zinc is essential for a number of physiological functions and plays a significant role in many enzyme actions in the living systems [25]. Zn contents in samples were found to be 19–39 mg kg−1 dw from China and 27–39 mg kg−1 dw from Peru, which were in accordance with the values for maca in literature available (25–89 mg kg−1 dw from China and 16–58 mg kg−1 dw from Peru) [10, 11, 13, 18–22].

4. Conclusions

The contents of eight elements of maca collected from China and Peru were determined. Cu contents in all of the maca samples from China, as well as Na contents in two samples from China, were remarkably higher than those values in other samples.

Answer: Contents of eight mineral elements in maca (Lepidium meyenii Walp.) from China and Peru were determined by inductively coupled plasma optical emission spectroscopy. Cu contents in maca samples from China (2.5–31 mg kg−1 dry weight, dw) were higher than the samples from Peru (<2.1 mg kg−1 dw). Na in two samples from China was found to be significantly of high content (2400 and 2600 mg kg−1 dw). The contents (mg kg−1 dw) of B, Co, Cr, Li, Ni, and Zn were, respectively, 8.1–21, <0.023, <1.1~3.5, 0.020–0.17, 0.085–4.5, and 10–39 for the samples from China, while being 6.6–12, <0.023, <1.1~2.3, 0.035–0.063, 0.68–1.7, and 27–39 for the samples from Peru.


Question:
You will be presented with the full text of a biomedical research paper. Please write an abstract for this paper. Your response should include the abstract and no additional text.

