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:
diabetic retinopathy ( dr ) is a common complication of diabetes mellitus ( dm ) and is a leading cause of blindness worldwide .
diabetic macular ischemia ( dmi ) is an important category of diabetic retinopathy ( dr ) [ 2 , 3 ] . during the imaging study of the normal macula ,
an important hallmark is the capillary - free region called foveal avascular zone ( faz ) .
it is recognized that the faz can enlarge and can become irregular in dr and seems to get larger as the stage of retinopathy advances [ 2 , 3 ] .
dmi is characterized by the occlusion and loss of the macular capillary network or capillary dropout [ 4 , 5 ] .
this condition results in upregulation of growth factors , which contributes to the development of diabetic macular edema ( dme ) , the most frequent sight - threatening disorder in individuals with dr .
dmi is an irreversible category of diabetic maculopathy , and its presence limits the potential benefits of treatments for dr [ 3 , 5 , 6 ] .
the early treatment of diabetic retinopathy study ( etdrs ) established dmi standards that were determined using fluorescein angiography ( fa ) . according to the etdrs report 11 , clinically , there is a correlation between dmi and poor prognosis that varies according to the severity of the macular ischemia .
the anatomy of the retina appears to be altered in dmi , with thinning of retinal nerve fibre layer ( rnfl ) and outer retina and thickening of outer choroid . in retinal microcirculation
, blood supply is divided mainly into superficial capillary plexus ( scp ) and deep capillary plexus ( dcp ) .
choroidal circulation appears to be the most important blood supply to the central macula , including photoreceptor inner segment ( is ) band , which is most likely the most important consumer of oxygen .
it is likely that the dcp is responsible for up to 15% of the blood supply to the photoreceptors , especially during dark adaptation [ 8 , 10 , 11 ] .
fa has been the gold standard imaging modality since it was introduced in 1961 [ 7 , 12 , 13 ] however , it requires venipuncture , and reports of anaphylaxis and death related to contrast injections , despite being rare , have been documented .
in addition , the technique is costly and time - consuming , requiring up to 10 minutes for framing acquisition [ 4 , 8 , 10 , 11 ] .
spectral domain ( sd ) oct has emerged as a potential alternative for detecting macular nonperfusion in diabetic patients , but results are contradictory [ 1 , 2 , 13 ] .
macular ischemia may disrupt the normal flow of nutrients to the outer retina , but photoreceptor status on sd - oct remains controversial . in diabetic patients , the disruption of photoreceptors in sd - oct can indicate a manifestation of underlying dcp nonperfusion in patients with a relatively healthy macula [ 1416 ] .
oct angiography ( octa ) has been used for 3d mapping at microcirculation level [ 9 , 1316 ] .
it allows detection of retinal and choroidal structures via motion contrast imaging and high speed scanning , which detect blood flow by analysing signal decorrelations between scans [ 7 , 11 , 14 , 15 ] .
both inner and outer retinal capillary plexuses are imaged in contradistinction in conventional angiography , which does not effectively image the outer plexus .
this study aimed to compare the use of fluorescein angiography and octa in the diagnosis and quantification of dmi when applying a split - spectrum amplitude - decorrelation angiography algorithm ( ssada )   to improve the detection of flow signals in angiography with the purpose of offering a suitable  sparing  contrast alternative using 3  mm  3  mm octa for clinical investigations of diabetic patients [ 8 , 1520 ] .
this was a retrospective cross - sectional comparative study analysis conducted at federal university of goias and approved by ethics committee of the same institution . signed informed consent was obtained from each subject prior to enrolment .
imaging data were collected from patients who underwent fa and octa on the same day in a tertiary referral retina center in the period between january 1 , 2015 , and july 30 , 2015 .
exclusion criteria included significant cataract ( without surgical indications at the time of examination ) , previous retinal arterial or venous occlusion , inherited macular dystrophy , posterior segment inflammation , and macular degeneration or scarring of any cause .
subjects that presented motion artifacts during octa or poor signal strength were also not included in this study .
all subjects underwent a comprehensive ophthalmologic examination , including best - corrected visual acuity ( bcva ) on etdrs charts , + 78  d noncontact lens slit - lamp fundoscopy , color fundus photography , and fa and oct angiography , on a single day .
color fundus photographs were obtained and fa was performed using a digital retinal camera ( topcon trc 50dx ; topcon , paramus , nj ) .
the central macular nonperfusion area , faz boundaries , and the maximum height and area were manually delineated .
the faz area was measured using a single fa photograph , such as the octa angiogram of the scp and then divided into
( superior to 0.32  mm ) and  small  ( inferior to 0.32  mm ) groups .
group comprised both  moderate  and  severe  etdrs established grading groups [ 3 , 19 , 20 ] .
the quantification of macular nonperfusion in both fa and oct angiography captured images was completed using imagej software ( imagej , national institutes of health , bethesda , md ) .
octa instrument used was the rtvue xr avanti with angiovue software for octa ( optovue , inc . ,
fremont , ca ) , and imaging data were obtained using the split - spectrum amplitude - decorrelation angiography ( ssada )   software .
the algorithm was employed to improve the signal - to - noise ratio [ 8 , 1521 ] .
this instrument operates at ~840  nm wavelength , 70,000 a - scans per second , and a bandwidth of 50  nm .
the tissue resolution is 5  m axially and there is a 15  m beam width .
each b - scan consisted of 304 a - scans for a total of 2  304  304 a - scans per acquisition , with a total acquisition time of approximately 3 seconds .
the scanning area was captured in 3  mm  3  mm sections , and the acquired oct volumes were centered on the fovea [ 811 ] .
a 3  mm  3  mm octa image was used primarily because this size is the one that provides images with higher resolution in the used octa device [ 10 , 11 ] .
the ssada algorithm was used to generate a volumetric rendering of blood flow from the internal limiting membrane ( ilm ) to the choroid , and it allowed the direct visualization of normal and abnormal blood circulation [ 1821 ] . for the octa angiograms ,
it was used the automatic segmentation of the retinal layers at the level of the scp generated by the angiovue software in an orthogonal view . in order to correct for automated segmentation error and projection artifacts ,
the segmentation slab was manually adjusted , using corresponding structural oct b - scans as a guide for the placement of 2 segmentation lines : the inner located at 3  m beneath the internal limiting membrane and the outer boundary at 15  m beneath the inner plexiform layer .
this semiautomatic method permitted the readers to select images that pictured the largest extent of scp for subsequent quantitative analysis .
the dcp image was segmented with an inner boundary 15  m beneath the inner plexiform layer and an outer boundary at 70  m beneath the inner plexiform layer .
this section captured both layers of the outer capillary plexus , which sandwiched the inner nuclear layer .
all statistical analyses were performed using statistical package for social science 22.0 ( ibm spss ; ibm , armonk , ny ) .
fa and octa images were independently reviewed by two independent masked readers retina specialists ( jg and di ) who reached an agreement regarding the area ( mm ) of macular nonperfusion that was obtained using both methods according to etdrs report 11 .
intraclass correlation coefficient ( icc ) was used to estimate the agreement between individual measurements from both readers .
since the icc was consistently > 0.9 between the 2 readers , nonparametric wilcoxon signed - rank test was used to compare area measurements performed by 1 reader on fa and octa 3  mm  3  mm scans .
thirty - four eyes from 34 patients , including 20 ( 58.82% ) females and 14 ( 41.18% ) males , were enrolled and separated according macular status ( table 1 ) .
twenty - four eyes from 24 patients were placed in the group of patients with dmi , including 15 ( 62.5% ) females and 9 ( 37.5% ) males .
the mean ( sd ) age of the dmi population was 61.20  6.95 years .
the group without dmi comprised 10 patients , including 5 ( 50% ) females and 5 ( 50% ) males .
four eyes from 2 patients were excluded from the study due to a high quantity of motion artifacts .
patients with dmi presented a mean fa of 0.68  0.53  mm ( 95% ic , 0.460.90 ; p < 0.05 ) .
octa angiogram analysis demonstrated a mean of 0.58  0.35  mm ( 95% ic , 0.430.73 ; p < 0.05 ) .
the wilcoxon signed - rank test did not identify a significant difference between fa and octa in patients diagnosed with dmi ( p = 0.1374 ) .
patients without dmi presented a mean fa of 0.19  0.67  mm ( 95% ic , 0.140.24 ; p < 0.05 ) .
octa angiogram analysis demonstrated a mean of 0.20  0.79  mm ( 95% ic , 0.150.26 ; p < 0.05 ) .
the wilcoxon signed - rank test did not identify a significant difference between fa and octa in patients without dmi ( p = 0.9594 ) ( figure 1 ) .
the iccs for faz area measurements between 2 observers with respect to fa and octa were 0.96 ( ic : 0.360.71 ) and 0.92 ( ic : 0.350.79 ) , respectively , demonstrating the reproducibility and consistency of the methodology .
this result highlights the notion that the faz is most often clearly demarcated by a distinct foveal vascular ring and that its abnormalities , in addition to its capillary dropout , can be lucidly obtained from octa images .
etdrs research group connected the severity of macular nonperfusion to the potential for progression in dr .
in fact , in dr , the advanced deterioration of macular perfusion is the basis for macular ischemia , and developing a method to perceive perfusion maps may allow correlations between central ischemia and the different stages of dr .
however , it is an invasive method requiring venipuncture and contrast infusion ; it is a time - consuming test and provides only 2-dimensional images .
therefore , reports of anaphylaxis and death related to contrast injections have been documented , despite being rare .
octa is a noninvasive method , obtains highly detailed 3-dimensional images without requiring injection of a contrast dye , and allows faster acquisition of images ( figure 2 ) [ 9 , 10 , 1214 ] .
octa performed using a split - spectrum amplitude - decorrelation angiography ( ssada ) algorithm has already been shown to be useful for imaging microvascular changes in dr [ 811 , 14 , 20 ] .
cole et al . also observed macular nonperfusion in a diabetic patient in a 3  mm  3  mm octa that was centered on the fovea by applying a similar technology .
the 3  mm  3  mm octa central sections obtained using sd - oct allowed us to obtain a higher resolution over a small area ( figure 3 ) .
this area was sufficient for detecting central dmi , but it was not large enough to identify peripheral retinal nonperfusion .
high - resolution oct imaging allows measuring thickness of segmented retinal layers in angiographically apparent ischemic dr .
future octa devices improvements may provide clinicians the ability to obtain wider field images with better resolution . in the present study , statistical analysis
also did not indicate significant difference between area measurements obtained with fa and octa in patients diagnosed with dmi .
the same has occurred among patient without dmi regarding the measurement of normal faz area .
the icc for the zaf area between the 2 observers on fa and octa demonstrated the reproducibility and consistency of used methodology .
these results highlight the notion that faz is most frequently demarcated by a distinct foveal vascular ring and its abnormalities , in addition to its capillary dropout , can be lucidly obtained from octa imaging device .
first , all of the results were obtained during a single appointment , and there was no follow - up .
the present study demonstrates that fluorescein angiography and octa provide similar results when used to diagnose macular ischemia in diabetic patients . with further improvements
in summary , octa may provide images with higher details regarding macular status , becoming a novel imaging technique for the diagnosis of dmi , and may become an alternative to fa for this purpose .
the results also offer improved quantification of faz area in diabetic patients without dmi when compared to diabetic subjects with established macular ischemia .

