Cyt b) genes from the GOBASE database were compiled and the evolutionary dynamics of the Cyt b gene introns was assessed. Cyt b gene introns were frequently present in the fungal kingdom and some lower plants, but generally absent or rare in Chromista, Protozoa, and Animalia. Fungal Cyt b introns were found at 35 positions in Cyt b genes and the number of introns varied at individual positions from a single representative to 32 different introns at position 131, showing a wide and patchy distribution. Many homologous introns were present at the same position in distantly related species but absent in closely related species, suggesting that introns of the Cyt b genes were frequently lost. On the other hand, highly similar intron sequences were observed in some distantly related species rather than in closely related species, suggesting that these introns were gained independently, likely through lateral transfers. The intron loss-and-gain events could be mediated by transpositions that might have occurred between nuclear and mitochondria. Southern hybridization analysis confirmed that some introns contained repetitive sequences and might be transposable elements. An intron gain in Botryotinia fuckeliana prevented the development of QoI fungicide resistance, suggesting that intron loss-and-gain events were not necessarily beneficial to their host organisms.In this study, all available cytochrome b ( Introns are widely distributed in numerous genes of viruses, prokaryotes and eukaryotes Introns Late theory argues that introns could have expanded in a fashion similar to transposable elements (TEs), have been the results of tandem duplications into exons Introns Late theory where introns might be able to spread horizontally between phylogenetically distant species of different kingdoms Intron density in eukaryote genomes varies by more than three orders of magnitude, suggesting that there must have been extensive intron gain and/or loss events during evolution Introns Early theory suggests that introns were abundant in ancestral genes and mainly evolved through intron loss Introns Early vs Late was concerned with the origin of nuclear spliceosomal introns, the details of evolution of mitochondrial group I or group II introns could provide insights.In contrast, via a double-strand breaks (DSBs)-repair process in trans to promote splicing of other group I introns in the same genome via an RNA-based mechanism known as ‘retrohoming’. The proteins encoded by group II introns are multifunctional, containing maturase, reverse transcriptase and DNA-binding functions as well as DNA endonuclease activity. The maturase activity facilitates intron splicing by stabilizing the catalytically active RNA conformation, while other functions aid in the retrohoming to allelic target sites Most group I introns harbor genes encoding so-called homing endonucleases, initiating intron mobility Monilinia spp. (anamorphs in Monilia), Cyt b genes, the targets of the Quinone outside inhibitor fungicides (QoIs), were isolated and analyzed Cyt b genes in M. fructicola, M. laxa, M. fructigena and M. yunnanensis have seven, whereas the Cyt b gene in M. mumecola has six large (>1 kb) introns. The intron/exon organization varies considerably among these species. In contrast to the large introns, the exons of Cyt b are usually small, ranging from 11 to 397 bp in length. The combined intron sequences of the Cyt b gene in M. fructicola, M. fructigena, M. laxa, M. yunnanensis and M. mumecola are 10,751, 12,380, 12,329, 11,456 and 13,027 bp, respectively. The large intron number and size make the Cyt b genes in Monilinia spp. the longest reported to date in all kingdoms of organisms. In contrast, the Cyt b gene in Blumeria graminis f.sp. tritici, the causal agent of wheat powdery mildew, is intronless, even though Monilinia and Blumeria species belong to the same fungal class Leotiomycetes. It will be interesting to know what caused the Cyt b genes in Monilinia species to harbor the largest ones in eukaryotic organisms.The number of mitochondrial introns or intron sequences may vary dramatically in different species. In recent studies on peach brown rot fungi Cyt b genes have been sequenced and annotated from thousands of species including distantly related species from different kingdoms and widely used in systematic studies or evolutionary studies Cyt b genes vary dramatically among different species. However, the dynamics of Cyt b gene introns, has not been systemically studied. In this study, we compiled all available Cyt b genes from the GOBASE database and assessed the evolutionary dynamics of Cyt b gene introns, and some introns in Monilinia and Botryotinia spp. were confirmed by Southern blot analyses.The Cyt b genes were previously isolated from peach brown rot fungi M. fructicola, M. fructigena, M. laxa, M. yunnanensis and M. mumecolaCyt b gene was isolated from the rice false smut fungus Ustilaginoidea virens. Briefly, degenerate primers were designed based on Cyt b gene sequences from phylogenetically close species, and PCR reaction was carried out to amplify the conserved region from U. virens isolate UV-8a. Once a conserved fragment was isolated, the flanking sequences were obtained using Tail-PCR or Inverse-PCR amplifications and sequenced. For cDNA amplification, total RNA was isolated using the RNeasy Plant Mini Kit  according to the manufacturer’s instructions. First-strand cDNA was synthesized using an oligo-dT primer and Superscript III reverse transcriptase  according to the manufacturer’s recommendations. Amplification of Cyt b-specific cDNA was performed using the primers designed to amplify the Cyt b sequence from translational start codon to translational stop codon. Intron numbers and precise locations were identified by comparing genomic DNA (gDNA) and cDNA sequences.The Cyt b gene and intron sequences were obtained directly from the GOBASE database  using the gene search and intron search options with Cyt b gene (cob) as gene name http://gobase.bcm.umontreal.ca/) is invalid currently. However, the accession nos. in GenBank (http://www.ncbi.nlm.nih.gov/) of all Cyt b genes showed in this study were indicated either in the table or figure legends.Cyt b gene is indicated by its “position” and “phase”. Intron position in this study refers to the position of codon at which the intron is inserted. Intron phase refers to the nucleotide position in a codon at which the intron is inserted. An intron is considered as phase “0” if it is inserted between two consecutive codons, “1” if inserted between the first and the second nucleotide of a codon, “2” if inserted between the second and the third nucleotide of a codon. The number, position and phase of introns in Monilinia spp. and U. virens were identified by comparing the gDNA and cDNA sequences. For Cyt b genes retrieved from the GOBASE database, the intron number, position and phase were determined as described below. To determine the locations of introns of the Cyt b genes, the deduced amino acid sequences of all Cyt b genes were aligned using the MegAlign program in the Software package DNASTAR . For the locations of introns to be comparable in different Cyt b genes, their relative locations corresponding to the Cyt b gene in U. virens were considered as their locations. If the locations of two introns vary by fewer than 6 nucleotides, they are called “sliding introns”. The annotation for all sliding introns were checked manually as described in rd intron of the Cyt b gene from M. mumecola.Information about intron location is important and the base for elucidating the dynamics of introns. In the present study, the relative location of an intron in the Cyt b gene cDNA sequences or intron sequences at position 164 were aligned with the program Clustal W in the software MEGA 4.0. The following settings were used to construct the phylogenetic trees: heuristic search using close neighbor interchange  with initial trees generated by random addition (100 reps).Maximum parsimony (MP) method was used to construct phylogenetic trees. Cyt b introns was identified by using a reciprocal BLAST to calculate the pairwise nucleotide identity of introns. Algorithm BLASTN was used for the identity search in program BioPerl with default parameters and an E-value cutoff of 1e-05. To obtain an accurate estimate of global hit statistics, a tiling of high-scoring segment pairs (HSPs) onto either the subject or the query sequence was performed. Two introns were considered as homologues if coverage (the sum of HSP length/longer one in paired introns) >30% and average pairwise identity >80%.The homology of EcoR I or Hind III and separated in 1.0% agarose gels in 0.5×TBE buffer and transferred to Hybond N+ membranes . Intronic fragments were amplified by PCR reactions with the intron-specific primer pairs listed in Southern hybridization analysis was performed mainly as described previously Cyt b gene from Ustilaginoidea virens was 9216 bp in size and contained six introns. They were located at cDNA positions (5′–3′) 201, 393, 490, 506, 562 and 823 with sizes of 1594, 1180, 1223, 1775, 1325 and 949 bp respectively. The exon and intron sequences of isolate UV-8a are available in GenBank under accession number JN204426. In other organisms, relative locations of introns corresponding to the Cyt b gene in U. virens were considered as their locations.The entire Cyt b genes were obtained from 3,276 species of four eukaryotic kingdoms. Only 92 species, including six fungal pathogens (Monilinia spp. and U. virens), had introns  and 16 had unique introns (present in only one gene). The intron numbers at individual positions varied from a single representative at 16 different positions to 32 different introns at position 131  have a certain chance of being homologous. However, the sum of HSPs of many hits only covered a limited part of either introns. Such introns should not be real homologues but just contained certain conserved regions. Thus, in the present study, only introns meeting additional criteria (coverage >30% and average pairwise identity >80%) were considered homologues.To understand the relationship between Cyt b genes had occurred. For example, at position 169, homologous introns were frequently detected from phylum Ascomycota , Eurotiomycetes  and Leotiomycetes , indicating that these introns were vertically inherited from a common ancestral intron before the divergence of these classes. These highly homologous introns were detected in all of the investigated species of Monilinia genus but not from the species B. fuckeliana of the same Sclerotiniaceae family, indicating that the intron at position 169 was recently lost in B. fuckeliana (after the divergence of Monilinia and Botryotinia genera). Intron loss events were also observed at position 67: homologous introns were detected in species from the Leotiomycetes  and Sordariomycetes , but not in the G. zeae species which is in the same order Hypocreales with U. virens under Sordariomycetes class. Besides positions 67 and 169, similar intron loss events were observed at least at positions 131, 143, 164, 260  of the intron . In the present study, most introns at common positions contained the LAGLIDADG endonuclease coding sequence  or the GIY-YIG endonuclease coding sequence  and thus should be able to move by lateral transfer. Homing endonuclease genes (HEGs) are disproportionately common in mitochondria and chloroplasts of eukaryotes. The first HEG discovered was ω (I-SceI) in the large subunit rRNA gene in the mitochondria of the yeast Volvox carteri (homologue 9), where the homologous introns Vc1 and Vc2 were located at different positions in the same gene. These observations suggested that the frequent gain-and-loss of introns occurred during evolution of Cyt b genes and some of the Cyt b introns might be TEs because they can move to new sites and undergo deletion or amplification in genomes.As shown in Cyt b intron sequences in the genome of Monilinia species. Intron My3 from M. yunnanensis is located at position 143, southern hybridization using My3 as probe showed expected bands  and M. mumecola  and M. fructicola, both species did not have