We conclude that prolonged hypoxia down-regulates BDNF-TrkB signaling leading to an increase in TNF-α in the cortex, while DOR activation up-regulates BDNF-TrkB signaling thereby decreasing TNF-α levels in the hypoxic cortex.We investigated whether δ-opioid receptor (DOR)-induced neuroprotection involves the brain-derived neurotrophic factor (BDNF) pathway. We studied the effect of DOR activation on the expression of BDNF and other proteins in the cortex of C57BL/6 mice exposed to hypoxia (10% of oxygen) for 1–10 days. The results showed that: (1) 1-day hypoxia had no appreciable effect on BDNF expression, while 3- and 10-day hypoxia progressively decreased BDNF expression, resulting in 37.3% reduction ( Evidenctrindole .Based on the facts from above mentioned studies, we speculate that the mechanism of DOR neuroprotection against hypoxic injury could involve regulation of BDNF-TrkB signaling pathway and its interaction with the regulation of TNF-α. As an initial step towards testing this possibility, we have performed this investigation to determine if DOR activation affects the expression of BDNF, TrkB and TNF-α in the brain following hypoxic exposure for varying durations.p > 0.05) (p < 0.05) (p > 0.05) (p > 0.05) (We first examined the expression of BDNF and DOR in mouse brain. As shown in  > 0.05) , and fur < 0.05) . In cont > 0.05) , suggest > 0.05) . The DOR > 0.05)  did not p < 0.05) in the hippocampus after 1-day hypoxic exposure as shown in p > 0.05) observed after 3 and 10 days of hypoxic exposure   .vs. 100% of the control level, p > 0.05)  , suggestSimilarly as in the cortex, the DOR expression  showed np > 0.05) after 1, 3 and 10 days of hypoxic exposure   . The DOR > 0.05) .p < 0.05)) (p > 0.05) suggesting that UFP-512 attenuated the hypoxia-induced reduction of 140 KDa TrkB expression after 3 days of hypoxic exposure. Although UFP-512 did not significantly affect the hypoxia-induced reduction in 140 kDa TrkB expression after 10 days of hypoxic exposure, the expression of 140 kDa TrkB in UFP-512  was higher compared to that without hypoxic exposure  (We examined the expression of both full-length and truncated TrkB receptors. As shown in < 0.05)) . In cont < 0.05) .p < 0.05) (p > 0.05) suggesting that UFP-512 attenuated the hypoxia-induced truncation of 140 kDa TrkB.The production of truncated isoform of TrkB (90 kDa) receptors is induced in response to an injury . Our res < 0.05) . In contp > 0.05) (p > 0.05), and was significantly upregulated after 10 days of hypoxic exposure  (p < 0.05)  , but the < 0.05) . In cont < 0.05)  suggestip > 0.05), but gradually decreased after 3 days , and 10 days of hypoxic exposure  (p < 0.05)  . In cont < 0.05)  suggestip > 0.05; pCREB/CREB: 106.3% ± 3.7%, 99.1% ± 2.8% and 97.7% ± 2.8% of the control level respectively, p > 0.05). DOR activation also had no appreciable effect on the expression of these proteins (p > 0.05)  .We report three major observations in the cortex exposed to hypoxia: (1) Hypoxia-induced damage is dependent on hypoxic duration, since prolonged (3- and 10-day) hypoxia caused a significant reduction in BDNF expression in the cortex when compared to short-term (1 day) hypoxia, and this reduction was partially reversed following DOR activation; (2) DOR activation partially reversed the hypoxia-induced reduction in functional TrkB (140-kDa) expression and attenuated the hypoxic increase in truncated TrkB (90-kDa) in the cortex exposed to 3- or 10-day hypoxia; (3) prolonged hypoxia (10 days) significantly upregulates the expression of TNF-α in the cortex but down-regulates the CD11b expression that was completely reversed following DOR activation. In addition, we observed that the levels of pCREB and pATF-1 were not significantly altered in the hypoxia-exposed cortex. These results support our hypothesis that the DOR-induced neuroprotection is mediated by an underlying BDNF-TrkB signaling pathway. Prolonged hypoxia causes a down-regulation of BDNF-TrkB signals leading to an increase in TNF-α in the cortex, while DOR activation upregulates BDNF-TrkB signals thereby decreasing the level of TNF-α in the hypoxic cortex.This is the first study that demonstrates partial reversal of hypoxia-induced reduction of BDNF and functional TrkB expression as well as hypoxia-induced increase in truncated TrkB (90-kDa) and TNF-α expression in the cortex following DOR activation without any change in DOR density. DOR expression plays an important role in protection against cerebral hypoxia. Our previous studies have demonstrated that cortical DOR activation is protective against hypoxia and glutamate excitotoxicity in neurons ,24. DOR in vitro and in vivo studies. A blockade of endogenous BDNF activity exacerbates the effects of cerebral ischemia [BDNF is an important neuroprotective factor that has been proven to increase neuronal tolerance against hypoxia/ischemia in both ischemia . Furtherischemia . BDNF biischemia . TrkB haischemia . The proischemia . Our stuBrain injury is influenced by multiple factors, including stress duration and neuronal age ,28. The In a sharp contrast, the subcortical regions and cerebellum did not show any significant alteration in BDNF expression under hypoxic exposure for various periods . This is partially because the subcortical and cerebellar regions may be relatively less sensitive to hypoxia and their BDNF can be compensated by transportation along cortical, nigral and brainstem neurons during hypoxia –32. In fAlthough neurons are the chief source of BDNF, it may also come from the microglia. Microglia are immune cells present in the central nervous system and activated in response to injury and diseases and are known to produce neurotrophic factors such as BDNF, glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), which provide trophic support to neurons during injury . PioneerHowever, activated microglia exerts both beneficial and deleterious effects on neurons. For example, activated microglia following cerebral ischemia express a variety of proinflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which induce neuroinflammation and neurotoxicity . The avaThe BDNF gene is one of the targets of CREB pathway . The DORTo summarize, we propose a potential neuroprotective mechnism involving interactions among DOR, BDNF-TrkB and TNF-α that is schematically illustrated in ad libitum. All animal procedures were approved by the University of Texas medical school at Houston Animal Care and Use Committee  and conformed to the National Institutes of Health Guide for the Care and Use of Animals in Research.Male C57BL/6 mice  at the age of 30-day old (P30) were maintained under a 12 h light/12 h dark cycle and 22 °C with standard rodent chow n = 15); (2) Hypoxia with saline (n = 15); (3) DOR activation with UFP-512 treatment under normoxic conditions (n = 15); and (4) DOR activation with UFP-512 treatment under hypoxic conditions (n = 15). Saline or UFP-512 (1 mg/kg) was daily administrated by intraperitoneal injection. In the hypoxic mice, the first injection was administered at 30-min prior to hypoxia induction. The numbers of the animals used for 3 time points  were 4, 5, and 6 respectively in each group under 4 experimental conditions .In total, 60 mice were randomly divided into 4 groups: (1) Normoxia control with saline (2 levels and humidity in the chamber were kept constant as the ambient levels. O2 levels in the chamber were strictly kept at (10 ± 0.5)% by constantly flushing with nitrogen that was automatically controlled by a ProOx P110 Oxygen Controller with E702 Oxygen Sensor . The ProOx O2 controller has an extremely wide range from 0.1% O2 all the way up to 99.9% O2 with the variation of 0.1%, which provides a very important tool for our research in terms of accurate control of O2 level. The mice were exposed to hypoxia for 1, 3 or 10 days. For both the saline control and UFP-512 control groups, mice were treated in the similar fashion as the hypoxic exposure mice, but under normoxia (21% of O2 level) instead of hypoxia.To expose mice to hypoxic stress, 2 cages of mice (2 or 3/cage) were placed in a plexiglass chamber (30″W x 20″D x 20″H) . The chamber was connected with the outside environment via holes in the wall of the chamber; therefore, COThe chamber used was custom designed, based on the experimental demands and was fitted with two eight-inch iris ports to access the animals during a prolonged hypoxic exposure in order to keep exposure changes to a minimum. Our daily saline or UFP-512 administration was carried out in the hypoxic chamber by accessing the animals with hands stretching through the iris ports in the front wall of the chamber. Therefore, all the procedures were uniform and strictly controlled for hypoxic exposure.UFP-512 (H-Dmt-Tic-NH-CH(CH2–COOH)-Bid), a specific and potent DOR agonist –46, was 133) were obtained from Millpore . Rabbit monoclonal anti-CREB and TrkB was obtained from Cell Signaling Technology . Mouse monoclonal anti-TNF alpha and rabbit polyclonal anti-CD11b was obtained from Abcam Inc . Rabbit polyclonal anti-BDNF was purchased from Alomone Labs Ltd (Cat: ANT-010). Mouse monoclonal anti-β-actin and RIPA buffer were obtained from Sigma-Aldrich . Horseradish peroxidase conjugated goat anti-rabbit/mouse IgG (H + L) were purchased from Invitrogen . Laemmli sample buffer, Precision Plus Protein™ Dual Color Standards and mini-protean precast gels were obtained from Bio-rad .Rabbit polyclonal anti-DOR and anti-phospho-CREB . The supernatants of tissue homogenates  of the cortex, subcortex, cerebellum and hippocampus from each mouse were boiled at 100 °C in laemmli sample buffer (Bio-Rad) for 5 min. After, the samples were electrophoresed on 4%–15% mini-protean precast gels (Bio-Rad), and transferred to 0.20 μm nitrocellulose membrane (Whatman). Membranes were blocked with 5% (m/v) nonfat dry milk in 0.1% Tween 20  for 1 h at room temperature and subsequently incubated overnight at 4 °C in the blocked buffer with the p-CREB (1:1000), CREB(1:2000), CD11b(1:800), TrkB (1:1000), TNF alpha (1:1000) and BDNF (1:200) antibodies, respectively. The membranes were washed with 0.1% Tween 20, and then treated with horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG  for 1 h at room temperature. Peroxidase activity was visualized with a super signal West Pico Chemiluminescent Substrate . Stripping filters and reprobing for β-actin were carried out for normalization. Controls for nonspecific binding were determined by omission of the primary antibody. The proteins were quantified by measuring optical densities of immunostained bands using an image analysis system .Mice were sacrificed at day 1, 3 or 10, and the brain was removed quickly. The cortical, subcortical , cerebellar and hippocampal tissues were dissected on ice, frozen immediately on dry ice, and then stored at −80 °C until use. The tissues were ultrasonically homogenized in a RIPA buffer and protease inhibitor cocktail, and the homogenates were centrifuged at 12,000× p values < 0.05.Two independent and blinded investigators examined the end point assessments. Data was expressed as mean ± SEM. One-way ANOVA with the post hoc test of Least Significant Difference (LSD) was used to analyze the difference between various groups. Statistical significance was determined based on versus the downregulation of the pro-inflammatory cytokine, TNF-α, suggests their role in the mechanism of DOR-mediated neuroprotection against hypoxia. Targeting the DOR-BDNF-TrkB pathway provides a potential therapy for neuroprotection against hypoxic/ischemia encephalonpathy. However, further studies are needed to ascertain the mechanisms of DOR-mediated regulation of BDNF-TrkB and TNF-α under hypoxic conditions.Our data suggest that the cortical neurons and microglia lose their ability to maintain normal levels of BDNF and functional TrkB after a prolonged exposure to hypoxia. Therefore, both BDNF and functional TrkB protein levels decrease, along with a significant increase in the level of TNF-α expression following a severe hypoxic injury. DOR activation can partially reverse the hypoxia-induced decrease in the BDNF and functional TrkB levels and protect the brain from hypoxic/ishcmic injuury. The upregulation of BDNF along with its TrkB receptor and CD11b