Answer:
purpose . to compare fluorescein angiography ( fa ) and optical coherence tomography angiography ( octa ) images of foveal avascular zone ( faz ) in patients with diabetic retinopathy ( dr ) with and without diabetic macular ischemia ( dmi )
 . methods . the wilcoxon signed - rank test was used to compare area measurements and p values of < 0.05 were considered statistically significant .
 fa and octa images were independently graded by 2 observers that reached agreement regarding quantitative dmi according established protocols .
 the ischemic area was divided into
 large  macular ischemia ( superior to 0.32  mm2 ) and  small  ( inferior to 0.32  mm2 ) groups .
 quantitative analyses of the faz were performed using custom software .
 results .
 thirty - four eyes from 34 diabetic patients were enrolled .
 subjects with dmi presented a mean area on fa and octa of 0.68  0.53  mm2 and 0.58  0.35  mm2 , respectively ( p = 0.1374 ) .
 patients without dmi presented a mean area on fa and octa of 0.19  0.67  mm2 and 0.20  0.79  mm2 , respectively ( p = 0.9594 ) .
 the icc for the faz measurements between the 2 observers on fa and octa was 0.96 and 0.92 , respectively .
 conclusion .
 octa represents a novel technique for the diagnosis of dmi and it may become an alternative to fa for this purpose .


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:
lymphomatoid granulomatosis ( lyg ) is a rare angiocentric and angiodestructive lymphoproliferative disease with granulomatous reaction involving the lungs most frequently , however , it may also involve the kidneys , skin and especially the central nervous system4,6,9,11 ) .
lyg is characterized by an infiltration of atypical lymphocytoid and plasmocytoid cells , with granulomatous inflammation in an angiocentric and angiodestructive polymorphic cellular infiltrate6,8 ) .
therefore the purpose of this study is to submit the first report about the case with thoracic spinal lyg after diagnosis with b - precursor childhood acute lymphoblastic leukemia ( all ) .
after surgical decompression with biopsy on the spinal lesion , diagnosis of grade ii lyg was made and rituximab was administered .
the patient is in good neurological condition without recurrence at the 6-year follow - up .
a 4-year - old girl was diagnosed with b - precursor all with the involvement of kidneys and pancreas in november , 2004 .
treatment was initiated according to the pediatric oncology group ( pog ) protocol 900612 ) .
on april 12 , 2006 , the patient was admitted to pediatric clinic with a complaint of intermittent fever for 3 days . on april 19 , 2006 , 7 days after the admission , the patient developed a gait disturbance .
neurological examination revealed paraparesis of grade 3 on medical research council ( mrc ) muscle strength grading scale1 ) with increased deep tendon reflex in both ankles , knee jerks , and bilateral positive babinski sign , but her senses were intact .
cerebrospinal fluid ( csf ) findings were as follows : elevation of protein ( 243.3mg / dl ) , and decrease of glucose ( 44mg / dl ) .
the condition was suspected to be an acute inflammatory demyelinating polyneuropathy and was treated with intravenous immunoglobulin ( 400 mg / kg / day ) for 5 days .
however , her paraparesis was aggravated to grade 1 on mrc muscle strength grading scale from april 26 , 2006 , 14 days after the admission , and spinal mri was performed .
t2 weighted mri demonstrated heterogeneous high signal intensity infiltration on t4 vertebral body and subligamental spreading soft tissue mass on t3 - 5 epidural space from t4 posterior vertebral body , causing spinal cord compression ( fig .
1 ) . on april 27 , 2006 , laminectomy of t3 , 4 with open biopsy were performed .
the epidural mass was encircling the spinal cord mainly on ventral surface and was slightly grey to yellow , solid and compact nature .
a histopathologic examination showed an angiocentric distribution of polymorphous lymphoid infiltrate , with extensive necrosis .
immunohistoche mical staining for cd3 and cd20 showed the predominance of mature t cells and atypical b cells ( fig .
hybridization revealed positivity for epstein - barr virus ( ebv ) encoded rna in a few lymphocytes , confirming the diagnosis of grade ii lyg .
one - week post - operative neurologic examination showed paraparesis grade 3 on mrc scale , and bare independent walking but as she still had gait impairment , she received rehabilitation .
after four weeks of the surgery , she restored her normal state and could walk quite well . at that time
, there were no pulmonary lesions , so we suspected the spinal lesion as the primary involvement site .
two weeks after , chest and abdomen ct revealed multiple low density nodular mass on the liver and the lower lobe of the left lung .
histopathologically , lesions were not blastic in appearance and in immunophenotype , and the lesions were negative for cd10 , tdt , and cd22 , so we ruled out the relapse of all .
she was treated with rituximab ( 275mg / m / day , 4 times per week ) for 4 weeks and intravenous high dose methylprednisolone ( 30mg / kg per day for 3 days , 20mg / kg per day for 2 days and 10mg / kg per day for 2 days ) .
chest and abdomen ct showed marked improvement of the lesions 3 months after the treatment compared with the lesions in the images obtained before the treatment .
a follow - up mri study performed 4 months after the operation documented no evidence of residual mass or recurrence ( fig .
3 ) . chest and abdomen ct performed 1 year after the operation showed no demonstrable focal lesion in lung field and mediastinum and unremarkable abdominal organs .
her weakness improved and she was able to walk independently and had no gait impairment .
but unfortunately we failed to take additional follow - up of mri for the patient , as she was terrified to take mri and full sedation was not possible .
moreover the parents of the patient did not agree with the idea of taking mri one more time because she was in good state at that time . to date ,
lyg is a rare lymphoproliferative disease characterized by angiocentric and angiodestructive ebv - positive b - cell proliferation of uncertain malignant potential , which might be associated with exuberant reactive t cell infiltration2,9 ) .
lyg affects men more than women with ratio 2 to 1 and presents mainly in adults between the fourth and sixth decades of life , although patients of all ages can be affected .
its occurrence in children is quite rare4 ) . there were three cases of lymphomatoid granulomatosis with onset after all .
two of these patients died of the disease , only one obtained benefit from acyclovir therapy7 ) .
lyg usually affects lungs primarily , and it may also present several significant extrapulmonary manifestations , affecting skin , kidney , spleen , and the central nervous system .
involvement of the spine has been rarely reported . to my best knowledge , this is the first report of thoracic spinal lyg presenting with unique progressive myelopathy .
however , numerous granulomatous lesions in the central nervous system and lungs are highly suggestive of lyg .
lyg with central nervous system involvement , observed in approximately 30% of the affected patients , indicates poor prognosis4,9,11 ) .
there was only one case report about lyg with spinal involvement . in that case ,
lyg involve cervical spinal cord , and the patient submitted four cycles of adjuvant cyclophosphamide , doxorubicin , vincristine and prednisone ( chop ) .
but the patient died 5 months after the treatment due to systemic progression of the disease7 ) .
a grading system of lyg is based on the number of atypical lymphocytes , ebv - positive b - cells and amount of necrosis5 ) .
pathophysiologically , grade i lesions consist of a polymorphous angiocentric and angiodestructive infiltrate of lymphocytes , plasma cells , and histocytes , with or without eosinophils .
large lymphoid cells or immunoblasts were less than five in high power field ( 400 ) or absent . if large lymphoid cells or immunoblasts were present , there would be no cellular atypia and minimal - to - absent necrosis .
grade ii lesions consisted of large lymphoid cells with less than twenty ebv - positive cells in high power field with some cellular atypia in small lymphoid cells , and necrosis was more common .
grade iii lesions , angiocentric lymphomas , consisted of large atypical cells with more than twenty ebv positive cells in high power field with the marked cytologic atypia in both small and large lymphoid cells4,13 ) . to date , no standard treatment has been established for lyg and treatments approach to systemic lyg include drugs acting on the immune system(corticosteroids , interferon alfa-2b ,
cyclosporine a , anti - cd20 monoclonal antibody - rituximab ) , mono- or combined chemotherapy , radiotherapy , and autologous stem cell transplantation .
several treatments with rituximab have been reported , with or without chemotherapy or radiotherapy3,9,10,15 ) .
rituximab was approved for the treatment of recurrent , poorly differentiated refractory or follicular cd20 positive b - cell non - hodgkin 's lymphoma14 ) . in this case , immunohistochemical staining for cd3 and cd20 showed the predominance of mature t cells and atypical b cells .
in situ hybridization revealed positive ebv encoded rna in a few lymphocytes , confirming the diagnosis of grade ii lyg .
the patient experienced rapid worsening of neurological symptoms and absence of systemic involvement on pre - operative laboratory findings and radiological imaging , and she therefore was treated with surgical decompression and biopsy on the t3 - 5 epidural lesion and improved neurologic symptoms .
surgical decompression can improve neurological morbidity of the patient . however , appropriate chemotherapy is highly needed as systemic progression of lyg make poor prognosis .
the patient responded to rituximab therapy well without adverse effects , and her condition remained stable for 6 years .
therefore it was suggestive that decompression and rituximab is suitable as the initial therapy for symptomatic spinal lyg .
this case described an unusual occurrence of thoracic spinal lyg in a 4-year - old girl .
childhood lyg is a rare disease but should be considered in the nodular pulmonary infiltrates and central nervous system manifestations , especially in the past diagnosis of childhood acute lymphoblastic leukemia .
awareness of the features of lyg can lead to earlier diagnosis and prompt appropriate therapy .

