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:
in canada , fish contribute to over 50% of the dietary intake of mercury ( hg ) by adults ( dabeka et al .
2003 ) , and fish containing the highest concentrations of hg are predatory fish , such as shark , swordfish and tuna ( health canada 2007 ) .
hg , present in fish as both methyl - hg and inorganic hg , is a potent neurotoxicant . on the other hand , fish are one of the best food sources of vitamin d and omega-3 fatty acids , dha and epa , and it is important to weigh the benefits of eating fish against the potential toxicity of hg present in the fish ( health canada 2007 ) . to promote consumption of fish while at the same time providing advice to vulnerable population groups about limiting consumption of predatory fish , and to evaluate the risk - to - benefit ratio of eating fish , it is important to know the levels of hg in predatory fish sold at the retail level in canada . in 2002 ,
2004 ) which found that predatory fish , such as shark , swordfish and tuna , contained high concentrations of hg , averaging 1820 ng g for shark , 1430 ng g for marlin and 930 ng g for fresh or frozen tuna .
this survey is a supplement to the previous work and provides levels of hg found in retail fish purchased in 2005 .
marlin , sea bass , red snapper , orange roughy , fresh water trout , grouper , black cod ( sablefish ) , arctic char , king fish ( king mackerel ) and tilefish are included in this survey .
, samples were purchased as cleaned fillets or whole fish at the retail level in toronto , vancouver and montreal in 2005 .
the retail stores included four supermarket chains and four specialty fish seafood outlets in each city .
each sample was shipped frozen to maxxam analytics inc . for cleaning ( if necessary ) , homogenization and bottling in pre - cleaned polyethylene bottles supplied for total hg and glass jars with teflon cap liners for methyl hg ( i - chem brand , thermo fisher scientific ) .
the samples were then frozen and shipped to health canada 's food research division in ottawa for analysis .
information provided with each sample included , where available , the species name as provided by the retail outlet , the state of the sample when purchased ( frozen , fresh or previously frozen ) , the city of sample pickup , name of store where purchased , country of origin and date of collection .
the countries of origin for the samples were usa ( 7 ) , argentina ( 1 ) , australia ( 5 ) , canada ( 26 ) , chile ( 13 ) , hungary ( 2 ) , mexico ( 1 ) , new zealand ( 3 ) and taiwan ( 1 ) , with the remaining samples being of unknown origin .
analyses were performed using the reagents , instrumentation and methodology described in dabeka et al .
briefly , after a low - temperature nitric / hydrochloric acid and hydrogen peroxide digestion of roughly 1 g fish tissue and dilution to 50 ml with water , measurements were made using a cetac-6000a dedicated hg analyser equipped with an asx-500 autosampler and adx autodilutor .
quality control measures for each analytical batch included three reagent blanks , two reagent blank spikes ( 200 ng hg ) , one sample spike of 400 ng hg ( in duplicate for both the unspiked and spiked 400 ng sample ) and duplicates of three different standard reference materials ( srms ) with certified hg concentrations .
two cross - check standards ( from a different manufacturer ) were included in each batch for standard verification during the run .
the solution limit of detection ( lod ) was estimated for each analytical batch by multiplying the standard deviation of the three reagent blanks by 3 .
sample lods were calculated by multiplying the solution lod by the dilution volume and dividing by the weight of the actual sample taken for analysis .
solution lods averaged 0.045 ng ml and sample lods averaged 1.9 ng g. recoveries from spiked blanks and samples averaged 97 and 101% , respectively .
one of the sample spike recoveries was high ( 155% in batch 8) , and this was due to the high concentration of hg in the sample selected for spiking in the batch compared with the spike added .
agreement with certified levels in the national research council of canada ( nrcc ) dolt-2 and dorm-2 srms and the national institute of standards and technology ( nist ) oyster tissue 1566a srm was generally satisfactory , although results for batches 9 , 10 and 11 were unusually high .
blank spaces in the tables denote that the particular test was not included in the batch .
note : blank spaces denote that the particular test was not included in the batch .
, samples were purchased as cleaned fillets or whole fish at the retail level in toronto , vancouver and montreal in 2005 .
the retail stores included four supermarket chains and four specialty fish seafood outlets in each city .
each sample was shipped frozen to maxxam analytics inc . for cleaning ( if necessary ) , homogenization and bottling in pre - cleaned polyethylene bottles supplied for total hg and glass jars with teflon cap liners for methyl hg ( i - chem brand , thermo fisher scientific ) .
the samples were then frozen and shipped to health canada 's food research division in ottawa for analysis .
information provided with each sample included , where available , the species name as provided by the retail outlet , the state of the sample when purchased ( frozen , fresh or previously frozen ) , the city of sample pickup , name of store where purchased , country of origin and date of collection .
the countries of origin for the samples were usa ( 7 ) , argentina ( 1 ) , australia ( 5 ) , canada ( 26 ) , chile ( 13 ) , hungary ( 2 ) , mexico ( 1 ) , new zealand ( 3 ) and taiwan ( 1 ) , with the remaining samples being of unknown origin .
analyses were performed using the reagents , instrumentation and methodology described in dabeka et al .
( 2002 ) . briefly , after a low - temperature nitric / hydrochloric acid and hydrogen peroxide digestion of roughly 1 g fish tissue and dilution to 50 ml with water , measurements were made using a cetac-6000a dedicated hg analyser equipped with an asx-500 autosampler and adx autodilutor .
quality control measures for each analytical batch included three reagent blanks , two reagent blank spikes ( 200 ng hg ) , one sample spike of 400 ng hg ( in duplicate for both the unspiked and spiked 400 ng sample ) and duplicates of three different standard reference materials ( srms ) with certified hg concentrations .
two cross - check standards ( from a different manufacturer ) were included in each batch for standard verification during the run .
the solution limit of detection ( lod ) was estimated for each analytical batch by multiplying the standard deviation of the three reagent blanks by 3 .
sample lods were calculated by multiplying the solution lod by the dilution volume and dividing by the weight of the actual sample taken for analysis .
solution lods averaged 0.045 ng ml and sample lods averaged 1.9 ng g. recoveries from spiked blanks and samples averaged 97 and 101% , respectively .
one of the sample spike recoveries was high ( 155% in batch 8) , and this was due to the high concentration of hg in the sample selected for spiking in the batch compared with the spike added .
agreement with certified levels in the national research council of canada ( nrcc ) dolt-2 and dorm-2 srms and the national institute of standards and technology ( nist ) oyster tissue 1566a srm was generally satisfactory , although results for batches 9 , 10 and 11 were unusually high .
blank spaces in the tables denote that the particular test was not included in the batch .
note : blank spaces denote that the particular test was not included in the batch .
a summary of the results ( table 2 ) found that arctic char , red snapper and fresh water trout contained the lowest concentrations of hg , averaging 37 , 148 and 55 ng g , respectively .
average levels in the other fish types varied from 384 ng g for black cod to 854 ng g for marlin .
note : designated fish subject to a 1000 ngg regulatory limit , others to a limit of 500 ngg average concentrations of hg in the different species were in general agreement with those found in other canadian and international surveys ( table 3 ) .
differences among the surveys can be attributed to the size of the individual fish from which the sample was taken , as fish mercury concentrations vary directly with fish size .
additionally , regional differences in hg concentrations may be a function of the food source .
for example , tilefish caught in the atlantic ocean contained 144 ng g hg , whereas those sampled in the gulf of mexico averaged 1450 ng g ( fda 2009 ) .
it is uncertain whether this difference is due to different food sources or to the situation that the tilefish in the two areas are different species .
comparison of hg levels ( ngg ) found in this study with those found previously in canada and other countries .
average of three tilefish - two samples purchased in 2008 containing 128 and 3000 ng g , and one purchased in 2005 contained 80 ng g. fda ( 2009 ) ; environmental protection agency ( 2000 ) ; national marine fisheries service ( 1978 ) .
the primary reference for the gulf tilefish and sablefish data in the fda report is a national marine fisheries service report ( hall et al .
1978 ) , while that for the kingfish or king mackerel is an epa report ( ache et al .
144 ng g in atlantic tilefish and 1450 ng g in those caught in the gulf of mexico ( fda 2009 ) .
the current canadian regulatory limit for total mercury in the edible portion of commercially sold fish is 500 ng g , with the exception of a 1000 ng g limit and accompanying consumption advice ( health canada , 2008 ) for specific species of piscivorous fish : shark , swordfish , marlin , orange roughy , escolar , and fresh and frozen tuna ( health canada , 2010 ) .
the proportion of individual samples exceeding the relevant standards was : 13% of black cod , 24% of grouper , 40% of king fish , 28% of marlin , 20% of sea bass and 50% ) of tile fish ( table 2 ) .
however , the uncertainty in these percentages is high due to the small numbers of each species tested . the mean and
median concentrations of mercury in black cod , grouper , king fish , marlin and sea bass were all below the standards for the respective species .
additional data would aid in characterizing the typical mercury concentrations in some types of fish , in particular king fish and tilefish .
for example , the us fda has reported a median mercury value in tilefish ( atlantic ) of only 99 ng g ( n = 32 ) ( fda , 2009 ) .
it should also be noted that the occasional consumption of infrequently consumed fish containing mercury levels greater than the regulatory limits would not be expected to pose a health risk to consumers .
thanks are due to elizabeth elliott and mark feeley of the chemical health hazard assessment division , bureau of chemical safety for review and helpful suggestions .