Alu elements are actively amplified in the human genome through retrotransposition of their RNA intermediates by the ∼100 still retrotranspositionally fully competent L1 elements. Retrotransposition can cause inherited disease if such an element is inserted near or within a functional gene. Using direct cDNA sequencing as the primary assay for comprehensive NF1 mutation analysis, we uncovered in 18 unrelated index patients splicing alterations not readily explained at the genomic level by an underlying point-mutation or deletion. Improved PCR protocols avoiding allelic drop-out of the mutant alleles uncovered insertions of fourteen Alu elements, three L1 elements, and one poly(T) stretch to cause these splicing defects. Taken together, the 18 pathogenic L1 endonuclease-mediated de novo insertions represent the largest number of this type of mutations characterized in a single human gene. Our findings show that retrotransposon insertions account for as many as ∼0.4% of all NF1 mutations. Since altered splicing was the main effect of the inserted elements, the current finding was facilitated by the use of RNA–based mutation analysis protocols, resulting in improved detection compared to gDNA–based approaches. Six different insertions clustered in a relatively small 1.5-kb region (NF1 exons 21(16)–23(18)) within the 280-kb NF1 gene. Furthermore, three different specific integration sites, one of them located in this cluster region, were each used twice, i.e. NM_000267.3(NF1):c.1642-1_1642 in intron 14(10c), NM_000267.3(NF1):c.2835_2836 in exon 21(16), and NM_000267.3(NF1):c.4319_4320 in exon 33(25). Identification of three loci that each served twice as integration site for independent retrotransposition events as well as 1.5-kb cluster region harboring six independent insertions supports the notion of non-random insertion of retrotransposons in the human genome. Currently, little is known about which features make sites particularly vulnerable to L1 EN-mediated insertions. The here identified integration sites may serve to elucidate these features in future studies.Long interspersed (L1) and  Alu elements accounting for more than one fourth of the human genome, are still actively amplifying. It is widely believed that retroelements insert randomly in the genome. Retroelements newly inserted in the germ line may cause genetic disease, if a functional gene is disrupted. Up to now, only ∼65 well-characterized pathogenic retroelement insertions in 31 different human genes have been reported. Therefore, retrotransposition is suspected to be underdiagnosed as disease-causing mutation mechanism. Reporting 18 novel insertions in the NF1 gene, all identified by a comprehensive RNA–based mutation analysis protocol, we show that L1 and Alu insertions represent 0.4% of all NF1 mutations. Strikingly, we found three integration sites within this 280-kb gene that were used twice independently to insert a retroelement. One of these sites was located in a 1.5-kb “hotspot” region where four additional integration sites clustered. These findings, together with three additional integration sites used multiple times independently to insert retroelements in other genes, indicate that some genomic sites may be especially prone to host newly retrotransposed elements. As some of these sites are embedded in “hotspot” regions, larger flanking sequences may play a role in making these sites particularly vulnerable.Repetitive retrotransposable elements, including LINE1 and  Alu sequences belonging to the family of short interspersed nuclear elements (SINEs) still actively amplify in the human genome, by a process called retrotransposition. L1 elements comprise ∼17% of the human genome sequence Alu elements comprising more than 10% of the human genome can retrotranspose in a non-autonomous process, using proteins encoded by L1 elements to mediate their mobility Alu elements are named master or source Alu elements Alu elements. Both elements are transcribed from an internal promoter Alu transcripts may be encoded directly from the genomic site of transcription Alu transcripts are then terminated at the 3′ end with a short run of U's 3′-AA/TTTT-5′ (and derivates thereof) Alu elements are reverse transcribed by the same process called “target primed reverse transcription” (TPRT), but they need to “borrow” the factors for TPRT from L1 elements Alu element including the poly(A) tail flanked by short direct repeats of the duplicated target site. For a detailed description of the mechanisms of autonomous and non-autonomous retrotransposition see Long interspersed nuclear elements (LINE-1 or L1 elements) and Alu elements dimorphic with respect to presence/absence at a given site contribute significantly to structural variation of the human genome. Furthermore, these studies show that current activity of mobile elements is likely to be higher than previously appreciated.Although retrotransposable elements have no immediate function in the cell, their motility can be important for genome plasticity and the creation of genetic variation Alu elements are believed to insert randomly into the human genome. Hence, de novo transposition of an element may occasionally cause an inherited disease when the element is inserted into (or at proximity of) the coding region of a functional gene de novo L1 EN-mediated insertions of retrotransposable elements causing genetic diseases have been reported Alu, 15 L1, four SINE/VNTR/Alu (SVA) composite element insertions as well as three simple poly(A) insertions de novo insertions were found in 31 different genes including one insertion in the NF1 gene NF1 gene: one poly(T), three L1 and 14 Alu insertions were all identified using an RNA-based core assay as the starting point for comprehensive NF1-gene analysis illustrating the strength of this approach in identifying also this complex type of mutations. Of note, six of the integration sites were located within a relatively small genomic region of 1500 bp between NF1 exons 21 (16) and 23 (18) . Even more striking, three of the integration sites within the NF1 gene were used twice. Our results indicate and confirm that some genomic locations may be especially prone to L1 EN mediated retrotransposition.L1 and NF1 mutation analysis in the two centers (UAB and MUI) uncovered heterozygous (∼50% of the transcripts affected) splicing alterations in 18 NF1 index patients (Alu elements inserted within the exonic sequences. In addition to the approximately 280-bp long Alu elements a 60–120-bp long poly(A) tail -or a poly(T) tail- was inserted depending on whether the Alu element was inserted in the sense or antisense orientation with respect to the NF1 reading frame . We reasoned that the substantial increase in size of the mutant exons containing the Alu/L1 insertions, compared to the wild type exons caused allelic drop-out under standard PCR conditions. We increased the extension time of the PCR reactions and/or increased the size of the PCR amplicons to enhance the amplification of the larger mutant alleles and, hence, facilitate the detection of possible de novo Alu insertions. Amplification of all exons with so far unexplained splicing alterations using PCR conditions optimized to amplify larger PCR products , 23 (18) and 9 (7) did not allow resolution of the cause of their mis-splicing in the patients UAB-R01429, UAB-R316001 and UAB-R91409, respectively. Use of a Taq polymerase enabling amplification of extremely long PCR products (up to >6 kb) was needed to amplify the mutant exons which had 10- and 17-times increased in size compared to their wild type normal allele in patients UAB-R316001 and UAB-R01429, respectively. A 6-kb full-length L1 element inserted in sense orientation into exon 39 (30) was identified in patient UAB-R01429 . Full seAluY element into intron 10 (8) cannot be determined due to a 71-bp deletion associated with this insertion that causes also lack of a TSD. Nevertheless, the most likely insertion site matching a L1 EN cleavage site is tentatively given in AluY and AluYb8 element inserted into the splice acceptor site of intron 14 (10c). The same L1 EN cleavage site was used once at the anti-sense strand to insert the AluY element in sense orientation with respect to the coding sequence of the NF1 gene (integration site according to HGVS nomenclature c.1642-1_1642) and in an unrelated patient once at the sense strand to insert the AluYb8 element in anti-sense orientation (integration site according HGVS nomenclature c.1642-12_1642-11). Furthermore, we found in two non-related patients . Both Alu elements were inserted in sense orientation and flanked by the same sized TSD . The length of TSDs flanking the two insertions differed between both patients (de novo insertions were distributed over the entire NF1 gene from exon 6 (4c) to intron 48 (39).Alignment of the sequences at the integration sites  shows thized TSD . Finallyized TSD  -one of patients  confirmiAlu elements , two insertions at different integration sites in exon 22 (17) and one insertion in exon 6 (4c) , 30 nucleotides downstream of the intron-exon 21 (16) border, resulted in the use of a cryptic 3′ splice site downstream of the integration site and loss of the first 229 bp of the exon 21 (16) (mutation UAB-R39428 in AluYa5 element into exon 47 (38) resulted in at least two different splicing effects, i.e. skipping of the exon and use of an exonic cryptic 5′ splice site upstream of the integration site  also resulted in a more complex splicing defect. This element lacks the first 39 bp at its 5′ end , leading to transcripts containing the first 61 nucleotides of this Alu element but lacking the 3′ half of exon 12 (10a), downstream of the Alu integration site (type 5 effect in Four out of 14 Insertion of the 5′-truncated L1 element into exon 23 (18) also caused simple exon skipping in the transcripts of the patient UAB-R316001 (type 2 splicing effect in Alu and L1 elements as well as the insertion of a poly(T) element lead to altered splicing of the transcripts from the mutant allele. None of the inserted Alu or L1 elements was fully included into the transcripts.Taken together, all NF1 gene. This is the largest number of disease causing mutations of this type found in a single human gene. Sixteen of these insertions were found in a cohort of 4300 unrelated NF1 mutation-positive patients analyzed at UAB and two in a cohort of 200 unrelated NF1 mutation-positive patients analyzed at MUI. Taken together, this suggests that retrotransposon insertions account for ∼0.4% (18/4500) of NF1 mutations identified. This frequency is two to four times higher than previously anticipated Here we report the identification of 18 pathogenic L1 EN-mediated insertions into the Alu, L1 and poly(T) stretch insertions, it was necessary in most of the cases to modify the PCR conditions to prevent allelic drop-out of the substantially larger mutant allele. Hence, most of these mutant alleles would be missed by an exon by exon-sequencing approach. Similar findings were reported for the BRCA1 and BRCA2 genes Firstly, mutations due to retrotransposition have long been thought to be underestimated, since they may be missed by mutation detection methods relying on PCR amplification of small amplicons, i.e. exons, from genomic DNA NF1 gene is particularly susceptible to retrotransposon integration. It is striking that three specific sites within the NF1 exons 21 (16) and 33 (25) and the splice acceptor site of intron 14 (10c) hosted each two independently retrotransposed elements. Moreover, one of these sites is located in a small region of approximately 1.5 kb within the ∼280-kb NF1 gene where six of the 18 insertions cluster. Together, these finding strongly support the notion of an non-random de novo insertion of retrotransposons in the human genome F8 and F9 mutated in hemophilia B, may have hotspot regions for retrotransposon integration APC, F9 and BTK, have been reported to be used twice independently for L1 EN-mediated retrotransposon insertions NF1 gene, a tenth (6/66) of the well characterized sites that harbor disease causing retrotransposon insertions have been used multiple times with half of them identified in the current study. It