homologous introns of My3 in their Cyt b genes. These results indicate that multiple copies of introns My3, Ml3 and Mm3 exist in the genomes of M. yunnanensis, M. laxa and M. mumecola, as well as in M. fructigena and M. fructicola. These introns were repetitive elements, a characteristic for TEs. Mfg2 was also confirmed to be a repetitive intron  . The presence of an intron at the position 143 has been shown to prevent the formation of the G143A mutation in some pathogens, because mutations in this codon would interfere with intron splicing and consequently prevent the formation of an active Cyt b gene S. cerevisiae, where an exon mutation at the second nucleotide upstream of the splice site of intron 4 in the Cyt b gene (COB4) did not permit correct splicing of the pre mRNA Single amino acid changes in the Cyt b alleles were discovered, one with and one without an intron at position 143 in B. fuckeliana. Southern hybridization using intron Bf3 as probe showed that this intron was absent from the entire genome of isolates lacking the intron at position 143 , the Bf3 gained event directly influenced the development of the QoI fungicide resistance. Once the isolates gained the Bf3, they lost the potential ability to develop the fungicide resistance based on the G143A mutation. Pathogens did not get any visible benefit from such an intron gain event but had negative influence for the development of fungicide resistance, suggesting that it was just one of many intron gain-and-loss events occurring neutrally and the intron gain-and-loss events were not necessarily beneficial to their host organisms.Two different tion 143 . Homolognia spp. . TherefoTable S1Primers used for intron-specific PCR amplification(DOC)Click here for additional data file.Figure S1Modification of the sliding intron position or phase. (A) The phase of intron Sc5 was modified based on one nucleotide shifting of the exon/intron sequence. (B) The phase of intron Gi3 was modified based on two nucleotide shifts that altered one codon. (C) Sliding introns can’t be modified to same position or phase. Lowercase letters indicate intron sequences, and uppercase letters represent the flanking exon sequences, the larger uppercase letters represent the codon at which individual introns located. If introns have different positions or phases, they are considered as different locations. The locations of introns were modified slightly from original information for some sliding introns. For example, the intron Sc5 at position 270 was a phase 0 intron based on the database information, but one nucleotide shifting of exon/intron sequence could make it have same phase with other introns at this position (such as Cbs2(2), Cc(2) etc. in (TIF)Click here for additional data file.Figure S2Cyt b gene introns in Monilinia spp and B. fuckeliana. Two restriction enzymes EcoR I and Hind III were used to generate the restriction profiles. Digested genomic DNA was separated in 0.8% agarose gel, and the blot was hybridized with the Mfg2 (A), Ml4 (B) and Bf3 (C) fragments. Lanes 1, 11 and 2, 12: M. yunnanensis isolates YKG10-64a and SM09-7c; 3, 13 and 4, 14: M. fructigena isolates SL10 and Mfg2-GE-A; 5, 15 and 6, 16: M. laxa isolates BEK-SZ and EBR ba11b; 7, 17 and 8, 18: M. mumecola isolates HWL10-11a and HWL10-20a; 9, 19 and 10, 20: M. fructicola isolates MPA14 and BM09-4a in A and B. For B. fuckeliana in C, 1, 5, isolate RhosimBC-4; 2,6, isolate PeachBC-1; 3,7, isolate GarlicBC-78; 4,8, isolate ViotriBC-1. The sizes (in kilobases) of marker DNA fragments are indicated on both sides  Co., Ltd).Identification of the (TIF)Click here for additional data file.


An intermediate higher refractive index overlay, deposited through Electrostatic Self-Assembly, is placed between the fiber cladding and the humidity sensitive film in order to increase the total effective refractive index of the coating. With this intermediate design, a three-fold improvement in the sensitivity was obtained. Wavelength shifts up to 15 nm against 5 nm were achieved in a humidity range from 20% to 80%.This work addresses a new configuration that improves the sensitivity of a humidity sensor based on a long-period fiber grating coated with a SiO Several sensing fiber architectures such as hollow core fibers, tapered optical fibers, side-polished fiber, U-bends, nano-Fabry-Perot cavities etc., have been reported for humidity measurements induced by refractive index changes of the external medium –4. Also,In previous works, humidity sensors based on polymeric overlays have been developed –8. Howev2 nanospheres, as a porous hydrophilic material, can be used as a humidity sensitivity film. In previous work [2 nanospheres coated on a LPG could provide a fast response humidity sensor. However, due to its low index of refraction, lower than the fiber itself, the sensitivity is quite low and should be improved in order to avoid expensive equipment. This can be done by increasing the total effective refractive index of the sensitive coating using an intermediate overlay with higher refractive index.It was proved that SiOous work  it was a2.01β and 0jβ are the propagation constants of the core and the j cladding modes respectively, and Λ is the period of the grating. Results obtained using this approximation present appreciable variation when compared with those values calculated with rigorous coupled mode differential equations [01,01ζ and 0j,0jζ are the self-coupling coefficients of the core and the j cladding modes, 0s is the coefficient of the first Fourier component of the grating and N is the refraction order. If the grating function is sinusoidal, 0s simplifies to unity. In the case of arc-induced LPG, this last approximation can be used without lacking rigor because the margin of error is compared with the fabrication tolerances of the own LPG.In order to analyze the spectrum evolution in structures based on LPG with a sensing overlay when the aim is only to detect the displacement of the resonance wavelengths, it is possible to use either the Bragg or the Bragg modified conditions . The firquations . Howeverquations :(2)β01 is halfway between its minimum value and the wavelength of the next lower cladding mode before deposition. More theoretical details can be seen in .3.The LPG used in this study was an arc-induced LPG  with Λ =® SM-30 SiO2-water colloid. In this case, the PAH and PDDA acted as polycations, and PolyR-478 and SM-30 were the anionic species. The number of overlays of PDDA/PolyR-478 was 14 layers and the number of PAH/SM30 overlays was 14 layers, which confer a total film thickness of less than 300 nanometers. Specifically, although the film thickness is difficult to measure due to the fibre geometry, in previous works it has been demonstrated that each PDDA/PolyR-478 and PAH/SM30 layers had thicknesses of approximately 12 nm and 7 nm, respectively [The polymeric layers were deposited using the ESA layer-by-layer method . In thisThe thickness overlay is chosen to guarantee that the attenuation band is located where there is good sensitivity and where it does not vanish at the same time. In this work, the good sensitivity band corresponds to ∼1,520 nm. In fact, the same effect noticeable when the ambient humidity increases, can be appreciated when the thickness of the coating is getting increased see . If the In 4.HR in percentual values):HR is the Relative Humidity and Δλ the wavelength shift. These relations allow obtaining the sensing heads sensitivity at different relative humidity levels. For example, in the range 20–50%, the sensitivity is ≈ 0.04 nm/%RH and 0.06 nm/%RH for the PAH/SM30 coating and the PDDA/PolyR + PAH/SM30 structures, respectively, while around a relative humidity level of 70% those values are improved to ≈ 0.13 nm/%HR and ≈ 0.48 nm/%HR for the PAH/SM30 coating and the PDDA/PolyR + PAH/SM30 structures, respectively. These results indicate that the configuration using an intermediate overlay of higher refractive index improves the sensitivity of the sensor by a factor of 1.5 at a HR ≈ 30%, which is improved to a value of 3.5 when dealing with HR around 70%.HR time response of the sensing head in the range 100–200 ms, far better than what is achieved using standard electrical humidity sensorsPreliminary experiments performed indicate a 5.2-nanospheres thin film was experimentally demonstrated. An intermediate higher refractive index overlay is placed between the fiber cladding and the humidity sensitive SiO2-nanospheres film in order to increase the total effective refractive index of the coating. In this way, compared with the situation of using only the layer sensitive to humidity, a sensitivity improvement factor larger than three was obtained. This result indicates a high potential of utilization of this sensing structure in the context of chemical and biochemical applications.In summary, a new configuration that improves the sensitivity of a humidity sensor based on a LPG coated with a SiO


Most bacterial group II introns harbour an open reading frame (ORF), coding for a protein with reverse transcriptase, maturase and occasionally DNA binding and endonuclease activities. Some ORF-containing group II introns were shown to be mobile retroelements that invade new DNA target sites. From an evolutionary perspective, group II introns are hypothesized to be the ancestors of the spliceosome-dependent nuclear introns and the small nuclear RNAs  that are important functional elements of the spliceosome machinery. The ability of some group II introns fragmented in two or three pieces to assemble and undergo splicing in Lactococcus lactis to be fragmented into three pieces in vivo. Trans-splicing tripartite variants of Ll.LtrB were selected using a highly efficient and sensitive trans-splicing/conjugation screen. We report that numerous fragmentation sites located throughout Ll.LtrB support tripartite trans-splicing, showing that this intron is remarkably tolerant to fragmentation.We used a transposon-based genetic screen to explore the ability of the Ll.LtrB group II intron from the Gram-positive bacterium trans-splice their flanking exons in vivo. The selected introns represent the first evidence of functional tripartite group II introns in bacteria and provide experimental support for the proposed evolutionary relationship between group II introns and snRNAs.This work unveils the great versatility of group II intron fragments to assemble and accurately  Self-splicing group II introns were initially discovered interrupting various mitochondrial and chloroplastic genes of higher plants and algae, as well as mitochondrial genes of lower eukaryotes Despite a lack of sequence similarity, group II introns fold into a conserved secondary structure composed of six domains (DI-DVI) radiating from a central wheel in vivo and in vitro. In vivo however, folding of these large ribozymes into their catalytically active structure depends on the maturase activity of their IEP or free-standing maturases. Group II introns self-splice from pre-mRNA transcripts through two consecutive transesterification reactions  that constitute the heart of the nuclear intron splicing machinery called the spliceosome trans-acting RNAs trans . Relaxase initiates conjugation by nicking the conjugative element at the origin of transfer (oriT) and delivers the DNA to recipient cells trans-splicing/conjugation assay in L. lactis, showing that Ll.LtrB is able to splice in trans when fragmented at natural group II intron fragmentation sites trans-splices when fragmented at various locations throughout its structure, revealing its trans-splicing versatility The Ll.LtrB intron from the Gram-positive bacterium trans-splicing Ll.LtrB variants were randomly generated and selected using an in vivo trans-splicing/conjugation assay. We discovered that Ll.LtrB can assemble and accurately trans-splice when fragmented into three pieces at many locations throughout the majority of its structure. Moreover, some of the Ll.LtrB tripartite variants studied were found to trans-splice only 11- to 35-fold less efficiently than their bipartite counterparts. This is the first demonstration of the tripartite trans-splicing ability