Answer:
total mercury was analysed in 188 samples of predatory fish purchased at the retail level in canada in 2005 .
 the average concentrations ( ng g1 , range ) were : sea bass 329 ( 381367 ) , red snapper 148 ( 36431 ) , orange roughy 543 ( 279974 ) , fresh water trout 55 ( 20430 ) , grouper 360 ( 81060 ) , black cod 284 ( 71651 ) , arctic char 37 ( 2854 ) , king fish 440 ( 42923 ) , tilefish 601 ( 791164 ) and marlin 854 ( 1252346 ) .
 the canadian standard for maximum total mercury allowed in the edible portions of fish sold at the retail level is 1000 ng g1 for shark , swordfish , marlin , orange roughy , escolar and both fresh and frozen tuna .
 the standard is 500 ng g1 for all other types of fish . in this study , despite the small number of samples of each species , the 1000 ng g1 maximum was exceeded in five samples of marlin ( 28% ) .
 the 500 ng g1 maximum was exceeded by six samples of sea bass ( 20% ) , four of tilefish ( 50% ) , five of grouper ( 24% ) , six of king fish ( 40% ) and one of black cod ( 13% ) ,


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:
mutations in codons 12 and 13 of kirsten rat sarcoma viral oncogene homolog ( kras ) and b - raf murine sarcoma viral oncogene homolog b1 ( braf ) genes are frequently present in tumors of patients with metastatic colorectal cancer ( crc ) ; they are observed in 30 - 45% and 5 - 20% of cases respectively 1 .
less commonly detected crc tumor mutations are codon 61 , 146 of the kras oncogene , and at other sites in the braf gene .
kras and braf mutations have been frequently described as mutually exclusive in crc , in that tumors usually have neither , or only one , of these specific mutations .
rarely patients may have crc with coincident kras and braf mutations ; herein we report three such cases and review of the literature .
although highly uncommon , identification of this dual tumor genotype is important , as current clinical trials are being designed enriching for patients whose tumors harbor either a kras or braf mutation , but haven't addressed those patients with coincident kras and braf mutations . due to the infrequent rate of coincident mutation , it is not known if these patients have a tumor biology distinct from kras or braf mutant tumors .
under an institutional review board approved retrospective chart review protocol ( dr-11 - 0113 ) , we reviewed samples tested at a clinical laboratory for tumor kras and/or braf mutations from 2008 - 2011 .
dna was extracted from microdissected paraffin - embedded tumor and analyzed by a polymerase chain reaction ( pcr)-based dna sequencing method to examine codons 12 , 13 and 61 of the kras proto - oncogene .
the sensitivity of detection of this assay is approximately 1 in 10 mutation - bearing cells in microdissected area .
dna was also analyzed by pcr and a pyrosequencing method for codons 595 to 600 of exon 16 of braf oncogene ; the methods for these assays have been described elsewhere 2 .
a total 6,633 crc patient tumors were screened for mutation analyses and of those 1,483 case were tested for both kras and braf mutations .
644 were kras - mutant ( 43% ) and 3 ( 0.2% ) of these were bearing a concomitant braf ( v600e ) mutation .
the first patient is a 65 year - old woman who presented with small bowel obstruction .
the patient underwent a biopsy of the metastatic liver lesion that was sent for kras mutation and braf mutation analysis ; a tumor mutation was detected in codon 12 ( ggt to gat ) of the kras gene that would change the encoding amino acid from glycine to aspartic acid ( g12d ) and codon 600 ( gtg to gag ) in exon 15 of the braf gene that would change the encoding amino acid from valine to glutamine ( v600e ) .
the second case identified was a 72 year - old woman with rectal cancer metastatic to the lungs ; similar tumor mutations were detected in kras ( g12d ) and braf ( v600e ) proto - oncogenes . in both cases
the tumors were microsatellite stable ; concomitant braf and kras mutations were not identified among patients samples ' whose tumors were microsatellite instable .
the third case is 51 year - old man who presented with abdominal pain and was found to have a descending colon cancer with liver metastasis . in specimen from liver lesion ,
coincident kras and braf mutations appear to be rare entity ( present in 3 out of over 1,928 samples tested ) . due to the infrequent observation of this phenomena , it is not clear whether or not these tumors have a different biology and natural history than kras or braf mutant tumors , or which of the two mutations is the dominant oncogene driving tumor proliferation .
a prior study has demonstrated that concomitant tumor mutations have been relatively frequently observed human crc cell line ; in this study , of 24 human crc cell lines , one cell line ( gp2d ) demonstrated mutations in both kras and braf oncogenes ( however the braf mutation was in the less frequently observed at the codon 529 site ) 3 . in an analysis of 250 patients tumor specimens with microsatellite stable disease , kras mutations were identified in 45.2% of primary tumors , and that concomitant kras and braf mutations were found in 10 lymph nodes ( 35.7% ) from a total 28 samples whereas only 3 of primary tumor with negative lymph nodes were positive for coincident mutation ( 2.3%)4 . there was also noted to be a higher proportion of concomitant mutations based on the degree of transmural penetration of the tumor ; 1/36 ( 2.8% ) for t2 tumors and 3/32 ( 9.4% ) for t4 tumors , suggesting activation of both genes is associated with progression of disease .
since kras mutation testing became standard - of - care for determining lack of treatment efficacy with antibody anti - epidermal growth factor ( egfr ) monoclonal antibody therapy in patients with advanced crc , large studies involving kras and braf mutation analysis crc screenings have been undertaken ; the results of studies with over 200 patients is demonstrated in table 1 .
unlike study by olivera et al 4 , all of these large data sets demonstrated that in crc kras and braf mutations were mutually exclusive , except in one case 5 .
in one other series , two patients had tumors with two distinct kras mutations 6 .
molecular profiling demonstrates that kras and braf mutant tumors have very different gene signatures suggesting different signaling pathways are activated 12 .
in addition early data suggest outcomes with the use of anti - egfr monoclonal antibody therapy appear to be different depending on the site of kras mutation 13 . at this time
it is not known which gene expression profile pattern of concomitant kras and braf tumors is , and whether it more represents a kras tumor gene signature or braf signature .
given fact of tumor heterogeneity and the large variety of mutations observed in this disease , as well as epigenetic changes observed , it is likely that in the future , tumor gene expression profiling will provide a better indication of the activation of tumor signaling , and be a better biomarker of treatment efficacy .
strengths of our study are screened large patient population and available detailed clinical and pathological information .
the main limitation , however , were retrospective nature of study and possible artefactual mutation detection .
further multi - center prospective studies are necessary to understand true frequency and the role concomitant kras and braf mutations .
concomitant kras and braf tumor mutations are rare enough to be considered virtually ( albeit not entirely ) mutually exclusive .
since kras and microsatellite instability analysis are the only validated negative biomarkers of therapy efficacy in crc , routine analysis for braf mutations in kras wild type tumors is not recommended .
however if patients are being considered for clinical trial , especially those in which the eligibility require the presence of a kras mutation , braf mutation analysis would be highly advisable .
future enriched clinical trials for kras or braf should specifically address eligibility of patients whose tumors harbor a concomitant kras and braf mutation .

Answer:
kras mutations occur frequently in colorectal cancers ( crc ) and predict lack of response to anti - epidermal growth factor receptor ( egfr ) monoclonal antibody therapy .
 crc braf mutations , most commonly at v600e , occur less than 10% of the time , and occur usually in kras wild - type tumors , and more frequently in microsatellite instable tumors .
 concomitant kras and braf mutant crcs are rare ( occurring in 0.001% ) ; braf mutations should not be routinely tested in patients with kras mutant tumors , unless the patients is participating in a clinical trial enriching for the presence of a kras or braf tumor .
 clinical trials treating patients with either kras or braf mutant tumors should address eligibility of patients with concomitant kras and braf mutations .