remains to be elucidated which features make these sites particularly vulnerable to L1 EN-mediated insertions. The fact that some of these sites are embedded in a larger sequence context that appears to be a hotspot region for insertions may indicate that flanking sequences and possibly also the chromatin structure in these regions may play a role.Secondly, it is possible that the Alu and L1 elements have a similar retrotransposition efficiency via-à-vis the molecular retrotransposition machinery NF1 gene also in previously published disease causing retrotranspositional insertions a preponderance of Alu over L1 insertions can be observed. As a possible explanation Dewannieux et al. trans mobilization of Alu elements even if they are unable to promote their own transposition. In other words, there appear to be more potential “drivers” for Alu retrotransposition than there are for L1 retrotransposition Alu over L1 insertions It has been suggested that Alu, L1 and simple poly(T) insertions. Hence, our data confirm and extend several observations deduced from 48 previously analyzed pathological insertions NF1 coding sequence. All Alu insertions belong to the evolutionary youngest “Y” Alu family  sequence inserted into the NF1 exon 25 (19b) may represent a severely truncated Group III Alu insertion AluY element inserted into intron 10 (8) was accompanied by a 71-bp deletion and lacked a TSD. Different possible mechanisms for the loss of genomic sequences in association with EN-dependent retroelement insertions have been discussed AluY insertion into NF1 intron 10 (8). In both models the first-strand L1 EN-mediated nick would have occurred at position c.1186-85_1186-86 in intron 10 (8)  element F8 gene in a hemophilia A patient NF1 gene switched and continued from the anti-sense strand. The presence of a 2-bp micro-homology that may have promoted strand switching due to secondary binding between the RNA-template and the 3′-end of the 5′-overhang at the antisense strand supports the model of twin priming According to Kojima Alu insertions identified here directly affect splice sites. Nevertheless, all L1 EN-mediated insertions in this study affected NF1 splicing in the patients. The predominant effect of the 13 exonic Alu, L1 and poly(T) insertions was exon skipping (6/13 cases) or use of a cryptic splice site -either a cryptic 5′ splice site upstream (3/13 cases) or a cryptic 3′ splice site downstream of the integration site (1/13 cases). In one of 13 cases both of these splice effects were observed in the same patient. In only two cases part of the Alu or L1 element were contained in the mRNA transcripts, inserted into exon 12 (10a) and 39 (30) respectively. Overall, we do not believe that ascertainment bias can explain why exon skipping and/or use of cryptic splice site are the main effects of the de novo Alu and L1 insertions in exonic sequences. Firstly, because we have no evidence that the long-range RT-PCR reactions used in the applied assays would miss Alu insertions in exons or introns that lead to full or partial exonisation of the inserted sequences in the mRNA transcripts. On the contrary, duplications of single or multiple exons which lead to a similar size increase of the RT-PCR products were readily detected by our assays Alu and L1 insertion is weakening of the exon definition resulting in altered splicing of the affected exon. Currently it is not fully evaluated by which mechanisms the inserted sequences reduce exon recognition by the splicing machinery. It has been proposed that inserted sequences may disrupt specific cis-acting exonic splice elements, such as exonic splicing enhancers (ESEs)  weakens exon definition simply by increasing the size of the affected exon and/or by disrupting the exonic structure of cis acting elements in a more general sense, e.g. by increasing the distance of exonic cis regulatory elements and the splice sites of the exon. In this respect it is of note that the use of cryptic 5′- and 3′- splice sites in exons 21 (16) and 25 (19b) is also observed in transcripts from mutant NF1 alleles that carry single nucleotide alterations destroying the respective natural 5′- and 3′- splice sites of these exons  caused a complex splicing effect, i.e. insertion of a cryptic exon embedded in the inserted L1 element and skipping of the preceding exon 9 (7). A similar splicing defect was described in a chronic granulomatous disease patient who carried a truncated L1 element in intron 5 of the Alu insertions. In addition, the RNA-based comprehensive NF1 mutation detection approaches unambiguously identify and functionally characterize other classes of mutations usually missed by DNA-based mutation detection strategies, such as intronic alterations outside the canonical splice site dinucleotides (GT-AG) (including deep intronic mutations), as well as silent and missense mutations with an effect on splicing De novo retrotransposon insertions within the 3′- or 5′-UTR can lead to reduced expression by the disruption of gene regulatory elements NF1 mutation analysis assay.Our results clearly show that RNA-based mutation analysis strategies have the potential to detect disease-causing L1 and NF1 testing to two centers, i.e. the Medical Genomics Laboratory at the University of Alabama in Birmingham (UAB) and the Division of Human Genetics, Medical University Innsbruck (MUI). Informed consent was obtained from all patients. This study was approved by the ethical committee from both institutions.All mutations reported were uncovered in samples from unrelated index patients sent for clinical NF1 mutation analysis is a direct cDNA sequencing approach that is based on the amplification of the entire NF1 coding region in three  or five (Innsbruck) overlapping RT-PCR fragments and subsequent sequencing of the entire PCR products with 18 (20) internal primers. Details on the RT-PCR reactions and primers can be found in In both laboratories the primary assay for comprehensive NF1 nucleotide numbering is based on GenBank reference sequence NM_000267.3 with the A of the ATG start codon being nucleotide position c.1. The integration sites are given according to the HGVS nomenclature after the first duplicated sequence regardless of whether the first nick occurred at the sense or the anti-sense strand and the thereof resulting orientation of the insertion with respect to the NF1 coding sequence. Exons are numbered according to the reference sequence with the widely known legacy numbering given in parenthesis.Alu/L1 insertion within or in close vicinity of the exon  or Taq DNA Polymerase . To amplify the mutant alleles containing a truncated 1753-bp and a full-length 6-kb L1 element, respectively, Expand Long PCR Taq  was used. To determine the precise sequence of the inserted Alu element as well as the duplicated sequence at the insertion site the PCR products showing a band of increased size were cloned according to the manufacturer's instructions into the TOPO-TA cloning vector pCR 4-TOPO  and individually sequenced. Alternatively or in addition, primers were designed to specifically amplify only the mutant alleles containing the Alu insertions. These primers contained at their 5′-end the exonic sequences immediately upstream/downstream of the insertion site and at the 3′-end a few nucleotides of either the Alu or the poly(A/T) stretch. All primers are listed in Alu sequences with the consensus sequences of the different Alu families  with the current SALSA-MLPA-kit P081-B1 and/or P082-B1 . When MLPA results were negative for a genomic deletion of the respective exon and no alteration was identified explaining the observed splicing defect, sequences were meticulously reanalyzed to uncover a possible exon see . To dimiFigure S1AluYa5 insertion in exon 47 (38). The sequence generated with a reverse primer from a RT-PCR product from the patient shows the border of NF1 exons 47 (38) and 48 (39). In addition to the wild type transcript two aberrantly spliced transcripts can be deduced from the sequence. One aberrant transcript lacks the entire exon 47 (38) and the other the last 62 nucleotides of exon 47 (38) due to the use of an exonic cryptic 5′-splice site upstream of the integration site.Detection of two aberrant splice products due to an (TIF)Click here for additional data file.Figure S2AluYa5 insertion in NF1 exon 47 (38). A) Agarose gel showing PCR products generated from gDNA from a control individual (C) and the patient (P) harboring an AluYa5 insertion in NF1 exon 47 (38), . PCR product of the patient shows a faint extra band of larger size (arrow) that is not present in the control. B) Sequences of the PCR products from the control individual and the patient. The sequence of the patient shows a faint background sequence (a poly(T) stretch) starting at nucleotide c.6952 . This indicates the insertion of a retrotransposon, in this case an AluYa5 element, in anti-sense direction with respect to the NF1 coding sequence at this site.Detection of an (TIF)Click here for additional data file.Figure S3AluYa5 insertion in NF1 exon 47 (38). A) Scheme showing the strategy to amplify the AluYa5 insertion. Exons are shown as boxes and introns as lines. The Alu-insertion is shown as an arrow in anti-sense orientation. In order to specifically amplify the mutant allele containing the Alu sequence two Alu insertion specific primers (Alu_47f and Alu_47r) spanning the exon 47 (38)-Alu insertion border were designed. These primers were used together with their respective regular exon primer (47r or 47f) at the opposite site of the exon resulting in fragments of 600 bp and 1000 bp, respectively, each containing the Alu insertion. C) Agarose gel showing the result of the Alu-insertion specific PCRs derived from the patient (P) and a control individual (C). B) Sequence analysis of the specific Alu-insertion product generated by the Alu insertion-specific reversed primer and the respective regular forward primer, shows the anti-sense orientation of the Alu insertion.Specific PCR for the (TIF)Click here for additional data file.Figure S4Alu sequences with the consensus sequence of the AluY, AluYa5 and AluYb8 subfamilies. Identical nucleotides between all sequences are indicated by capital letters. In order to maximize the alignment gaps were introduced (dashed). The reference sequences of the Alu subfamilies were taken from Repbase Giri Sequence alignment of the 14 (TIF)Click here for additional data file.Figure S5NF1 exon 39 (30) of a patient (R01429). The sequence of the hot L1 element is taken from Alignment of the full length L1 sequence with the consensus sequence of the hot L1 element. The element was found inserted in sense orientation into the (TIF)Click here for additional data file.Figure S6NF1 coding sequence at least 1088 bp from the center of the L1 ORF2 and ends with the 3167th nucleotide of the 3825-bp L1 ORF2 suggesting that during the process of retrotransposition the orientation of the reverse transcription from the L1-RNA template that started at the sense strand of the NF1 gene switched and continued from the anti-sense strand. The duplicated nucleotides of the TSD are underlined. The 130-bp cryptic exon embedded in the L1 element is indicated in a darker shade of green. The sequence used to design L1 insertion-specific primers is underlined. The splices site scores of the cryptic exon as calculated by Splice Site Prediction by Neural Net (http://www.fruitfly.org/seq_tools/splice.html) is 0.62 for the 5′ splice site and 0.86 for the 3′ splice site.Truncated L1 element inserted into intron 9 (7). Genomic sequence of intron (IVS) 9 (7) (black letters) with truncated L1 element (green letters) inserted at position c.1062+195_1062+196 and the flanking exons 9 (7) and 10 (8) (red letters). The inserted sequence contains at the 5′-end a poly(T) stretch indicating that the poly(A) tail of the L1 transcript had annealed to the sense strand where reverse transcription from the template started. However, the 3′-end of the inserted sequence contains in sense orientation with regard to the (TIF)Click here for additional data file.Table S1NF1 exons, Alu and L1 sequences. (B) List of PCR programs.(A) List of primers for amplification of (PDF)Click here for additional data file.