Paper text:
over the last few years , the number of people infected with multidrug - resistant bacteria has been rising in a number of countries , among them germany . as a characteristic feature ,
this inhibits the efficacy of antibiotic treatment , with serious consequences for the health of infected individuals , the utilization of resources in the health care sector and the availability of resources in the economy in general . following an infection with multidrug - resistant bacteria ,
a number of effects may arise , each representing an economic cost : first , an individual will suffer from the risk of severe impairment of her health , including premature death .
next , appropriate treatment will take more time to produce the desired health outcome or will require more resources which must be diverted from other uses in the health care sector .
in addition , the economy will incur losses in productivity whenever infected individuals belong to the workforce .
finally , aggravating these already serious consequences , infected individuals may spread multidrug - resistant bacteria , thus inflicting harm on other individuals and increasing the economic cost to society .
a first objective of the present contribution is to work out in more detail the economic effects which an infection or a colonization with multidrug - resistant bacteria may bring about .
building on this account , it will be argued that the status quo is inefficient .
next , an approach will be outlined which can be used to determine whether strategies to prevent or to reduce infections with multidrug - resistant bacteria provide a net benefit to society . in order to be able to implement a strategy that has been shown to be advantageous in this sense ,
in essence , both objectives require a regional management of multidrug - resistant bacteria . by taking into account the entire health care sector within a region
in order to carry out the tasks outlined above , it is important to be clear about how to assess the costs and benefits associated with strategies to prevent or reduce infections with multiresistant bacteria .
furthermore , the range of effects which need to be taken into account will depend on the perspective of the analysis . more specifically , while the ( in-)efficiency of the status quo or of an intervention can only be evaluated from the perspective of society , the issue of ( in-)appropriate incentives must be judged from the individual perspective .   first , it is helpful to distinguish three types of costs .
direct costs represent the monetary value of resource utilization that takes place due to the production and consumption of health care .
apart from the cost of health care itself , this also includes any cost incurred by the patient in order to receive care , e.g. , travel costs .
the next type is given by indirect costs which cover the productivity losses , also valued in monetary units , due to illness or premature death .
finally , intangible costs capture the loss in utility or welfare for the patient which results from a reduction in her health status ( including death ) . in comparison with the other two types , intangible costs are more difficult to measure because it is necessary to develop a valuation from scratch .
intangible costs can be measured either in monetary units ( i.e. , as willingness - to - pay ) or in other units such as quality - adjusted life years ( qalys ) .
next , strategies which aim to prevent infections with multidrug - resistant bacteria or to control their impact will usually involve a reduction in the associated costs . relying on a similar categorization as on the cost side
, these benefits may turn up either as direct benefits , or as indirect benefits , or finally as intangible benefits . whereas direct benefits ( or indirect benefits ) accrue as reductions in direct costs ( or indirect costs ) , intangible benefits arise from improvements in health status or an extension of length of life . from the individual perspective of an economic unit such as , e.g. , a physician , an insurer , or a patient
, it makes sense to take into account only those costs and benefits which accrue to the unit itself .
thus , a decision is individually rational if it maximizes benefits over costs for the unit , thereby reaping the greatest net benefit .
in contrast , the societal perspective takes into account the costs and benefits of all decision units .
hence , a decision is rational from the perspective of society , or efficient for short , if it maximizes the net benefit that accrues to society . otherwise , it is inefficient and it is at least possible to make everyone better off by moving to an efficient decision .
it is important to realize that both the perspectives are necessary to explain why inefficient decisions involving a waste of resources are taken and how they can be improved upon . according to a basic assumption that will also be imposed below
, economic units always act individually rational , i.e. , they always maximize their own net benefit .
simple though it is , this assumption nevertheless can be quite helpful in explaining inefficient decisions .
more precisely , such decisions are likely to occur whenever the associated costs and benefits do not fall exclusively on the unit who takes them .
e.g. , patients infected with multidrug - resistant bacteria are likely to receive inefficient health care if the providers have no incentive to take into account all the costs and benefits associated with the treatment they provide . applying the societal perspective
provides the only way to identify those strategies to prevent or control infections that are efficient . in order to promote their implementation , it is essential to modify the incentives which the providers of health care face such that applying an efficient strategy is in their own interest , i.e. , becomes individually rational . as claimed above , this demonstrates that both the societal perspective and the individual perspectives of the relevant players must be taken into account in order to arrive at an efficient provision of health care to patients infected with multidrug - resistant bacteria .
individuals who are colonized with multidrug - resistant bacteria but not yet infected usually do not suffer from any immediate consequences . however , there are substantial risks involving serious medical problems and economic costs , both for the individuals themselves as well as for other individuals who get in touch with them .
first , there is the risk of developing an infection , imposing direct and intangible costs and possibly also indirect costs as outlined above .
the available studies , usually focusing on one type of bacteria and on a single disease , clearly demonstrate a considerable increase in length - of - stay and in direct hospital costs , , , , .
even though the reductions in health status due to an infection with multidrug - resistant bacteria are less well documented , the associated rise in mortality indicates that intangible costs may well turn out to be very high , , .
at the same time , individuals colonized with multidrug - resistant bacteria put other individuals at risk because their presence increases the probability that further colonization will take place .
an analysis of the costs of multidrug - resistant bacteria from the societal perspective must take these costs into account as well .
economically speaking , they represent external costs because the colonization of one individual may exert a negative impact upon the health status of other individuals , .
in fact , the external effects may reach very far , both with respect to the number of individual s concerned and over time .
consider the use of antibiotics : even with proper use in treatment , eventually resistant bacteria will turn up .
however , antibiotic misuse on the part of either health care providers or patients substantially accelerates the evolution of resistant strains .
thus , a flawed treatment of patients today imposes a cost on future patients . in economic terms ,
efficacy of antibiotic treatment can be viewed as a common social good which , due to wrong incentives for the major players in the health care sector , suffers from the tragedy of the commons . in order to move towards a solution of the current problems with multidrug - resistant bacteria , it is necessary to identify strategies for improving the provision of health care . in a further step
, care must be taken to make sure that promising strategies will also be implemented by the relevant actors . applying both steps in a systematic manner regularly and based on appropriate empirical data to check available strategies to prevent or control infections , one obtains a management of multidrug - resistant bacteria .
given the problems of the status quo as outlined above , a change from simply dealing with multidrug - resistant bacteria ad hoc to a proper management can be expected to result in a substantial reduction in the associated costs for society . as a first step ,