of Ll.LtrB, as well as the tripartite trans-splicing versatility of a bacterial group II intron. This study further illustrates the great plasticity of group II intron to fragmentation and supports the theory suggesting that spliceosomal snRNAs were derived from group II intron fragments.Here, we took advantage of our previously engineered Tn5-based genetic screen to explore sites throughout Ll.LtrB that would tolerate fragmentation into three pieces. Tripartite trans-splicing/conjugation assays in L. lactis. In one assay, trans-splicing of Ll.LtrB fragmented in two pieces at natural group II intron fragmentation sites was monitored by conjugation of a relaxase-deficient chromosomal sex factor (SF) (107-fold detection range) trans-splicers fragmented at various positions throughout the intron by the conjugation of their carrying pLE12 mobilizable plasmid (104-fold detection range) We previously established two very sensitive group II intron trans-splicing efficiency in three pieces by the conjugative transfer of its carrying pLE1 mobilizable plasmid (oriT) of the L. lactis SF so that it could be mobilized upon relaxase production from tripartite trans-spliced Ll.LtrB transcripts   . PrevioultrB::tet was used as the donor strain for plasmid conjugation  (ltrB::Tn bank (ltrB exon 1 (PvuI) and spans domains I through III of the first intron fragment including a portion of the ltrA gene in DIV (RT domain) (BsaI) (The engineered Tn5 transposon harbors a promoter  [17]. ThTn bank) , step 1 :Tn bank , step 2.ltrB L. lactis strain and the transformants were pooled and used as donor cells for the conjugation assay , only 11- to 35-fold lower than their bipartite counterpart, S3 (4.2×10−5) (−7 range (−9 (ltrA (data not shown). Expression of LtrA from a second plasmid (pDEltrA) led to a significant increase in trans-splicing efficiency, the tripartite intron being as efficient as its bipartite counterpart, S3 (Twelve tripartite introns were chosen for further investigation (.2×10−5) . One iso−7 range , S3-447,ange   and only 3.5- to 11.5-fold over background . However, even though the conjugation efficiencies of these tripartite variants were significantly lower compared to their bipartite counterparts we were able to detect ligated exons by RT-PCR for the majority of them , and the transposon insertion sites were identified by sequence analysis. The Tn5 fragmentation sites were found again scattered throughout the regions subjected to the screen . Next, wn assays . All the.5×10−8) , 100- to of them .trans-splicing conjugation assays we previously demonstrated the bipartite trans-splicing versatility of the Ll.LtrB group II intron. Ll.LtrB was shown to support fragmentation at natural fragmentation sites trans-splicing of various bipartite introns in vivoUsing psaA gene of C. reinhardtiinad5 gene of O. berterianatrans-splicing ability of Ll.LtrB when fragmented into three pieces. The power and reliability of this experimental approach designed to select fragmented introns that can assemble and splice in trans from a bank of fragmented introns was previously demonstrated trans-splicing variants were selected from bipartite introns using sensitive trans-splicing/conjugation assays in L. lactis . Although LtrA is not detectable by Western blot, six potential start codons downstream of the fragmentation site are found to be in the correct reading frame. The use of one of these initiation codons may lead to low levels of expression of a N-terminal truncated LtrA protein with maturase activity. Accordingly, the expression of LtrA in trans from a second plasmid rescued splicing of this tripartite intron inducing conjugation efficiencies at levels similar to the bipartite S3 intron. This shows that the additional fragmentation site within DIV does not significantly affect Ll.LtrB trans-splicing efficiency. This is reminiscent of the previously selected bipartite introns fragmented within ltrA but downstream from the maturase domain. We proposed that these introns expressed C-terminal truncated versions of active maturases. Similarly, the expression of full-length LtrA from a second plasmid significantly increased the trans-splicing efficiency of these introns The lack of fragmentation observed within DIV confirms the importance of LtrA as a co-factor for Ll.LtrB As previously observed in our screens for bipartite introns trans-splicing efficiencies. Although we hypothesize that the location of the fragmentation site determines the assembly and trans-splicing efficiency, the stability of the first two fragments may not be a significant factor, especially if the third fragment is available in limiting amounts. Nevertheless, the three RNA fragments retain the resilience to fold, assemble and trans-splice accurately in vivo.Through Northern blots we clearly detected the three RNA fragments of the twelve Ll.LtrB tripartite variants chosen. All three fragments seem to be expressed to similar levels while some degradation products can be seen. The third fragment appears to be the least stable when compared to the first two fragments. In identifying the first intron fragment of the tripartite variants, we observe an increase in the intensity of the bands when fragmentations occur downstream of DI. Since DI serves as a docking site for the remaining domains, its folding as a complete module possibly contribute to the increase in stability of the fragments extending into DII to DIV. Even though certain tripartite variants appear to have fragments more stable than others this does not seem to significantly affect their trans-splicing respectively 11- and 28-fold less efficiently. In addition, significantly less transconjugants were obtained following the trans-splicing conjugation screens. Accordingly, the analysis of independent transconjugants from the pLES4::Tn and pLES10::Tn screens showed conjugation efficiencies 100- to 600-fold lower than their bipartite counterparts only 11.5- to 3.5-fold over background. This is in contrast to the pLES3::Tn transconjugants that trans-splice only 11- to 35-fold less efficiently than S3 and 2,900- to 920-fold over background. Similarly to what was previously observed following bipartite screens, when the end of DIV was subjected to transposon insertion (S4), introns fragmented within the ltrA gene downstream of the maturase domain were selected suggesting the expression of truncated LtrA proteins with maturase activity trans-splicing introns fragmented in DI are tripartite introns also fragmented in DIV. While the great majority of bipartite introns were found fragmented in DIV, no bipartite introns were ever found fragmented in DI. Taken together, this suggests that the two natural tripartite introns probably underwent fragmentation in DIV before being further fragmented in DI.In a complementary approach to fragmenting the first piece of a bipartite intron (S3), we submitted the second fragment of bipartite introns severed in DI at either position 172 (S4) or 44 (S10) to our fragmentation screen. We first noticed that the S4 and S10 bipartite introns were not as proficient as S3, psaA gene of C. reinhardtiinad5 gene of O. berterianaL. lactis is quite tolerant to tripartite fragmentation as it can be fragmented into three pieces not only in DI and DIV like the two organellar tripartite introns but at various locations throughout its structure. The selected Ll.LtrB tripartite introns retained the ability to fold correctly, assemble and accurately splice in trans. This suggests that following fragmentation, trans-splicing group II introns would be under significant pressure to improve their splicing efficiency in order to restore the expression level of the gene they interrupt. Such adaptation would lead to fragmented introns that trans-splice more efficiently and that can be further fragmented. Since no functional bipartite or tripartite group II introns were found yet in bacterial genomes the selected introns represent the first evidence of functional tripartite group II introns in bacteria. Assembly of three intron RNA fragments during trans-splicing in vivo probably involves a complex combination of base pairing, long-range tertiary interactions and the potential involvement of the splicing co-factor LtrA The discovery of naturally occurring group II introns fragmented into two or three pieces supports the theories proposing that self-splicing group II introns are the progenitors of nuclear introns and that the spliceosomal snRNAs were derived from fragments of group II introns Demonstration that a bacterial group II intron can be fragmented in three pieces at multiple sites throughout its structure and still be functional provides additional experimental support to the theory proposing that the five snRNAs of the eukaryotic spliceosome were derived from group II intron fragments.Escherichia coli DH10β strain was used for both cloning and plasmid amplification, and was grown in LB broth at 37°C with shaking. The methylase-free E. coli GM119 strain was used to isolate non-methylated DNA since methyl-free PvuI digested DNA fragments are ligated more efficiently. L. lactis strains NZ9800ΔltrB::tet (NZΔL-b) (TetR) R) The oriT) from L. lactis SF was PCR-amplified  under the control of P23 right (BssHII) and the second piece (3′-intron–ltrBExon2) under the control of P23 left (NotI). These constructs were shown to be mobilizable by conjugation  resistance, and the P23 constitutive promoter  and 2.8 times in NZ9800ΔltrB following transformation. The Tn5 transposon was therefore inserted between each nucleotide of the PvuI/BsaI fragment of the intron 2.8 times immediately preceding the trans-splicing/conjugation assay. The same procedure was done to subject the second intron fragment to the screen in pLE-S4 (BsrGI/AatII) and pLE-S10 (EagI/BsaI). The statistical representation of the generated banks was calculated by dividing the number of Cam/Spc colonies recuperated following transformation of the Tn5-containing plasmids by twice the size of the fragments . This method of calculation takes into consideration that Tn5 can insert randomly in both orientation within the target plasmid The Tn5 transposon variant used in this study was previously generated promoter . With thConjugations were performed on milk plates as previously described ltrB cells harbouring various selected tripartite introns as previously described 32P labelled oligonucleotide probe (Total RNA was isolated from NZ9800Δde probe  and visu