Patients with idiopathic pulmonary fibrosis (IPF) frequently develop a dry, irritating cough which often proves refractory to anti-tussive therapies. The precise pathogenetic mechanisms responsible for this cough are unknown. We hypothesised that changes in nerves modulating mechanical sensitivity in areas of interstitial fibrosis might lead to enhanced cough response to mechanical stimulation of the chest in IPF.We studied 27 non-smoking subjects with IPF , mean (SD) age 71.7 (7) years and 30 healthy non-smokers. Quality of life (Leicester Cough Questionnaire), cough symptom scores and cough severity scores  were recorded. Percussion stimulation was applied over the posterior lung base, upper anterior chest and manubrium sternum at sequential frequencies (20 Hertz (Hz), 40 Hz and 60 Hz) for up to 60 seconds and repeated twice at two minute intervals. The number of subjects achieving two and five-cough responses, total cough counts and cough latency were recorded. In separate experiments, the effect of mechanical stimulation on the pattern of breathing was determined in eight IPF subjects and five control subjects.In patients with IPF, we demonstrated strong correlations between subjective cough measurements, particularly the cough symptom score and Leicester Cough Questionnaire . Mechanical percussion induced a true cough reflex in 23/27 (85%) IPF subjects, but only 5/30 (17%) controls (p < 0.001). More patients with IPF reached the two-cough response at a lower frequency (20 Hz) posteriorly than at other positions. Highest mean cough totals were seen with stimulation at or above 40 Hz. Mechanical stimulation had no effect on respiratory rate but increased tidal volume in four (50%) subjects with IPF, particularly at higher frequencies. It was associated with increased urge to cough followed by a true cough reflex.This study demonstrates that patients with IPF show enhanced cough reflex sensitivity to mechanical stimulation of the chest wall whilst normal individuals show little or no response. The observation that low frequency stimulation over the lung base, where fibrosis is most extensive, induces cough in more patients than at other sites supports the hypothesis that lung distortion contributes to the pathogenesis of cough in IPF. Idiopathic pulmonary fibrosis (IPF) is a disease characterised by lung parenchymal distortion by fibroblastic proliferation with extracellular matrix deposition and an inflammatory cell infiltration. Patients typically present with progressive breathlessness but the majority develop an irritating cough during the course of the disease,2. This The majority of respiratory diseases associated with cough, such as chronic bronchitis, asthma and acute viral infections, predominantly affect the airways or upper respiratory tract where sensory innervation is dense. By contrast, pathological changes in IPF principally affect the lung parenchyma and alveoli, where innervation is sparse. It is therefore surprising that cough is so common in this disorder. The mechanisms which cause cough in IPF are unknown but several theories have been proposed. These iCough reflex sensitivity to chemical stimulation from inhaled capsaicin and substance P has been shown to be increased in patients with IPF, suggesting functional upregulation of pulmonary c-fibres,6. HowevCrystal et al.  reportedMechanical stimulation of the throat and trachea has been shown to induce cough in patients with upper respiratory tract infection but little or no cough in healthy subjects,10. In oIn this context, the present study was devised with the following aims:• To examine whether mechanical stimulation of the chest wall can induce cough in patients with IPF and if so, whether this response is reliable and reproducible.• To assess whether varying the frequency of vibration and the site of stimulation induces different patterns of cough in patients with IPF.• To correlate measures of any cough induced by mechanical stimulation with subjective measures of cough assessed by validated questionnaires.• To determine whether mechanical stimulation of the chest wall has any effects on the pattern of breathing in patients with IPF or controls.Twenty seven patients fulfilling the American Thoracic Society/European Respiratory Society criteria for the diagnosis of IPF were recruited. All patThree separate measures of cough were undertaken. All subjects were asked to grade their cough severity from 0 (no cough) to 100 (worst cough ever) using a 100 mm linear visual analogue scale (VAS). The impSubjects underwent mechanical stimulation of the chest wall using a percussor . A soft sponge rubber applicator  was used to apply the percussor to the chest wall. Use of a right angle directional stroking adaptor converted the oscillatory effect produced by the face plate to an optimal percussive effect with stimulation frequencies from 20 to 60 Hz , was recorded as the six minute cough frequency. The number of subjects who achieved a two-cough and a five-cough response were recorded as C2 and C5 cough thresholds respectively. For determination of C2 and C5 responses, only coughs occurring during or within 15 seconds of cessation of mechanical stimulation were counted, in accordance with guidelines for other cough challenges. Cough lTo determine any effects of mechanical stimulation on the pattern of breathing, the above protocol was repeated in eight patients with IPF  and five normal volunteers using a portable recording device designed for the diagnostic assessment of cardio-respiratory sleep disorders . This equipment provides measurement of: airflow from a nasal pressure cannula and oral thermistor; movements of abdominal and chest wall from effort belts, oxygen saturation and pulse using pulse oximetry. A built-in microphone also records associated sounds simultaneously. At the end of the recording period, all subjects were asked to describe subjective feelings of urge to cough, change in pattern of breathing or feelings of breathlessness.t test was also used to compare baseline variables between groups. Non-parametric data are expressed as median (IQR) and compared by the Mann-Whitney U test and Wilcoxon's signed rank test. When analysing multiple comparisons of mean cough counts at different stimulation frequencies within individuals, a repeated measures one-way analysis of variance (ANOVA) was applied to normally distributed data with Tukey's pairwise analysis for determining true differences. Fisher's exact test was used to analyse categorical data and Spearman's rank correlation coefficient to assess association between variables[Total cough counts during six minutes of stimulation are expressed as mean (SEM) and compared by the unpaired t-test. The unpaired variables. p valuevariables. All datBaseline demographic and pulmonary function data for patients and control subjects is shown in table Compared with controls, IPF patients had significantly greater median cough symptom scores than control subjects as assessed by VAS  and CSS  and lower median LCQ scores . In patients with IPF, there were strong negative correlations between the LCQ score and both the CSS and VAS scores, with a further strong positive correlation between the VAS and CSS  exhibited no cough at any frequency at any site of stimulation. By comparison, 23 out of 27 patients (80%) with IPF coughed on percussive stimulation (p < 0.0001). Of the four patients with IPF who did not cough at all, three were receiving prednisolone.Of the IPF patients, 17 out of 27 reached a two-cough response during an individual period of mechanical stimulation. This was detected in eleven subjects at 20 Hz, four at 40 Hz and two at 60 Hz Figure . A five-Nineteen subjects with IPF achieved a two-cough response. This was achieved by nine subjects at 20 Hz, six at 40 Hz and four at 60 Hz Figure . In fiftOf the IPF group, 13 subjects demonstrated a two-cough response. This occurred in five subjects at 20 Hz, seven at 40 Hz and one at 60 Hz Figure . In eighThe mean total cough counts (corrected for background cough) for the total three stimulation episodes at different sites of stimulation and different frequencies are shown in Figure In IPF subjects who coughed in response to mechanical stimulation, the mean time to first cough (cough latency) ranged from 32.7 to 57.3 seconds at each stimulation frequency in each location tested. No clear pattern emerged in relation to site of chest wall stimulated, frequency of stimulation or subjective measures of cough (data not shown).1, FVC, TLC or DLCO).The correlation between individual IPF patients' total cough counts at different sites and frequencies of percussion and the three subjective cough symptom scores are shown in table Repeat experiments under identical conditions in six patients with IPF studied at least one week apart suggested there was some variability in mean total cough counts and C2 cough threshold Figure . HoweverIn healthy subjects, there was no change in rate or depth of respiration at any stimulation frequency. Similarly, in patients with IPF, no change in respiratory rate was observed. However, an increased tidal volume was clearly demonstrated in four patients (50%) in response to mechanical stimulation and this became more pronounced at higher frequencies Figure . It was This is the first study to investigate the effects of mechanical chest wall percussion on the cough reflex and patterns of breathing in patients with IPF. The technique appears to induce a true cough rather than an expiratory reflex in the majority of subjects with IPF, but has little or no effect on healthy controls in whom any cough was minor and short-lived. The latter observation is consistent with previous studies in healthy humans,19,20. IRARs terminate in the intrapulmonary airways of all mammalian species and are exquisitely sensitive to mechanical stimulation-23. TheyIn this study, we excluded patients with chest wall deformities in order to minimise this as a confounding factor. However, stimulation over different areas of the chest wall may result in variability in the percussive stimulus exerted on the underlying lung due to differences in chest wall anatomy or thickness. As a group, the Body Mass