it is necessary to evaluate the costs and benefits to society arising from strategies to prevent infections with multidrug - resistant bacteria .
strategies that confer a net benefit to society improve on the current provision of health care and contribute to attaining the goal of an efficient management .
observe that the term strategy is defined in a narrow sense : in particular , it refers to a specific group of patients receiving care from a specific provider at a specific place .
thus , if the benefits of , e.g. , a screening strategy exceed the associated costs , it is nevertheless possible that a further modification , for example by focusing on a different group of patients , may provide an ever higher net benefit to society .
there are good reasons to tackle these tasks within a regional management of multidrug - resistant bacteria .
as its name suggests , a regional management has a clear focus on a specific region but implies no further restrictions otherwise .
thus , it represents a comprehensive approach which is not confined to a single branch of the health care system . furthermore , in view of the problems outlined above it seems necessary to adopt a regional management because such an approach offers a unique opportunity to apply the perspective of society to strategies to prevent or control infections with multidrug - resistant bacteria .
this includes the setup and the continuous maintenance of a database relating to multidrug - resistant bacteria in order to record the prevalences of colonized individuals or infected patients as well as other relevant data .
a major objective would be to take into account the entire process of the development and also the spread of colonization and infections with multidrug - resistant bacteria . in some contrast to the prevailing approach with its focus on hospitals and on the control of infections , a regional management offers the prospect to recognize both the contribution of other health care providers and of strategies to prevent infections in order to deal with infections with multidrug - resistant bacteria in a manner that provides a maximum net benefit to society .
at present , a major problem is that appropriate data on the costs of colonization with multidrug - resistant bacteria are not available . to be sure ,
nevertheless , there are still no or at least no systematic data on some of the cost effects explained above . referring to infections with multidrug - resistant bacteria ,
this is true for the direct costs outside of hospitals as well as for the intangible costs due to a reduction in length of life or a deterioration of health status .
in addition , there is no evidence on the probabilities with which colonized individuals develop an infection or cause other individuals either to be colonized or infected with multidrug - resistant bacteria .
these data would need to be collected with respect to the type of bacteria ( pathogen ) and possibly also with respect to individual attributes such as , e.g. , age or prevailing chronic diseases .
note that the lack of data on some important cost effects also has further implications . since the benefits of strategies to prevent or control infections arise primarily as reductions in the direct , indirect or intangible costs of multidrug - resistant bacteria , the full benefits to society are not known either . more specifically
, there is a lack of data on the impact of efforts to prevent infections , and also on the valuation of the corresponding health effects by the affected individuals . in order to explain the present scale of infections with multidrug - resistant bacteria in germany , it is necessary to investigate the incentives of health care providers to employ strategies to prevent or control infections .
consider a health care provider who applies a screening test to patients deemed at risk such that patients will be isolated until the result becomes available and will be sanified if the test yields a positive result . in many instances , for the provider
this means incurring a certain cost with the prospect of only a small expected benefit : first , the remuneration may be insufficient ( perhaps even nil ) and/or the strategy may not generate enough cost savings for the provider .
the latter reason applies , e.g. , to ambulatory physicians because the higher cost of treatment due to an infection will occur primarily in hospitals .
furthermore , the additional benefits due to averted infections of other individuals will have no direct effect on the provider under consideration either .
a similar statement applies to the careful and cautious therapy with antibiotics which may also contain the development and the spread of multidrug - resistant bacteria : again , the cost falls primarily upon the prescribing physician whereas the benefit turns up elsewhere in society . even though it is not possible currently to describe the economic impact of colonization with multidrug - resistant bacteria in full , the lack of sufficiently strong incentives for health care providers
can safely be taken to imply that too little effort is devoted to the prevention or the control of infections . as a gauge ,
consider the costs and benefits to society of strategies to prevent or control infections with multidrug - resistant bacteria : as argued above , health care providers implementing such strategies will usually reap only part of the associated benefit while bearing the full cost .
therefore , the incentives facing health care providers are too weak to induce them to provide efficient care with respect to the protection from infections with multidrug - resistant bacteria . in turn
, this implies that , from the perspective of society , there will be too many infections imposing an overall cost that is unnecessarily high .   if a strategy to prevent infections with multidrug - resistant bacteria conferring a net benefit to society has been identified , the next task is to make sure that it will be implemented .
in essence , this means setting appropriate incentives : the net benefit to society needs to be distributed among the relevant actors in a manner such that it is individually rational for everybody to apply the strategy .
more specifically , this implies an appropriate remuneration of health care providers , covering at least their own cost due to the effort to prevent or control infections with multidrug - resistant bacteria .
moreover , it may make sense to promote the cooperation among different sectors of the health care system , given that multidrug - resistant bacteria turn up not only in hospitals but also in ambulatory care or in nursing homes . applying
a regional management in a suitable manner also offers the prospect of overcoming the tragedy of the commons problem that besets the current approach to multidrug - resistant bacteria . by implementing the perspective of society in an entire region
, it will be possible to take into account most of the substantial cost effects that are now external . improving surveillance as well as providing reliable information on the appropriate use of antibiotics may help to improve the efficacy of treatment for future patients . in this sense
, a successful regional management may also contribute to save costs in the health care sector and to improve the health of patients in the future .
the current provision of health care to individuals who are colonized or infected with multidrug - resistant bacteria suffers from a number of shortcomings due to , e.g. , a lack of data and inappropriate incentives for providers .
implementing suitable strategies to control or prevent infections offers the prospect of reducing the costs associated with multidrug - resistant bacteria substantially , either by improving the health status of patients or by easing the financial burden on the health care system . for several reasons ,
a regional management represents an approach that can be trusted to improve on the status quo and to reap substantial net benefits for society by better dealing with the problems caused by multidrug - resistant bacteria .
it is true that building up and maintaining a regional management of multidrug - resistant bacteria will involve a considerable cost .
however , given the high costs of the status quo which will continue to rise unless precautionary measures are taken , this cost will be more than offset by the benefits that can be reaped by implementing efficient strategies to prevent or control infections .
a regional management of multidrug - resistant bacteria represents the first step towards a better integration of the health care system .
if this step is taken successfully , it may also act as a role model for dealing with other problems in the provision of health care . at any rate
, the potential benefits appear to be sufficiently high to more than justify embarking on this first step .