Tbx5 and Tbx4, two genes expressed in forelimb and hindlimb-forming regions respectively, play crucial roles in the initiation of limb outgrowth. Evolution of regulatory elements that activate Tbx5 in rostral LPM was essential for the acquisition of forelimbs in vertebrates. We identified such a regulatory element for Tbx5 and demonstrated Hox genes are essential, direct regulators. While the importance of Hox genes in regulating embryonic development is clear, Hox targets and the ways in which each protein executes its specific function are not known. We reveal how nested Hox expression along the rostro-caudal axis restricts Tbx5 expression to forelimb. We demonstrate that Hoxc9, which is expressed in caudal LPM where Tbx5 is not expressed, can form a repressive complex on the Tbx5 forelimb regulatory element. This repressive capacity is limited to Hox proteins expressed in caudal LPM and carried out by two separate protein domains in Hoxc9. Forelimb-restricted expression of Tbx5 and ultimately forelimb formation is therefore achieved through co-option of two characteristics of Hox genes; their colinear expression along the body axis and the functional specificity of different paralogs. Active complexes can be formed by Hox PG proteins present throughout the rostral-caudal LPM while restriction of Tbx5 expression is achieved by superimposing a dominant repressive (Hoxc9) complex that determines the caudal boundary of Tbx5 expression. Our results reveal the regulatory mechanism that ensures emergence of the forelimbs at the correct position along the body. Acquisition of this regulatory element would have been critical for the evolution of limbs in vertebrates and modulation of the factors we have identified can be molecular drivers of the diversity in limb morphology.Tight control over gene expression is essential for precision in embryonic development and acquisition of the regulatory elements responsible is the predominant driver for evolution of new structures.  Tbx5. Activation of Tbx5 in the forelimb-forming region of the developing embryos is essential for forelimbs to form and disruption of human TBX5 causes limb abnormalities. We show that activation of Tbx5 in a restricted territory is achieved through a combination of activation inputs that are present broadly throughout the embryo flank and dominant, repressive inputs present only in more caudal regions of the flank. The sum of these inputs yields restricted activation in the rostral, forelimb-forming flank. Our results explain how the regulatory switches that were harnessed for the acquisition of limbs during evolution operate and how they can be turned off during the evolution of limblessness in species such as the snake.The acquisition of limbs during vertebrate evolution was a very successful innovation that enabled this group of species to diversify and colonise land. It has become clear recently that the primary driver behind the evolution of new structures, such as limbs, is the acquisition of novel regulatory elements that control when and where genes are activated rather than the proteins encoded by the genes themselves acquiring novel functions. We have identified the regulatory element from a gene,  Tbx5 in nascent forelimbs and Tbx4 in hindlimbs Fgf10 in the limb mesenchyme Fgf8 expression in the apical ectodermal ridge (AER) and Fgf8 produced from the AER, in turn, maintains Fgf10 expression in mesenchyme to establish a positive feedback loop of Fgf signalling that maintains limb growth. Mutations in human TBX5 cause Holt-Oram Syndrome (HOS OMIM142900), a disorder characterised by upper limb and heart abnormalities TBX4 cause Small Patella Syndrome (SPS OMIM 147891), a disorder characterised by knee, pelvis and toe defects Tbx5 is the earliest marker of presumptive forelimb mesenchyme and because activation of this factor within a defined region of the LPM ultimately dictates the position at which the forelimbs will arise, identifying the factors that control activation of this Tbx5 expression domain will reveal the mechanisms employed that allowed the acquisition of limbs in vertebrates and that dictate forelimb position in the embryo.Forelimbs and hindlimbs are derivatives of the lateral plate mesoderm (LPM) that arise at fixed positions along the vertebrate body axis. Limb formation is initiated by limb induction signals from axial tissues Tbx5 is initially expressed in the forelimb-forming region of LPM prior to the emergence of a bud and it is subsequently restricted to the forelimb region as development proceeds. Tbx5 is essential for forelimb formation and this exclusive requirement is limited to a short time window when limb bud initiation occurs Tbx4, the paralog of Tbx5, is able to rescue forelimb formation following conditional deletion of Tbx5Tbx4/5 gene represented by AmphiTbx4/5 of the limbless cephalochordate, amphioxus, can fully compensate for the loss of Tbx5 in the mouse Tbx5 expression in the LPM was a critical step in the acquisition of limbs during vertebrate evolution.Hox genes are conserved homeodomain-containing transcription factors that are arranged in clusters in the genome. The chromosomal organization of the genes in the complex reflects their expression pattern along the rostro-caudal body axis to determine positional identity Tbx5 regulatory element sufficient for early forelimb expression Tbx5. However, since the ability to activate Tbx5 is not strictly restricted to Hox genes expressed only at forelimb level, the mechanism by which a rostro-caudal Hox code establishes forelimb-restriction of Tbx5 remained unknown. Here, we demonstrate how Hox paralogous group members act cooperatively to restrict expression of Tbx5 in the LPM, which ultimately determines the positions the forelimbs will emerge from the flank of the embryo. We show that mutations of a single Hox binding site in the Tbx5 forelimb regulatory element cause expanded reporter gene expression in caudal LPM. Rostral restriction in Tbx5 expression through repression in the caudal LPM is mediated by Hoxc8/9/10 genes and this repressive function is limited to Hox genes that are expressed in Tbx5-negative caudal LPM. We further map the Hoxc9 protein domains required to confer transcriptional repression that distinguishes these paralogs from other Hox proteins expressed throughout the flank of the embryo. Our results demonstrate how a nested, combinatorial code of Hox protein transcriptional activation and repression along the rostro-caudal embryo axis restricts Tbx5 expression to the forelimb and ultimately determines forelimb position.We have previously identified a Tbx5 gene that recapitulates the dramatic forelimb-restricted expression of this gene Tbx5 expression, we generated a series of constructs in which each individual Hbs site1-6 is mutated and tested their ability to activate a LacZ reporter gene in transgenic mice. While the Tbx5 int2(361) reporter construct drove forelimb-restricted expression of LacZ (Tbx5 int2(565)) . To distinguish the requirement for Hbs2 and 6bp3′, we next mutated either of these sites )  that cont2(565)) . We haveess Tbx5 . These rse sites . Mutatioreporter , while mreporter  equivalereporter . These rThese results demonstrate that the binding sites within this regulatory element can be divided into 2 distinct functional groups. Hbs1 and 3-6 act as ‘on’ switches important for the amplitude of activation, whereas Hbs2 determines spatial resolution by hosting repressive complexes that restrict the domain of activation. This element can therefore have a binary function, serving as a site for the formation of transcriptional activation or repression complexes.Hox genes can activate this regulatory element Hox genes in chick and mouse embryos at stages when Tbx5 is first expressed in the forelimb-forming region. Tbx5 is first expressed at the level of somites 13–20 in chick and somites 4–11 in mouse embryos. As previously reported Hox4 and Hox5 paralogs overlap with that of Tbx5 in both mouse and chick embryos   construct  reporter, LacZ expression is repressed in the forelimb region   LacZ repficiency ), β-gal ficiency  indicatireporter  demonstrTbx5 expression, we co-electroporated Hoxc9 with a Tbx5 int2(361) reporter in which Hbs2 is mutated  showed clear repression of the reporter demonstrating that within the limits of this assay the entire N-terminus or the domain overlapping the junction between N1 and N2 is required for repressive activity  can reduce LacZ expression , suggestTbx5 as an assay, we have been able to distinguish the opposing transcriptional activities of different Hox paralogous group proteins. Hoxc9, as well as Hoxc8 and Hoxc10, that are normally expressed in the LPM caudal to the forelimb, can repress Tbx5 to restrict its expression to the forelimb level region of the LPM  is required for its repression in caudal LPM (Our results demonstrate that five of the six Hox binding sites (namely Hbs1 and Hbs3-6) within the udal LPM . One posudal LPM , suggesthttp://www.genomatix.de) is the presence of a 6-bp sequence named Pbx1-Meis1 complexes site located 3′ of Hbs2 (6bp3′). Pbx is a Hox co-factor that can attenuate Hox-mediated gene transcription by recruiting histone deacetylases (HDACs) Tbx5 forelimb regulatory element.An alternative model is that the transcriptional activity of the Hox complex bound at Hbs2 is determined by a co-factor(s). The sequence of Hbs2 is identical to the sequences of Hbs1 and Hbs3, therefore we compared the sequences surrounding these Hox binding sites. One distinguishing feature of Hbs2 identified using Mat Inspector  in the protein. The hexapeptide of AbdA represses dpp expression by inhibiting the function of a glutamine (Q)-rich C-terminal activation domain Tbx5 transcription, we suggest the model that Hoxc9 supresses Tbx5 expression by interaction with co-repressor(s) (in vitro translated proteins or nuclear extract from LPM (data not shown). Other potential collaborators are Smad proteins. In the Drosophila haltere, a Mad/Med/Shn complex works in combination with Ubx to repress Sal expression Tbx5 expression . Other candidate repressors are engrailed (En) and sloppy paired (Slp) since, in Drosophila, they form a complex with Hox, Exd and Hth to repress transcription Engrailed1 and Engrailed2, are expressed in LPM at pre-limb bud stages As it is unlikely that Hox protein itself directly represses essor(s) . One canTbx5 expression or that they share similar 3D structure domains in spite of their distinct amino acid sequences.We have shown that Hoxc8 and Hoxc10 have transcriptional repression ability similar to Hoxc9 . To gainTbx5 forelimb regulatory element reveals a direct link between patterning of the rostro-caudal axis of the embryo by Hox genes and the programme that controls positioning of the forelimb forming territory. A clear correlation between Hox expression and establishment of the forelimb territory of the LPM has previously been suggested Tbx5Hoxc9 is reduced Tbx5 (and the subsequent forelimb programme) being essential for emergence of an ectopic wing bud from this region. In the limbless python, Hoxc8 expression is rostrally expanded to the anterior limit of the trunk Tbx5-negative caudal LPM at pre-limb bud stages in chick and mouse , however, we did not detect expression of Hoxc9 in the forelimb-forming region, in contrast to the strong staining in caudal tissues. We therefore conclude that Hoxc9 is not present in the forelimb-forming region at stages when Tbx5 expression is first initiated. Later expression of Hoxc9 is not sufficient to cause detectable repression of the domain of Tbx5 already activated by Hox4/5 paralogous genes.A previous study has demonstrated the presence of and a function for Tbx5 expression, our study does not exclude the possibility that other caudally-expressed Hox genes have a similar repressive ability. We favour a model in which other caudally-expressed Hox paralogs have redundant functions in repression of Tbx5. Hoxc cluster null mice have no defects in the limb skeleton Tbx5 in these mutants have not been reported and we predict that the ectopic expansion of Tbx5 in caudal LPM would not cause any skeletal defects. Further analysis will be required to uncover the requirement of caudally–restricted Hox paralogs, such as Hox8, Hox9 and Hox10 for Tbx5 repression in caudal LPM.While we have shown that Hoxc8, Hoxc9 and Hoxc10 can repress Tbx5 in the LPM, a similar Hox protein code is present in axial tissues  that do not express Tbx5. The activity of the forelimb regulatory element of Tbx5 is restricted to LPM and this LPM restriction is maintained following mutation of Hbs2 that leads to caudal expansion in expression. One possible explanation for LPM restriction is the presence of unknown repressors in axial tissues or alternatively additional factors, which are active exclusively in LPM, are required for Tbx5 expression. Odd-skipped related (Osr) genes are candidates as they are expressed in LPM, but excluded from axial tissues such as neural tube and somites Tbx5 forelimb regulatory element to test if reporter activity was lost. The activity of the element was unaffected, however, suggesting Osr genes are not required for Tbx5 LPM expression  complex that ultimately determines the caudal boundary of Tbx5 expression. Thus, the regulation of Tbx5 expression in the LPM represents an excellent system to understand the interactions between neighbouring Hox binding sites and how the consequent output is integrated.Our analysis of the http://www.genomatix.de). Transgenic embryos were generated by the Procedural Service section, NIMR by standard pronuclear microinjection techniques. Mouse embryos were staged according to LacZ transgene were identified by PCR using specific primers . Sequences surrounding putative Hox binding sites and the mutations induced are as followings, binding sites are shown in bold; Hbs1, ATTATTGGAAC; mut Hbs1, ATGCTTGGAAC; Hbs2, ATTATCGACTCTCA; mut Hbs2, ACGATCGACTCTCA; mut 6bp3′, ATTATCGACTGCAA; mut Hbs2+6bp3′, ACGATCGACGCTTA; Hbs3, TAATTCAGA; mut Hbs3, TCGTTCAGA; Hbs4, TTATTAAGGCC; mut Hbs4, TTGGCAAGGCC; Hbs5, TTATCTTGCCAT; mut Hbs5, TCGTCTTGCCAT; Hbs6, TTATTTTG; mut Hbs6, TCGTTTTG.For reporter analysis in chick and mouse, we used the BGZA reporter vector in situ hybridizations were carried out essentially as previously described Pitx1, Tbx5 and mouse Hox genes have been described previously Whole mount Fertilized chick embryos  were incubated at 38°C and staged according to Hamburger Hamilton (HH) In vitro translated proteins were produced using a TnT Coupled Reticulocyte Lysate System (Promega). Proteins were labelled with 35S-Methionine (PerkinElmer) to verify and quantify translation. LPM strips adjacent to somites 5–10  and lateral to somite 14 to its caudal extreme  were dissected from E9 mouse embryos. Nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce) following manufacturers instructions.32P by incubating with T4 polynucleotide kinase (NEB) for 30 minutes. 