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:
retinitis pigmentosa ( rp ) was defined as a group of hereditary disorders that diffusely involve photoreceptor and pigment epithelial function characterized by progressive visual field loss and abnormal electroretinogram .
rp also has changes at vitreoretinal interface , such as vitreoretinal traction and preretinal membrane , which causes macular disorders such as cystoids macular edema and macular hole .
we report a case of vitreoretinal traction syndrome associated with rp , which spontaneously resolved after posterior vitreous detachment ( pvd ) .
a 81-year - old woman presented with bilateral loss of peripheral vision and defective dark adaptation 15 years before .
the best - corrected visual acuity was 20/28 and 20/28 in the right and left eyes , respectively .
slit - lamp examination showed bilateral retinal arteriolar narrowing and intraretinal pigment deposition ( bone spicule pigmentation ) in both eyes ( figure 1a ) .
the visual fields of the patient were markedly constricted to the central 10 in both eyes ( figure 2 ) .
on june 2 , 2006 , retinal detachment was detected in the nasal periphery in the right eye ( figures 1b and 1c ) . at that time , pvd was confirmed with weiss ring ( figure 3 ) .
fluorescein angiography showed a hyperfluorescence due to atrophy of retinal pigment epithelium but no active leakage in the detached area ( figure 1c ) .
the detached area was within the area where the visual field was lost , and we observed a clinical course of this eye without any treatment . retinal detachment was kept localized and not extended .
five months later , pvd further progressed beyond the arcade area , accompanying retinal hemorrhage around the detached retina .
in this case , an eye with rp was complicated with tractional retinal detachment , possibly associated with increased vitreoretinal traction secondary to the incidence of pvd .
the retinal detachment was limited within the area where the visual field was lost and was not progressive .
eight months later , spontaneous resolution of tractional retinal detachment resulted from progression of pvd , which was suggested by transiently increased traction - related retinal hemorrhage .
eyes with rp may have abnormal liquefaction of vitreous and altered distribution of vitreoretinal adhesion .
the incidence of pvd in the posterior pole might result in the focal remarkable traction in the peripheral retina and tractional retinal detachment .
eight months later , the retinal detachment was resolved because local vitreoretinal traction was weakened with extention of pvd beyond the arcade vessels .
rp has vitreomacular traction - related pathologic events involving vitreomacular traction syndrome , cystoids macular edema , and macular hole.13 such changes in this patient with rp might result in a tractional retinal detachment .
rani et al4 reported that a 50-year - old man underwent a tractional retinal detachment involving the posterior pole with a taut posterior hyaloids surface . in this study ,
vitrectomy was not performed because the patient opted for no surgical intervention in view of poor prognosis . on the other hand ,
it was reported that vitrectomy was effective for vitreous opacity,5 cystoid macular edema,6 exudative retinal detachment,7 and macular holes8 in rp . however , regarding the indication to perform a vitrectomy , the balance of the expected gain in visual function and usual risks of vitrectomy and rp - related risks , such as phototoxicity worsening of visual field loss , and cystoid macular edema .
it might be a choice that a patient with tractional retinal detachment in rp is observed without surgical treatment , if the detached area is not progressive .

Answer:
we present a case of retinitis pigmentosa with vitreoretinal traction - associated retinal detachment .
 the retinal detachment was detected in the nasal periphery .
 no retinal breaks and no active vascular leakage were observed by fundus scopy and fluorescein angiography , respectively .
 however , 8 months later , the tractional retinal detachment was spontaneously resolved with posterior vitreous detachment .


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:
Cell surface display yields evolvable, clickable antibody fragments

Paper sections:

The introduction of non-canonical amino acids (ncAAs) into recombinant proteins enables enhancement or alteration of protein function.[1,2] Replacement of canonical amino acids by ncAAs, in response to either sense codons or nonsense codons, can lead to marked changes in reactivity, stability, and other properties.[3–9] In either case, introduction of ncAAs may lead either to loss of protein function or to beneficial changes in behavior. High-throughput screening methods and directed evolution[10,11] can be used to isolate ncAA-modified protein variants with novel and desirable characteristics.

Here we present a cell surface display method for evolving binding proteins that contain reactive, "clickable" ncAAs. Specifically, we replace the methionine (Met; 1; Figure 1A) residues in single chain variable fragment (scFv) mutants of the anti-digoxigenin 26-10 antibody[12] with the Met surrogates homopropargylglycine (Hpg; 2), azidohomoalanine (Aha; 3) or norleucine (Nrl; 4). The display platform[13,14] enables identification of variants with improved expression levels, solubility, and antigen-binding properties. Several of the isolated Aha variants exhibit improved binding kinetics relative to their Met counterparts, and the azide side chains of Aha can be chemically modified under mild conditions without affecting binding function. Antigen binding and protein modification can be investigated concurrently for scFvs displayed on the E. coli cell surface.

We first used flow cytometry to investigate the function of the 26-10 anti-digoxigenin scFv displayed on the surface of E. coli cells in the Lpp-OmpA' display system (see Supporting Information for display details).[13,14]ScFv function was assessed in multiple amino acid contexts using BODIPY FL digoxigenin 5 as a probe of binding behavior (Figure 1). Cells displaying the Met form of the scFv could be readily labeled with 5 (Figure 1B), while cells displaying the Hpg form exhibited low fluorescence levels (Figure 1C). Fluorescence levels of cells displaying the Aha-substituted scFv were reduced relative to the cells expressed in Met (Figure 1D), while cells displaying the Nrl-substituted construct exhibited unchanged fluorescence levels (Figure 1E).

The loss of function of the Hpg form of the scFv led us to investigate whether point mutations could enable recovery of digoxigenin binding. We developed an error-prone PCR library (Library 1) in which the antibody fragment and the majority of the display anchor were subjected to mutation. In the first round of sorting, Library 1 was expressed and sorted under conditions leading to partial substitution of Hpg for Met (Figure S1).[15] Sorting this population led to enrichment of clones exhibiting Hpg tolerance. Using expression conditions leading to near-complete replacement of Met, the Hpg-tolerant population was further enriched for functional clones in two additional rounds of sorting (Figure S1).

Individual clones from the thrice-sorted Library 1 were isolated, tested for their ability to bind antigen when expressed in Hpg form on the E. coli cell surface, and sequenced. All ten clones bound 5 when Met was replaced with Hpg, and each variant contained at least one amino acid mutation in the scFv (Table S1). While some display-anchor mutations were identified during screening of Library 1 and Library 2 (see below), none appeared to affect scFv properties in any significant way. We performed additional analysis of Mut2, a clone recovered from five of the ten colonies (Figure 1, Table 1, Table S1). In flow cytometry experiments, cells displaying Mut2 bound more 5 than cells displaying the parent 26-10 scFv, irrespective of whether the scFv was expressed in its Met, Hpg, Aha or Nrl forms. The effect was most striking for the Hpg and Aha forms of the protein (Figure 1G, H).

Seeking to improve the binding behavior of the scFv in multiple ncAA contexts, we prepared Library 2 from DNA isolated from thrice-sorted Library 1 via error-prone PCR, and attempted to isolate variants with improved binding after replacing Met with ncAAs. Four rounds of sorting under increasingly stringent conditions were performed, using kinetic competitions in the last two rounds to isolate clones with the lowest off rates (Figure S2, Table S2).[16] Individual clones were picked after three (Hpg and Aha contexts) and four (Hpg, Aha, and Nrl contexts) rounds of enrichment, sequenced (Table S3), and subjected to on-cell off-rate measurement (Table S4).[16] Most clones exhibited off rates equal to or lower than the off rates of the parent construct or Mut2 when expressed in the same amino acid context.

We examined the sequences of individual clones and the sorted populations in detail (Table 1, Figure 2, Table S5). Mutations at Met codons were common; the Met residues at positions H20 and H80 (Kabat numbering) were replaced in the majority of variants. On the other hand, the Met residues at positions H34 and H100B were conserved. The preservation of M(H100B) is especially striking in view of the fact that it is in direct contact with the antigen.[12] The data also reveal the frequent addition of a Met codon at position H82C; Met replaces a leucine at this position in more than 50% of all sequenced variants. Of the mutations not involving Met, S(H24)F, S(H24)Y, and F(H29)S occurred most frequently. Quantitation of mutational frequencies in the different populations indicated that ncAA context changes mutational frequency only modestly (Table S5).

We produced and characterized several antibody fragments in soluble form. Each scFv of interest was produced in its Met form and in the ncAA form in which it was isolated. In addition, attempts were made to produce the parent 26-10 scFv and Mut2 in Met and all ncAA contexts. The parent construct could be expressed and purified only in its Met and Nrl forms, while Mut2 could be expressed and purified in all four forms. MALDImass spectrometry indicated replacement of 80–93% of Met by the ncAA in each isolated variant (Table S6, S7). Table 1 and Figure 2 summarize the mutations found in the selected scFv variants.

Variation in the antigen-binding behavior of the variants was dominated by changes in koff, the dissociation rate constant. Surprisingly, the kinetic data indicate that replacement of Met by Aha in a given variant consistently decreased koff by a factor of two. Replacement of Met by Hpg increased koff by a factor of four, while replacement of Met by Nrl caused negligible changes in koff. It seems most likely that changes in koff are most sensitive to the identity of the residue at position H100B, which is in direct contact with the antigen.[12] Modest reductions in koff were achieved through further screening in both the Hpg and Nrl contexts, with the Hpg form of Hpg3x3 and the Nrl form of Nrl4x11 exhibiting two-fold lower dissociation rate constants as compared to Mut2 in the same ncAA contexts.

The Aha and Met forms of several scFv variants were subjected to strain-promoted azide-alkyne cycloaddition (SPAAC) with dibenzocyclooctyne dye 6 (Figure 3A, Figure S3). In-gel fluorescence experiments confirmed selective labeling of the Aha forms; minimal background labeling of the Met forms was detected. Mass spectrometry and the lower reactivity of the Aha form of Aha4x4 (which lacks Aha at H34) implicated position H34 as a major site of modification (Figure S4, Table S8). Our attempts to modify Hpg- and Aha-containing scFvs by the copper-catalyzed azide-alkyne cycloaddition were unsuccessful, possibly because of the limited surface accessibility of the reactive ncAA side chains in the proteins (Figure S5).[17]

SPAAC-modified scFv fragments retain antigen-binding behavior, as shown by western blotting assays. Samples for western blotting were prepared by adding digoxigenin-modified bovine serum albumin (BSA) in varying amounts to E. coli cell lysates. The samples were transferred to nitrocellulose membranes, and the membranes were probed with the Aha or Met forms of Aha4x5 fragments previously treated with 6. Scanning the membranes for fluorescence emission from 6 (Figure 3B) revealed that only the Aha form of the protein enabled detection of digoxigenin-modified BSA. To confirm that treatment with 6 did not adversely affect the Met form of the protein, the blots were further probed for the hexahistidine tags on the scFvs (Figure 3C). These experiments confirmed that both forms of Aha4x5 remained capable of binding to digoxigenin after SPAAC. Biacore assays further indicate that SPAAC conditions did not seriously alter antibody kinetic properties; kon values were reduced slightly, while koff values remained unchanged (Table S9).