Answer:
the increasing number of people who are colonized or infected with multidrug - resistant bacteria imposes a high economic burden on society which includes the negative impact on health status as an intangible cost .
 an economic analysis leads to the conclusion that currently too little is done to prevent or control infections .
 the reasons include insufficient incentives for health care providers , a lack of reliable data on both the prevalence and the effects of infection , and a lack of coordination among the different branches of the health care sector .
 a regional management of multidrugresistant bacteria which does not focus on a single branch but rather on the health care sector overall can achieve a substantial reduction in the number of infected people and of the associated economic cost for society .


Question:
You will be presented with the full text of a biomedical research paper. Please write an abstract for this paper. Your response should include the abstract and no additional text.

Paper text:
a 69-year - old woman with a transitional cell carcinoma in the renal pelvis underwent a laparoscopic nephroureterectomy .
the patient had a history of hypertension , and had no history of allergy or previous exposure to indigo carmine .
anesthesia was induced with thiopental 400 mg and vecuronium 10 mg , and maintained with 2% sevoflurane after tracheal intubation .
a 20-gauge catheter was inserted into her left radial artery to monitor continuous arterial pressure . during the operation ,
the patient 's vital signs were stable , with blood pressure ( bp ) 100 - 130/70 - 95 mmhg , heart rate 62 - 88 bpm , and spo2 98 - 100% .
two hundred minutes after the induction of anesthesia , she received a bolus injection of 40 mg indigo carmine ( sodium indigotindisulfonate ; korea united pharm . ,
one minute later , her bp suddenly decreased from 120/85 mmhg to 50/30 mmhg , but heart rate remained at 80 - 90 bpm .
there was no evidence of massive blood loss or electrocardiographic changes prior to the hypotensive episode , and there were no skin rashes , angioedema , or any sign of bronchospasm during the hypotensive episode .
she was immediately administered ephedrine 20 mg and phenylephrine 0.3 mg , which restored her bp to 120/75 mmhg .
ten days later , a 73-year - old man who underwent a radical retropubic prostatectomy under general anesthesia experienced severe hypotension after administration of indigo carmine .
ninety minutes after anesthesia induction , he received an injection of 40 mg indigo carmine , and then bp decreased from 110/65 mmhg to 60/35 mmhg without any skin rash or urticaria .
he was administered ephedrine 40 mg and phenylephrine 0.3 mg , which restored his bp to the pre - indigo carmine level .
following these adverse events , we decided to dilute indigo carmine in normal saline and to administer it slowly over five minutes .
the patient had a drug allergy to saridon - a ( roche korea , seoul , korea ) , which contains acetaminophen , anhydrous caffeine , and isopropylantipyrine .
two hundred minutes after induction , she was administered 40 mg indigo carmine diluted in 20 ml of saline over five minutes . a sudden drop in bp
was observed ; from 125/72 mmhg to 45/30 mmhg , and injection of ephedrine 30 mg restored her bp within three minutes . following this third episode of hypotension , we suspected a manufacturing error and checked the lot number of indigo carmine .
we discarded all of the indigo carmine with lot number 001 , used in our third patient , and decided to use another lot number , 002 .
two weeks later , two additional patients experienced severe hypotension following administration of indigo carmine .
the first , a 68-year - old man , underwent transurethral bladder tumor resection under general anesthesia .
five minutes later , his bp dropped from 128/75 mmhg to 65/43 mmhg , and injection of phenylephrine 0.3 mg restored his bp .
another patient was a 77-year - old man who underwent transurethral resection of the bladder .
spinal anesthesia was induced with hyperbaric bupivacaine 11 mg at the l4 - 5 level .
after forty - five minutes , he received indigo carmine , and then his bp decreased from 110/65 mmhg to 60/35 mmhg .
after administration of 15 mg ephedrine , his vital signs remained stable throughout the rest of the procedure .
the company 's director informed us that lot numbers 001 and 002 were manufactured with the same raw materials , and the manufacturer provided us with indigo carmine of lot number 906 , which to date has not been associated with any such adverse events .
the raw materials used to manufacture indigo carmines were analyzed , including their crystalline status , thermo - physiochemical properties and particle size distribution , all of which are required analytical criteria for drug manufacture , as determined by the korea food and drug administration .
a comparison of the raw materials used to manufacture lot numbers 002 and 906 showed no differences .
further analysis of the raw materials by liquid chromatography ( lc ) and mass spectrometry ( ms ) showed that , when compared with lot number 906 , lot number 002 had a separated peak on lc and an abnormal fragment peak on ms , suggesting the presence of impurities ( fig .
all indigo carmine numbered as 001 and 002 were recalled and its usage was restricted .
the term anaphylaxis has been used for all types of acute life - threatening , generalized hypersensitivity reactions due to abnormal sensitivity to a triggering agent .
although indigo carmine has no known activity in the human body , it has been found to directly activate alpha - adrenergic receptors , which may lead to reflex bradycardia and/or hypertension .
hypotension after indigo carmine administration is an unexpected adverse effect and has been reported in fewer than 10 patients over 80 years of use .
sudden , severe hypotension after indigo carmine administration was first reported in a series of four patients with no known drug allergies who underwent radical prostatectomy under epidural anesthesia . due to the temporal association between indigo carmine injection and the hemodynamic change ,
usually , indigo carmine might cause both stimulant and depressant effects simultaneously , however , those cases could be induced by unopposed depressant component under sympathetic blockade .
severe hypotension , bronchospasm and urticaria after administration of indigo carmine were observed in a patient under general anesthesia .
that patient , who was undergoing transurethral resection of a bladder tumor , manifested the complete spectrum of anaphylaxis five minutes after injection of indigo carmine , including hemodynamic collapse , cutaneous changes , laryngeal edema and bronchospasm . due to a lack of prior allergies or prior exposure to indigo carmine
, their case was thought to be caused by histamine release such as an anaphylactoid reaction .
life - threatening adverse reaction associated with indigo carmine presenting as even asystole was reported .
severe hypotension , bradycardia , hypoxia were observed after indigo carmine injection , and then the patient became asystolic .
following chest compression and intraaortic balloon pump support , the patient 's blood pressure was stabilized .
the patient 's cardiac arrest event was believed to be due to an anaphylactoid reaction in response to indigo carmine .
a diagnosis of anaphylaxis requires evidence of involvement of three or more organs and the detection of specific ige or a positive skin test to the alleged agent .
however , since these reports did not verify these findings , it would be reasonable to diagnose these events as idiosyncratic reactions , including anaphylaxis . compared with the previous reports ,
our patients had somewhat bizarre clinical manifestations . during the period described for all five patients , we used lot numbers 001 and 002 of indigo carmine on 15 patients .
hypotensive episodes in some of the other patients may have been overlooked because their symptoms were mild and they recovered immediately . in all five patients described above , saline replacement was adequate and there was no massive blood loss .
there were no symptoms or signs suggesting anaphylactoid reactions , such as cutaneous changes or wheezing .
two patients who experienced hypotensive episodes were tested for hypersensitivity to indigo carmine of lot number 002 by skin prick tests and intradermal skin tests , and both were negative . although a negative response to a suspected agent is unreliable in ruling out drug hypersensitivity , the clinical manifestations of these patients indicate that drug hypersensitivity or anaphylaxis was unlikely causes .
we focused on the five patients who experienced hypotension serially within a one month period , and suspected that flaws in production were responsible for these adverse reactions . during the manufacturing process
, indigo carmine molecules may be fragmented by accelerated electron beams , and these fragmented ions can be classified by their mass to charge ( m / z ) ratios on ms ( fig .
analysis of the raw materials by lc / ms showed an abnormal peak in lot number 002 that was not present in lot number 906 .
this additional peak contained abnormal amounts of fragmented ions , with m / z = 340 , which had not been filtered out during the manufacturing process .
raw materials , reagents , and degradation during manufacture and storage are all possible sources of impurities in drugs
. these impure drugs may cause various adverse reactions , regardless of their pharmacological effects . determining the lot number of the drug may be helpful in investigating the causes of adverse reactions .
usually , physicians tend to ignore or miss symptoms and signs if they are not critical .
however , practitioners should always pay attention when using drugs and prevent further adverse drug reactions by meticulous monitoring and prompt reporting .

Answer:
indigo carmine has been used for eight decades with few adverse effects .
 several of our patients , however , experienced severe hypotensive episodes after indigo carmine administration within a period of one month .
 analysis of the raw materials used to formulate the preparation of indigo carmine we used showed that they contained impurities .
 following recall of these impure materials , none of our patients experienced further hypotensive episodes .


Question:
You will be presented with the full text of a biomedical research paper. Please write an abstract for this paper. Your response should include the abstract and no additional text.