Answer:
lymphomatoid granulomatosis ( lyg ) is a lymphoproliferative disease involving the lungs most frequently ; however , it may also involve the kidneys , skin and especially the central nervous system .
 unique initial presentation of spinal involvement is extremely rare and epidural lesion of thoracic spine has not been reported .
 the prognosis for lyg has been reported to be poor , and there currently exists no satisfactory established treatment protocol . the purpose of this study is to report a case of successful treatment with surgery and rituximab combination therapy in thoracic spinal lyg .


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:
Sequential C–O decarboxylative vinylation/C–H arylation of cyclic oxalates via a nickel-catalyzed multicomponent radical cascade

Paper sections:
Introduction

In the last decade, the nickel-catalyzed cross-coupling reaction has emerged as a powerful technique to construct C-C bonds in chemical synthesis. 1 Notably, nickel-catalyzed multicomponent reactions, that allow for the formation of multiple C-C and/or C-heteroatom bonds thus enabling the streamlined synthesis of complex molecular scaffolds in a single operation, are particularly attractive due to their good compatibility and unique selectivity. 2 Signicant progress has been achieved in the area of catalytic multicomponent difunctionalization of unsaturated systems, enabling the simultaneous, one-pot installation of two functionalities over double bonds or triple bonds via nickel catalysis (Fig. 1). 3,4 In contrast, there has been a lack of reports in which saturated hydrocarbons have been manipulated for cross-coupling at more than one reaction site in one single operation. 5 This catalytic strategy would simultaneously install two sp 3 C-C bonds in saturated hydrocarbons, readily available and abundant building blocks in organic synthesis; nevertheless, the realization of such a strategy requires the ability to overcome the relative inertness of saturated hydrocarbons and to control the selectivity in the presence of multiple similar chemical bonds.

Alcohols have been widely employed as salient synthetic building blocks in chemical synthesis. Notable advances have been made in selective functionalization of sp 3 C-H bonds of alcohols through the hydrogen-atom-transfer (HAT) process. 6,7 Moreover, alcohols can function as latent alkylating agents through transition-metal-catalyzed ipso-C-O activation 8 or homolytic C-O cleavage. 4j,k,9,10 These catalytic approaches enable the facile construction of diverse structural motifs from abundant alcohols. Nevertheless, one single transformation that combines ipso-C-O functionalization and remote C-H functionalization of alcohols, which would provide an attractive platform for the synthesis of highly functionalized sp 3 -rich scaffolds, 11 has not been reported yet. We herein report a selective, cascade C-O bond vinylation/C-H bond arylation of alcohols that achieves site-selective dual carbofunctionalization of simple abundant aliphatic alcohols via a photoredox/nickel 12enabled multicomponent radical cascade process. This strategy relies on a synergistic combination of alkyl oxalate decarboxylation, 1,5-hydrogen atom transfer (1,5-HAT) of the vinyl radical, and C(sp 3 )-C(sp 2 ) cross-coupling. Our original design for this radical cascade strategy is outlined in Fig. 1. Specically, we envisioned that a radical addition of alkylalkyne would give rise to a s-type vinyl radical I, which would prefer to undergo an intramolecular 1,5-HAT process to yield a nucleophilic alkyl radical species II. 13 Subsequent nickel-mediated coupling of alkyl radial II with aryl halides would forge a C(sp 3 )-Ar bond, 12 and nally lead to a sequential two-site functionalization of oxalates, i.e. C-O decarboxylative vinylation & C-H arylation (Fig. 1). We are particularly interested in exploring cyclic alkyl oxalates to construct highly functionalized cycloalkanes, 14 due to two considerations: (i) this synergistic cascade process would undoubtedly expedite the synthesis of challenging aliphatic rings, and more importantly, would provide a novel retrosynthetic disconnection for complex saturated ring systems from simple starting materials; (ii) conformation of cyclic substrates might be benecial for the intramolecular 1,5-HAT migration of vinyl radical species. 15

Results and discussion

We began our investigations by employing tertiary cyclic oxalate 1 as a model substrate to test the possibilities (Table 1).

Pleasingly, we found that irradiation of a DMSO solution of 1, aliphatic alkyne 2, and 4-bromobenzonitrile 3 did afford the desired 1,3-vinylarylation product 4 in 84% yield in the presence of catalytic Ir[dF(CF 3 )ppy] 2 (dtbbpy)PF 6 , NiCl 2 (Py) 4 , 4,4 0 -di-tertbutyl-2,2 0 -dipyridyl (dtbbpy), and bis(4-methoxyphenyl) methanone (Table 1, entry 1). The countercation of oxalates impacted the reaction efficiency to a small extent, while the use of cesium oxalates afforded higher yields than the corresponding Li, Na, K salts (entries 2-4). Further evaluation indicated that both Ni(II) and Ni(0) catalysts were able to promote the desired transformation, with the precatalyst NiCl 2 (Py) 4 proving optimal (entries 5-9). Control experiments demonstrated that the photocatalyst, the nickel catalyst, and visible light were all essential to this synergistic cascade process, as the desired 1,3-vinylarylation products were not observed in the absence of any of these components (entries 10-12). Nevertheless, low conversion (27% yield) was still observed in the absence of the bipyridine ligand (entry 13). The use of bis (4methoxyphenyl)methanone as an additive proved to be slightly benecial to the reaction efficiency (entry 14). 13 With the optimal conditions in hand, we next turned our attention to evaluating the applicability of substrates as well as the potential limitations of this dual functionalization protocol. As shown in Scheme 1, a number of tertiary cyclic oxalates, readily prepared from the corresponding alcohols, could undergo the sequential C-O vinylation/C-H arylation with excellent site-selectivity, installing both vinyl and aryl functionalities onto the skeletons of alcohols under redox-neutral and mild conditions (products 4-14, 36-77% yields). A number of substituents including alkyl, ketal, ketone, and alkene on the cyclohexyl alcohols were tolerated in this dual catalytic cascade system, furnishing multi-substituted cyclic alkanes in a single operation under mild conditions (products 5-9, 36-77% yields). Nonetheless, steric hindrance of the substituents was found to have a considerable effect on the reaction efficiency: installing a methyl group at the aor gposition, or replacing methyl with ethyl at the ipso position of cyclohexyl oxalates resulted in decreased efficiency (products 6-8, 36-54% yields). Pleasingly, saturated O-, S-, and Nheterocyclic oxalates turned out to be viable substrates, yielding the vinyl/aryl-disubstituted saturated heterocycles with good efficiency (products 10-13, 50-76% yields). Moreover, cyclopentyl oxalates were also competent substrates, delivering the 1,3-difunctionalized cyclopentane products in synthetically useful yields (product 14, 40% yield).

Unfortunately, cyclic oxalates with larger or smaller ring sizes (e.g. 4-and 7-membered cyclic oxalates), bicyclic oxalates, and linear oxalates were unsuccessful substrates for this cascade protocol, probably due to the less favorable conformation (for unsuccessful oxalates, see Section 5, page S62 in the ESI †). Interestingly, the reaction of 4-methylenecyclohexyl oxalate with aryl bromides under optimal conditions afforded the 1,5-vinylarylation products in moderate yields (products 15-18, 58-64% yields). Excellent chemoselectivity was observed in this case, with no observations of 1,3-difunctionalized products. We reasoned that allyl-Ni species, generated via intramolecular 1,5-HAT followed by nickel trapping, underwent a selective coupling with aryl bromide  at the terminal position, probably due to steric hindrance, to afford the cyclohexene product. This protocol represents a new and efficient platform to construct highly functionalized saturated heterocycles, important structural scaffolds for bioactive molecules, from simple starting materials. Next, we examined the scope with respect to the alkyne component (Scheme 1). Pleasingly, a wide range of terminal alkylalkynes could be efficiently employed in this cascade protocol, yielding the corresponding trans-alkenes in moderate to good yields (products 19-29, 45-74% yields). Notably, aliphatic alkynes tethered with complex molecules, exemplied by oxaprozin and dehydroabietic acid, also worked with moderate efficiency, further demonstrating the amenability of this synergistic strategy for late-stage manipulations (products 27 and 29, 53% and 50% yield, respectively). Nevertheless, internal alkynes proved to be inefficient substrates, most of which remained intact under the standard conditions.

Finally, we explored the scope of aryl halides in this multicomponent transformation (Scheme 1). Aryl bromides containing electron-withdrawing substituents, including aldehydes, ketones, esters, nitriles, sulfones, and tri-uoromethylates, are competent coupling partners under optimal conditions, delivering the desired products with good efficiency (products 30-35, 53-70% yields). The mild conditions allow for the good tolerance of these important functionalities. This reaction is amenable to heteroaryl halides, selectively installing pyridines and benzothiazoles into cyclohexanes with moderate yields (products 36-38 and 41-42, 50-77% yields). Additionally, (hetero)aryl iodides/chlorides also participated in this sequential C-O/C-H dual functionalization process smoothly (products 39-42, 52-61% yields). Electron-rich aryl halides were applicable coupling partners, albeit with decreased efficiency (products 39-40).

To further demonstrate the synthetic application of our cascade protocol, we have performed several transformations by utilizing the alkene functionality (Scheme 2). The double bond of compound 41 readily underwent selective hydrogenation with H 2 in the presence of a Pd/C catalyst (product 43). Epoxidation of compound 30 with m-CPBA gave epoxide 44 in 65% yield. Furthermore, ozonolysis of 30 led to the formation of aldehyde 45, which could subsequently be oxidized to the corresponding carboxylic acid 46, or be reduced to the related alcohol 47 in good yield.