We extended our functional analysis of tagged conjugates to scFvs displayed on the E. coli cell surface. We subjected E. coli cells displaying the Aha or Met forms of Aha4x5 to 16-hour labeling reactions with 7 (Figure 3D).[18,19] We then labeled the cells with 5 and streptavidin-phycoerythrin and performed flow cytometry. Cells displaying the Aha form of the scFv exhibited substantial fluorescence in both channels (Figure 3E), confirming that the Aha form of Aha4x5 retains binding function after chemical modification on the cell surface, while cells displaying the Met form bind 5 but exhibit low reactivity toward the cyclooctyne (Figure 3F). Additional experiments indicated that treatment of cells with unlabeled digoxin prior to treatment with 5 blocks the binding of 5 to scFvs regardless of chemical modification state (Figure S6). These experiments also revealed that some binding activity appears to be lost upon treatment of cells displaying the Aha form of Aha4x5 with 7; these losses in binding function may reflect an increase in crowding and steric hindrance on the surface of cells after treatment of 7 or denaturation of some scFvs during the click reaction. Finally, experiments verified that 7 reacts primarily with Aha side chains in scFvs, not Aha in other newly synthesized proteins; fluorescence values suggest that approximately 75 percent of 7 detected on the cell surface has reacted with displayed scFvs, with the remaining 25 percent reacting with other membrane proteins.

Having shown that both antigen-binding and modification of displayed scFvs can be monitored in a single flow cytometry experiment, we used a subset of samples to demonstrate the separation of various cellular populations via fluorescence-activated cell sorting (FACS). We mixed together cells exhibiting antigen binding only, chemical modification only, simultaneous binding and modification, and neither binding nor modification (Figure 4A). Four-way sorting of this mixture resulted in the recovery of clean populations exhibiting all combinations of binding and modification (Figure 4B–E). These results suggest that this display platform should be amenable to simultaneous, high throughput evaluation and isolation of proteins based on chemical reactivity and binding function.

This work demonstrates how high-throughput screening of antibody fragments containing ncAAs can yield proteins with novel properties. The screening methods described here yielded scFvs with enhanced expression and antigen-binding behavior. Fragments containing Aha exhibited improved dissociation rate constants and susceptibility to chemical modification under mild conditions. These properties were demonstrated both in solution and on the E. coli cell surface. Our approach is complementary to the screening of libraries of antibody fragments via phage display with ncAAs[20] and to the introduction of reactive amino acids into antibody fragments site-specifically.[21] In the future, incorporating multiple ncAAs into recombinant proteins may enable re-design of antibody complementarity determining regions[22] with unconventional side chains to enhance molecular recognition. Cell surface-display of ncAA-modified proteins should also enable direct screening of libraries of chemically modified proteins of interest in the engineering of bi- or multivalent proteins,[23] switchable sensors,[24] or cyclized peptides.[25]

Answer: Non-canonical amino acids (ncAAs) provide powerful tools for engineering the chemical and physical properties of proteins. However, introducing ncAAs into proteins can affect protein properties in unpredictable ways, necessitating screening efforts to identify mutants with desirable properties. In this work, we describe an E. coli cell surface display platform for evolving clickable antibody fragments. This platform enabled isolation of antibody fragments with improved digoxigenin binding and modest affinity maturation in several different ncAA contexts. Azide-functionalized fragments exhibited improved binding kinetics relative to their methionine counterparts, facile chemical modification via the azide-alkyne cycloaddition, and retention of binding properties after modification. The results described here suggest new possibilities for protein engineering, including modulation of molecular recognition events by ncAAs and direct screening of libraries of chemically modified proteins.


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:
it is characterised by the production of abnormal hb referred to as sickle hb or hbs.123 the prevalence of sca is high in sub - saharan africa with nigeria having the highest burden.45 sca has been associated with hyperhaemolysis , cerebrovascular disease , acute chest syndrome , vaso - occlusive crisis , pulmonary hypertension and premature death among others.67 relatively , individuals with sca enjoy a compensated state of ill health interspersed with periods of acute exacerbation characterised by hyperhaemolytic ( anaemic ) or vaso - occlusive ( voc ; painful crisis ) with infection , tissue hypoxia and micro - vascular occlusion as important predisposing events.68 abnormal lipid homeostasis has been reported in sca as well as other haematological disorders such as -thalassemia and this has been suggested to have the potential to alter membrane fluidity and function of red blood cell ( rbc ) in individuals with sca.91011 earlier studies reported significant increase in plasma triglyceride ( tg ) levels and concurrent significant decrease in plasma levels of total cholesterol ( tc ) , high - density lipoprotein - cholesterol ( hdl ) and low - density lipoprotein - cholesterol ( ldl ) in sca subjects.91112 several inconclusive mechanisms such as heightened erythropoiesis ( causing increased cholesterol utilization ) , defective liver function ( due to iron overload ) and defects in postabsorptive plasma homeostasis of fatty acids have been put forward to explain the pathogenesis of this sca - associated lipid abnormalities.913 however , it is worthy of note that this lipid phenotype is generally recognized as a risk factor for cardiovascular diseases .
zorca et al.11 reported that elevated plasma tg is a potential risk factor for pulmonary hypertension ( ph ) in sca subjects . the impact of disordered lipid metabolism on the course of sca and its numerous complications are not yet clearly defined . also , there is little information on the lipid profile of sca subjects in voc . due to the present dearth of knowledge
; this study determined the lipid profile of adult nigerians with sca in vaso - occlusive crisis ( voc ) and in steady state ( ssca ) .
eighty - two participants comprising 58 adults with sca ( 30 in steady state and 28 in voc ) and 24 age - matched healthy individuals with hbaa genotype were recruited into this study .
the sca ( hbss ) subjects were recruited from the hematology day care unit , department of hematology , university college hospital , ibadan after approval by university college hospital ( ui / uch ) joint ethics review committee , and informed consent by participant .
steady state ( ssca ) and vaso - ooclusive crisis ( voc ) were defined as earlier reported.14 subjects with other forms of genotype apart from hbss and hbaa , diabetes mellitus , hypertension , human immunodeficiency virus ( hiv ) , hepatitis , cancer and with established endocrine dysfunctions were excluded from the study .
blood pressure ( bp ) was obtained using a mercury sphygmomanometer after at least 10 minutes of rest .
after an overnight fast of about 10 hr , 5 ml of venous blood was obtained from each sca subject in steady state ( ssca ) and the controls .
samples were collected upon admission in the voc group as voc is an acute clinical condition hence ; could not have been predicted for possible overnight fast .
most subjects in voc would probably be anorexic because of the acute pain they were going through .
blood samples were dispensed into edta - containing samples bottles and after determining the packed cell volume ( pcv ) and total white blood cell count ( wbc ) , plasma was appropriately obtained and stored at 20c until analyses were done .
haemoglobin phenotype of each subject was determined using standard electrophoretic method at ph 6.8 while pcv and wbc were determined as described by cheesbrough.15 plasma lipid profile was determined using enzymatic method while ldl was calculated using friedwald equation.16 the distribution of the data was assessed using histogram with normal curve .
results are presented as mean  standard deviation or as median ( interquartile range ) for gaussian and non - gaussian distributed data , respectively .
analysis of variance ( anova ) or kruskal - wallis test was used to compare all the three groups while differences between two groups were determined using independent student 's t - test or man - whitney u as appropriate .
results are presented as mean  standard deviation or as median ( interquartile range ) for gaussian and non - gaussian distributed data , respectively .
analysis of variance ( anova ) or kruskal - wallis test was used to compare all the three groups while differences between two groups were determined using independent student 's t - test or man - whitney u as appropriate .
pcv , tc , hdl and ldl were significantly lower while wbc was significantly higher in sca compared with the control subjects .
there was slight , but insignificant elevation of tg in sca compared with the control subjects . in table 2 , all the components of the lipid profile between the three groups ( ssca , voc and controls ) were significantly different .
other components of the lipid profile had no specific pattern of differences . characteristics of the subjects in table 3 , tc and hdl were significantly lower while tg / hdl was significantly higher in sca subjects in steady state ( ssca ) compared with the control subjects .
similarly , tc and ldl were significantly lower in sca subjects in vaso - occlusive crisis ( voc ) compared with controls . however , tc , tg , ldl and tg / hdl were significantly lower while hdl was significantly higher in voc compared with ssca .
comparison of lipid profile in ssca , voc and control subjects using anova to find out if there is any interaction between wbc and lipid profile , sca subjects were classified into two groups based on the mean wbc value ; 11.97 ( 10/l ) [ table 1 ] into 11.97 ( 10/l ) and > 11.97 ( 10/l ) groups .
as shown in table 4 , the two groups had similar lipid profile but they exhibited a similar pattern to that observed when ssca were compared with voc .
there was insignificant reduction in the levels of tc , tg , ldl , tg / hdl and insignificant elevation of hdl level in sca subjects with > 11.97 ( 10/l ) wbc compared with sca subjects with 11.97 ( 10/l )   lipid profile in ssca , voc and control subjects pattern of lipid profile based on mean total white blood cell count ( wbc ) in sca subjects
despite intense research for over 4 decades , mechanism of lipid homeostasis alteration in sca subjects is not yet fully understood.11 the observed lower levels of tc , hdl and ldl in the combined sca subjects ( ssca and voc ) are not novel findings .
hypocholesterolemia has been widely reported in sca subjects1112 and was thought to be due to increased cholesterol utilization consequent to increased erythropoiesis of sca .
however , the reports of westerman17 and ngogang et al.,18 showed that hypocholesterolaemia is a common feature of both haemolytic and non - haemolytic anaemia and that serum cholesterol is in equilibrium with the cholesterol content of total red cell mass .
it was , therefore , suggested that sca - associated hypocholesterolemia is a consequence of anaemia itself and not increased erythropoiesis.1117 the interaction between sca complications such as voc and disturbed metabolic homeostasis in individuals with sca has been reported.1419 in this study , tc and ldl decreased progressively from control - to - ssca - to - voc .
ssca had lower tc and hdl while voc had lower tc and ldl compared with the control subjects .
this observation further confirms that sca - associated hypocholesterolemia might be anaemia dependent as intense haemolysis has been associated with various complications of sca.20 also , tg / hdl was higher in ssca than the control groups .
the ratio of tg to hdl has been reported to be relevant in determining the risk of clinical vascular disease .
it has been used to identify diabetic patients with an atherogenic lipid profile and has been found suitable in selecting patients needing earlier and aggressive treatment of lipid abnormalities.21 our observation is not surprising as the ssca group had slightly higher tg with concurrent lower hdl compared with the control subjects .
zorca et al.11 reported that high tg / hdl is associated with endothelial dysfunction and suggested that high tg / hdl is a potential risk factor for pulmonary hypertension .
although ldl is usually low in sca subjects , belcher et al.22 showed that ldl from sca subjects is more susceptible to oxidation and cytotoxicity to endothelium .
our observation , together with earlier reports , indicates that anaemia - associated lipid homeostasis disturbance could predispose sca subjects to various vascular diseases .
unfavorable plasma fatty acid composition has been associated with clinical severity of sca.23 similarly , nouraie et al.20 reported that intensity of haemolytic anaemia is an independent risk factor for the development of sca complications such as ph and hypoxaemia . in this study ,
plasma levels of tc , ldl and tg / hdl were lower in voc compared with ssca .
this observation could be as a result of possible intense haemolytic anaemia in voc which would facilitate the attainment of a new equilibrium between the serum cholesterol and cholesterol content of total red cell mass.1117 the observed higher hdl in voc compared with ssca supports the report of darbari et al.3 which showed that higher hdl is independently associated with frequent voc .
the observed elevated plasma hdl could be a marker of less marrow activity in sca subjects since formation of erythroid cell membrane requires cholesterol.1124 the reason for the observed lower tg in voc compared with ssca is presently unclear
. however , intake of drugs and/or reduced food intake ( due to possible anorexia ) before presentation may be responsible for this observation .
further research work is still required to properly understand the disturbance in lipid homeostasis following voc as standard fasting period could not be ensured in our voc subjects .
also , the small sample size used in this study could limit proper data interpretation .
our study further confirms the widely reported defective lipid homeostasis in adults with sickle cell anaemia .
it also showed that the alteration in the lipid metabolism becomes pronounced with vaso - occlusive crisis .