Paper text:
kienbck s disease , defined as avascular necrosis of the lunate , is a relatively rare condition with a poorly understood etiology .
the disease is a relatively rare condition in which the etiology and natural history of this condition are poorly understood and even more poorly documented in the literature .
a wide array of treatment recommendations is available , and reported results vary , which may hinder consistent treatment recommendations .
conservative and invasive treatments for kienbck s disease include the following : wrist immobilization with a splint during early disease stages , surgical joint - leveling procedures to attempt to reduce loading on the joint and to promote revascularization and increased blood flow , vascularized bone grafting , proximal row carpectomy , and total wrist arthrodesis .
proximal row carpectomy and total wrist arthrodesis attempt to alleviate arthritis pain due to carpal collapse ; however , these 2 interventions greatly limit range of motion .
none of these treatments effectively address the chronic pain , loss of range of motion , and decreased grip strength associated with kienbck s disease .
the staging distinguishes only the  state of collapse  in an ordinal classification scheme and does not allow localization or indicate partial involvement of the lunate , which the image contrast from mri may provide .
osteochondral transfer for treatment of kienbck s disease may be possible for individuals with segmental or incomplete avascular necrosis of the lunate .
previous investigators have acknowledged the importance of donor site cartilage thickness to aid in minimizing postoperative abnormal stresses and resulting poor function .
selection of donor cartilage with an appropriate thickness may be difficult due to source availability and donor site location relative to the load - bearing region of a joint , which may lead to donor site morbidity .
an alternative to matching cartilage thickness is the matching of curvature of the articular surface . matching the curvature of donor and recipient sites
has been shown to maintain preoperative contact stresses for a given loading regimen . in this short communication , we report the treatment of a patient s kienbck s disease by combining mri with mathematical modeling to optimize the congruency between the curvature of donor and recipient sites of an autologous osteoarticular plug transfer .
the purpose of this study was to describe the feasibility of a novel surgical technique .
the results indicate that donor site selection for autologous osteoarticular transfer using a quantitative evaluation of articular surface curvature may be beneficial for optimizing the likelihood for restoring the radius of curvature and thus joint articulation following cartilage repair .
a 48-year - old , right hand  dominant woman had left wrist pain associated with decreased wrist flexion since september 2009 . in november 2009 , the patient experienced severe wrist pain after falling on her hand . after acquiring standard radiographs ,
the woman was diagnosed with kienbck s disease , and a proximal row carpectomy was recommended by an initial orthopedic surgeon .
the patient sought a second opinion after understanding that the carpectomy would not only reduce pain during activities of daily living but would also limit range of motion . upon presentation to our institution ,
physical examination of the left wrist revealed no focal swelling , dystrophic changes , or evidence of muscular atrophy .
the left wrist range of motion was 20 of flexion and 30 of extension ; the patient had full , painless range of motion in her right wrist .
grip strength in her left hand was 111.2 n as compared with 333.6 n in her right hand .
mri of the wrist was performed utilizing a clinical superconducting 3-t imaging unit ( sigma hdx , general electric healthcare , waukesha , wi ) and a dedicated wrist coil .
pulse sequencing included a 3-dimensional gradient - recalled sequence with tr / te of 39/14 milliseconds , field of view of 9 cm , slice thickness of 1 mm with no gap , flip angle of 10 , and band width of 41.7 khz .
coronal , sagittal , and axial cartilage  sensitive fast spin echo sequences were obtained with a long tr ( 4,000 - 5,500 milliseconds ) , moderate te ( 31 - 38 milliseconds ) , field of view of 8 to 9 cm , slice thickness of 2 to 3 mm , acquisition matrix of 512  512 ( 320 - 352 ) , 2 excitations , a band width of 14.7 to 41.7 khz , and echo train length of 10 to 12 .
coronal fast inversion recovery sequence was performed with a tr of 5200 milliseconds , te of 23 milliseconds , inversion time of 170 milliseconds , field of view of 9 cm , slice thickness of 2.5 mm , matrix of 288  192 , 2 excitations , band width of 31.2 khz , and echo train length of 12 .
the examination demonstrated collapse of the proximal pole of the lunate , with subchondral sclerosis and a mild bone marrow edema pattern ( fig .
1 ) . foci of completely devitalized marrow were noted in the proximal pole with an associated intense synovitis .
moderate partial wear of cartilage was seen over the collapsed lunate with preservation over the radial side of the lunate fossa .
a best - fit circle was manually placed on the lunate with its articulation with the distal radius to determine the joint curvature .
preoperative coronal ( a ) and fat - suppressed ( b ) fast spin echo images of the wrist demonstrate sclerosis and partial collapse of the lunate ( arrows ) with hyperintensity in the adjacent cartilage .
one - year postoperative sagittal ( d ) and fat - suppressed ( e ) fast spin echo images and computerized tomographic evaluation ( f ) demonstrate partial bony incorporation expected for the time interval .
computerized tomographic examination was performed utilizing axial acquisition with a slice resolution of 0.8 mm , subsequently reformatted into sagittal and coronal reformations .
concurrent computerized tomography was performed to quantify the extent of subchondral collapse , which measured up to 1 mm .
mri of the left knee was subsequently acquired to provide a digital template for the planned osteochondral transplantation .
a 3-dimensional t1-weighted spoiled gradient echo data set with frequency - selective fat suppression was acquired for cartilage segmentation : tr of 13.6 milliseconds , te of 3.0 milliseconds , field of view of 16 cm , flip angle of 20 , slice thickness of 1.5 mm , matrix of 512  512 , receiver bandwidth of 41.7 khz , and 1.2 excitations .
the cartilage was segmented from the image data using custom - written semiautomated software ( general eelectric healthcare ) , with minimal manual editing performed as needed .
a 3-dimensional mesh representation of the segmented lateral condyle image data was constructed and smoothed for curvature calculations .
curvature calculations were performed on the 3-dimensional surface mesh using a custom - written matlab ( mathworks , natick , ma ) program .
2a ) , and a best - fit paraboloid ( z(x , y ) = ax + bxy + cy ) was fit to each vertex and the surrounding 2-ring neighborhood ( fig .
2b ) . the analytical solutions for maximum curvature ( max ) and minimum curvature ( min ) were determined from the best - fit paraboloid ( fig .
this process is repeated for each vertex on the 3-dimensional surface mesh to generate max and min maps .
regions with large values of min display concavities on a surface , and regions with large values of max display convexities of a surface .
the model of a paraboloid was chosen due to the computational efficiency as well as the verified accuracy of the curvature calculation as compared to other numerical methods .
( a ) each mesh vertex ( red point ) had a 2-ring neighborhood ( blue points ) calculated .
( b ) the coefficients of a best - fit paraboloid through the vertex and 2-ring neighborhood were used to calculate the maximum and minimum curvatures .
the directions of maximum curvature ( red arrow ) and minimum curvature ( green arrow ) were also calculated .
( c ) the direction and magnitude of the maximum curvature and minimum curvature at the vertex are shown by the orientation and radius of the pink and green circles , respectively .
the radius of each circle (  ) is the inverse of the calculated curvature (  = 1/ ) . at the indicated vertex ( red dot ) ,
the anterolateral femoral condyle had the maximum curvature in the mediolateral direction and minimum curvature in the anteroposterior direction .
the patient underwent osteochondral transfer from her left femoral condyle to her left lunate . using a tourniquet to control bleeding , the necrotic area of the lunate , which measured approximately 8 mm  1 mm  1 cm , was debrided .
after thorough debridement of the avascular necrotic bone , intraoperative fluoroscopy was used to identify and better assess the defect in the lunate . the donor site was filled with a synthetic acellular biphasic copolymer plug ( trufit , smith and nephew , andover , ma ) . using the digital template acquired from the algorithm ,
an osteochondral plug from the left femoral condyle that closely matched the radius of curvature of the lunate , 12.7 mm from the manual measurement , was harvested during knee arthroscopy ( fig .
the location of the plug was assessed from the previously constructed 3-dimensional model of the articular surface , relative to the local anatomy . a 10 blade and rongeur
were used to fit the dimensions of the osteochondral plug with those of the lunate .
2 fiberwire ( arthrex , naples , fl ) stitch was placed through the scapholunate ligament .
internal fixation was performed with a 1-mm drill , drilled antegrade through the lunate to fix the osteochondral plug ; a 1.5-mm  8-mm screw was then compressed across the plug with fixation in the distal aspect of the lunate .
there was no block in flexion , extension , or radial deviation , and the osteochondral fragment remained stable through a full range of motion .
the patient was placed in a sling and discharged with instructions to return for regular follow - up visits .
maximum ( left ) and minimum ( right ) principal curvature maps of the anterior portion of the lateral femoral condyle .
postoperative radiographs demonstrated that the wrist was in good alignment and the hardware was intact .
follow - up radiographs and physical examinations were performed at 2 , 4 , 6 , 8 , and 10 weeks ; 3 , 4 , 7 , and 10 months ; and 1 year postoperatively .
we treated kienbck s disease with the aid of a 3-dimensional model of femoral articular surface and calculated the principal curvatures of the surface .
this was performed to determine the optimal congruency of the chondral donor and recipient sites .
surgical challenges for autologous osteoarticular transfer include restoration of the articular surface as well as the tidemark .
koh et al . have shown in a preclinical model that plugs left elevated or angled result in increased contact pressures , thus presenting potential risk to the cartilage on the opposite side of the joint . the mesh model created in the knee allowed for objective assessment of the articular surface to ensure more optimal plug alignment ,
provide reconstitution of joint architecture , and promote biologic incorporation of the plug into the recipient site .
we did not seek to evaluate the functional outcome of the surgical procedure in this short report but to introduce the feasibility of this novel surgical planning technique for osteochondral repair .
this case report presents the unique application of mri and mathematical modeling when applying the autologous osteoarticular transfer procedure to the lunate .
these techniques require only an additional limited 3-dimensional model of the knee with minimal scan time beyond the routine examination of the wrist for assessment of marrow viability and the integrity of the articular surfaces .
additional applications would include osteochondral lesions of the distal femur , capitellum , talus , and potentially , over the femoral head .
future studies will refine the image acquisition and 3-dimensional modeling processes for optimal selection of the osteochondral plug based on local tissue morphology .