Index of our patients with IPF was significantly greater than controls. Thus, any reduced transmission of the vibration due to increased chest wall thickness would favour a null hypothesis.We cannot be certain that direct stimulation of RARs by mechanical vibration contributed to the cough response seen in patients with IPF. Indeed, the precise distribution and structure of RAR terminations in the intrapulmonary airways of patients with this condition are unknown ,22. FurtThis implies the mechanical vibration applied in the subsequent experiments was a true stimulatory impulse.In contrast to the C2 response, fewer patients with IPF achieved the C5 threshold at any frequency. These results are similar to those of a previous study which used chemical stimulation with capsaicin to induce cough in IPF patients. This obOur measurements of chest and abdominal wall movement and airflow at the mouth together with audio-visual recordings demonstrated that the cough we induced by vibration was indeed a true cough and not an expiratory reflex. Interestingly, it appears to be preceded by an increase in tidal volume but not rate of respiration and then followed by an urge to cough. The explanation as to why this should occur is uncertain but may relate to variation in density or sensitivity of RARs between individuals. In a previous study on chest wall vibration in five patients with asthma, the subjects described a sensation of breathlessness similar to an acute exacerbation of asthma but no cough was reported . To our It is noteworthy that a previous study reported an increased cough threshold to citric acid in healthy volunteers during simultaneous vibratory stimulation of the chest wall anteriorly in the right second intercostal space. The autThe total cough response (six minute cough count) in patients with IPF was greater when higher frequency stimulation 40 and 60 Hz) was applied to all three areas of the chest wall, whilst again normal controls showed little or no cough response. This may indicate there is a threshold frequency for induction of cough which is lowered in patients with IPF. Interestingly, the total cough count correlated with some subjective assessments of cough severity, particularly the CSS. A recent study demonstrated strong correlations between objective 24 hour cough frequency and both the VAS and LCQ scores in patients with IPF. These f0 Hz was In the present study, patients were asked to provide three different subjective measure of cough severity, all of which have been previously validated. The median VAS score of 38 mm was similar to results from previous studies,6,28,29.Previous studies in IPF have been unable to demonstrate a correlation between the VAS cough score and measures of disease severity assessed by pulmonary function,6. The pIn summary, this study demonstrates that patients with IPF show an enhanced cough reflex sensitivity to mechanical stimulation of the chest wall. It also shows there is a good correlation between cough induced by mechanical stimulation and subjective measures of cough severity, particularly the CSS. The observation that low frequency stimulation of the postero-basal lung base, where fibrosis is often most extensive, induces cough in more patients than at other sites is consistent with the hypothesis that lung distortion is a contributory factor to the pathogenesis of cough in IPF, possibly by activating RARs or destruction of inhibitory c-fibres. The use of chest wall percussion to induce a true cough reflex may prove a useful additional method for assessing novel anti-tussive therapies for cough in patients with IPF.The authors declare that they have no competing interests.RMJ contributed to the design of the study, data collection, analysis and interpretation and drafting/revising of the manuscript. SH was also involved in the data collection. RMJ, NKH and RE were involved in drafting and revising the manuscript. NKH, SH, BDMHG and RE were all involved in the study design and conception and provided substantial input into the analysis and interpretation of the data. All authors have read and approved the final version.


Conventionally, SPPs have been studied in noble-metal structures, where the SPPs are intrinsically bound to a 2D metal-dielectric interface. Meanwhile, recent advances in the growth of hybrid 2D crystals, which comprise laterally connected domains of distinct atomically thin materials, provide the first realistic platform on which a 2D metal-dielectric system with a truly 1D metal-dielectric interface can be achieved. Here we show for the first time that 1D metal-dielectric interfaces support a fundamental 1D plasmonic mode (1DSPP) which exhibits cutoff behavior that provides dramatically improved light confinement in 2D systems. The 1DSPP constitutes a new basic category of plasmon as the missing 1D member of the plasmon family: 3D bulk plasmon, 2DSPP,  Low-dimensional collective electron excitations at metal surfaces, the so-called surface plasmon-polaritons (SPPs), provide a route towards tremendous electric field enhancement4567891011121314151617181920568910111214816Meanwhile, at the cutting edge of materials science are hybrid 2D crystals222324222324231DSPP, and 0D localised SP).In this study, we show that 1DMD interfaces in general 2D metal-dielectric systems support a fundamental and unique 1D plasmonic mode (1DSPP). Through an illustrative example on a hybrid graphene/graphene platform, we show how unique cutoff behaviour of 1DSPPs could allow for a dramatic improvement in the electric field confinement of plasmons in 2D systems, exceeding that of previously predicted quasi-1D and 1D plasmonic excitations in graphene891011121314y = 0 plane consisting of two semi-infinite domains that are laterally connected along the z axis with sheet conductivities of σ(L) = σ(L)′ + iσ(L)″ (x < 0) and σ(R) = σ(R)′ + iσ(R)″ (x > 0) ″ > 0) and dielectric (σ(R)″ < 0) domains, respectively. At this point, to simplify our discussion, we omit ohmic and interband losses in both domains ′ = 0; the lossy case is considered below and in the 26z = 0) in the quasi-static limit can be shown to be (see Methods): σ(R)″ = Kσ(L)″ (K < 0), the normalised effective index N = n/n2D  is the effective index of the 1D interface plasmon with wavenumber q normalised to that of the planar 2D transverse magnetic (TM) plasmons of region L, n2D = 2cεε0/σ(L)″  system in the L)″ Ref. , and m iN(K) is plotted in N is purely real) exist strictly in the window 0 ≤ −K < 1. At K = 0, the plasmon wavenumber takes its minimum value as that of the bare edge plasmon21K| → 1, that is, when the electric susceptibilities on the two sides of the interface become equal (|σ(R)″| → σ(L)″), the plasmon wave number diverges. Interestingly, this divergent behaviour reveals a strong connection to the conventional 2D surface plasmon polaritons (SPPs) localised to bulk metal-dielectric interfacesqSPP = nSPPk0) diverges at the surface plasmon (SP) frequency ωSP given by the non-retarded SP conditionεd + εm(ωSP) = 0; εd > 0 and εm < 0 are the relative permittivities of the dielectric and the metal, respectively. Likewise, at the 1DMD interface, we note the cutoff condition K = −1, or σ(R)″ + σ(L)″ = 0 ′ = 0). This reveals that the plasmonic mode of a 1DMD interface is a 1D manifestation of SPPsin 2D systems (1DSPP). An important consequence of the reduced dimensionality can be observed in the respective divergence behaviours: the SPP behaviour takes the form ε = εd − |εm|), which diverges much less rapidly than that of the 1DSPP, n~1/Δσ (Δσ = σ(L)″ − |σ(R)″|).While σ ″ > 0 i.e., where both domains (L and R) are metallic. In that case, while there is no solution to N, there is a solution for complex N which corresponds to a so-called inter-edge plasmon that is damped as it leaks into the extended 2D plasmons supported by the domain with the smaller (positive) imaginary part of conductivityK < 1) is entirely non-leaky, and completely bound to the 1DMD interface. It is also important to note a clear distinction between the 1DSPP, and the recently predicted quasi-1D plasmonic excitations of graphene p-n junctions1414σ″), much like the multiple modes of a graded-index waveguide, and the graphene is everywhere metallic. In contrast, the 1DSPP manifests exclusively as a consequence of the metallic-to-dielectric transition across the 1DMD interface, either side of which the conductivity is spatially uniform. In this regard, we predict that the IR edge wave observed graphically in Vakil et al. ′, σ(R)′ ≠ 0) is readily obtained on introducing the complex parameter K defined in terms of the complex conductivities K = σ(R)/σ(L) = K′ + iK″, and solving for the complex normalised effective index N = n/n2D = N′ + iN″ (where n2D = i20cε/σ(L) – Ref. σ(L)′ = 0, σ(R)′ ≠ 0), we obtain the convenient and meaningful expressions K′ = σ(R)″/σ(L)″ , and K″ = −σ(R)′/σ(L)″. Thus we may imagine that fixing K′ and simultaneously increasing |K″| corresponds to fixing of the imaginary part of conductivity of the L and R domains, and linearly increasing the loss (σ(R)′) in the dielectric domain. Conversely, fixing |K″| and increasing K′ is equivalent to fixing σ(R)′ and linearly increasing σ(R)″. Meanwhile, the quantity N′/(2πN″) reduces to the propagation length defined as the number of optical cycles within one exponential decay length (l = Re(q)/[2πIm(q)]). We note that this physical scenario is particularly relevant to a hybrid graphene/graphene metal-dielectric system at finite temperature . To illustrate and quantify the onset of 1DSPP damping, the main window of K″ dependence of the 1DSPP propagation length at several fixed values of K′. At a given value of K″, the propagation length decreases with an increase in K′, which is typical of plasmons as their wave number increases . We note that, at each value of K′, the curve is cutoff at the x-axis corresponding to the propagation length of just one wavelength . It is illustrative to compare the K″ cutoff with a particular K′ e.g., when K′ = −0.6, the cutoff is K″ ~ −0.06, corresponding to K″/K′ = −σ(R)′/σ(R)″ ~ 0.1. Obviously, the loss is more forgiving for smaller K′, e.g. when K′ = −0.2, the cutoff is K″ ~ −0.12, corresponding to −σ(R)′/σ(R)″ ~ 0.6.The green and red curves in the inset of μ is the Fermi energy of domain L or R, respectively, and details at finite temperatures are given in the σ″ at the interface  provides convenient closed-form expressions for the optical conductivity in both domains567face see , so hybre see 222432.n2D) is completely determined by the dimensionless conductivity K, this behaviour suggests a flexible tunability of n through the variation of either σ(R)″ (μ(R)) or σ(L)″ (μ(L)) with the doping of either graphene domain. We demonstrate this tunability and simultaneously verify our quasi-static analytical results by employing a mode-solver tool in the numerical finite element method (FEM) package COMSOL, which includes retardation effects. In n on μ(R) for several fixed values of μ(L) (see respective curves) and the spatial evolution of the 1DSPP electric field components xE and yE. Note the normalisation of μ in terms of f = 20 THz and 80 THz. One can observe excellent agreement between the dispersion analytically predicted by n as μ(L) decreases (at fixed μ(R)) can be readily understood in terms of the corresponding decrease in σ(L)″and thus Δσ . Each curve diverges asymptotically towards a cutoff value of μ(R) given by the condition σ(L)(μ(L)) + σ(R)(μ(R)) = 0. The cutoff value of μ(R) approaches μ(L) increases. When the plasmon energy μ(L) are of approximately the same order of magnitude , the cutoff is very close to 0.5 because of the relative strengths of the interband and intraband terms in the graphene conductivity (see Methods).The conductivities on either side of the interface are completely determined by the respective normalised frequenciesσ(R) = 0) demonstrate the nature of the cutoff dynamics at the 1DMD interface. The net power flow parallel to the interface is given by the integral of zS = xHy*E − yHx*E over the xy plane. zS is symmetric about the x axis, although the terms xHy*E and yHx*E both exhibit odd sign parity about the y axis; thus, cumulative integration on either side of the interface leads to their respective partial cancellation. This cancellation is weak in the case of GEPs because of the strong field asymmetry, and therefore, the net power flow is significant. Conversely, as the cutoff condition (σ(R)″ → −σ(L)″) is approached, the symmetrisation of the field components caused by equal charge screening on either side of the interface ; a two-order-of-magnitude improvement over the graphene bare edge plasmon .K. 1DMD interfaces in graphene/graphene hybrid 2D systems at finite temperature are intrinsically lossy because of the presence of interband transitions, which are essential to achieving the dielectric character in one domain ″ < 0; K < 0), and further exhibit a temperature-dependent upper limit on |σ(R)″| (and |K|). Nevertheless, we predict the propagation lengths of 1DSPPs on hybrid graphene systems to be several plasmon wavelengths at room temperature (T = 300 K) and ~100 plasmon wavelengths at the temperature of liquid nitrogen (T = 80 K); for example, at a free space wavelength λ = 1.8 μm we find l ~100nm (μ(L) = 1 eV, μ(R) = 0.42 eV, T = 300 K) and l ~ 2.6 μm (μ(L) = 1 eV, μ(R) = 0.40 eV, T = 80 K), respectively . The effective index of 1DSPPs diverges asymptotically towards a cutoff as the magnitudes of the susceptibilities on the two sides of the interface become equal, in striking analogy to conventional SPPs at bulk metal-dielectric interfaces. On a sample 2D metal-dielectric graphene/graphene platform, highly sensitive tunability of the 1DSPP dispersion was demonstrated via the doping of either graphene domain, which further allowed for the achievement of spatial confinement of electric-field intensity to regions orders of magnitude smaller than that of the plasmonic excitations of a bare graphene edge (GEP). The unique and tuneable cutoff behaviour of 1DSPPs thus presents a means for dramatic enhancement of light confinement in 2D systems over other 1D or quasi-1D excitations, such as GEPs or plasmonic excitations of graphene p-n junctions. At a frequency of 50 THz, we predicted tremendous localisation of the electric-field intensity of the 1DSPP to a modal area more than one million times smaller than the diffraction limit. As a new member of the existing plasmon family of bulk plasmons, surface plasmons, localised plasmons, etc., we foresee a new field of low-dimensional plasmonics based on 1DSPPs, particularly branching out into various multi-material 2D systems222324y = 0 plane comprising two semi-infinite domains laterally connected along the z axis with sheet conductivities of σ(L) = σ(L)′ + iσ(L)″ (x < 0) and σ(R) = σ(R)′ + iσ(R)″ (x > 0) (see inset in ε. In the quasi-static theory of Volkov and Mikhailov26φ(r) = φ0exp(iqzz−iωt), the dispersion relation of plasmons propagating along a 1D junction between two adjoining 2D electron gases (2DEG) is given byWe consider a hybrid 2D system in the βγδσ = σ(R)βγ − σ(L)βγ, where β and γ are tensor indices , and R,Lε are the effective dielectric permittivities of the left (L) and right (R) 2DEGε0 is the permittivity of free space. For electrically isotropic 2DEGs in the absence of an external magnetic field, we have σxz = 0 and δσxz = 0. Taking δσxx = δσ = σ(R) − σ(L), we obtain δσ ≠ 0, the dispersion relation reduces to when the hyperbolic cosine of the argument in the above equation is zero. To simplify the analysis we start by setting σ′ = 0, which neglects ohmic and interband losses −1/2, we arrive at f(KN) − f(N), where C, its numerical solution combined with its asymptotic behaviour is revealing. We find that Im[f(C)] = 0 when C ≤ 0, Im[f(C)] = π2/2 when C ≥ 1, and Im[f(C)] increases monotonically as C increases within the range 0 < C < 1  when K > 0  because 0 < f(KN), f(N) ≤ π2/2, which constrains −π2/2 < Im[f(KN) − f(N)] < π2/2, so that the imaginary part of m. Indeed, this was earlier pointed out by Mikhailovσ″).Here, K < 0; σ(L)″ > 0, σ(R)″ < 0)]. This case has not been previously considered26σ″ in conventional 2DEGs. New opportunities to realise 1DMD interfaces in hybrid 2D systems prompted us to investigate fundamental solutions to K<0 when N > 1 (and m = 0), provided that Re[f(−|K|N)] = Re[f(N)] ] = 0 for NK < 0 , and Im[f(N)] = π2/2 for N > 1, thus satisfying the imaginary parts of f(C), we empirically find the relation Re[f(C + Δ)] ≈ Re[f(−C)] (C > 0) (see the inset of C = |K|N leads to Re[f(|K|N + Δ)] ≈ Re[f(−|K|N)], and recalling the solution condition Re[f(−|K|N)] = Re[f(N)], we must have (comparing the positive arguments of the function Re[f(C)] which monotonically increases for C > 1, and noting Δ > 1) |K|N + Δ = N. This indicates a dispersion relation of the form N = eN/(1 − |K|), where we have identified Δ = eN as the bare edge plasmon dispersion , or recalling the definitions of N and K and takingN,N|K| ≫ 1; writing f(−|K|N)−f(N) and Taylor-expanding the respective arguments to the first order, we find K| → 1 of N ≈ (2/π)(1 + |K|)/(1 − |K|), which is consistent with the (1 − |K|)−1 dependence in the empirical relation obtained above. Solution to m = 0 naturally precludes the existence of higher-order, multipolar modes because of the step-like transition of the conductivity across the junction; this result is expected, considering that multipolar modes of a bare edge plasmon manifest only when the conductivity decreases monotonically from a constant to zero over a non-zero length a beyond some cutoff26K = 0, given by the root of eN = N ≈ 1.217…N(K) as determined from the numerical solution of K| > 0.5 .Now, we turn to the pertinent case of the 1D metal-dielectric (1DMD) interface [www.comsol.com). In our example of the graphene/graphene hybrid 2D system, the conductivities on either side of the junction are calculated using the random phase approximation (RPA) in the local and zero-temperature limitδ = 0.2 nm, an effective relative dielectric permittivityε = 1+iσ/ωε0δ, and σ given by δ → 0 limit (at δ = 0.2 nm).The 1DSPP dispersion was obtained using the mode analysis tool in the COMSOL RF module (x > 0) as (Ω(R))−1 >0.6, thus confirming the previous assertionK > 0. At values of (Ω(R))−1 < 0.5, the dispersion must be solved for a complex value of N because of the introduction of non-zero σ(R)′; nevertheless, considering that we would likely have σ(R)′ > |σ(R)″| ″| see , the 1DSD. M. and S. M. contributed equally to this work. D. M. and S. M proposed and verified the existence of the 1DSPP. D. M. and S. M. performed theoretical and numerical calculations, S. Y. assisted in data interpretation, and N. P. supervised the work and added physical interpretations of the results. All authors participated in discussions and in writing the manuscript.1DSPP Supplementary Information