Answer:
background : abnormal lipid homeostasis has been reported in sickle cell anaemia ( sca ) as well as in other haematological disorders
 . however , there is little information on the lipid profile of sca subjects in vaso - occlusive crisis ( voc ) .
 this study determined the lipid profile of adult sca subjects in voc and in steady state ( ssca).materials and methods : fifty - eight ( 58 ) adults with hbss ( 30 in steady state and 28 in vaso - occlusive crisis ) and 24 age - matched healthy individuals with hbaa genotype were recruited into this study .
 standard methods were used for the determination of blood pressure ( bp ) , packed cell volume ( pcv ) , total white blood cell count ( wbc ) and haemoglobin phenotype .
 after an overnight fast , 5 ml of venous blood was obtained from each ssca and the controls while samples were collected upon admission in the voc group .
 plasma lipid profile was determined using enzymatic method .
 differences between two groups were determined using independent student 's t - test or man - whitney u as appropriate .
 p - values less than 0.05 were considered significant.results:plasma total cholesterol ( tc ) and high density lipoprotein ( hdl ) were significantly lower while the ratio of triglyceride ( tg ) to hdl ( tg / hdl ) was significantly higher in ssca compared with the controls .
 low density lipoprotein ( ldl ) and tc were significantly lower in sca subjects in voc compared with controls .
 however , tc , tg , ldl and tg / hdl were significantly lower while hdl was significantly higher in voc compared with ssca.conclusion:sickle cell anaemia subjects have defective fasting lipid metabolism which becomes pronounced with voc .


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:
angiomyxomas are benign , locally infiltrative mesenchymal neoplasms with a predilection for the female pelvis and perineum in their reproductive age group .
these tumours often reach too large dimensions before becoming clinically symptomatic and they usually tend to recur after excision . accurate preoperative diagnosis should alert the surgeon to the need for wide excision , which is essential for prevention of local recurrence .
this is easily done with robotic assistance due to its advantages like three dimentional vision and advanced degree of freedom within the pelvic cavity .
we report here a similar case presenting as a retrovesical tumor in a male patient , which was excised with robotic assistance .
a 62-years - old male presented to us with mild obstructive lower urinary tract symptoms ( luts ) since last 1 year , with a history of acute retention of urine 3 month back with failed catheter free trial .
he underwent holmium laser enucleation of prostate ( holep ) elsewhere with histopathology reporting as benign prostatic hyperplasia .
patient failed to void after catheter removal and on further evaluation he was found to have pelvic mass .
digital rectal examination revealed a cystic mass simulating benign prostatic hyperplasia ( grade 3 ) .
magnetic resonance imaging ( mri ) pelvis revealed a well - marginated lesion within the prostate gland resulting into significant prostatomegaly with no extra prostatic extension [ figure 1 ] .
transrectal biopsy was done , which was inconclusive with no identifiable prostatic tissue . in view of discrepancy between mri report and biopsy report we performed cystoscopy which showed a hump in the prostatic urethra extending into the trigone , with prostatic lobes not visible ( because of previous holep ) .
tumor was ~6 cm  4 cm  2.5 cm in size [ figure 2 ] .
we were not able to separate the mass from bladder due to difficulty in entering into proper plane .
hence we opened bladder deliberately and resected from within the bladder , closed cystostomy and drained bladder .
histo - pathological examination [ figure 3 ] revealed moderately cellular tumor composed of sheets of spindle to stellate shaped cells with vascular channels in between . on immunohistochemistry cells were positive for desmin , focal positivity with smooth muscle antibody ( sma ) , s100 and ki-67 .
magnetic resonance imaging showing a well - marginated lesion within the prostate gland resulting into significant prostatomegaly , with no extra prostatic extension .
also gross specimen of the tumor with gelatinous appearance and size of ~6 cm  4 cm  2.5 cm .
histopathology revealed moderately cellular tumor composed of sheets of spindle to stellate shaped cells with vascular channels in between . on immunohistochemistry cells were positive for desmin , focal positivity with smooth muscle antibody , s100 and ki-67 .
aggressive angiomyxoma was first described in 1983 by steeper and rosai , and fewer than 150 cases have been reported in the world medical literature .
the recurrence rate is high , and often extensive resections are performed with considerable morbidity . in men ,
the tumour involves analogous sites including the scrotum and inguinal area and usually appears at an older age .
there might be a relation with hormonal status that might explain a female to male ratio of slightly more than 6:1 .
these lesions are characterized as soft , non - encapsulated tumours with finger - like projections infiltrating the surrounding soft - tissues .
the tumour grows slowly and is benign as suggested by the histology and by the fact that it shows no tendency to metastasize .
the low attenuation at unenhanced computed tomography ( ct ) and high signal intensity at t2-weighted mri are consistent with a myxoid matrix , high water content within the mass , or both .
the enhancement of t1-weighted mri after administration of gadolinium is related to the vascularity of aggressive angiomyxoma .
there is no evidence of gross fat within the mass , which distinguishes this tumor from other fat - containing tumors that also occur in the pelvis of women .
surgery is usually the first line of treatment ( open , laparoscopic or robotic assisted ) , radical surgery with wide margins and long - term follow - up is advised .
hormonal suppression seems to be a plausible treatment option because these tumours occur predominantly in premenopausal women of reproductive age , may grow rapidly during pregnancy and have been shown to express immunohistochemical positivity for oestrogen and progesterone receptors .
their surgery is challenging because of the infiltration and the difficult dissection . in the past , most authors advocated wide excision along with genito - urinary and digestive tract resections if necessary .
most recently robotic assisted laparoscopic excision has been promising , as seen in our case .
male angiomyxoma , athough a rare benign tumour , should be considered in the diferential diagnosis of pelvic mass in a male patient presenting with severe obstructive luts , and robotic assisted excision should be utilised wherever available .