2 µl of in vitro translated protein or nuclear extract were blocked with 200 ng poly-dIdC, 2 µg of poly-dGdC or 2 µg of poly-dAdT in binding buffer  in a total volume of 22 µl for 15 minutes on ice. For super-shift, 2 µl of the antibody recognising flag epitope  was added to the binding reaction and incubated for a further 15 minutes. Then, 1 µl of 32P -labelled double-stranded oligonucleotides were mixed and incubated for 30 minutes. The protein∶DNA hybrids were resolved on 6% PAGE in 0.5xTBE.Double-strand oligonucleotides were labelled with Figure S1Tbx5 forelimb regulatory element. A–C. The Tbx5 int2(361) LacZ reporter construct was electroporated with pCAβ-dsRed-Express in the presumptive forelimb region of HH14 chick embryos. 24 hours later, the efficiency of electroporation was assessed by dsRed expression (A–C) and embryos were stained for LacZ to analyze the enhancer activity (A′–C′). Co-electroporation of pcDNA-mHoxc8 (B and B′) and pcDNA-mHoxc10 (C and C′) reduced LacZ expression.Hoxc8 and Hoxc10 can repress activity of the (TIF)Click here for additional data file.Figure S2LacZ reporter expression. A, Schematic representation of the Tbx5 intron2 regulatory element. A putative smad binding site and Hox binding sites are shown as green and blue boxes, respectively. B–C, E9.5 embryos were stained for β-galactosidase for a mutated construct of Tbx5 int2(565) reporter.Disruption of a putative smad binding site does not expand (TIF)Click here for additional data file.Figure S3LacZ reporter expression. A, Schematic representation of the Tbx5 intron2 regulatory element. A putative Osr binding site (purple box) locates proximal to Hbs5 (blue box). B–C, E9.5 embryos were stained for β-galactosidase for mutated constructs of Tbx5 int2(565) reporter.Disruption of a putative Osr binding site does not affect (TIF)Click here for additional data file.


Objective: The purpose of the study was to identify technical item flaws in the multiple choice questions submitted for the final exams for the years 2009, 2010 and 2011.Methods: This descriptive analytical study was carried out in Islamic International Medical College (IIMC). The Data was collected from the MCQ’s submitted by the faculty for the final exams for the year 2009, 2010 and 2011. The data was compiled and evaluated by a three member assessment committee. The data was analyzed for frequency and percentages the categorical data was analyzed by chi-square test.Results: Overall percentage of flawed item was 67% for the year 2009 of which 21% were for testwiseness and 40% were for irrelevant difficulty. In year 2010 the total item flaws were 36% and 11% testwiseness and 22% were for irrelevant difficulty. The year 2011 data showed decreased overall flaws of 21%. The flaws of testwisness were 7%, irrelevant difficulty were 11%.Conclusion: Technical item flaws are frequently encountered during MCQ construction, and the identification of flaws leads to improved quality of the single best MCQ’s. Altering the mode of examination to single best question type represents a major challenge for a faculty in any medical college. With change in the curriculum, the modality of assessment also changes. The new system of examination focuses on application, problem solving and integration of the different concepts taught. In Islamic International Medical College the mode of examination based on one best question is being practiced since 2009.1 Properly made MCQs leads to impartial testing of the student that can measure knowledge, comprehension, application and analysis.2,3MCQs or single best questions are difficult and time-consuming to construct, even for those who have been formally trained in the construction of MCQs.4 Questions that aim to assess really important topics cannot do so unless they are well-structured i.e. avoiding flaws that benefit the testwise examinee; those students who answer questions alone on their test taking skills and not on their amount of expertise on the subject that is being covered.5 Also avoiding irrelevant difficulty are prerequisites that must be met in order for test questions to generate valid scores.5Characteristics of effective MCQs can be described in terms of the overall item, the stem, and the options. The stem generally consist of a clinical case presentation and a lead-in question, followed by a series of choices, typically one correct/best answer and four distractors.3,4 Flawed MCQs interfere with accurate and meaningful interpretation of test scores and negatively impact student pass rates. One aspect where many MCQs fail is in having effective distractors.5 Teachers often spend a great deal of time constructing the stem and much less time on developing plausible options to the correct answer.5Outlines regarding effective item-writing have been documented; however manipulations of these principles are very common in medical education with resultant flawed item questions.4,5 Flaws related to irrelevant difficulty make the question difficult for reasons unrelated to the trait that is the focus of assessment. The increased test and item difficulty associated with the use of flawed items lead to artificial difficulty to the test scores.6Two types of technical item flaws: testwiseness and irrelevant difficulty are described in literature. Flaws related to testwiseness make it easier for some students to answer the question correctly, based on their test-taking skills alone.The purpose of this study was to examine structural concerns which are important for the formation of high-quality test questions. Thus the main objective of this study was to identify common technical flaws in assessment items encountered during the paper setting of examinations of 2009 to 2011.This descriptive study was conducted at Riphah University Rawalpindi after the completion of assessment for the year 2011. There were no human subjects involved in the study. Therefore it was exempted for obtaining an ethical approval certificate.The assessment data for the years 2009, 2010 and 2011 was collected and reviewed. These items were reviewed by a three member assessment committee. The original single best choice questions that had been submitted to the assessment committee for the purpose of exams were grouped according to the year and were then analyzed for technical item flaws.Inclusion criteria: All questions submitted for the years 2009, 2010 & 2011. During analysis intrinsic structure of the question was checked for technical accuracy. Items were classified as 'flawed' if they contained one of the flaws. Frequently observed flaws were grouped into:1- Issues Related to TestwisenessGrammatical Cues and errorsLogical cuesoften, sometimes in MCQ) terms.5Use of absolute  is 46.2% which is a study conducted on nursing curriculum.9 However, the evaluation of the data from 2011 showed that overall the total item flaws were 21%. This is substantially less as compared to data seen from the year 2009 and 2010.While much has been written in context of developing a good MCQ, there is very little actual data concerning the analysis of a MCQ. The results in this study show that the frequency of item flaws encountered in the year 2009 were 67% which is comparable to flaws encountered in a study conducted by Ellsworth et al in psychology test banks.4,5In the present, study a number of violations were found that help students correctly answer questions based on cues given in the stem or the options, rather than knowledge. Item writing flaws (IWFs) such as longest correct option, logical cues, word repeats, use of ‘‘all of the above,’’ and use of absolute terms make MCQs easier by providing helpful cues to students as to what is the correct answer. MCQ’s with heterogenous options apparently increase the difficulty of a question and deal with miscellaneous facts.10 Though that study was analysis of data from module exams that were conducted during the academic year, the proportion of the flaws related to testwiseness steadily decreased in 2010 and 2011.This study analyzed these item flaws encountered during the analysis of item submitted across three years. The frequency of flaws related to testwiseness were 21% and those which were correlated to irrelevant difficulty were 40% for the year 2009. This percentage of item flaws is comparable to a study by Danish in 2010 which had similar percentage of item flaws.11 Research in other disciplines has shown that training improves the quality of MCQs developed by teaching faculty.12 Despite the fact that in the year 2009, the faculty had been trained for the MCQ writing, lack of practice was a recognizable factor contributing to the high number of errors in items analyzed for the year 2009 and 2010. As the faculty preceded from 2009 into 2010 and then 2011, the level of MCQ construction and item writing skills of the faculty improved. Moreover, relevant feedback regarding removal of errors give good results in improving the quality of items prepared by the faculty members.The identification of these item writing flaws also highlights the fact that the faculty members preparing these items should be trained in a faculty development workshop.It is also suggested to the faculty members that in order to combat the issues of irrelevant difficulty, while preparing for lecture the relevant MCQ be prepared at the same time. Most often important assessment are written and assembled at the last moment. While the faculty members take lecturing seriously few make an effort to prepare the assessment. Furthermore, by the time items are collected from half a dozen or more lecturers who may have been involved in teaching there is inadequate time or opportunity to review before being submitted to the assessment committee.For planning an effective assessment it is emphasized that the items prepared by the faculty members should be carefully analyzed before they are put in an evaluation paper. The current research points in detail the types of mistake that are mainly committed in the construction of MCQs. It also gives guidelines for authors of the MCQ items about the common error committed during the preparation of MCQ’s.If an MCQ is going to be used to assess higher order cognitive skills, there needs to be a process in place where adequate instruction and feedback is given to the item authors. The results verify that with repetition and practice the standard of MCQ for assessment paper’s can be improved. To ensure better quality of MCQ it is suggested that the items before being submitted to the assessment committee should be evaluated at inter departmental level and then submitted to the finalizing committee. This will lead to better written items and save time as well.Technical item flaws are frequently encountered during MCQ construction, and the identification of these flaws leads to improved quality of the single best MCQ’s. In order to rectify these flaws the faculty should be trained in item writing skills. While on the spot training can be done at the time of assessment but better results can be obtained if the faculty is trained prior to the final exams.