To shed some light on the potential reaction pathway, we have conducted several mechanistic experiments (Scheme 3). Reaction of ethynylcyclopropane with oxalate 1 and aryl bromide 3 gave the allene product 48, presumably generated via a ring-opening/cross-coupling process, indicating the involvement of vinyl radical species (Scheme 3A). Initially, we assumed that the 1,5-HAT process could be related to the subtle conformation of oxalates. We also found that the expected 1,5-HAT process is reliant on the nature of the vinyl radicals (s-type vs. ptype). For instance, competitive experiments between aryl and aliphatic alkynes showed that arylalkynes exhibited higher reactivity to afford the exclusive formation of 1,2-alkylarylation products, 4j and the 1,5-HAT/coupling product was not observed in this case (Scheme 3B). Regarding the coupling step, we prepared the ligated Ar-Ni(II) complex 50, 16 and found that the stoichiometric reaction of the Ar-Ni(II) complex with alkyne and oxalate didn't form the desired 1,3-disubstituted cycloalkane product 51, suggesting that Ar-Ni(II) might not be a reactive intermediate for this cascade transformation (Scheme 3C). Nevertheless, the Ar-Ni(II) complex was able to catalyze the synergistic cascade reaction, giving the 1,3-disubstituted cyclohexane 33 in 30% yield in acetone (Scheme 3D).

On the basis of these experimental results as well as literature precedents, 12,17 a plausible mechanism for this photoredox/ nickel-catalyzed dual functionalization is depicted in Scheme 4. A thermodynamically feasible SET event between the photoexcited Ir-catalyst B and oxalate C would generate a tertiary alkyl radical D via decarboxylation, followed by a radical addition of alkyne to give rise to a s-type vinyl radical E. The resulting vinyl radical E would go through an intramolecular 1,5-HAT to selectively activate the sp 3 C-H of oxalates and to afford a nucleophilic, secondary alkyl radical species G. Subsequent interception of alkyl radical G by Ni(0) H would generate an alkyl-Ni(I) species I, which then undergoes an oxidative addition with aryl halide J to yield an (alkyl)(aryl)Ni(III) intermediate K. 17 This high-valent Ni(III) complex K would undergo a feasible reductive elimination to forge the C(sp 3 )-Ar bond and furnish the nal product L as well as Ni(I) species M. Finally, a SET event between Ni(I) M and the reduced Ir(II) F would regenerate Ni(0) H and the ground state Ir(III) A to close these two catalytic cycles. At this stage, we could not preclude another pathway that proceeds via oxidative addition of aryl bromide to Ni(0) followed by interception of Ar-Ni(II) N by alkyl radical species to afford the same (alkyl)(aryl)Ni(III) K.

Conclusions

In summary, we have developed a sequential C-O decarboxylative vinylation/C-H arylation of cyclic oxalates via photoredox/nickel dual catalysis. This synergistic protocol enables efficient and selective assembly of both vinyl and aryl functionalities onto saturated cyclic hydrocarbons in one single operation under mild and redox-neutral conditions, providing a new and complementary retrosynthetic method for densely functionalized saturated cyclic hydrocarbons. The mild conditions allow for excellent compatibility of functional groups and substrate scope in the oxalates, alkynes, and (hetero)aryl halides.

Conflicts of interest

There are no conicts to declare.

Answer: A selective, sequential C-O decarboxylative vinylation/C-H arylation of cyclic alcohol derivatives enabled by visible-light photoredox/nickel dual catalysis is described. This protocol utilizes a multicomponent radical cascade process, i.e. decarboxylative vinylation/1,5-HAT/aryl cross-coupling, to achieve efficient, site-selective dual-functionalization of saturated cyclic hydrocarbons in one single operation. This synergistic protocol provides straightforward access to sp 3 -enriched scaffolds and an alternative retrosynthetic disconnection to diversely functionalized saturated ring systems from the simple starting materials.


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:
Single point activation of pyridines enables reductive hydroxymethylation†

Paper sections:
Introduction

Nitrogen-containing heterocycles play an essential role in organic chemistry. They are found in a wide range of pharmaceuticals, drug candidates, agrochemicals, natural products, functional materials, and catalysts.1 Despite considerable synthetic advances, the pharmaceutical industry still requires processes that allow the facile preparation of aliphatic nitrogen-containing heterocycles along with methods that install small but essential functional groups such as –CH2OH and methods which enable the rapid and robust preparation of such useful molecules are of great interest to the synthetic chemistry community (Scheme 1).2

Dearomatisation reactions have garnered considerable interest because they represent an expedient way to prepare complex three-dimensional molecular scaffolds from flat and readily available aromatic compounds.3 Given the particular relevance of piperidines, and the commercial availability of pyridines, the dearomative pathway represents an attractive approach to preparing saturated azacycles.4 In particular dearomative methods that form both C–H and C–C bonds represent an important class of reaction that are particularly versatile and efficient. However, despite their importance,5–12 these types of reactions continue to be underrepresented in the literature.

Recently our own laboratory has reported a new iridium-catalysed interrupted transfer hydrogenation process that leads to the reductive hydroxymethylation of pyridines.13–16 In addition to forming two new C–H bonds, this process also forms a new C–C bond in a single synthetic step. Mechanistic work has shown that the metal catalyst oxidises formaldehyde in methanol to methyl formate, forming a metal hydride in the process.13 Addition of the metal hydride to the C-2 position of the pyridine then forms a key enamine intermediate A. Reaction of the enamine with excess formaldehyde, and subsequent iminium ion reduction then completes the transformation.

As with many dearomative reactions, it is necessary to activate the pyridine substrates to facilitate the initial aromaticity-breaking step. We found that activation could be achieved through a two-point strategy: (i) quaternisation of the pyridine nitrogen to form a highly electron deficient pyridinium salt and (ii) the presence of an electron-withdrawing substituent in the 4-position of the pyridine to further enhance the electron deficiency. The 4-substituent also ensures the initial reduction happens at C-2 exclusively, and prevents over reduction in situ.

While our initial work reported 3-hydroxymethylation of pyridines, the requirement of a strongly electron withdrawing group in the 4-position is a significant limitation. Groups such as esters and ketones work well, however any less electron-withdrawing substituents result in either low yielding or completely inactive pyridines. For example 4-phenylpyridine was poorly active under the reaction conditions (giving product 2a in 15% yield using an iridium catalyst and 27% yield using a rhodium catalyst, Scheme 2) and pyridines with alkyl and heteroatom substituents were completely inactive. 4-Aryl substituents are of particular interest as this motif can be found in molecules such as the anti-depressant drug Paroxetine (Scheme 1).

Results and discussion

In order to address this limitation, we set about developing a single point activation strategy that would enable the use of more electron-rich pyridines, such as 4-phenyl pyridine. From analysis of crude reaction mixtures, it was apparent that the initial dearomative addition of a metal hydride to the pyridine C-2 position was the troublesome step when using less electron-poor substrates. Therefore, our first approach was to utilise metals which have a stronger reductive profile while using N-benzyl-4-phenylpyridinium iodide 1a as a test substrate (Scheme 2).17 In this case, both rhodium (27%) and ruthenium (17%) were found to give higher yields of 2a than iridium (15%).

To further increase the electron deficiency of the heteroaromatic system, while maintaining a single point of activation (i.e. quaternisation), analogous substrates bearing electron-deficient benzyl groups were then prepared and subjected to the metal-catalysed conditions. Using [Ru(p-cymene)Cl2]2 as the pre-catalyst, pleasingly the 2-nitrobenzyl substrate 1b was found to give the product 2b in a higher 40% yield, as did the 2,4-bistrifluoromethyl substrates 1c (2c, 65%) and 3,5-bistrifluoromethyl substrates 1d (2d, 32%). [IrCp*Cl2]2 was found to generate a far less proficient catalytic species, delivering 2c in only 25% yield. Finally, [RhCp*Cl2]2 was also found to deliver 2c in a good yield (65%). After deciding that the 2,4-bistrifluoromethyl activating group gave the best yield, substrate 1c was taken as a model substrate and subjected to further optimisation and screening (Table 1). Ru catalysis was studied further because it is significantly less expensive than Rh.

1H NMR yield using trimethoxybenzene as internal standard.

Using [Ru(p-cymene)I2]2 as catalyst.

Using the bromide salt of 1c.

Isolated yield.

Results and discussion

Initially, the role of KI additive, thought to improve the reducing ability of the metal hydride, was investigated; relative to our initial conditions using 4 equivalents,18,19 the absence of any KI (40%) or the presence of 2 equivalents (60%) led to a reduced yield of 2c (entry 2 and 3). However, increasing KI loading to 6 equivalents (50%) also led to a decrease in yield (entry 5). Using the bromide salt of 1c as starting material, instead of the iodide salt, also led to a slight decrease in yield (61%, entry 6).

Changing the base to potassium methoxide resulted in a drop in yield to 24%. Decreasing the loading of magnesium methoxide to 0.5 equivalents had a limited effect (64%) but increasing the loading to 1 equivalent increased the yield to 70%; a further increase of base equivalents did not increase the yield (entry 8, 9 and 10). Finally, decreasing the paraformaldehyde equivalents to 10 led to a reduction in yield (65%) while increasing to 30 equivalents increased the yield to 72%, giving an isolated yield of 69% (entries 11 and 12). Note that it was necessary to add excess formaldehyde in order to ensure complete reaction of the enamine formed in situ. Although the optimised conditions differ from our initial iridium catalysed work, we believe that mechanistically the reaction process proceeds in a similar fashion (see ESI†).13 With the optimal N-activating group and conditions in hand, a range of 4-aryl substituted pyridinium salts were subjected to the reaction conditions (Scheme 3).