Answer:
angiomyxoma is a rare tumour found predominantly in pelvis of young females .
 less than 150 cases have been reported , more than 90% in females and only few cases in males .
 its surgical excision is a big challenge and usually leads to recurrence due to incomplete excision .
 we report a case of retrovesical angiomyxoma in an elderly male .
 the aim of this report is to highlight the rarity of this disease , especially in males , and robotic assisted excision as an evolving option of treatment .


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:
currently , nanoparticles ( nps ) are used in several scientific applications , but among these silver nanoparticles ( ag - nps ) are predominant .
this is because of several characteristic features of this type of metal np.13 antimicrobials and nanocomposite fabrication are some of the highly useful applications , among others , of applications that were attributed to ag - np.4,5 in the fabrication of nps , it is very important to control particle size , shape , and morphology .
laser ablation in liquid media is a simple and clean method for synthesis of nps.69 in this technique , there is no need to use any chemical reagents ( such as nabh4 ) in the fabrication process or any purification techniques to characterize the produced nps .
for example , nps using fatty acids ( oleic and lauric acid ) and vegetable oils as stabilizers have been synthesized.1012 it was found that these materials consist of amphiphilic molecules with polar carboxylic groups which are able to adsorb nps , and their nonpolar long carbon chain prevents nps agglomeration through steric repulsion . castor oil is a vegetable oil obtained by cold pressing the seeds and subsequent clarification of the oil by heat .
it has high viscosity , high polarity , and very low vapor pressure and optical activity in comparison with other oils .
castor oil is classified as a safe and effective stimulant laxative by the us food and drug administration ( fda ) .
it is a triglyceride containing fatty acid chains , 90% of which are ricinoleic acid .
oleic and linoleic acids are the other significant components.12 with the above characteristics , it is expected that castor oil can be used as a good stabilizer in nps fabrication . in this study
, we report fabrication of ag - nps in castor oil . to the best of our knowledge ,
fabrication of ag - nps in castor oil using laser ablation has not been reported previously .
for the fabrication of ag - nps , a silver plate was ablated by laser in castor oil for 10 min .
as shown schematically in figure 1 , the plate with high purity ( > 99.99% ) was located 5 mm behind a glass cell wall filled with 10 ml of castor oil .
a nanosecond - pulsed q - switched nd : yag laser ( brilliant ; lambda photometrics ltd , hertfordshire , uk ) with 10-hz repetition rate , 5-ns pulse duration , and 360 mj / pulse energy at its original wavelength ( 1064 nm ) was applied .
a lens with focal length of 250 mm was used to focus the laser pulses onto the silver plate . during plate ablation ,
an atomic absorption spectrometer ( aas - s series ; thermo scientific , san jose , ca ) was used to measure concentration of ag - nps in castor oil .
the obtained concentration of ag - nps in castor oil was 0.13 mg / l . due to the high viscosity of castor oil
the prepared sample was characterized using a uv - vis double beam photospectrometer ( shimadzu , columbia , sc ) , a transmission electron microscope ( tem , hitachi h-7100 ; hitachi , tokyo , japan ) , and a fourier transform - infrared ( ft - ir ) spectrometer ( 1650 ; perkin elmer , waltham , ma ) .
figure 2a indicates the absorption spectrum of the castor oil immediately after laser ablation of the silver plate inside it .
the appearance of the maximum absorption peak around 400 nm confirms that the solution contained ag - nps , and that this peak originated from the oscillation of collection of free conduction electrons of ag - nps , which is called surface plasmon resonance .
the ultraviolet - visible spectrum of the ag - nps in castor oil was measured after a period of 2 months in order to check the ability of castor oil as a stabilizer . as shown in figure 2b
, the spectrum does not show any significant change in comparison with the spectrum of the freshly prepared sample ; however , there is a small reduction in absorption .
this showed that the ag - nps produced in castor oil using the laser ablation technique were stable and did not agglomerate during storage of the sample at room temperature for quite a long period of time .
the slight decrease in absorption intensity was possibly due to the sedimentation of larger particles .
the spherical shape nps can be very appropriately used for drug loading and for most biological applications such as antibacterial properties.13 figure 3 shows an approximate 5.06 nm average diameter for freshly prepared ag - nps obtained from two areas .
nps are formed via nucleation , transition , and crystal growth of materials such as silver atoms , clusters , and droplets that were emitted from the silver plate upon laser ablation.14 castor oil can adsorb the produced ag - nps and long - chain hydrophobic moieties protect them through a steric stabilization mechanism .
ft - ir spectroscopy of castor oil and castor oil containing ag - nps ( figure 5 ) shows absorption bands at 3448 , 2923 , 2857 , 1739 , and 1160 cm related to the vibrational - stretching mode of o  h ( intermolecular hydrogen bond ) , c  h ( sp ) , c  h ( sp ) , co , and c  o , respectively .
in addition , the appearance of the absorption band at 3780 cm can be attributed to nonhydrogen - bonded o
we believe that the existence of ag - nps prevents intermolecular hydrogen bonding , and therefore , the vibrational stretching modes of free o  h appear at a higher wave number.15
ag - nps have been successfully prepared in castor oil by laser ablation from a silver plate . the results obtained showed that the produced ag - nps were well dispersed and also stable for quite a long period of time .
this ability of castor oil to prevent agglomeration of ag - nps is due to the presence of long - chain hydrophobic moieties .

Answer:
silver nanoparticles were fabricated by ablation of a pure silver plate immersed in castor oil .
 a nd : yag - pulsed q - switch laser with 1064-nm wavelength and 10-hz frequency was used to ablate the plate for 10 minutes .
 the sample was characterized by ultraviolet - visible , atomic absorption , fourier transform - infrared spectroscopies , and transmission electron microscopy .
 the results of the fabricated sample showed that the nanoparticles in castor oil were about 5-nm in diameter , well dispersed , and showed stability for a long period of time .


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:
cutaneous hyperpigmentation is a recognized adverse effect of chronic minocycline use occurring in up to 50% of patients , . in a recent study performed at the mayo clinic , 54% of 291 patients receiving long - term minocycline suppression for orthopedic infections developed some degree of hyperpigmentation after a mean follow - up of 4.8 years . in this cohort ,
factors associated with minocycline - induced cutaneous hyperpigmentation ( mich ) include a history of vitamin d deficiency , presence of a shoulder prosthesis , noncirrhotic liver pathology , and use of a concurrent medication ( e.g. , calcium channel blocker ) also known to cause hyperpigmentation .
mich is not associated with adverse clinical effects , and it is mostly cosmetic in nature .
we herein present a rare case of extensive skin hyperpigmentation involving both lower extremities in a patient receiving long term minocycline .
in june 2016 , a 76-year - old male with a past medical history significant for nephrolithiasis and diverticulitis , presented to the authors institution with extensive hyperpigmentation involving both lower extremities .
he underwent a left total knee arthroplasty in 1988 at an outside institution and underwent revision surgery in 1992 . in 2001
, he underwent a second revision for fractured patella that was complicated by an infection with a coagulase - negative staphylococcus .
he received six weeks of treatment with parenteral vancomycin followed by oral trimethoprim - sulfamethoxazole .
operative cultures from the knee grew enterococcus sp . , prevotella sp , viridans group streptococcus , as well as candida parapsilosis . after completing antimicrobial treatment with vancomycin , ertapenem and fluconazole
, he underwent reimplantation using a rotating hinged knee arthroplasty in september 2005 ( fig .
since that time , the patient was maintained on oral minocycline chronic suppression . in 2010
he uses a brace as well as a cane to ambulate . at a follow - up in february 2012
when he was seen again in june 2016 , there was extensive blue - gray pigmentation in both cheeks and in the lower extremities ( fig .
2 ) , as well as sub - ungual blue - gray pigmentation in both hands ( fig .
there are three types of pigmentation patterns that can result from taking minocycline for long periods of time .
type i is a blue - gray pigmentation occurring around areas that were previously inflamed .
type ii has the same appearance as type i and covers areas of normal skin such as the anterior shins , arms , and ankles .
type iii is a muddy brown pigmentation usually occurring on areas of the skin that are exposed to the sun .
the blue - gray pigmentation is due to the deposition of iron within the dermal macrophages .
the diffuse hyperpigmentation seen in the patient presented in this case report includes both types i and ii . blue - gray pigmentation is clearly seen around the scar on the left knee from his knee replacement surgery ( type i ) .
hyperpigmentation is also distinctly seen on the shins of both legs and around the ankles of the patient ( type ii ) .
hyperpigmentation is predominantly seen with minocycline and less likely to occur with other tetracyclines such as doxycycline .
minocycline is five times more lipophilic than doxycycline ; hence , central nervous system adverse events are more common with minocycline .
although doxycycline may have a higher incidence of gastrointestinal upset and photosensitivity , minocycline has an increased likelihood of severe and permanent cosmetic adverse events and central nervous system adverse events . in the study of orthopedic patients on long - term minocycline suppression ,
these side effects are an addition to gastrointestinal adverse effects that can also occur with minocycline . in the absence of data to show that minocycline is superior to doxycycline for long - term suppression of infections including orthopedic infections , the authors propose that doxycycline be looked upon favorably when chronic use is indicated .