Answer:
kienbck s disease , defined as avascular necrosis of the lunate , is a relatively rare condition with a poorly understood etiology .
 conservative and invasive treatments for kienbck s disease exist , including wrist immobilization , surgical joint - leveling procedures , vascularized bone grafting , proximal row carpectomy , and total wrist arthrodesis .
 staging kienbck s disease using radiography assumes near complete avascularity of the lunate .
 the staging distinguishes only the  state of collapse  in an ordinal classification scheme and does not allow localization or indicate partial involvement of the lunate , which the image contrast from mri may provide . in this short communication , we report the treatment of a patient s kienbck s disease by combining mri with mathematical modeling to optimize the congruency between the curvature of donor and recipient sites of an autologous osteoarticular plug transfer . follow - up mri and radiographs at 1 year postoperatively demonstrated gradual graft incorporation and bone healing .
 the purpose of this study was to describe the feasibility of a novel surgical technique .
 the results indicate that donor site selection for autologous osteoarticular transfer using a quantitative evaluation of articular surface curvature may be beneficial for optimizing the likelihood for restoring the radius of curvature and thus joint articulation following cartilage repair .


Question:
You will be shown the title of a chemistry paper, together with sections excerpted from the body of the paper. The paper sections may include HTML tags like `<p>` and `</p>`; please ignore these. Your task is to write an abstract for the paper. Your response should include the abstract and no other text.