Eyelid involvement in sarcoidosis is very rare. A search of the medical literature indicates one previous report of sarcoidosis with destructive eyelid lesions. We describe the case of a 50-year-old woman with severe systemic sarcoidosis, which included her eyelids. To our knowledge, the case presented herein represents the first to show the full-thickness histopathology of destructive eyelid lesions in sarcoidosis. Sarcoidosis is a multisystem inflammatory disease of unknown etiology characterized histopathologically by noncaseating granulomas. The condition has a female predilection with a peak incidence in the 3rd and 5th decades of life. Pulmonary involvement is the most common manifestation. Common extrapulmonary manifestations include the skin, central nervous system, liver, kidney, and musculoskeletal system. Furthermore, sarcoidosis can affect the heart, peripheral nervous system, salivary glands, eye, and ocular adnexa. Ocular involvement occurs in 25%–60% of patients with systemic sarcoidosis , 2. WhilA 50-year-old female with severe systemic sarcoidosis, including pulmonary, skin, and joint disease, presented for evaluation of ocular involvement. The patient had noted eyelid nodules for over 5 years; however, she recently developed eyelid deformities, tearing, and eye irritation.On examination, she had notching of her upper and lower eyelids and extensive scarring of the posterior lamella leading to entropion and focal trichiasis . Slit laThe patient subsequently underwent full-thickness wedge resections in both upper eyelids and her right lower eyelid resulting in symptomatic improvement and elimination of the localized areas of trichiasis. Histopathologic evaluation of all three full-thickness wedge resections showed typical “naked,” noncaseating granulomas composed of epitheliod histiocytes and multinucleated giant cells . Serial  Early reports have limited discussion of the incidence of eyelid involvement in sarcoidosis , 5. Eyelhttp://www.ncbi.nlm.nih.gov/pubmed/, accessed 10 March 2013) of “eyelid and sarcoid” indicates one previous case [A Pubmed literature search (ous case  of involous case . The autous case . To our


Here, we report from the Afrotropical greater honeyguide the first detailed observations of honeyguides killing host chicks with their specially adapted bill hooks, based on repeated video recordings . Adult greater honeyguides puncture host eggs when they lay their own, but in about half of host nests at least one host egg survived, precipitating chick killing by the honeyguide hatchling. Hosts always hatched after honeyguide chicks, and were killed within hours. Despite being blind and in total darkness, honeyguides attacked host young with sustained biting, grasping and shaking motions. Attack time of 1–5 min was sufficient to cause host death, which took from 9 min to over 7 h from first attack. Honeyguides also bit unhatched eggs and human hands, but only rarely bit the host parents feeding them.The most virulent avian brood parasites obligately kill host young soon after hatching, thus ensuring their monopoly of host parental care. While the host eviction behaviour of cuckoos (Cuculidae) is well documented, the host killing behaviour of honeyguide (Indicatoridae) chicks has been witnessed only once, 60 years ago, and never  While mthe nest . Second otropics  and the otropics , the parotropics . The repotropics  .Prodotiscus show bill hooks and host young have never been found alongside them [Nestlings of only five of the 17 honeyguide species have ever been observed, but since all including the basal genus ide them , it is aIndicator indicator is remarkable for its interactions with other species. It shows a unique mutualism with human honey-gatherers whom it guides to bees' nests [The Afrotropical greater honeyguide s' nests , as wells' nests ; (ii) th2.ca 5000 ha area centred on 16°45′′ S, 26°54′′ E. The habitat is mixed miombo woodland, seasonally flooded depressions and tobacco cultivation. Here, the little bee-eater Merops pusillus [ca 0.5 m long and 0.2–0.5 m underground) typically dug into the roofs of aardvark (Orycteropus afer) holes, or sometimes earth banks or bushpig (Potamochoerus larvatus) diggings. Clutch size is three to six eggs.We conducted fieldwork in the Choma region of southern Zambia during September–November 2008–2010, in a pusillus  is the cWe filmed chick behaviour by inserting an infrared camera at the end of the bee-eaters' access tunnels. Cables led to the surface where a digital recorder was concealed nearby. Bee-eaters ignored this equipment. Killing behaviour was filmed at five nests of three host species , involving four honeyguide chicks and six host chicks. In two nests, we had moved the honeyguide from another nest, matched in developmental stage, but attacked by ants or predators; foster nests are excluded from mass analyses. Data on honeyguides' impact on hosts all refer to the commonest host species, the little bee-eater.3.n = 194) of host eggs in parasitised nests were punctured. In 30 of 55 parasitised nests (55%), at least one (range one to four) host egg was unpunctured and could have hatched; proportions were similar for other host species. Hence, egg puncturing only partially removed the scope for subsequent chick killing.Of 172 little bee-eater nests followed, 113  were visited by a honeyguide, as revealed by a honeyguide egg and/or punctured host eggs. However, of these visited nests, 64 (56.6%) were deserted by hosts at this early stage. Therefore, a parasitic egg was incubated by hosts in only 49 (28.5%) of all host nests. Honeyguide oviposition occurred any time from the start of host egg-laying to late into the host incubation period. Greater honeyguides did not remove host eggs, but  to hatch, compared with 18–20 days for hosts [ca 2–4) days old when the first host chick hatched. At hatching, honeyguides weighed ca 3 g, possessed fully developed bill hooks (a) and immediately attempted to bite our fingers when handled. Honeyguides had attained substantially greater weight by the time host chicks hatched and were killed: 6.06–11.08 g  for the first-hatched host and 10.22–13.29 g  for the last-hatched. Hosts were attacked soon after hatching , when little bee-eaters weighed less than 2 g .Honeyguides always hatched before host young, despite sometimes asynchronous laying . Honeygll hooks a and imma–c and electronic supplementary material, videos S1–S4). They typically held onto hosts for a period of time  during which they repeatedly opened and closed their mandibles, and often shook the host chick, either from side to side or up and down by rocking on their hindlegs . Honeyguides reached out haphazardly rather than targeting particular body regions. They most commonly bit the back (n = 20 bites), head (14) and abdomen (14), and occasionally the neck (5), wings (4) and legs (3). Biting rarely caused open wounds, but rather haemorrhaging underneath the skin and heavy bruising (figure 2b). As previously noted [figure 2c). Host chicks sustained an average of 177 s of direct attacks , but from the time of first attack, hosts took from 9 min to over 7 h to die. Hosts did not attempt to avoid attacks and after a first bout of attack soon ceased any attempts to beg for food. Host corpses usually remained and decomposed in the nest, but in one nest a parent removed two of three corpses (figure 2f).Honeyguide chicks attacked host chicks using their bills (figure 2d). Only once did a honeyguide bite a host parent (d), despite being frequently brooded by them. A host chick was once attacked while host parents attended the nest (e).Honeyguides occasionally bit unhatched host eggs, but failed to puncture their shells d. Only ot parent d, despitthe nest e.e), corresponding to potentially late host encounters in large and asynchronously hatching host clutches. Hooks do not appear to be shed (cf. [ca one week before fledging (which occurs at about 30 days).The bill hook is retained for at least 14 days e, corres4.Virulence in the greater honeyguide results from a succession of three specialised adaptations which together ensure the death of all the host young: (i) egg puncturing by the laying female , which wHost killing is carried out by blind hatchlings in total darkness, and the proximal cues used by honeyguides to start and stop biting remain unclear. Honeyguides sometimes lay alongside host young for tens of minutes before suddenly starting to attack, locating the host chick by repeatedly biting into mid-air while rapidly moving the head from side to side. Attacks often ceased when host chicks lay maimed and largely immobile, suggesting that movement may be a more important cue for attack than body heat. We only once witnessed a honeyguide biting a host parent, in spite of regularly being brooded by them; perhaps either the parents' food calls, or their feathered texture, serve as cues to prevent honeyguides from biting the hand that feeds them.The degree of virulence shown by brood parasites should be determined by a trade-off between the costs and benefits of chick-killing, just as pathogen virulence is shaped by trade-offs . What arSecond, the fitness costs versus benefits of honeyguide chicks′ sole occupancy of the nest remain to be quantified. When parasitizing host species that they are easily able to outcompete in sibling competition, such as little bee-eaters, parasites may do better to reduce virulence and co-opt host nestling begging to their own advantage . We migh