Answer:
cutaneous hyperpigmentation is a recognized adverse effect of chronic minocycline use occurring in up to 50% of patients . in this report
 we present a rare case of extensive skin hyperpigmentation involving both lower extremities in a patient receiving long term minocycline .
 the patient was receiving minocycline as suppression for chronic prosthetic joint infection .
 risk factors associated with minocycline - induced cutaneous pigmentation ( mich ) will be reviewed .


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:
, individuals can live for 80100 years , much longer than in past generations . according to the who , the proportion of people over 60 years of age is increasing quickly and is expected to exceed 2 billion people worldwide by the year 2050 .
immune senescence comprises a set of changes occurring to the innate and adaptive immune responses that accompany human aging .
age - associated immune senescence is a catch - all phrase that has been used to describe a plethora of changes to the immune system over the lifespan .
immune aging is a complex process that comprises many reconstructions and regular changes rather than being a simple one - way reduction in all immune functions ; thus , all parts of the immune system in immune senescence are not affected equally .
for instance , it has been observed that natural immunologic structures that are common to all living things are less affected than are acquired immune system structures .
active participation of inflammation , which forms the most basic defense mechanism in the aging process , is also an indicator of this .
the decline in immune function with age is unanimously recognized and supported by epidemiologic and clinical studies [ 46 ] .
many studies have demonstrated that immune functions and cells in the immune system are affected by aging .
some studies reported that differences in immune system due to aging vary between males and females .
sex - related differences in immune system susceptibility have also been observed in several mouse models and may be related to differences in the expression patterns of immune response genes .
understanding the basis of sex and age differences in immune response genes is important for developing new approaches to prevention , diagnosis , and treatment of diseases .
our aim in this study was to investigate how lymphocyte subgroups in peripheral blood are affected by aging among males and females .
study participants were 70 healthy individuals from 3 different age groups , observed from january 2010 to january 2012 .
participants were divided into 3 different groups according age : group 1 ( n=20 ) was 2545 years old ( 10 males , 10 females ) , group 2 ( n=25 ) was 4565 years old ( 12 males , 13 females ) , and group 3 ( n=25 ) was older than 65 years old ( 13 males , 12 females ) .
we determined the average levels of cd3 + , cd4 + , cd8 + , cd19 + , cd16+/cd56 + , cd3+/cd69 + , and cd19+/cd69 + by age group and sex .
individuals who were smokers or who had a chronic disease were excluded from the study .
collected data were statistically analyzed by kruskal - wallis - anova , with p<0.05 considered to be statistically significant .
we found a significant reduction in the rate of cd3+t cells related with age , but no significant change in cd19 + b cell rates ( p<0.005 ) .
another noteworthy result was that the level of cd8+t cells was lower in males compared to females and varied by age group ( p<0.005 ) .
level of activated t and b cells did not differ by age group , but levels of activated b cells ( cd19+/cd69 + ) decreased with age in males ( p<0.005 ) ( figure 1 ) .
projections indicate that by 2025 the world population over age 65 will be increasing 3.5 times as rapidly as the total population and the proportion of individuals age 60 years and older , which accounted for 10% of the world population in 2000 , will increase to approximately 22% of the world population by 2050 .
aging is a highly complex and continuous process that affects almost all organ systems , causing molecular and physiological changes , both qualitatively and quantitatively .
the aging of the immune system is a dynamic process that may at least partly reflect adaptation of the response to the evolving pathogen surroundings [ 16 ] .
aging is increasingly recognized as being associated with a pro - inflammatory state that plays an important role in the development of chronic diseases .
immune senescence comprises a set of changes occurring to the innate and adaptive immune responses that accompany human aging . these result in complex manifestations of still poorly defined deficiencies in the elderly population . in the evaluation of t and b cell activations ,
no change due to aging was observed in level of activated t cells . on the other hand ,
our results show that levels of activated t and b cells did not differ by age group .
we found that that aging reduces activated b cell ( cd19+/cd69 + ) levels of males .
males have shorter life expectancy than females due to the effects of aging making them more sensitive . the immune system in males
, we observed the lower cd8+t cell rates of males compared to those of females , especially when compared by age .
therefore , we especially wanted to observe the changes in peripheral blood lymphocytes of males and females who belong to different age groups .
recent studies have revealed that changes in the number of lymphocytes and a decrease in cell activation and proliferation appear with aging . however , the difference between males and females in immune senescence is still a hotly debated topic because many different factors may be involved .
differences between males and females in susceptibility to infection and differences in prevalence of autoimmune diseases by sex may help understand these differences in aging and health [ 25,12 ] .
in general , it has been demonstrated in human and mouse studies that females produce stronger humoral and cellular immune responses against varied antigens than males do .
this difference also plays a role in female allograph rejection , which has been demonstrated in mouse studies .
for instance , it is reported that female estrogen stimulation , which leads telomerase induction by c - myc , has an  anti - aging  effect .
another theory that has been put forward about this topic in recent years suggests that depending on the development needs mitochondria in female cells have a better adaptation than male cells have [ 1113 ] .
the immune response to infections , immunizations , and tumors in the elderly is quite different from that in young people .
our study shows that there may be differences between males and females in terms of immune senescence .

Answer:
backgroundage - associated immune senescence is a catch - all phrase that has been used to describe a plethora of changes to the immune system across the lifespan .
 aging is associated with a decline in immune function .
 our aim in this study was to investigate how lymphocyte subgroups in peripheral blood are affected by aging among males and females.material/methodsstudy participants were 70 healthy individuals from 3 different age groups , observed from january 2010 to january 2012 .
 the average levels of cd3 + , cd4 + , cd8 + , cd19 + , cd16+/cd56 + , cd3+/cd69 + , and cd19+/cd69 + were determined for each group and compared in terms of age and sex.resultswe found significant reduction in the level of cd3+t cells related with age , but no significant changes in cd19 + b cell levels ( p<0.005 ) .
 aging significantly reduces activated b cell ( cd19+/cd69 + ) levels in males ( p<0.005).conclusionsour results show that there may be differences between males and females in terms of immune senescence .