The starting materials were prepared in one step from 2,4-bistrifluoromethylbenzyl iodide and the corresponding pyridine. While the model 4-phenylpyridine 1c gave the product 2c in 69% yield, scaling this reaction to 3 mmol, led to the formation of 2c in an increased yield of 73%. Pleasingly we found that a number of both electron-donating (methyl, tert-butyl, and methoxy) and electron-withdrawing (trifluoromethyl, fluoro, chloro, methyl ester, and sulfone) substituents were well tolerated on the C-4 aromatic group, giving the products in a range of yields (39–78%). Within the C-4 aromatic group, substitution was tolerated at the ortho, meta, and para positions, together with 3,5-disubstitution and 3,4,5-trisubstitution. Of note, substrate 2j bearing a 4-fluorophenyl group was formed in 78% yield and was used as the key step in the synthesis of (±)-Paroxetine, vide infra. In addition, substitution at the 3-position of the pyridine is also tolerated (2r–2u), giving the hydroxymethylated product in reasonable yields (37–55%). In this position aromatic groups bearing ortho, meta, and para substitution along with a n-butyl group were all shown to be viable substrates. The activation strategy utilising the 2,4-bistrifluoromethylbenyl group was then extended to enable the use of pyridines substituted at the 4-position with strongly electron-rich heterocycles. These substrates are completely inactive when using the standard N-benzyl activating strategy which shows the importance of the electron withdrawing N-benzyl derivative described here. Carbazole (2x 52%), indole (2w, 67%), and benzofuran (2v, 49%) C-4 substituents were all used successfully to give the hydroxymethylated products in good yields. These results highlight the ability of this methodology to selectively activate and functionalise pyridines in the presence of other heteroaromatic rings. Furthermore, the N-2,4-bistrifluorobenzyl salt of Rosoxacin ester, a commercially available quinoline antibiotic, was subjected to the reaction conditions and the desired product 2y was isolated in 23% yield, showing that late stage derivatisation of important pharmaceutically relevant molecules can be achieved with this methodology. Moreover, the highly electron-rich 4-methoxypyridine was successfully activated using our strategy to give enol ether 2z in 50% yield. While the corresponding 4-alkyl products 2aa and 2ab, bearing a methyl and tert-butyl group respectively, were also formed, the isolated yields were only 20% and 27% respectively. However, despite these lower yields, they do demonstrate a proof of concept that simple C-4 alkyl-substituted pyridines can now be utilised in this methodology for the first time. Unfortunately substrates lacking a 4-substituent did not undergo hydroxymethylation successfully and gave complex mixtures of products (2ac); we plan to tackle this issue in our future work.

To highlight the versatility of the products, further synthetic transformations were undertaken on a representative example 2c (Scheme 4). The newly installed CH2OH group was converted to the corresponding alkyl iodide 4via an Appel reaction in good yield (66%). Importantly, removal of the activating group was achieved easily through protection of the free –OH group with a benzyl group followed by either treatment with 1-chloroethyl chloroformate and subsequent reaction of the intermediate carbamate with methanol to furnish the free amine 5 in 56% yield over two steps. Alternatively treatment with methyl chloroformate, allowed a protecting group switch to form carbamate 6 in good yield (66%) over two steps. A Mitsunobu reaction of 2c using sesamol, followed by cleavage of the activating group delivered the corresponding amine 7 in 43% yield over two steps. These synthetic manipulations, together with the easy removal of the benzyl derived activating group, enables the wide array of products to be converted into highly useful small molecules.

In order to show the utility of this methodology, we chose to prepare Paroxetine, which is a selective serotonin reuptake inhibitor used as a pharmaceutical drug to treat a variety of conditions.20 Starting with commercially available 4-bromopyridine hydrochloride we prepared (±)-Paroxetine using our methodology in only six steps and 20% yield (Scheme 4). Following the preparation of 2j, three simple and efficient transformations gave Paroxetine. The first of these was a key substrate controlled hydrogenation using Crabtree's catalyst to deliver the piperidine 8 (3 : 1 dr, from which the pure trans isomer of 8 was isolated in 65% yield). Finally, the synthesis was completed with a Mitsunobu reaction to install the electron rich aryl group (84%) and removal of the activating group using ACE-Cl and methanol (55%). In order to probe the mechanism of the reaction we have subjected 1j to the reaction using [CD2O]n and observed deuterium incorporation on C-2, C-6 and the exocyclic hydroxymethyl group. This was consistent with our proposed mechanism and also provided a convenient method to access deuterated Paroxetine if so desired (see ESI pp56†).

Conclusions

In conclusion, we have developed a new catalytic system and shifted the boundaries of the dearomative hydroxymethylation reaction, expanding the scope far beyond what was possible in our initial work. Ranging from 4-arylpyridines to the electron-rich 4-methoxypyridine the hydroxymethylation proceeded in good yields. The key aspect of the work involves the use of a ruthenium catalyst in conjunction with a highly electron-deficient 2,4-bistrifluoromethylbenzyl activating group. This bespoke activating group can be readily removed to furnish the free amine. Finally, we have utilised this highly useful methodology in the synthesis of (±)-Paroxetine and plan to employ it further to make more medicinally relevant molecules and natural products.

Conflicts of interest

There are no conflicts of interest.

Answer: The single point activation of pyridines, using an electron-deficient benzyl group, facilitates the ruthenium-catalysed dearomative functionalisation of a range of electronically diverse pyridine derivatives. This transformation delivers hydroxymethylated piperidines in good yields, allowing rapid access to medicinally relevant small heterocycles. A noteworthy feature of this work is that paraformaldehyde acts as both a hydride donor and an electrophile in the reaction, enabling the use of cheap and readily available feedstock chemicals. Removal of the activating group can be achieved readily, furnishing the free NH compound in only 2 steps. The synthetic utility of the method was illustrated with a synthesis of (±)-Paroxetine.


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 well known that genes can be silenced by antisense rna oligonucleotides called small interfering rna ( sirna ) ( 1,2 ) . in order to design an efficient sirna sequence , empirical rules based on the features of the sirna sequence
have been discovered , including , for example , low g / c content , lack of self - structure , preference of a at position 3 , absence of g or c at position 19 and asymmetry in the stability of the terminal base pairs ( 310 ) .
the self - structure of the target and oligonucleotide is also an important consideration for the effective binding ( 1115 ) .
it is desirable to select an oligonucleotide having high accessibility to the target - binding site and low duplex stability . here
, the oligowalk server , which predicts efficient sirna sequences using an accessibility calculation with a convenient web interface , is described .
overall , the positive predictive value of the server is 0.786 , meaning that 78.6% of the sirnas selected by the server will be efficient at silencing ( 16 ) .
the positive predictive value was determined by testing against a database of sirna experiments conducted under diverse experimental conditions ( 17 ) . in the calculation of the oligowalk server ,
unimolecular and bimolecular self - structures for the sirna are considered along with unimolecular self - structure in the target at the oligonucleotide binding region ( 16 ) .
oligowalk predicts the free energy changes ( g ) involved in these equilibrium states ( 18 ) .
the predicted thermodynamics ( g ) , plus the oligonucleotide sequence features ( 19 ) , are then utilized to predict sirna efficacy for candidate sirna sequences ( 16 ) , which are generally 19 nucleotide duplexes with 3 dinucleotide dangling ends ( 7 ) .
a support vector machine ( svm ) program ( 20 ) is embedded in the server to take the thermodynamic and sequence features as input .
the svm classification model used in the server has been proven to be able to predict efficient sirna ( greater than 70% inhibition of the target mrna expression ) with high accuracy ( 16 ) .
the svm was trained on a sirna database that contains 2431 experimental results conducted in human cells at 37c ( 10 ) .
the output of the oligowalk server is a table of sirna candidates , showing the sirna sequences and the probabilities of being efficient ( having silencing efficacy larger than 70% ) .
each of the free energy change terms for each candidate is also listed in a separate table .
the oligowalk server uses the cgi ( common gateway interface ) module of perl for taking user input and submitting calculations from the homepage .
the input of oligowalk server is the rna sequence of the target gene . only a , u , t , g and c are the acceptable types of nucleotides in the sequence ( the server will replace the nucleotide t with u for calculations ) , and the maximum sequence length is 10 000 nucleotides .
an email address is required because the server sends an email to the user when the calculation is completed .
submit query , the server generates a list of efficient sirna candidates for the target gene .
jobs are submitted by the server to a cluster of seven nodes with 3.2 or 3.4 ghz pentium 4 processors running fedora linux ( http://fedoraproject.org/ ) , managed by sun grid engine ( http://gridengine.sunsource.net/ ) .
when the calculation is complete , an html ( hypertext markup language ) page is generated with links to tables containing predicted sirna efficacy data and thermodynamic binding data . in the sirna efficacy table ( figure 1 ) , the sequences of sirna candidates are ranked in the output list by their probabilities of being efficient sirna .
the probabilities are predicted by a svm embedded in the web server for selecting efficient sirna .
the classification model ( 16 ) used in the svm was trained with a publically available database ( 10 ) , using thermodynamic and sequence features of sirna candidates . the position number of each sirna candidate is also listed in the table as the index of the 5 most base in the target - binding region .

the sequences of sirna are ranked from top to end by their probabilities of being efficient ( antisense efficacy larger than 70% ) . a table of sirna candidates generated by oligowalk server .
the sequences of sirna are ranked from top to end by their probabilities of being efficient ( antisense efficacy larger than 70% ) .
in addition , the predicted equilibrium thermodynamics table is generated as a reference for advanced users . in the table
 overall ( in kcal / mol ) is the overall free energy change of oligonucleotide - target binding ,   when all contributions are considered , including breaking target and oligonucleotide self - structures ( 18 ) .
 duplex ( in kcal / mol ) is the free energy change of hybridized duplex between oligonucleotide and target ( antisense  sense duplex ) , .
the value is independent of oligonucleotide concentration because it is a standard free energy change .
tm - dup ( in c ) is the melting temperature in degrees for the duplex formation of oligonucleotide and target .
break - targ. ( in kcal / mol ) is the free energy cost to open the intramolecular target base pairs for oligonucleotide binding , .
a more negative number indicates higher free energy cost , which is unfavorable for oligonucleotide - target binding .
intraoligo ( in kcal / mol ) is the free energy change of intramolecular oligonucleotide structure , .
it usually has a negative value or , if there is no favor - able intramolecular structure , it is zero .
interoligo ( in kcal / mol ) is the free energy change of intermolecular oligonucleotide structure , .
antisense bimolecular structure , which decreases the oligonucleotide - target ( antisense  sense ) binding affinity .  end_diff. ( in kcal / mol ) is the free energy difference between the 5 and 3 end of the antisense strand of sirna , with windows of two base pairs .
functional sirna prefer to have an unstable 5 end ( 3 ) , which means a positive end_diff .
 prefilter_score is the score calculated with a method based on the empirical rules by reynolds et al . ( 7 ) .
( 7 ) , except for the melting temperature of intramolecular oligonucleotide self - structure because the free energy ( 21 ) and enthalpy parameters ( 22 ) used by oligowalk are more recent .
when calculating the prefilter score , 57c is used as the cutoff of the intramolecular oligonucleotide melting temperature , as suggested in another study ( 23 ) . as an example
, the prediction of the webserver is compared with experimental results in figure 2 . in the experiment ( 3 ) , sirna were tested for efficacy against the target mrna , human cyclophilin ( genbank i d : m60857 ) , at 37c .
the inhibition efficacy of each sirna is defined as 100% minus the percentage of mrna level after sirna application as compared to matched control .
the prediction result is the probability of being efficient ( having inhibition efficacy larger than 70% ) , which is calculated with the server . in figure 2 , most of the sirna with high inhibition efficacy are predicted to have high probability of being efficient .