The removal of non-coding sequences, introns, from the mRNA precursors is an essential step in eukaryotic gene expression. U12-type introns are a minor subgroup of introns, distinct from the major or U2-type introns. U12-type introns are present in most eukaryotes but only account for less than 0.5% of all introns in any given genome. They are processed by a specific U12-dependent spliceosome, which is similar to, but distinct from, the major spliceosome. U12-type introns are spliced somewhat less efficiently than the major introns, and it is believed that this limits the expression of the genes containing such introns. Recent findings on the role of U12-dependent splicing in development and human disease have shown that it can also affect multiple cellular processes not directly related to the functions of the host genes of U12-type introns. At the same time, advances in understanding the regulation and phylogenetic distribution of the minor spliceosome are starting to shed light on how the U12-type introns and the minor spliceosome may have evolved. © 2012 John Wiley & Sons, Ltd. U12-type introns were initially described as a handful of unusual introns containing non-consensus AT–AC termini and a high degree of conservation at the 5′ splice site (5′ss).The significance of having two parallel intron types and two machineries for their removal was initially perplexing, especially since the U12-type introns are present at very low frequencies. The function and significance of the U12-dependent spliceosome are still far from being fully understood, but there are several lines of evidence indicating that U12-type splicing has essential functions. These are related to the conservation of U12-type introns in distantly related organisms and their presence in specific types of genes, as well as to the slow kinetics of their removal, which may regulate the expression of the genes containing them. Here, we review the current knowledge on U12-type introns and the function of the U12-dependent spliceosome. In particular, we discuss the recent findings on the regulation of the U12-dependent splicing, its function in development and disease, and the impact of these findings on understanding the evolution of the two spliceosomes.Although the U12-type introns were first identified on the basis of terminal AT–AC dinucleotides  it was soon realized that these termini are not exclusively present in U12-type introns.6Naturally occurring U12-type introns mostly belong to either the AT–AC or GT–AG subtypes. A few natural examples of U12-type introns with other combinations of terminal residues have been reported, and non-canonical termini have also been shown to support U12-dependent splicing experimentally, although often with reduced efficiency.17Caenorhabditis elegans and SaccharomycescerevisiaeArabidopsis thaliana, while in Drosophilamelanogaster only 19 U12-type introns have been described.A.thaliana.22Currently, U12-type introns have been identified in all major eukaryotic taxa, including plants, fungi, and animals, as well as a few deep-branching single-celled eukaryotes, but are nonetheless absent in many species, including such common model organisms as The genes containing U12-type introns are enriched in certain functional classes and pathways as described originally by Burge et al.65 and U2AF35, respectively, together forming the spliceosomal commitment (or E) complex. During the formation of the pre-spliceosome, or A complex, U2 snRNP replaces SF1 at the BPS. At later stages, the U4/U6.U5 tri-snRNP stably associates with the spliceosome (B complex). Rearrangements in RNA and protein interactions lead to the formation of the catalytically active spliceosome (B* complex) that catalyzes the first transesterification reaction, followed by the second transesterification in the C complex  and additional non-snRNP protein components. complex .The U12-dependent spliceosome contains four specific snRNPs, U11, U12, U4atac, and U6atac, each of which contains a specific snRNA component that is equivalent to but distinct from its U2-type counterpart, i.e., U1, U2, U4, and U6, respectively. U5 snRNP is shared between the two spliceosomes. Although the sequences of the snRNAs with equivalent function are quite divergent in the two spliceosomes, they share a common overall secondary structure . U4atac,The overall assembly pathways of the two spliceosomes are similar, and the main difference is the absence of a separate commitment complex in the minor spliceosome. Instead, the preformed U11/U12 di-snRNP binds the intron as a unit, and the 5′ss and BPS are recognized in a cooperative manner within the A complex, although U11/5′ss basepairing still precedes the formation of stable U12/BPS basepairing.4935, Urp,45Interaction of the U12 snRNA with the BPS is similar to the U2/BPS basepairing in the major spliceosome, resulting in the exclusion of the branch point (BP) adenosine from the U12/BPS helix.In the major spliceosome the U1 and U2 snRNPs recognize the 5′ss and BPS independently, and non-snRNP proteins are required for their association to each other.The formation of the catalytically active spliceosome is thought to follow a pathway similar to that of the major spliceosome. After initial recognition of the splice sites by the U11/U12 di-snRNP, the U4atac/U6atac.U5 tri-snRNP associates with pre-spliceosome to form complex BAlthough the formation of the catalytic core appears to be somewhat more flexible in the U12-dependent spliceosome,in vitro splicing experiments documented a splicing rate for U12-type introns that was three- to fivefold slower than that of U2-type introns.in vivo experiments, where approximately twofold higher levels of unspliced U12-type introns were detected in the steady-state transcript pools isolated from insect and mammalian cells.72Both intron types are spliced co-transcriptionally within the nucleus Ref  and mighin situ hybridization studies, mostly with zebrafish, as well as cell fractionation and reverse transcription polymerase chain reaction (RT-PCR) analyses. The publication contradicted many of the earlier studies on minor spliceosome component localization and function  can be extremely long, such that it can take an hour for an entire intron to be transcribed. Therefore, initial splice site definition typically takes place over the considerably shorter exons, with 3′ss-recognizing factors in the upstream intron interacting with the 5′ss-recognizing factors in the downstream intron  contain overlapping U12 and U2-type introns or ‘twintrons’.dUrp mRNA isoform is apparently destroyed by NMD,prospero alternative splicing results in distinct developmentally regulated protein isoforms with amino-acid differences in the homeodomain region.86Given that AS is extremely prevalent in humans, and the extensive interactions of the minor spliceosomes with other splicing factors, it is somewhat surprising that AS events involving U12-type introns appear to be almost entirely absent. This may be because of the more rigid sequence and distance constraints on the U12-type 5′ss, BPS, and 3′ss. AS events involving the use of an alternative 3′ss have been observed, but it is not entirely clear whether they are the result of true regulated AS or of splicing errors, especially since the selection of the U12-type 3′ss has been shown to be prone to errors.D. melanogaster,A. thaliana also led to abnormal growth of the plant, but only after the bolting stage.prospero transcripts in D. melanogaster. However, it is likely that another, even more significant factor affecting developmental processes is the slower kinetics of minor splicing, which can limit not only the expression of the genes containing themD. melanogaster, likely underlying the developmental arrest.A. thaliana was also associated with downregulation of a specific set of genes related to gibberellic acid metabolism.Knockdown of protein components specific to the minor spliceosome leads to reduced proliferation of cultured cells,LKB1 and SEDL genes cause Peutz-Jeghers syndrome and spondyloepiphyseal dysplasia tarda, respectively,From a medical point of view, it would be interesting to know to what extent the activity of the minor spliceosome contributes to regulating the expression of genes in different human tissues. This question remains mostly unanswered. Some protein components specific to the minor spliceosome (31K and 65K) have been observed to be differentially expressed in different tissues,D. melanogaster carrying mutations in U6atac snRNA.D. melanogaster.A more complex class of diseases are those arising from defects in minor spliceosome components of which the TALS/MOPD1  is a recent example. This severe developmental disorder is caused by recessive mutations in the U4atac gene Refs . Unlike It is clear that the U12-dependent spliceosome is crucial for the viability and development of many multicellular organisms. However, intriguing questions about the minor spliceosome remain: Where did it originate? Why is it not present in all eukaryotes, and do its function and importance vary in different evolutionary lineages? We do not currently have clear answers to these questions, but recent advances in large-scale sequencing and bioinformatics are paving the way toward deeper understanding of the significance of the minor spliceosome.D. melanogaster has only 19 U12-type introns, almost all of them have orthologous U12-type introns in human.A. thaliana.67The two spliceosomes have been proposed to originate from group II self-splicing introns, but it is not clear whether they arose simultaneously or sequentially, and whether this took place in one ancestral eukaryote or separate lineages that later fused.Diptera, which typically only have 15–20 U12-type introns in total in their genomes, several fold less than in most other insects.21Despite the high overall conservation of U12-type introns, they are entirely absent from many eukaryotes whose relatives nonetheless have them, suggesting repeated loss of U12-dependent splicing during evolution. The loss seems to have been most prevalent among diverse eukaryotic microbes, but some animals, including some but not all nematodes, have also lost their U12-type introns Refs . The losdUrp twintron).In most cases, the mechanism of U12-type intron loss involves a simple deletion of the intronic sequence, apparently through the homologous recombination of reverse transcribed mRNAs.Diptera, where both the overall numbers and interspecies conservation of U12-type introns are low.Drosophila dUrpMany splicing factors are regulated through a feedback mechanism, in which they bind to regulatory elements within their own pre-mRNA, resulting in alternatively spliced, unstable mRNA isoforms. Interestingly, all canonical SR and hnRNP proteins, as well as a number of core spliceosomal factors, are regulated in this manner, and in many cases the regulatory elements are also evolutionarily conserved.Historically, insight into the function and significance of the U12-dependent spliceosome has come primarily from the biochemical characterization of splicing factors and spliceosome assembly, and has recently been complemented by the increasing number of bioinformatic analyses. However, our understanding of the significance of minor splicing is far from complete in both regards. The function of many of the U12-specific components is still unknown, and more components might still remain undetected. Importantly, although there are many independent lines of evidence indicating that U12-type introns are important for the regulation of gene expression, definitive large-scale evidence on the connection between the activity of the minor spliceosome and gene regulation is still lacking. Are there more regulatory elements that respond to U12-type factors? In what other ways is the minor spliceosome regulated, and how does this correlate with the expression of U12-type intron-containing genes in different tissues? Could there be U12-type introns with more degenerate splice sites, and could they be involved in more extensive alternative splicing? An exciting way of studying these questions is now offered by the rapidly developing high-throughput sequencing and cross-linking methods, and will hopefully deepen our understanding on the significance of minor splicing in the coming years.