Title:
The AT-Hook motif as a versatile minor groove anchor for promoting DNA binding of transcription factor fragments

Paper sections:

Transcription Factors (TFs) are specialized proteins that, upon recognizing specic DNA sequences, play a key role in the regulation of gene expression. 1 Therefore, alterations in their activity are at the origin of many diseases, including cancer. 2 Owing to this relevance, there is a great interest on the development of non-natural DNA binding peptides that can somewhat mimic the DNA binding properties of naturally occurring TFs. 3 It is well known that although the DNA-binding of TFs is mediated by relatively small peptide motifs, high-affinity DNA recognition requires the full protein domain and in many cases, the concerted action of multiple DNA-binding components. 4 Therefore, isolated monomeric DNA binding fragments of bZIP or zinc nger TF families do not interact with their target sites with signicant affinity. We have previously shown that the DNA binding of these monomeric regions can be restored by conjugation to small molecule minor groove binders, such as distamycin or pentamidine derivatives. 5 Although these hybrids exhibit interesting recognition properties, they suffer from relatively poor sequence selectivity as a consequence of the intrinsic high affinity of the minor groove binders for A/T-rich DNA sites. 6 Furthermore, the synthesis of these conjugates requires elaborate multistep procedures, combining solution and solid-phase methods.

In the search for more efficient and selective bivalent DNA binders, we were intrigued by the AT-Hook motif, a naturally occurring cationic short peptide present in HMG-I(Y) eukaryotic nuclear proteins. 7 It is known that monomeric AT-Hooks bind their target sites with weak affinity (in the millimolar range). 7,8 However, these proteins attain high DNA-binding affinity thanks to the cooperative action of three appropriately spaced AT-Hook repeats. 9 NMR and crystallography studies have provided a detailed structural picture of the interaction of the AT-Hook (RKPRGRPKK) with DNA, and have shown that the central Arg-Gly-Arg core of this oligocationic peptide is deeply inserted into the minor groove (Fig. 1, le), while the various lysines in the sequence (RKPRGRPKK) introduce additional electrostatic contacts with the phosphates of the DNA backbone. 10,11 The low intrinsic DNA affinity of monomeric AT-Hooks might discourage their application as minor groove anchors to promote the DNA binding of tethered TF fragments. However we envisioned that such low affinity might actually offer an opportunity for achieving greater sequence selectivity, because a high affinity, cooperative interaction should only occur in DNA sequences containing both targeting sites at adjacent positions (Fig. 1, right). Moreover, and importantly, the peptidic nature of the AT-Hook motif should considerably simplify the synthetic access to the conjugates.

Herein we report the design and synthesis of two peptide chimeras consisting of an AT-Hook motif linked to the DNA binding domains of GCN4 and GAGA, selected as representative members of the basic leucine zipper (bZIP) and Cys 2 His 2 zinc-nger (ZF) families of transcription factors, 1,12 and demonstrate that these conjugates exhibit excellent DNA binding affinity and selectivity for target specic sites of 8-9 base pairs. The GCN4/AT-Hook conjugate (brH) was based on a GCN4 peptide fragment comprising of residues Asp 226 to Gln 248 , which has been identied as the shortest peptide that, as a disulde dimer, retains the specic DNA binding properties of the full GCN4 DNA binding domain. 13 The design was based on the structures of the GCN4 dimer bound to the AP1 (5 0 -ATGA(c) TCAT-3 0 ) site, 14 and the third AT-Hook DNA binding domain of HMG-I(Y), RKPRGRPKK, bound to the PRDII sequence of the IFN-b promoter. 10 We built a qualitative computer model for the simultaneous interaction of both peptides bound to contiguous sequences of the DNA (see the ESI †). Inspection of this model suggested that the Arg 245 residue in GCN4, which is oriented towards the adjacent minor groove, was a good candidate for introducing the tether between the two peptides (ESI †). Thus, we synthesized the selected GCN4 basic region fragment containing the mutation Arg 245 / Lys following standard Fmoc/ tBu solid-phase peptide synthesis protocols (br, Scheme 1). 15 The Lys 245 residue was introduced with its side chain protected by an orthogonal alloc group, which could be selectively removed in the solid phase under Pd catalysis. Subsequent assembly of an O1Pen linker and the AT-Hook sequence, followed by standard deprotection/cleavage steps and reversephase HPLC purication, gave the expected peptide brH in a good overall yield (approx. 20%, Scheme 1). It should be remarked that the whole synthesis can be completed in one day.

Having at hand the desired peptidic chimera brH, we rst studied its DNA binding properties using standard non-denaturing EMSA experiments in polyacrylamide gels. 16 Thus, the incubation of the ds-oligonucleotide AP1 hs $AT, which contains a composite binding sequence including a AP1 half site (AP1 hs , TCAT) and an A/T-rich tract (AT, AATT), with increasing concentrations of brH led to a new, slow-migrating band, consistent with the formation of the desired brH/ AP1 hs $AT complex (Fig. 2a). Importantly, the conjugate brH does not elicit retarded bands when incubated with non-target sequences lacking the A/T-rich or the AP1 half site (Fig. 2b and  c). This contrasts with previous conjugates with small molecule minor groove binders that also displayed considerable interactions with DNAs containing just A/T-rich sites, 5 and supports our premise that using low-affinity DNA minor groove binders leads to better selectivities.

In agreement with the results obtained by the EMSA, circular dichroism (CD) experiments revealed that the addition of 1 equiv. of the target oligonucleotide AP1 hs $AT to a 5 mM solution of brH promotes a signicant increase in the intensity of the negative bands at 208 and 222 nm, which is consistent with the expected folding of the GCN4 basic region into an a-helix upon insertion in the major groove of the AP1 hs site (Fig. 2, bottom le). 17 Fluorescence anisotropy titrations using the TMR-labeled dsDNA AP1 hs $AT conrmed that brH recognizes the DNA with a high affinity, with an apparent K D of z28 nM at 20 C (Fig. 2, bottom right), which is considerably better than the binding constants corresponding to each of the isolated components for  5 0 -CGCGTCATAATTGAGAGCGC-3 0 ; AP1 hs $GC: 5 0 -CGCGTCATCAGCGAGAGCGC-3 0 ; and GC$AT: 5 0 -GACGGAATTTGA-GAGCGTCG-3 0 . Bottom left: circular dichroism of a 5 mM solution (phosphate buffer pH 7.5) of brH (dashed line) and the same solution after the addition of 1 equiv. of the target AP1 hs $AT dsDNA (solid lane). The contribution of the DNA to the CD spectrum has been subtracted for clarity. Bottom right: fluorescence anisotropy titration of a 25 nM solution of TMR-AP1 hs $AT in the presence of increasing concentrations of brH. The best fit to a 1 : 1 binding model is also shown.

their respective targets (in the high micromolar-low millimolar range). Taken together, these results support the formation of a cooperative, bivalent DNA complex at specic composite DNA sites of eight base pairs.