figure 2.a comparison between the prediction and experimental result . in blue is the experimental result ( inhibition efficacy ) for an mrna target , human cyclophilin ( genbank i d : m60857 ) , at 37c ( 3 ) .
the inhibition efficacy is defined as 100% minus the percent level of mrna expression after sirna application as compared to a control . in red
is the probability ( ranging from 01 ) of being efficient ( having inhibition efficacy larger than 70% ) , as predicted by oligowalk .
a comparison between the prediction and experimental result . in blue is the experimental result ( inhibition efficacy ) for an mrna target , human cyclophilin ( genbank i d : m60857 ) , at 37c ( 3 ) .
the inhibition efficacy is defined as 100% minus the percent level of mrna expression after sirna application as compared to a control . in red
is the probability ( ranging from 01 ) of being efficient ( having inhibition efficacy larger than 70% ) , as predicted by oligowalk .
advanced options ( figure 3 ) are available for users who understand the underlying calculations and would like to test novel hypotheses . the option form is written in html , embedded with javascript controlling the options so that they are context - aware .
the oligonucleotide concentration does not affect the result of sirna sequence design , because the inputs to the svm are standard free energy changes , g . concentration changes do , however , alter the overall free energy change ( ) , which is provided to the user with the thermodynamic details table .
the sense  antisense duplex free energy changes are calculated without considering the self - structures of the target . this mode is not recommended for an accurate sirna prediction .
it is more rigorous to consider the accessibility of the target and oligonucleotide self - structures because binding affinity is lost to open existing base pairs in the target and oligonucleotide .
the second mode is to break local structure . in this case , the structure of target is fixed and only the base pairs on the binding site will be broken without refolding the global structure of target . the final and most rigorous mode is to refold the target rna . in this mode ,
the rna target is folded before oligonucleotide binding and refolded afterwards for each possible oligonucleotide to consider complete equilibration .
the default setting of sirna design is shown in the figure . if the target rna secondary structure is considered , three different prediction methods are available to calculate the free energy change of target self - structure .
the first method is optimal structure prediction , where only the optimal structure ( lowest free energy structure ) of the target is considered to calculate the free energy cost of opening the base pairs of binding region .
the second method considers a set of suboptimal structures to determine the free energy cost .
each structure 's free energy cost is weighted according to the free energy change of the structure to arrive at the ensemble cost .
for this option , at most 1000 suboptimal structures ( within 10% free energy difference from the optimal structure ) are generated with a heuristic method ( 24 ) .
the number of suboptimal structures will be listed in the output table if the target is folded with suboptimal structure prediction method .
constrained target structure is the refolded structure where the binding region is forced to be single - stranded , so that the oligonucleotide can bind to it .
this is the most rigorous method because it considers every possible secondary structure in the folding ensemble , with boltzmann weight .
the structure prediction only folds a certain total number ( folding size ) of nucleotides centered at the binding region .
the user can define this number , but the largest folding size is 1000 nucleotides for the webserver in order to save compute time .
users can define longer folding sizes by downloading and installing the oligowalk program to a local machine .
( 7 ) can be used to rule out nonefficient sirna candidates before folding the target sequence , i.e. the sirna sequences having score less than six points will not be considered for the folding step .
it is suggested to turn on the prefilter option to save considerable computation time ( table 1 ) .
furthermore , the scan region can be redefined if the user is interested only in a specific region of the target .

table 1.calculation size and time of oligowalk server for different target sequencestarget mrna ( genbank id)sequence length ( nucleotide)time ( h : min : sec)memory ( mb)nm_0205487300:57:1793m608578513:55:53110nm_00287012116:09:25112nm_00246721896:36:42113aj27221234606:53:05117the benchmarks were performed with the default options : the oligonucleotide was a 19 base rna ; the folding size of the target was 800 nucleotides centering on the binding site ( full length if the whole target has less than 800 nt ) ; the partition function calculation was conducted ; the entire mrna was scanned ; and the prefilter was on . the time cost changes little for long sequence because the prefilter ( 7 ) is on and number of candidates being folded is limited to about the same number for each sequence.the calculations were submitted and benchmarked on the oligowalk web server ( http://rna.urmc.rochester.edu/servers/oligowalk ) .
each node has a 3.2 or 3.4 ghz pentium 4 processor running fedora linux.the calculation for sequences less than 800 nucleotides is relatively fast because the dynamic programming arrays are reused for calculations of short sequences ( 16 ) .
calculation size and time of oligowalk server for different target sequences the benchmarks were performed with the default options : the oligonucleotide was a 19 base rna ; the folding size of the target was 800 nucleotides centering on the binding site ( full length if the whole target has less than 800 nt ) ; the partition function calculation was conducted ; the entire mrna was scanned ; and the prefilter was on .
the time cost changes little for long sequence because the prefilter ( 7 ) is on and number of candidates being folded is limited to about the same number for each sequence .
the calculation for sequences less than 800 nucleotides is relatively fast because the dynamic programming arrays are reused for calculations of short sequences ( 16 ) .
the oligowalk web server predicts the hybridization thermodynamics of an oligonucleotide binding to a complementary target rna using the most recent rna folding parameters ( 21 , 22 ) .
it predicts efficient sirna with high accuracy using a transparent implementation of an svm ( 16 ) , which considers both sequence and thermodynamic features .
the calculation time and memory size of oligowalk are shown in table 1 for a sample of mrna sequences .
the prefilter ( 7 ) that uses local sequence information to narrow down the list of sirna candidates before calculating the equilibrium affinity is used by default .
for example , the server takes 3 h and 43 min for a complete scan of all possible sirnas on an mrna having 730 nucleotides using the partition function calculation . for the same sequence ,
the algorithm time scales o(mn ) and the memory use scales o(n ) ( table 1 ) , m is the number of candidates and n is the value of folding size .
the time and memory costs change little with sequence because the same folding size ( e.g. 800 nucleotides ) is used and the prefilter ( 7 ) is turned on , which limits the number of candidates to be folded in a way that is apparently independent of target length .
there is currently significant interest in using sirna for both basic science and medical research .
the fact that not all sirna duplexes will function in silencing means that there is a significant cost in trial and error for sirna design .

Answer:
given an mrna sequence as input , the oligowalk web server generates a list of small interfering rna ( sirna ) candidate sequences , ranked by the probability of being efficient sirna ( silencing efficacy greater than 70% ) .
 to accomplish this , the server predicts the free energy changes of the hybridization of an sirna to a target mrna , considering both sirna and mrna self - structure .
 the free energy changes of the structures are rigorously calculated using a partition function calculation . by changing advanced options , the free energy changes
 can also be calculated using less rigorous lowest free energy structure or suboptimal structure prediction methods for the purpose of comparison .
 considering the predicted free energy changes and local sirna sequence features , the server selects efficient sirna with high accuracy using a support vector machine .
 on average , the fraction of efficient sirnas selected by the server that will be efficient at silencing is 78.6% .
 the oligowalk web server is freely accessible through internet at http://rna.urmc.rochester.edu/servers/oligowalk .


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:
craniopharyngioma is an uncommon tumor of the nervous system ; it is well - known to recur even several years after surgery .
we are here with reporting a case of craniopharyngioma which recurred at a site removed from the original site 5 years after surgery and radiotherapy .
a 4-year - old girl was admitted for progressive deterioration of vision of 2 months duration . in addition
there was no history of endocrinopathy , fits or any symptom of raised intracranial pressure . on examination ,
visual acuity was questionable perception of light on the right side and counting fingers at 3 m distance on the left .
imaging revealed a solid and cystic craniopharyngioma in the sellar - suprasellar region [ figure 1a and b ] .
she underwent a right frontotemporal craniotomy and transsylvian exploration and almost total excision of the tumor .
postoperative mr showed a tiny residual tumor adherent to the pons [ figure 2a and b ] .
( a and b ) showing the preoperative images ( before first surgery ) ( a and b ) images after first surgery showing no residual tumour in the primary site but a small fragment adherent to the pons she was given a course of radiotherapy for this residue 54 gy in 30 fractions .
follow - up imaging at the end of 2 years did not reveal any residual tumor [ figure 3 ] .
two years after surgery and radiotherapy no recurrence imaging was being done periodically to check for recurrence .
the 5-year surveillance imaging showed a recurrence in the right sylvian fissure along the route taken during the first surgery .
there was no evidence of tumor in the sellar - suprasellar area [ figure 4a and b ] .
she underwent reexploration by the same route , and a tiny fragment densely adherent to the middle cerebral artery was left behind .
( a and b ) showing remote recurrence five years after first surgery and radiotherapy .
although craniopharyngiomas are benign tumors they are known to recur even after years and even after the administration of radiotherapy , recurrence rates ranging from 25% to 70% .
recurrences at a site removed from the original site are very rare < 25 cases have been reported .
these ectopic recurrences are not to be misinterpreted as ectopic primary occurrences since craniopharyngioma can occur anywhere along the obliterated rathke 's pouch
. these ectopic recurrences may occur along the surgical pathway or at a site , not along the surgical pathway .
the cells of the tumor may get implanted and may subsequently metamorphose into a fresh neoplasm .
these tumor cells may in turn give rise to the regrowth of the tumor . the usual time to recurrence is around 4 years . but why this has to be a peculiarity of craniopharyngiomas can not be explained . another way the tumor may get seeded at a distant site
the evidence for this is strong since tumor cells have been observed in the csf .
although most recurrences are along the surgical corridor an instance where the recurrence has occurred in the spine has been recorded .
when the transsphenoidal route is used , the csf spaces are not violated this may explain the absence of recurrences after transsphenoidal route .
it is not surprising to observe that the histologic examination of the primary and recurrent lesions are the same .
recurrences rates are said to be low after total surgical excision . but recurrences even decades after a quiescent period are well - known .
it can be assumed that radiotherapy would have  sterilized  the surgical corridor and ectopic recurrences will not occur .
but this was not the case in our patient , and in the few that have been reported .
total excision is not an assurance that recurrences at ectopic or primary site will not occur .
certain measures have been proposed to minimize these ectopic recurrences , protecting the operative field with patties to prevent seeding , emptying the cyst prior to removal of tumor , thorough irrigation of the field before dural closure .
probably , a higher mib-1 index and expression of p53 may predispose to these recurrences . but eternal vigilance and regular imaging are mandatory to detect recurrences . another point worthy of note
is why this phenomenon is not seen with respect to other benign tumors like meningiomas or even malignant tumors .