The as-produced graphene was characterized by X-ray photoelectron spectroscopy, ultraviolet-visible spectroscopy, scanning electron microscopy, and high-resolution transmission electron microscopy (HR-TEM). In particular, the HR-TEM demonstrated the layered crystallites of graphene with fringe spacing of about 0.32 nm in individual sheets and the ultrafine facetted structure of about 20 to 50 nm of Au particles in graphene composite. Scanning helium ion microscopy (HIM) technique was employed to demonstrate direct write deposition on graphene by lettering with gaps down to 7 nm within the chamber of the microscope. Bare graphene and graphene-gold nanocomposites were further characterized in terms of their composition and optical and electrical properties.Graphene nanosheets were prepared using a modified Hummer's method, and Au-graphene nanocomposites were fabricated by  Graphene, structurally known as a monatomic layer of allotropic-carbon atoms in a hexagonal honeycomb two-dimensional lattice system, has always been a potential candidate for various applications due to its remarkable structural, physical, and electronic properties -9. The z4- ions in graphene suspensions to fabricate gold (Au) nanoparticles-graphene hybrid assemblies. In yet another report, Goncalves et al. [). These graphene/Au nanocomposites act as potential substrates for surface-enhanced Raman scattering. Min et al. [The composites of metal nanoparticles on graphene sheets also provide a new way to develop catalytic, magnetic, and optoelectronic materials. Moreover, adhesion of such metal nanoparticles to the graphene prevents their aggregation in dry state . Recentls et al.  demonstrn et al.  have alsBut in comparison to these reports, the work presented here highlights a simpler route to obtain stable Au nanoparticles-graphene nanocomposites. It also demonstrates the capability of direct labeling on nanocomposite by use of scanning helium ion microscopy (HIM).in situ ion lithography, nano-patterning, device prototyping, fabrication of quantum dots, beam-induced chemistry, and milling at nanoscale [The demonstration of imaging by helium (He) ions is relatively a new technique to characterize the surfaces at sub-nanoscale with extraordinary additional advantages of anoscale ,16. Suchanoscale -22 that 4) was purchased from Aldrich . Sodium borohydride (NaBH4) was acquired from Merck . Inorganic transparent electrodes of SnO2:F-coated glass  were cleaned in a soap solution, 30% HCl solution, double-distilled water, acetone, and trichloroethylene (in that order) prior to use. Deionized water (resistivity ≈ 18.2 MΩ cm) obtained through Milli-Q system, nitric acid (HNO3) (Merck), sulfuric acid (H2SO4) (Merck), and toluene were used as solvents.Hydrogen tetrachloroaurate (HAuCl2SO4:HNO3 in a 3:1 volume ratio (12 ml H2SO4 and 4 ml HNO3) and 2 g graphite powder was made in a flask and refluxed at 40°C for 16 h. The resulting solution was washed with deionized water till the pH was reduced to 5 or 6. As a result, a black colored solution of acid-functionalized graphene was obtained.For acid functionalization of graphene, a solution with Hin situ in graphene suspension by the reduction of gold(III) complex by NaBH4. A concentrated aqueous solution of 0.4 M NaBH4 was first mixed with acid-functionalized graphene suspension in toluene. With continuous stirring, 30 mM of HAuCl4 was then introduced into this suspension. After continuously stirred for 1 h, the resulting Au-graphene composites were collected by centrifugation and washed with water for three times.To fabricate Au-graphene nanostructures, Au nanoparticles were synthesized I-V measurements of films were carried out on Keithley 238 high-current electrometer characterization system. Absorbance (A) spectra were recorded in the 200- to 800-nm wavelength range in a Perkin Elmer Lambda 25 spectrophotometer . X-ray photoelectron spectroscopy (XPS) spectra were recorded for the as-synthesized graphene samples using a Perkin Elmer 1257 model PHI, Maple Grove, Minnesota, 55311 U.S.A operating at a base pressure of 3.8 × 10-8 Torr at 300 K with a non-monochromatized AlKα line at 1,486.6 eV, an analyzer pass energy of 60 eV kept for core level spectra and a hemispherical sector analyzer capable of 25-meV resolution. The overall instrumental resolution was about 0.3 eV. The core level spectra were deconvoluted using a non-linear iterative least squares Gaussian fitting procedure. For all fitting doublets, the FWHMs were fixed accordingly.Fourier transform infrared spectra for the films were recorded in reflection mode with a Perkin Elmer GX2000 OPTICA spectrophotometer at 28°C, RH ≈ 50% to 53%. 2 F 30 STWIN FEI, Achtseweg Noord 5 5651 GG Eindhoven, Netherlands (operated at the electron accelerating voltage of 300 kV). HR-TEM specimens were prepared by dispersing the graphene films on copper grid of 3.05 mm in diameter having a 200-mesh pore size. Further, the surface topography of graphene and graphene-Au composite films was analyzed by HIM . The He ion capability of the microscope was used to perform the experiments of nanoscale patterning on the surfaces of graphene.Surface morphology of the graphene sheets was studied employing a variable pressure scanning electron microscopy (SEM), model: Zeiss EVO MA10 Carl Zeiss SMT AG, Germany. Nanostructural imaging at high magnifications was carried out using HR-TEM model: FEI-Tecnai Gn→π* transitions of C=O bonds [-1 to 545 nm at 0.2 and 0.4 g l-1. The quenching in the peak intensity was also observed which is clearly visible in the inset of Figure The successful synthesis of graphene and Au nanoparticles decorated graphene was confirmed by ultraviolet-visible (UV-Vis) spectroscopy Figure . The UV-=O bonds . When Au5/2) and Au(4f7/2) [+ state. Deconvolution was performed on C(1s), O(1s) and Au(4f) XPS core spectra are shown in Figure 0 (81.8 and 85.3 eV) exhibit the metallic nature of Au, while Au+ state (83.7 and 87.4 eV) probably due to the interaction with the negatively charged graphene around Au induces a positive charge. The contribution of various spices of core level spectra is listed in Table The formation of stabilized Au-graphene nanocomposite was further confirmed by the XPS spectra as shown in Figure u4f7/2)  due to tu(4f7/2)  for Ag/gu(4f7/2) ,27. Inteu(4f7/2) , and the  due to rmed on Cs, O(1s) c-axis growth of carbon lattice also justify a distinct orientation relationship and therefore a crystallographic compatibility between the carbon as matrix and the Au as second phase.Crumpled, folded, layers of bare graphene can be seen in the SEM image shown in Figure in situ examination of the microstructure and direct write of arbitrary patterns on the given nanomaterial. HIM is showing the capability to create smaller structures than possible with other technique [in situ nano-scaled structural buildings elucidates a new field which is so far relatively unexplored in fabrication and process control of fundamentally important nano-objects like graphene. In the present work, the text  was created by deposition of carbon. This pattern, in the form of a bitmap, was opened up in Orion software Carl Zeiss NTS LLC, 1 Corporation Way, Peabody MA 01960, U.S.A. Subsequently, the user defined the size of the overall pattern, and the pixel size was scaled accordingly. The ion dose per pixel was also variable, being set in proportion to the gray level in the bitmap (up to 256 levels). Lettering with gaps down to 7 nm was observed. In this process, the direction of scanning was also user selectable. In another set of experiments, HIM was used to study the distribution of Au in the matrix of graphene (Figure Nano-patterning by use of a high-resolution microscope is a fast developing method which facilitates echnique -17. In te Figure . We haveI-V characteristics of Au-graphene and functionalized graphene were recorded in the following configuration: SnO2:F/Au-graphene/aluminum as shown in Figure 2:F-coated glass substrates. The AuNPs decoration was having a beneficial effect on the electronic conductivity of graphene. Both the films show ohmic contact with the substrate in 0 to +1 V potential region as can be seen from the linear variation of current with applied bias. For the Au-graphene composite, the conductivity was determined to be 0.49 S cm-1 which is much higher than blank functionalized graphene film where the value was estimated to be 0.07 S cm-1 which validates the role of interaction with AuNPs in the enhancement of conductivity. In the literature, the value of pristine graphene has been reported to be 0.2 S cm-1 [The I-V characteristics between bare graphene and its Au incorporated nanocomposites has been noticed. An important in situ direct write deposition on nanosheets of graphene has been demonstrated by employing He ions inside the chamber of the microscope.A simple modified Hummer's method was used to fabricate graphene and graphene-Au nanocomposites. A significant change in The authors declare that they have no competing interests.SB carried out the graphene preparation and interpretation of results. MD did the lectrical measurements. AGJ carried out XPS measurements and interpretations. APS assisted in synthesis of material. AKS initiated the idea of working on present topic and subsequently compiled the data.