In order to conrm the general applicability of the AT-Hook motif as an effective minor groove DNA anchor for highly efficient and selective interactions, we designed a second chimera based on the DNA-binding domain of the GAGA protein: a monomeric Cys 2 His 2 zinc nger transcription factor. 18 As with GCN4, the design of the GAGA hybrid with the AT-Hook motif was based on the structure of the GAGA binding domain complexed with its target h3/h4 (GAGAG) DNA site. 18 Based on earlier reports that identied the minimal domain required for specic binding, 19 as well as on our own previous work with GAGA-distamycin hybrids, 20 we selected a fragment of the DNA binding domain from residues Ser 28 to Phe 58 that by itself is incapable of interacting with its target DNA site. The selected peptide sequence was modied to include an appropriate mutation (Arg 44 / Lys) for tethering the AT-Hook to the abb core of the transcription factor (see the ESI †). An inspection of the superimposed structures of the AT-Hook and the GAGA DNA binding domain bound to adjacent sites suggested that in this case the conjugate might be better assembled by a chemoselective ligation of fully deprotected peptides. In particular, we designed a synthetic scheme involving the coupling of a reactive bromoacetyl derivative of the AT-Hook with a nucleophilic cysteine attached to the side chain of the Lys 44 of the zinc nger moiety (Scheme 2). The chemoselective coupling to this thiol could be attained by blocking the other two natural cysteine residues (Cys 36 and Cys 39 ) by coordination to Zn(II) in the form of the folded zinc nger domain. 21 The core GAGA peptide containing a Lys 44 orthogonally protected with an alloc group in its side chain was synthesized following standard Fmoc SPPS procedures. The alloc group was removed under Pd catalysis, and a Cys residue was coupled to the side chain. Fmoc deprotection and acetylation, followed by standard deprotection and cleavage steps led to the expected peptide (SH)GAGA (Scheme 2). The AT-Hook sequence was assembled following standard methodology, and capped on its N-terminus with an electrophilic bromoacetyl handle (see the ESI †). Finally, the nucleophilic peptide (SH)GAGA and the AT-Hook bromide derivative were coupled in solution in the presence of Zn(II). The reaction took place at rt in a deoxygenated phosphate buffer (pH 7.5) in the presence of 1.5 equiv. of ZnSO 4 . Aer 1 h, HPLC-MS analysis showed the formation of the desired product, which was puried by HPLC and identied as the desired conjugate GAGAH (see Scheme 2 and the ESI †).

As in the previous case, we assessed the DNA binding of the conjugate using EMSA under non-denaturing conditions. The incubation of a double stranded oligonucleotide containing the target composite binding site for the AT-Hook motif and the GAGA domain (AT$GAGA), at rt, with increasing concentrations of the conjugate GAGAH, led to the appearance of a new retarded band consistent with the formation of the expected peptide-DNA complex (Fig. 3a). Other oligonucleotides mutated in the GAGA or in the AT-Hook binding site failed to give rise to stable DNA peptide complexes, and indeed barely show the formation of any nonspecic complexes (Fig. 3b and c, respectively). Moreover, uorescence anisotropy titrations using TMRlabeled dsDNA conrmed the high affinity interaction of the peptide chimera, with an apparent K D of 26 nM at 20 C (Fig. 3, Scheme 2 Strategy used for the synthesis of the AT-Hook/GAGA chimera GAGAH by the chemoselective modification of the GAGA Cys 2 His 2 peptide in solution. A limitation for the potential biological application of this type of DNA-binding peptide construct might derive from its presumable poor cell internalization. However, it is known that oligocationic peptides have a benecial effect in cellular transport, 22 and therefore we decided to check whether the presence of the AT-Hook could enhance the cellular entrance of these conjugates. Thus, as a preliminary test, we incubated mammalian Vero cells with a tetramethylrhodamine (TMR) derivative of brH, as well as with a control peptide that lacks the AT-Hook (TMR-br, see the ESI †). Remarkably, while TMR-br was essentially not internalized, 23 the incubation of the cells with TMR-brH led to a bright emission inside the cells, which was mainly localized in endosomes (Fig. 4). 24 Therefore it is clear that the presence of the AT-Hook enhances the transport across the cell membrane, which opens the door to the cellular applications of these peptide chimeras.

Conclusions

Taken together, these results demonstrate that the AT-Hook motif is a synthetically accessible minor groove binder that can be effectively exploited for the straightforward construction of functional DNA binding conjugates containing TF peptide fragments. The resulting bivalent chimeras display excellent DNA recognition properties in terms of affinity and selectivity, properties that rely on the cooperative action of two weakly DNA binding components. This work highlights the role of the hitherto overlooked AT-Hook motif as a DNA binding handle. The synthetic accessibility and nanomolar affinity and selectivity exhibited by these conjugates, together with their good cell transport properties, allows anticipation of potential applications to modulate gene transcription processes.

Answer: We report the development of chimeric DNA binding peptides comprising a DNA binding fragment of natural transcription factors (the basic region of a bZIP protein or a monomeric zinc finger module) and an AT-Hook peptide motif. The resulting peptide conjugates display high DNA affinity and excellent sequence selectivity. Furthermore, the AT-Hook motif also favors the cell internalization of the conjugates.