Answer:
the aim was to present a rare case of recurrent craniopharyngioma remote from the primary site of origin .
 a young girl was operated for sellar region craniopharyngioma . for a small residual tumor
 , she underwent radiotherapy .
 follow - up imaging did not reveal any residual tumor or recurrence .
 surveillance magnetic resonance imaging after 5 years revealed a recurrence in the right sylvian fissure .
 this tumor was totally excised .
 recurrence of craniopharyngioma is well - known , but recurrence at a site remote from the original site after radiotherapy is extremely rare .
 one such case is being presented .


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:
insulinoma is a rare neuroendocrine tumor of the pancreas , accounting for 2% of all pancreatic tumors .
its occurrence is 1:100,000 and it is more common in women over 50 years of age .
insulinomas are usually solitary benign tumors , and less than 10% of them are malignant2,3 ) .
malignant insulinomas are diagnosed when metastases occur , but distinguishing malignant insulinomas from benign insulinomas is difficult histologically .
focal nodular hyperplasia ( fnh ) represents a localized liver cell hyperplasia containing central and/or stellate fibrous scar4 ) .
children account for only 7% of cases of this tumor , and fnh comprises only 2% of all pediatric liver tumors5,6 ) .
we report a case of an insulinoma in an 11-year - old child who subsequently developed multifocal nodular lesions of the liver after resections of the insulinoma .
an 11-year - old girl was admitted for evaluation of recurrent seizures that had begun 5 months before admittance , especially in the morning , despite administration of oral antiepileptic medication .
although brain magnetic resonance imaging ( mri ) and electroencephalogram studies were all normal 2 months before admittance , she again had a seizure and was prescribed with oral levetiracetam to control her seizure attacks .
ten days prior to admittance , the patient had been brought to the emergency department due to an ongoing seizure .
her serum glucose level was 35 mg / dl and she was controlled seizure by intravenous infusion of 10% dextrose fluids .
after discharge , she experienced 3 additional attacks of seizures that had been relieved by grape juice .
upon admittance , her height was 161.3 cm ( > 97th percentile ) , and her weight was 47.6 kg ( 75th < percentile<90th ) and her body mass index was 18.8 kg / m ( 50th < percentile<75th ) . all vital signs and the physical characteristics of the organs were all normal .
initial laboratory results , including a serum glucose level of 104 mg / dl , were unremarkable .
a 72-hour fast test was initiated , and the patient demonstrated drowsiness , trembling , and tachycardia after less than 6 hours .
laboratory test results during the fast period showed hyperinsulinemic hypoglycemia without ketonuria : the serum insulin level was 50.1 u / ml ( normal range , 2.0 - 25.0 u / ml ) , serum c - peptide level was 1.81 ng / ml ( 0.59 - 1.56 ng / ml ) , and plasma glucose level was 21 mg / dl ( 70 - 100 mg / dl ) .
subsequent glucagon stimulation test results revealed an increase in insulin level to 85.8 u / ml .
the mri studies revealed a solid mass , 3 cm2.4 cm in size , with lobulated margins , in the pancreatic tail ( fig .
1 ) , with no metastasis , while abdominal ultrasonography ( us ) and computed tomography ( ct ) results were normal .
the tumor was easily detected due to its superficial position at the end of the pancreatic tail , and no enlarged lymph nodes were detected around the pancreas or metastatic lesions in the abdominal cavity .
the pathologic examination indicated that the resected tumor ( 3 cm3 cm2.5 cm in size ) was multilobulated and not encapsulated , and no necrosis was evident .
the tumor was of neuroendocrine origin , well differentiated , with an intermediate grade ( mitoses 4/10 high - power fields ) with angioinvasion .
the ki-67 index , a cellular marker for proliferation which < 3% means a low grade tumor , was 1% ( fig .
, fasting serum glucose levels were maintained between 110 to 140 mg / dl without any treatment .
follow - up mri studies were conducted 2 months after tumor removal , and revealed multiple hypointense nodular lesions in the hepatic segments s5 , s6 , and s8 ( fig .
us - guided needle biopsy of the liver yielded a diagnosis of the lesions as fnh , with no necrosis or mitotic activity was noted . since then
the patient has been free of hypoglycemia for 2 years , and there was not the learning problem in her school .
recent mri studies have shown that the fnh lesions have decreased in size , without any evidence of metastasis .
the classical diagnosis of insulinomas is based on whipple 's triad : hypoglycemia of serum glucose lower than 50 mg / dl , neuroglycopenic symptoms ( such as behavioral or personality changes and seizure ) , and prompt alleviation of symptoms after administering glucose .
malignant insulinoma is extremely rare in the pediatric population , with only a few cases reported in the english literature .
distinctions between benign and malignant insulinomas are based on metastasis or the invasion of the primary tumor into the lymph nodes , tissues , or other organs8 ) .
insulinomas larger than 3 cm are also more likely be malignant , with local invasion or metastases to the liver9 ) . recently ,
janem et al.3 ) reported a pediatric case of malignant insulinoma in the context of a literature review of 9 preceding cases . among the nine cases , five cases showed liver metastasis , two cases showed capsular invasion , and data for the remaining two cases were not available .
considering the criteria of malignant insulinoma , which require metastasis or local invasion of adjacent lymph nodes or tissues , these five cases with liver metastasis - together with the case reported by janem et al.3 ) involving metastasis to the liver , bone , and bone marrow - may be the only true reported malignant insulinomas to date .
however , sata et al.10 ) and janez11 ) reported cases of malignant insulinomas initially diagnosed as benign , that recurred as liver metastases 8 and 15 years , respectively , after initial removal .
neither of these studies confirmed metastasis or invasion during the initial surgery or in subsequent histologic exams of the initial specimens .
thus , insulinomas may be considered as tentatively malignant because these rare tumors have unpredictable features and progression through the body .
neuroendocrine tumors have recently been identified as risk factors that are strongly associated with aggressive tumor behavior leading to malignancy12 ) .
histopathologic grading of tumor cells based on mitosis may aid in prediction of future insulinoma outcomes , as the biologic behavior of low - grade tumors is often rather nonaggressive , whereas high - grade tumors are very aggressive12 ) .
our histopathologic findings showed a well - differentiated , intermediate neuroendocrine tumor of the resected insulinoma and our operation field findings revealed no evidence of metastasis .
nevertheless , the possibility of malignancy was included due to the size of the tumor and its histological angioinvasion . the unusual multifocal hepatic lesions following tumor removal
it has been infrequently documented in adults and children following chemotherapy , radiotherapy , and stem cell transplantation for different types of solid tumors13 ) .
the cause is unknown , although one hypothesis is that obstruction of hepatic vessels or abnormal vascularization could account for fnh , as suggested by the reported association with clinical and anatomic findings like hypoplasia or agenesis of the portal vein , vascular malformations , hemangioma and vascular dysplasia , budd - chiari syndrome , and hereditary hemorrhagic telangiectasia14 ) .
although ct and mri findings of fnh may commonly include the presence of a central scar and typical enhancement patterns , biopsy is necessary in difficult cases with differential diagnosis of liver adenomas and carcinomas .
treatment recommendations regarding fnh are based on longitudinal follow - up of a small series of patients . because fnh is a benign tumor with rare complications , most lesions are followed nonoperatively with serial us or mri , provided that a reliable diagnosis can be achieved using radiologic imaging3,15 ) .
hepatic lesions can induce diagnostic problems when metastasis must be ruled out . in our case ,
hepatic nodular lesions were identified as fnh by needle biopsy , but the association of an insulinoma with the fnh after surgery remains unclear . to our knowledge , this is the first pediatric case report of an insulinoma in which diagnosis was confused due to newly discovered hepatic lesions after tumor resection .
close observation and follow - up imaging studies are required in patients who show malignant potential on histopathologic findings .

Answer:
insulinoma , which arises from insulin - producing pancreatic beta cells , is a rare tumor in children .
 only 5%-10% of insulinomas are malignant and undergo metastasis .
 we report a case of an 11-year - old girl who experienced hypoglycemia - related seizures induced by an insulinoma ; after resection of the primary tumor , she developed hepatic focal nodular hyperplasia ( fnh ) .
 laboratory test results indicated marked hypoglycemia with hyperinsulinemia . abdominal ultrasonography ( us ) and computed tomography results were normal ; however , magnetic resonance imaging ( mri ) showed a solid mass in the pancreatic tail .
 therefore , laparoscopic distal pancreatectomy was performed .
 two months after the surgery , an abdominal mri revealed multiple nodular lesions in the liver .
 an us - guided liver biopsy was then performed , and histological examination revealed fnh without necrosis or mitotic activity .
 the patient has been free of hypoglycemia for 2 years , and recent mri studies showed a decrease in the size of fnh lesions , without any evidence of metastasis .
 even though no metastatic lesions are noted on imaging , close observation and follow - up imaging studies are required in a child with insulinoma that has malignant potential on histopathologic findings .