Valid sarcoidosis incidence assessment is contingent on access to medical care, thoroughness of reportage, assiduity of radiographic interpretation, employment and health care screening policies, misclassification, and population ethnicity. To diminish ambiguity and foster inter-population comparison, the term “sarcoidosis incidence” must be modified to convey the methodology employed in compiling the numerator. In age-delimited cohorts, valid comparison to population incidence requires age adjustment due to the age-dependency of incidence. The “true incidence” of sarcoidosis is a notional concept: more than 90% of cases are subclinical and radiographically inevident. Occupational causal inference based on incidence differential vs. populations has been undermined by methodological differences in ascertainment and computation. Assessment of sarcoidosis incidence requires dividing the annual number of newly identified cases (numerator) by the number in the source population (denominator). This apparent simplicity is illusory, for the numerator is a function of ascertainment methodology and ethnic composition.Ascertainment depends on access to medical care and thoroughness of reportage. Additionally, it reflects acuteness of recognition, which is influenced by the experience and assiduity of the interpreting radiologist. Sensitivity to subtle hilar adenopathy and pulmonary shadowing, easily overlooked in mass population screening, is enhanced (“ascertainment bias”) by awareness of a putative germane exposure e.g., among World Trade Center disaster responders. Increasingly frequent performance of chest CT and transbronchial mediastinal biopsy may increase ascertainment .Methodological differences are a major impediment to obtaining comparable incidence data. This is illustrated by differential incidence ascertainment in observable (see below) cases. To formalize its meaning, the term “incidence” must be preceded by an adjective defining the operation by which the numerator is acquired, thereby facilitating valid inter-population comparison. Observable cases are divisible into three operationally defined classes, each subsuming the prior class: Cases identified because of symptoms or an incidental chest radiograph are designated “clinically identified.” Addition of cases identified by health or employment screening chest radiographs  defines “clinically recognized.” Addition of cases identified by population screening defines “clinically ascertainable.” This classification scheme can be visualized as concentric circles in a Venn diagram.The incidence of clinically identified cases peaks at ages 20–49. It varies widely between ethnic and geographic populations; it is far higher in Blacks and Northern Europeans. The annual incidence of clinically identified sarcoidosis in Whites in the U.K is 3.4 . The latIn summary, among US Whites, 3 is a plausible provisional estimate of clinically identified sarcoidosis incidence; 4 may be considered an upper bound. The figure is slightly higher in the UK. The clinically identified incidence in Scandinavian populations is 5. The clinically ascertainable incidence is three-fold the clinically identified incidence.The clinically identified sarcoidosis incidence in Blacks in the two reporting, ethnically mixed, US health maintenance organizations were 36.4 and 35.5 . BecauseA requirement for histological verification is not typically stated in sarcoidosis incidence compendia. Clinical experience has verified the accuracy of clinical diagnosis in stage I; Hillerdal et al. accepted a clinical diagnosis of stage II .Individuals exhibiting a local or regional granulomatous response to a neoplasm are not infrequently classified erroneously as having sarcoidosis. The proportion of unascertained chronic berylliosis in sarcoidosis registries is unknown. A phenocopy of sarcoidosis, it can escape detection unless careful inquiry into industrial exposure, supported by beryllium-lymphocyte proliferation testing, has been undertaken .Lacking a diagnostic gold standard, autopsy prevalence is imprecise. Enumeration is contingent, in part, on the pathologist’s decision whether pulmonary involvement suffices, whether one or more than one organ demonstrating epithelioid granulomatous changes are required for confirmation, and whether historical or pathologic evidence of potentially confounding, co-existent diseases or exposures are present. Hägerstrand and Linell in a Swedish autopsy series of 6,706 cases reported a prevalence of “certain” (pulmonary or multi-organ epithelioid granulomas) of 640, while the prevalence of uncertain cases was 210. The 640 figure was 10-fold the point prevalence  sarcoidosis estimated by mass radiographic screening in a Stockholm population . Reid reEmploying as the denominator the number of persons at risk (rather than the population from which they are derived) in computing the incidence results in a spuriously elevated figure if the peak incidence is concentrated in the age range of the at risk cohort. This consideration is directly applicable to the occupational incidence of sarcoidosis because ~ 85% of cases are identified in the occupational age group, 20–49.Several investigators have inferred a causal connection between sarcoidosis and environmental or occupational exposures based on a marked incidence differential vs. population based, clinically identified figures. This inference is weakened by ascertainment bias, lack of an identified putative antigen, the incidence-trebling effect of cohort radiographic surveillance, and the employment of the surveyed, working age cohort as the denominator in the computation .1. Methodological identity is required to ensure valid inter-population incidence comparison.2. The clinically identified incidence of sarcoidosis in US Whites is 3; in UK Whites, 3.4; in persons of Scandinavian descent, 5. Population screening of Whites triples that figure. The clinically identified incidence in US Blacks is 36.3. “The” incidence of sarcoidosis is indeterminable. It is a notional concept due to a combination of ascertainment and methodological contingencies and the preponderance of unobservable cases.4. Judging from autopsy prevalence, sarcoidosis is common; clinically identified cases constitute a small minority of those that are histologically verifiable.5. Causal inference based on incidence differential is confounded by differences in case acquisition and incidence computation.The author declares that he has no competing interests.The Author has had an active interest in this subject for a number of yearsAm J Med 1985, 78:61–67.1. Reich JM, Johnson RE: Course and prognosis of sarcoidosis in a nonreferral setting: analysis of 86 patients observed for ten years. Cancer 1985, 55:366–369.2. Reich JM: Acute myeloblastic leukemia and sarcoidosis: implications for pathogenesis [case report]. J Roy Soc Med 1992, 85:306.3. Reich JM: Sarcoidosis and acute leukemia [letter]. Sarcoidosis 1993, 10:4–8.4. Reich JM: Acute myeloblastic leukemia and sarcoidosis [editorial]. Chest 1995, 107:605–613.5. Reich JM, Mullooly JP, Johnson RE: Linkage analysis of malignancy-associated sarcoidosis. J Intern Med 1994, 235:179–182.6. Reich JM: Sarcoidosis and agnogenic myeloid metaplasia [case report]. Chest 1995, 105:1474–1475.7. Reich JM, Johnson RE: Sarcoidosis and malignancy [letter]. Sarcoidosis Vasc Diffuse Lung Dis 1996, 13:173–177.8. Reich JM, Johnson RE: Estimated incidence of clinically identified sarcoidosis in a Northwest United States population. Am J Med 1997, 102:131.9. Reich JM: Sarcoidosis mortality in the United States [letter]. Thorax 1997, 52:102.10. Reich JM: BTS sarcoidosis study [letter]. Chest 1998, 113(1):147–153.11. Reich JM, Brouns MC, O’Connor EA, Edwards MJ: Mediastinoscopy in patients with presumptive stage I sarcoidosis: a risk/benefit, cost/benefit analysis. Am J Epidemiol 1998, 148(1):100–101.12. Reich JM: Racial differences in sarcoidosis incidence: a 5-year study in a health maintenance organization [letter]. Chest 1998, 113:559–160.13. Reich JM: Corticosteroid therapy and relapse in sarcoidosis [letter]. Chest 1998, 113:1143.14. Reich JM: Deciphering histoplasmosis, systemic noncaseating granuloma, and sarcoidosis in the literature [letter]. Thorax 1998, 53(7):625–626.15. Reich JM: Malignant neoplasms in pulmonary sarcoidosis [letter]. Sarcoidosis Vasc Diffuse Lung Dis 1999, 16:108.16. Reich JM: How common is sarcoidosis? [letter]. Eur Respir J 1999, 14(2) 482–483.17. Reich JM: Sarcoidosis and cancer revisited [letter]. Chest 2000, 118(6):1838.18. Reich JM: Clinical diagnosis of stage I sarcoidosis [letter]. Eur Respir J 2001, 17:833.19. Reich JM: Course and prognosis of sarcoidosis in African-Americans vs. Caucasians [letter]. Chest 2002, 121:32–39.20. Reich JM: Mortality of intrathoracicsarcoidosis in referral vs. population-based settings: influence of stage, ethnicity, and corticosteroid therapy. Chest 2003, 124:367–371.21. Reich JM: What is sarcoidosis? Sarcoidosis Vasc Diffuse Lung Dis 2003, 20(3):227–234.22. Reich JM: Adverse long-term effect of corticosteroid therapy in recent-onset sarcoidosis. Chest 2004, 125(3):1172–1173.23. Reich JM: Sarcoidosis cycle [letter]. Eur J Intern Med 2004, 15(5):269–273.24. Reich JM: Eight fundamental unsolved problems in sarcoidosis. Chest 2005, 27:1864–1866.25. Reich JM: Treatment outcome in M.avium pulmonary disease: a correction and comment [letter re. M. avium and sarcoidosis]. Eur J Intern Med 2006, 17(2):81–87.26. Reich JM: Neoplasia in the etiology of sarcoidosis. Chest 2006 April 12.27. Reich JM: Investigation of Löfgren’s syndrome [E-letter]. Sarcoidosis Vasc Diffuse Lung Dis 2007, 24:77.28. Reich JM: A requirement for histologic confirmation of stage I sarcoidosis is far more likely to prove harmful than helpful [letter]. Cytopathology 2007, 18:392–397.29. Reich JM: Tissue verification of stage I sarcoidosis [letter]. Chest 2008, 133(6);1529–1530.30. Reich JM, Patterson J, Asaph J, Brouns M: Tissue verification of stage I sarcoidosis: the question is if, not how [letter]. Chest 2009; 136:943.31. Reich JM: Concurrent sarcoidosis and lung cancer [letter]. BMJ Nov 10, 2009.32. Reich JM: Comments on Sarcoidosis (review) [Owen JD et al.] Rapid response Chest 2010, 137:235–236.33. Reich JM: Endobronchial Ultrasonography-Guided Transbronchial Needle Aspiration in Patients With Suspected Sarcoidosis. Respir Med 2010, 104:1778–1781 and 1782–1783. [Pro: Culver DA. Complete debate: 1775–1783.]34. Reich JM: Con: The treatment of the granulomatous response is beneficial in acute sarcoidosis [Debate]. Am J Ind Med 2011, 54(9):696.35. Reich JM: “Sarcoid like" granulomatous pulmonary disease in World Trade Center disaster responders: Influence of incidence computation methodology in inferring airborne dust causation [letter]. Eur J Intern Med 2012, 23:105–109. doi:10.1016/j.ejim.2011.09.01136. Reich JM: On the nature of sarcoidosis. J Occup Environ Med 2012, 54(1):2.37. Reich JM: Sarcoidosis and World Trade Center Disaster [letter]. Am J Respir Crit Care Med 2012, 185(4):461–462.38. Reich JM: Sarcoidosis mortality [letter—in response to Swigris and Baughman]. Am J Ind Med 2013, 56:496–500. doi:10.1002/ajim.22083.39. Reich JM: Shortfalls in imputing sarcoidosis to occupational exposures [editorial]. Am J Med 2013, 126(1):e17. doi:10.1016/j.amjmed.2012.05.03140. Reich JM: Neoplasia in the etiology of sarcoidosis [letter]. Am J Ind Med 2013, 56(4):503–504. doi:10.1002/ajim.2214941. Reich JM: Response to Dr. Michael Hodgson’s letter re. “Shortfalls in imputing sarcoidosis to occupational exposures.” J Bronchol Intervent Pulmonol 2013 (in press)42. Reich JM: Tissue confirmation of presumptive stage I sarcoidosis [editorial].