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:
despite of presentation of medical ethics as a new science in academic teaching , the ethical concepts have been alongside the medicine , and its antiquity back to the medicine history .
for instance , literatures such as hippocratic oath letter , liturgy of ibn maymun , and shirazi ethics ordinance are the old literatures in which the principles such as the necessity of patient preference defender on the physician and observing the principle of confidentiality have been emphasized . however , in the past literature using  physician s commitment and pledge
human being is a creature with physical , mental and spiritual dimensions which has rights during the health and illness .
patient rights are the very expectations he has from the health care services and must encompass his physical , mental , spiritual and social needs which are manifested as criteria , standards , rules and laws ( 4 , 5 ) .
emphasis on patient rights in the health care services particularly maintains patient dignity as a rank of a human , and is considered important especially when patient s vulnerability easily expose him to the violations and weaknesses of the health care system ( 6 ) .
considering that health is the most important existence aspect of every person and according to the article 29 of the constitution , providing health is the most important commitment of the government of islamic republic of iran ; because of this , in 2002 , for the first time the patients right charter was developed in iran and was notified by department of health , treatment and medical education ( 7 )
. the patients right charter of iran was approved by health policy council with a new and comprehensive viewpoint aimed to clarify the rights of the health services recipients and observance of moral standards in the treatment and medical fields in november 26th , and in december 1st it was communicated to the relevant centers ( 1 , 8 , 9 ) . in this charter patient satisfaction is considered as one of the characteristics of hospital effectiveness ( 9 ) .
today , the issues related to the quality of health care services , attention to the patients as customers and accomplishing their satisfaction are the main priorities and are of high importance .
one of the important factors in patient satisfaction is regarding their demands and observing their rights and providing care along with respect ( 6 ) .
awareness of the patients rights and observing them accomplishes more satisfaction of the patient , physician and other medical team and hospital staff and will lead to the spread of good morals among patients and medical team ; so eventually the moral status of all the individuals such as patients and medical team will be upgraded , but otherwise provided not observing these rights , it would lead to distrust to health care team . if there is no trust between medical staff and patients , it would lead to damages and losses for the patient and the medical team .
furthermore , it would lead to terrible and unpleasant occurrences which are difficult to compensate and would be followed by the legal prosecution ( 5 , 10 ) . protecting the patient rights by the nurses
only will be possible when they have gained necessary knowledge about it and suitable conditions be provided for respecting these rights ( 11 ) .
appropriate care and observing patient rights require nurses knowledge which would be possible through different ways such as side studies , retraining courses , and academic courses during education ( 5 ) . in the studied
researches about the nurses level of knowledge from patient rights , several aspects were mentioned .
nasiriani et al ( 5 ) and houshmand et al ( 11 ) reported good nurses level of knowledge in yazd hospitals and teaching public wards of tehran hospitals respectively .
observed moderate and parsinia et al obtained weak nurses knowledge in tehran and karaj hospitals respectively ( 10 ) .
the clinical researchers in different wards of teaching hospitals have reported that the patients rights have been ignored or have not been considered seriously due to shortage of nursing staff , high number of patients in the hospitals , psychological pressures and etc .
taken together , we decided to evaluate the nurses knowledge about patients rights in one of the teaching hospitals of tehran city .
this was a descriptive cross - sectional study in which the nurses knowledge about patient rights was determined .
the study samples consisted of 156 nurses who have been selected randomly from one of the teaching hospitals of tehran .
the data collection instrument was a two - part questionnaire ; the first part included the demographic information ( age , gender , marital status , work experience , educational level , etc . ) ; the second part consisted of 10 questions according to the 10 section of patients rights charter of iran ( 11 ) .
this part determined the following areas about patient rights : right to receive essential information about health care providers , rate of tariff , target insurance coverage if sent to the other medical centers .
right to receive respectful and quick treatment and care regardless of cultural and racial factors .
right to know about the probable complications , treatment options and participate in the ultimate treatment choosing .
right to make a decision about the presence of those who are not directly involved in the treatment process .
right to announce personal satisfaction from ending the treatment and referring to other centers , and right to preserve privacy and being ensured about confidentiality of all of the medical information .
the answers of the study subjects to the questions were quantified by measuring a three - score scale ;  good  ( 3 scores ) ,  moderate  ( 2 scores ) and  weak  ( 1 scores ) .
the maximum score in the questionnaire was 30 scores which were considered 2130 as high knowledge , 1020 as moderate knowledge and less than 10 as weak knowledge .
the validity was done using content validity i.e. the questionnaire was given to 10 faculty members of universities of tehran and after collecting the comments , the relevant comments were applied .
the validated questionnaire was given to 10 eligible study subjects for reliability and in both stages the questionnaire was completed with a 10-day interval and the required correlation of the first and second answers and confidence was obtained r = 0.90 and finally these people were excluded from the study population .
the questionnaires were completed during two weeks by direct referring of the researcher to the wards . in order to observe ethics , confidentiality and integrity ,
the mentioned questionnaires were anonymous and in all stages of the study the information were collected confidential and were kept by the researcher .
the review of the demographic variables indicated that the majority of the nurses were females ( 76.28% ) with mean age of 34.31  7.3 .
most of them ( 91.2% ) had bachelor degree and were married ( 62.82% ) . in terms of employment and work experience , almost half of nurses ( 47.43% ) were contractual , 46.79% had 610 years of work experiences , 30.99% never passed any course about  patient rights  and 30.99% of them had the simultaneous experience in public and private sectors .
no association between the variables of gender , age , degree and marital status and nurses knowledge about patient rights was found .
there was no significant difference between them in terms of work section , work experience and simultaneous work in the public and private sectors , however there was a significant difference between their level of knowledge about patient rights and simultaneous work in the public and private hospitals and work experience ( table 1 ) .
the findings of the study about the nurses level of knowledge in different areas indicated that the highest level of knowledge ( 95.51% ) was in the area of  right to preserve privacy and being ensured about confidentiality of all medical information  and the lowest level was  right to receive essential information about health care providers , rate of tariff , target insurance coverage if sent to the other medical centers  .
observing patients rights is the most important ethical issue in a hospital which should absolutely be considered . regarding patients rights and respecting them are two main factors for patients care .
observing patients rights means the accountability of all health care staff to the patients at the time of treatment and care giving ( 12,13 ) .
promoting patients rights is a multidimensional issue and in order to achieve it , comprehensive efforts should be done .
world health organization has offered some strategies such as active participation of health care recipients and providers policy making and extending educational programs for health care providers and entire community ( 11 ) .
the findings of the study showed that nurses level of knowledge about patients rights in terms of the variables such as type of the hospital , work experience , degree , age and gender was good , moderate and weak in 58.33% , 39.10% , and 2.56% of them respectively . in the studies conducted in karaj hospitals ( 10 ) and the public wards of teaching hospitals of tehran ( 11 ) also the nurses level of knowledge reported as weak .
today patients are more aware of their rights and physicians responsibilities and commitments and also hospitals managements regarding their rights ( 14 ) and they are more insisting on their principle rights ( 2 ) . therefore it is highly needed that nurses with low level of knowledge , and the other health care providers be aware of patients right charter and increase their knowledge .
determined the reasons of the nurses low level of knowledge as lack of institutionalization and regulation of the rights ( 12 ) ; lack of adequate time for studying and researching due to various obstacles such as poor economic conditions ; lack of positive vision in selecting nursing profession ; tough job conditions in the hospitals such as large number of the patients versus staff shortages , and lack of necessary facilities such as adequate and suitable libraries ( 5 ) .
the results indicated that there was a significant and direct association between level of knowledge and simultaneous jobs in the public and private hospitals ( p=0.01 ) i.e. the nurses who provided services simultaneously in the public and private hospitals had higher level of knowledge .
the reason might be due to regulations and rules which are performed more in the private hospitals compared to the public hospitals i.e. no implementation of patients right charter which may be followed by punishment .
the findings of the present study indicated that in the area of  right to preserve privacy and being ensured about confidentiality of all the medical information  , the nurses had the highest level of knowledge . in the patients
right charter codified in 2009 it says observance of the principle of confidentiality is necessary about all the information related to the patient except for the cases the law excluded ( 1 ) . in a comparative study about the patients right charter in the selected countries with iran in 2007 , it was indicated that regulations of iran for the right of the confidentiality of the patient information and medical records is similar to the other countries such as hungary , hong kong , new zealand , united states , south africa , european union and lithuania ( 14 ) . unlike the above results , in canada
it was indicated that 84% of the medical staff did not have enough knowledge about commitments and obligations of the law about confidentiality of the information and privacy of the patients information ( 15 ) .
our findings indicated that there was a direct and significant association between level of knowledge and work experience ( p= 0.008 ) and the level of knowledge of the study subjects from patients rights charter was increased by increasing work experience which was in accordance with the studies of nasiriani et al . and
in addition , the study indicated that there was no association between level of knowledge and degree and increasing degree had no effect on the level of knowledge . in a study in karaj
it was mentioned that the level of knowledge of the technicians was higher in comparison with supervisors and matrons and the most important reasons were sense of job security , professional status and disregardness of matrons and supervisors to the important tasks ( 10 ) .
the findings indicated that nurses in the area of  right to receive essential information about health care providers , rate of tariff , target insurance coverage if sent to the other medical centers  had the lowest level of knowledge .
the patients right charter codified in 2009 states that information must be given to the patient at the right time and appropriate condition by considering individual characteristics such as language , degree and perception ; however the patient has the right to access all the information recorded in his / her clinical records ( 16 , 17 ) . in the patients right charter of some countries such as canada and united states unlike the other countries such as iran , hong kong , new zealand , africa , european union , lithuania ,
 right to ask for explanation about the costs  has been discussed ( 14 ) .
the results indicated that 66% of the nurses was aware of the  right to access to the health care providers during hospitalization  , instead of 90% of turkish nurses ( 17 ) . nowadays ethical and legal concepts such as patient rights are included in the educational curriculum of greek and turkey ( 18 , 19 ) .
according to the considerable gap between development and realization of the patient rights ( 20 ) and the increased knowledge , claims and demands of the patients and also the results of this study , in order to observe patients right the following solutions are suggested : - encouraging the nurses to consider patients right charter seriously . - establishing and empowering the sanction of patients right charter - more emphasizing on concepts of professional ethics and patient rights in teaching nursing students - attending expertise meetings with presence of beneficiaries for evaluating barriers and presenting strategies in order to implement the patients right charter as soon as possible - providing periodic educational programs for health care providers and patients about patient rights
generally , since 41.6% of the nurses of the study had not a proper level of knowledge about patient rights and considering the fact that awareness and knowledge can be the base of nurses performance and also patients increasingly are getting informed about their rights , the implementation of patients right charter is highly recommended .
holding educational programs , seminars , workshops and academic panels for nurses , and nursing students help overcome difficulties .

Answer:
patients rights observance is one of the effective measures of patients satisfaction of health care services .
 we performed this study at the aim of evaluation of nurses awareness of patients rights in a teaching hospital in tehran.this cross - sectional study was conducted in 2010 . in this study 156
 nurses were randomly selected .
 two - part questionnaire was used for data collection .
 the validity and reliability of questionnaire was determined and then it was distributed between subjects .
 the data were analyzed by spss version 15 using descriptive and inferential statistics .
 our results showed that % 58.33 , % 39.10 and % 2.56 of nurses have good , medium , and poor levels of awareness respectively .
 we observed a significant relationship between nurses awareness and work experience ( p=0.008 ) and concurrent work in public and private hospitals ( p=0.01 ) .
 the most of the nurses ( % 95.51 ) were aware of  right to privacy protection and ensure confidentiality of information  and the least of them ( % 33.97 ) were aware of  right to receiving necessary information about the health care providers , the rate of tariff and insurance coverage.according to our survey it is concluded that implementation of patients right charter in this hospital is accompanied by some limitations which necessitates promotion of the nurses awareness about patients rights .
 taken together in order to enhance nurses awareness special measures and strategies should be considered .