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 humans , cryoglobulinemia can be responsible for a membranoproliferative glomerulonephritis ( mpgn ) , which is characterized by the formation and/or deposition of immunoglobulins ( igs ) and complement in the glomeruli , followed by an influx of inflammatory cells [ 1 , 2 , 3 ] .
since the early studies to investigate complement activation by immune complexes in experimental nephrotoxic nephritis , the complement system has been considered to play an important proinflammatory role in the glomerulus [ 5 , 6 ] . in cryoglobulinemia , however , there is no clear evidence that complement activation is central in mediating tissue damage by immune deposits .
results have been obtained in mice by trendelenburg et al . , who analyzed the role of complement in the pathogenesis of initial glomerular inflammation induced by murine 619 igg3 monoclonal cryoglobulins .
their results indicated that complement c5 has a predominant role in neutrophil recruitment as c5-deficient mice had significantly reduced glomerular infiltration by polymorphonuclear neutrophils .
this is of particular interest , as the monoclonal complement c5-blocking antibody eculizumab could offer a promising therapeutic option for patients with cryoglobulin - induced glomerulonephritis .
cryoglobulinemia is a rare disease and is characterized by igs that precipitate in the cold .
depending on the clonality of the precipitating igs , three types of cryoglobulinemia can be distinguished .
type i cryoglobulinemia consists of a single monoclonal cryoprecipitable ig , whereas type ii and iii cryoglobulins are mixed , i.e. composed of monoclonal ( type ii ) and polyclonal ( type iii ) antibodies with rheumatoid factor activity against other igs .
mixed cryoglobulinemia may be associated with numerous infectious and immunological diseases as well as lymphatic malignancies .
in particular , mixed cryoglobulinemia type ii is strongly associated with chronic hepatitis c , and nowadays rarely appears as an isolated disease , i.e. the so - called essential cryoglobulinemia .
clinically , cryoglobulinemia manifests itself with symptoms like weakness and arthralgias and variable cutaneous and visceral organ involvement [ 9 , 10 ] .
it is poorer in patients with renal involvement , liver failure , lymphoproliferative disease and malignancies .
steroids suppress inflammation with success in some patients , but additional plasmapheresis to remove circulating cryoglobulins and immunosuppressive treatment to inhibit the formation of new cryoglobulins are often necessary [ 11 , 12 , 13 ] .
, usa ) is a monoclonal antibody that targets complement c5 and prevents the cytolytic and proinflammatory effects of complement activation .
it effectively reduces intravascular hemolysis , thereby improving anemia and quality of life and decreases transfusion requirements [ 15 , 16 ] .
since the initial validation of inhibition of a complement component as a therapeutic target , the field for the use of eculizumab has been expanded . recently
, eculizumab has been approved from the fda for the treatment of patients with atypical hemolytic uremic syndrome , in whom the control of complement activation is insufficient .
furthermore , eculizumab has been successfully used preemptive prior to kidney transplantation [ 18 , 19 ] or postoperatively to prevent disease recurrence in the allograft [ 20 , 21 , 22 ] in several cases with complement disorder - associated atypical hemolytic uremic syndrome . here , we report on an exceptional case of mixed essential cryoglobulinemia with severe glomerulonephritis characterized by a prominent infiltration of the glomeruli by neutrophils . a 1-patient clinical trial of eculizumab was attempted to investigate whether complement inhibition would be sufficient to inhibit this relapsing glomerulonephritis .
the initial partial response was , however , not sustained , and the patient returned to intermittent plasmapheresis .
a 55-year - old woman was diagnosed with a mixed type ii cryoglobulinemia ( monoclonal igm - kappa and polyclonal igg ) and immune complex - mediated glomerulonephritis in may 2006 .
she developed an acute nephritic syndrome with pathological urinary sediment ( i.e. dysmorphic erythrocytes , cellular casts and leucocytes ) , high proteinuria ( 7 g / day ) , arterial hypertension , volume overload and moderate renal function impairment , which got worse over time .
further , she suffered from weakness and arthralgias , but other organ manifestations besides the kidney were not present .
the first renal biopsy showed endocapillary hypercellularity with infiltration of mononuclear cells , an unusual high number of neutrophils , and subendothelial immune deposits , which were positive for igm as well as complement c3 and c5b-9 , resembling acute poststreptococcal glomerulonephritis ; however , classical humps were missing ( fig .
extensive screening for an infectious disease ( i.e. serological testing and pcr for hiv , hcv , hbv , cmv and ebv ) and malignancy ( bone marrow biopsy , computed tomography of the chest and abdominal organs ) was negative .
further , autoantibodies were negative ( ana , anca , anti - dna , antiphospholipid antibodies ) except for rheumatoid factor , with a titer > 1/10,000 .
a second renal biopsy 1 year later showed early stage mpgn with a still high number of neutrophils , which was interpreted as a progression of disease .
the patient was unresponsive to high - dose steroids and subsequently received cyclophosphamide pulse therapy , mycophenolat mofetil and two courses of the monoclonal anti - cd20 antibody rituximab . these immunosuppressive therapies all failed to get the cryoglobulin - induced glomerulonephritis under control .
on october 2006 , once to twice weekly plasmapheresis was started with success . however , plasmapheresis was complicated by two catheter - related blood stream infections .
in addition , she suffered from headache and complained about unpleasant diaphoresis during the procedures .
at the request of the patient , plasmapheresis was interrupted several times with prompt disease relapses manifested by an increase in serum creatinine and proteinuria , and reinstitution of intensive plasmapheresis .
thus , we designed a protocol to test whether eculizumab could replace plasmapheresis at the time of a relapse .
the protocol was approved by the local ethical committee , and informed consent was provided in accordance with the declaration of helsinki .
criteria for treatment success were : ( i ) prevention of progression as determined by maintenance or improvement in renal function , ( ii ) reduction of proteinuria , and ( iii ) no appearance of new extrarenal disease manifestations . in june 2011 ,
the study was started after the last series of plasma exchange , with a continuous surveillance of renal function and clinical signs of relapse .
as expected from previous intervals between plasmapheresis series , it took 3 weeks until relapse .
eculizumab was dosed at 900 mg intravenously every 7 days for 4 weeks , at 1,200 mg 7 days later , and another dose of 1,200 mg 14 days later . at that time of initiating eculizumab therapy ,
mol / l ( egfr 13 ml / min ) , proteinuria was 8 g / day and the patient still had nephritic urinary sediment .
the patient had been vaccinated against neisseria meningitis 14 days prior to receive the first dose of eculizumab .
2 shows the clinical course and renal function ( i.e. serum creatinine and proteinuria ) , before and after initiating eculizumab .
blood samples were drawn to control blood concentrations of eculizumab and blockage of the complement system during the treatment .
complement activity was shown to be blocked using a hemolytic assay ( ch50 ) and by the drop to background of the high levels of soluble c5b-9 ( microvue complement sc5b-9 plus ; quidel corporation , san diego , calif .
the eculizumab infusions were well tolerated and the patient reported an improvement in her well - being .
nevertheless , without plasmapheresis the blockage of the terminal complement was not sufficient to control the disease .
proteinuria remained at high levels of 68 g / day , hematuria persisted and renal function remained stable for some weeks before deteriorating .
after 7 weeks of therapy , the patient was urgently admitted to hospital because of fluid overload .
to our knowledge , this is the first report of a clinical trial to investigate the monoclonal complement inhibitor eculizumab in a patient with mixed cryoglobulinemia and severe glomerulonephritis .
the unique features of the cryoglobulinemic mpgn in this patient were the resistance to treatments such as steroids , cyclophosphamide and rituximab , the presence of high numbers of neutrophils in two renal biopsies taken at 1-year interval ( on both occasions at the time of a severe relapse ) , and the prompt response to plasmapheresis .
all these features suggested that complement was a major player in the disease process : indeed complement was activated and deposited in the glomeruli , and complement factor c5a is known to be a highly active chemotactic agent for neutrophils .
in addition , recently macrophages have been shown to be central to the nephritis in a model of murine cryoglobulinemic mpgn .
the improvement felt by the patient and the stabilization of the creatinine level after initiation of eculizumab suggested that complement might be involved in the inflammation and tissue damage induced by cryoglobulins . however , it became evident after 7 weeks that there was no improvement , rather a slow deterioration of renal function with ongoing signs of active nephritis in the urinary sediment .
this observation does , however , not exclude a role for complement in cryoglobulin - induced glomerulonephritis .
for instance , we do not know whether complement blockade would prevent a relapse , i.e. whether complement might induce a cascade of inflammatory events , which then become independent of continuous complement activation .
further , one can argue that the blockage of c5 by eculizumab plays a central role to prevent the chemotactic function of c5a for the recruitment of neutrophils .
once they have invaded the glomerulus where the inflammatory processes take place , they act independently of complement c5a , which has no proinflammatory effects .
this would be an argument for a prophylactic and sustained use of eculizumab in patients with cryoglobulinemia when the disease is in remission and against its use as a salvage therapy in disease relapse , to induce a more prolonged remission or reduce the frequency of relapses .
, where the c5-deficient mice had impressively less influx of neutrophils , as their experimental model of cryoglobulin - induced glomerulonephritis rather resembles a model of preemptive blockage of c5a to prevent glomerular inflammation just at the beginning and not when the inflammatory process is fully developed as it is in case of a relapse .
in addition , eculizumab will not prevent the deposition of c3 fragments and release of c3a , which by themselves might interact with complement receptors on mesangial cells , macrophages / monocytes and neutrophils and activate these cells .
usually , the development of clinical therapy protocols relies on evidence - based guidelines that mainly focus on randomized , controlled trials ; however , for rare diseases , such as in the present case of essential cyoglobulinemia , treatment options rely on similarities to other diseases and the understanding of the pathological process on experimental data obtained in animal models . since
human data of cryoglobulinemia are scarce , the present 1-patient trial report demonstrating the response to eculizumab  whether considered partially positive or negative  might be of use for the medical community .

Answer:
cryoglobulinemia induces an immune complex - mediated glomerulonephritis that is characterized by the presence of large immune deposits , including complement c3 and c5b-9 , marked macrophage influx and mesangial cell proliferation .
 the precise role of complement in cryoglobulin - induced glomerulonephritis in humans remains unclear , whereas in mice there has been evidence that complement activation might be a central factor favoring glomerular inflammation , particularly by the recruitment of neutrophils .
 we report on an exceptional case of cryoglobulin - induced glomerulonephritis in a patient with mixed essential cryoglobulinemia type ii .
 the clinical features included relapsing proteinuria and renal function impairment that were controlled by plasmapheresis .
 complement was low in plasma and two renal biopsies at 1-year interval showed prominent immunoglobulin and complement deposits , with unusual high numbers of neutrophils . in a 1-patient clinical trial , we tested whether the monoclonal anti - c5 antibody eculizumab would be sufficient to control renal function at the time of a relapse .
 although during the initial weeks renal function was stabilized , slow increase in creatinine could not be controlled by this treatment , so that plasmapheresis was reinstituted .
 this result suggests that despite evidence for a role of complement in enhancing renal damage in this patient , other inflammatory processes dominated .