The humidity sensor consists of a sensing capacitor and a sensing film. The sensing capacitor is constructed from spiral interdigital electrodes that can enhance the sensitivity of the sensor. The sensing film of the sensor is polypyrrole, which is prepared by the chemical polymerization method, and the film has a porous structure. The sensor needs a post-CMOS process to coat the sensing film. The post-CMOS process uses a wet etching to etch the sacrificial layers, and then the polypyrrole is coated on the sensing capacitor. The sensor generates a change in capacitance when the sensing film absorbs or desorbs vapor. The ring oscillator circuit converts the capacitance variation of the sensor into the oscillation frequency output. Experimental results show that the sensitivity of the humidity sensor is about 99 kHz/%RH at 25 °C.This study presents the design and fabrication of a capacitive micro humidity sensor integrated with a five-stage ring oscillator circuit on chip using the complimentary metal oxide semiconductor (CMOS) process. The area of the humidity sensor chip is about 1 mm The humidity sensor was a capacitive type, and the polyimide was locally cured at a temperature over 350 °C for 1 hour by the MEMS micro hotplate. The humidity sensor exhibited a sensitivity of 0.77 pF/%RH and a hysteresis of 0.61 %RH. Miyoshi et al. [et al. [et al. [et al. [Humidity sensors that are used to measure and monitor environmental humidity are important devices in many industrial and biomedical fields. The micro electromechanical system (MEMS) technique has become popular for the miniaturization of sensors. Humidity sensors fabricated by the MEMS technique have the advantages of small size, high performance, easy mass-production and low cost . Severalet al.  presente [et al.  was fabrIn this study, we investigate the fabrication of a micro humidity sensor integrated with a ring oscillator circuit on chip using the CMOS-MEMS technique. The sensing material of the sensor is polypyrrole. The post-process uses wet etching to etch the sacrificial layers and the polypyrrole is coated on the humidity sensor. The sensor produces a change in capacitance when the sensing film absorbs or desorbs vapor. The ring oscillator circuit converts the capacitance of the humidity sensor into the oscillation frequency output.2.sensor represents the capacitance of the humidity sensor and Cload is the load capacitance. The ring oscillator circuit is used to convert the capacitance variation of the sensing capacitor into the frequency output. As shown in A five-stage ring oscillator is adopted as the sensing circuit owing to the fact its phase noise is smaller than that of three-stage ring oscillator, and it has a stable output signal . Figure sensor of the ring oscillator circuit is given by [inv is the delay time associated with the inverters, τsensor the delay time associated with the humidity sensor, Cload the load capacitance, Csensor the humidity sensor capacitance, and ΔV and Iave are the threshold voltage and average current, respectively. According to The ring oscillator circuit connects with odd inverters does not have a stable operating point. Therefore, the circuit will oscillate when any of the odd inverter input or output voltage separates from the unstable operating point. The oscillation frequency fgiven by (1)fsensThe professional circuit simulation software, HSPICE, was used to analyze the characteristics of the ring oscillator circuit. 3.The commercial 0.35 μm CMOS process of Taiwan Semiconductor Manufacturing Company (TSMC) was utilized to manufacture the integrated humidity sensor chip. The chip requires a post-process to etch the sacrificial layers and to coat the polypyrrole film after completion of the CMOS process. 4H5N) is mixed with 50 mL deionized water and stirred continuously; (2) 10 g anhydrous ferric chloride (FeCl3) is mixed with 30 mL deionized water and stirred continuously; (3) the solution of FeCl3 is added into the pyrrole solution with stirring for 48 h. The resulting product is filtered, dropped on the humidity sensor chip. Then, the chip is followed by calcination in air at 120 °C for 3 h.4.The performance of the humidity sensor was measured by a power supply, a LCR meter, a spectrum analyzer and a test chamber . The test chamber could supply a temperature range of 0–100 °C and a humidity range of 25–95 %RH. Temperature and humidity in the test chamber can be tuned separately and kept at constant levels. The spectrum analyzer was utilized to record the signal response of the sensor to humidity changes. The LCR meter was adopted to detect the capacitance of the sensor. The humidity sensor was set in the test chamber. The power supply provided a bias voltage of 3.3 V to the sensor. In order to characterize the capacitance variation of the sensor to humidity, the sensor without the ring oscillator circuit was tested. The test chamber supplied different humidity to the humidity sensor. When the humidity rose or dropped, the humidity sensor changed in capacitance. The capacitance variation of the humidity sensor was measured by the LCR meter. In order to understand the influence of temperature, the output frequency of the humidity sensor was tested at different temperatures. S represents the sensitivity of the humidity sensor and T is the temperature. According to Dai  proposed5.This humidity sensor integrated with a five-stage ring oscillator circuit has been implemented using the commercial 0.35 μm CMOS process and the post-process. The humidity sensor was composed of a sensing film and a sensing capacitor. The sensing film was polypyrrole, that was synthesized by the chemical polymerization method. The electrodes of the sensing capacitor were designed with a spiral interdigital shape that could increase the sensor capacitance. The post-process used wet etching to etch the sacrificial layers, and the polypyrrole was coated on the sensing capacitor. The ring oscillator circuit converted the capacitance variation of the sensor into the frequency output. The experiments showed that the ring oscillator circuit was normally operation after the wet etching post-process. Thereby, the post-process was compatible with the commercial CMOS process. The measured results depicted that the humidity sensor had a sensitivity of 99 kHz/%RH at 25 °C, and its sensitivity exceeded that of Dai . The hum


To the best of our knowledge, this is the first report of a case of conjunctival sarcoidosis that presented as limbal conjunctival hypertrophy and was controlled with topical cyclosporine.A 70-year-old Asian woman presented with painless limbal conjunctival hypertrophy. An incisional biopsy of the hypertrophic lesion showed noncaseating granulomas that consisted of multinucleated giant cells and epithelioid cells. The results of a systemic evaluation were normal except for a slight increase in serum angiotensin-converting enzyme to 58.9IU/L . Twice-daily 0.05% topical cyclosporine was prescribed for the treatment of remaining lesions. After two months, the lesions had completely resolved and her serum angiotensin-converting enzyme level had normalized.Sarcoidosis should be considered in the differential diagnosis of conjunctival hypertrophy. Topical cyclosporine can be useful in conjunctival sarcoidosis. Sarcoidosis is a multisystemic inflammatory process characterized by noncaseating granulomatous infiltration . Ocular We present the case of a patient with isolated conjunctival sarcoidosis presenting as limbal conjunctival hypertrophy who was successfully treated with 0.05% topical cyclosporine.A 70-year-old Asian woman was referred to our hospital for an evaluation of limbal conjunctival hypertrophy in her left eye. She reported mild discomfort but had no other symptoms, such as pain or itching. She had chronic angle closure glaucoma in her left eye and was on 0.1% brimonidine twice a day and 0.005% latanoprost four times a day. Her past medical history was unremarkable except for hypertension and hyperlipidemia. A slit-lamp examination showed multiple yellowish conjunctival nodules in both eyes and hypertrophy around the superior limbal area in her left eye Figure , and herAlthough conjunctival sarcoidosis mostly presents as multiple nodules resembling follicular conjunctivitis, other conjunctival manifestations including conjunctival deposit, large conjunctival tumor and multilobular limbal nodules have been reported -3,5-7. TCyclosporine is known to be useful in various ocular surface inflammatory diseases, such as ocular rosacea, dry eye disease, atopic keratoconjunctivitis, graft-versus-host disease and herpetic stromal keratitis, due to its anti-inflammatory and immunomodulatory action . AlthougOur experience suggests that sarcoidosis can manifest as limbal conjunctival hypertrophy, and should therefore be included in the differential diagnosis of sarcoidosis. Topical cyclosporine can be a useful option in the treatment of conjunctival sarcoidosis, especially when there are concerns about steroid side effects.Written informed consent was obtained from the patient for publication of this manuscript and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.ACE: angiotensin-converting enzyme; CD: cluster of differentiation.The authors declare that they have no competing interests.SBH and HKY acquired, analyzed and interpreted the patient data regarding the sarcoidosis. JYH and WRW performed the histological examination of the conjunctival tissue sample. SBH and HKY were major contributors in writing the manuscript, and JYH and WRW revised it critically. All authors read and approved the final manuscript.SBH, JYH and WRW work in the corneal and refractive surgery division at Seoul National University College of Medicine, and HKY works in the strabismus and pediatric ophthalmology division at Seoul National University College of Medicine.


A 1.1 μW power dissipation, voltage-output humidity sensor with 10% relative humidity accuracy was developed in the LFoundry 0.15 μm CMOS technology without post-processing. The sensor consists of a woven lateral array of electrodes implemented in CMOS top metal, a humidity-sensitive layer of Intervia Photodielectric 8023D-10, a CMOS capacitance to voltage converter, and the self-calibration circuitry. After n2.Humidity sensors using hygroscopic polymer films are the most suitable for integration in CMOS. A hard-cured polymer film absorbs moisture, increasing its effective dielectric constant with humidity in predictable and reversible process. But this approach has problems to solve. Firstly, hygroscopic films produce humidity-dependent capacitance, so a capacitance-to-voltage on-chip converter is necessary for simplified processing of the sensor output. Secondly, the implementation of the sensing element is not straightforward. To obtain a large relative capacitance change, electrical flux must be confined to the hygroscopic film. The simplest way to reach this objective is if the electrodes enclose the hygroscopic film from all sides. But in order for moist air to access the sensing film, there must be a large area of the polymer exposed to the air. In the implemented humidity sensor, electrical flux reaching the CMOS substrate was confined by a woven electrode mesh below the main sensing electrodes see . The wovFurthermore, the previously proposed comb-type electrodes have a metal density of 50% if sensitivity is maximized . For theThe design in  was compA C/V (capacitance-to-voltage) converter derived from  was deve3.Experimental results for manufactured humidity sensors are summarized in The accuracy mentioned in the 4.An ultra-low power, low-cost, self-calibrated humidity sensor in CMOS without post-processing was designed and evaluated. Simple control and readout of the proposed sensor allows its usage in low-end sensor nodes and embedded systems. The sensor can work with an RC-oscillator and a low-cost MCU to comprise a complete sensing system. The performance of the developed humidity sensor is summarized in