Comparative genome analysis of Pasteurella multocida strains of porcine origin.

Pasteurella multocida causes acute/chronic pasteurellosis in porcine, resulting in considerable economic losses globally. The draft genomes of two Indian strains NIVEDIPm17 (serogroup D) and NIVEDIPm36 (serogroup A) were sequenced. A total of 2182-2284 coding sequences (CDSs) were predicted along with 5-6 rRNA and 45-46 tRNA genes in the genomes. Multilocus sequence analysis and LPS genotyping showed the presence of ST50: genotype 07 and ST74: genotype 06 in NIVEDIPm17 and NIVEDIPm36, respectively. Pangenome analysis of 61 strains showed the presence of 1653 core genes, 167 soft core genes, 750 shell genes, and 1820 cloud genes. Analysis of virulence-associated genes in 61 genomes indicated the presence of nanB, exbB, exbD, ptfA, ompA, ompH, fur, plpB, fimA, sodA, sodC, tonB, and omp87 in all strains. The 61 genomes contained genes encoding tetracycline (54%), streptomycin (48%), sulphonamide (28%), tigecycline (25%), chloramphenicol (21%), amikacin (7%), cephalosporin (5%), and trimethoprim (5%) resistance. Multilocus sequence type revealed that ST50 was the most common (34%), followed by ST74 (26%), ST13 (24%), ST287 (5%), ST09 (5%), ST122 (3%), and ST07 (2%). Single-nucleotide polymorphism and core genome-based phylogenetic analysis clustered the strains into three major clusters. In conclusion, we described the various virulence factors, mobile genetic elements, and antimicrobial resistance genes in the pangenome of P. multocida of porcine origin, besides the rare presence of LPS genotype 7 in serogroup D.

Comparison of MRCP and ERCP in the evaluation of common bile duct and pancreatic duct pathologies.

Background: Magnetic resonance cholangiopancreatography (MRCP) is a non-invasive imaging modality that has high diagnostic accuracy for a wide range of bile duct and pancreatic duct pathologies. Endoscopic retrograde cholangiopancreatography (ERCP) is still the gold standard for the exploration of the biliopancreatic region. Aim: The aim of the study was to compare the diagnostic accuracy of MRCP with that of ERCP in the diagnosis of bile duct and pancreatic duct pathologies. Material and methods: A total of 60 patients with common bile duct (CBD) and pancreatic duct pathologies detected on MRCP were subsequently evaluated by ERCP in this observational study. A comparison of MRCP findings with ERCP was made. Results: MRCP had a sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of 88.1%, 94.4%, 97.3%, 72.7%, and 90%, respectively, in diagnosing choledocholithiasis in comparison to ERCP. For CBD dilation, the sensitivity was 90.91%, specificity was 93.75% and the PPV, NPV, and accuracy were 97.56%, 78.95%, and 91.67%, respectively, for MRCP. In CBD stricture, MRCP showed a sensitivity, specificity, PPV, NPV, and accuracy of 83.33%, 97.92%, 90.91%, 95.92%, and 95%, respectively. In pancreatic duct dilatation, the sensitivity, specificity, PPV, NPV, and accuracy were all 100%. Pancreatic duct stricture showed a sensitivity, specificity, PPV, NPV, and accuracy of 80%, 98%, 88.89%, 96.08%, and 95%, respectively. For the diagnosis of periampullary carcinoma, the sensitivity, specificity, PPV, NPV, and accuracy rate of MRCP were 80%, 98%, 88.89%, 96.08%, and 95%, respectively. Conclusion: No significant difference was found between MRCP and ERCP in diagnosing those six pathologies.

Expression of Claudin-4 and D2-40 and their significance in prostatic adenocarcinoma.

BACKGROUND: Claudins are a clan of proteins that are the most important component of tight junctions. The claudin-4 expression has been linked to tumour cell invasion and progression in a variety of primary malignancies. Evaluation of lymphovascular density (LVD) correlates with tumour aggressiveness and may correlate with prognosis. D2-40 is a highly specific marker of lymphatic vessels. AIMS: To evaluate the claudin-4 expression in relation to LVD by D2-40 expression and with clinicopathological parameters in prostatic adenocarcinoma. SETTINGS AND DESIGN: Prospective study. MATERIALS AND METHODS: 39 cases of prostatic adenocarcinoma were taken, the D2-40 and claudin-4 immunohistochemical stains were performed and correlation was done with clinicopathological parameters. STATISTICAL ANALYSIS USED: Statistical analyses such as mean, median, standard deviation, Mann-Whitney U test, Fischer exact test, Spearman's rank-order correlation coefficient, Chi-square test and T-test were used. RESULTS: The claudin-4 expression was seen higher in cases with higher Gleason score but it was statistically non-significant (P = 0.778). The claudin-4 expression did not correlate with any clinicopathological parameters. LVD in the peritumoral area was significantly higher as compared to the intratumoral area (P = 0.005). Intratumoral LVD and perineural invasion were found to be statistically significant (P = 0.048). CONCLUSION: The claudin-4 expression may correlate with adverse prognostic parameters. Higher lymphatic vessels can be responsible for the higher metastatic potential of prostatic adenocarcinomas.

Whole-genome sequence analysis of Clostridium chauvoei isolated from clinical case of black quarter (BQ) from India.

Black quarter (BQ) is an infectious disease affecting cattle and small ruminants worldwide caused by Gram-positive anaerobic bacterium Clostridium chauvoei. In this study, a draft genome sequence of C. chauvoei NIVEDIBQ1 strain isolated from clinical case of black quarter was analyzed. Sequence analysis indicated that genome had 2653 predicted coding DNA sequences, harbored numerous genes, mobile genetic elements for pathogenesis, and virulence factors. Computational analysis revealed that strain contained 30 virulence-associated genes. An intact genomic region highly similar to the Clostridium phage was present in the genome. Presence of CRISPR systems and the transposon components likely contribute to the genome plasticity. Strain encode diverse spectrum of degradative carbohydrate-active enzymes (CAZymes). Comparative SNP analysis revealed that the genomes of the C. chauvoei strains analyzed were highly conserved. Phylogenetic analysis of strains and available genome (n = 21) based on whole-genome multi-locus sequence typing (wgMLST) and core orthologous genes showed the clustering of strains into two different clusters suggesting geographical links.

Comparative genome analysis of Pasteurella multocida serogroup B:2 strains causing haemorrhagic septicaemia (HS) in bovines.

Pasteurella multocida, a Gram-negative bacterium with ubiquitous nature, is known to affect wide range of host species worldwide with varied clinical manifestations including haemorrhagic septicaemia (HS) in bovines. Although, HS causing P. multocida strains were identified and characterized by conventional tools and PCR assays, diverse strains are indistinguishable by these tools in the face of disease outbreaks. In this study, draft genomes of three virulent P. multocida serotype B:2 strains (NIVEDIPm32, NIVEDIPm34 and NIVEDIPm35) were analyzed following whole genome sequencing, assembly, annotation and compared them with existing global genomes (n = 43) of bovine origin in the database. Three draft genomes of NIVEDIPm strains consisted of 40-52 contigs with GC content of ∼40.4%. The genome size and predicted genes content was ∼2.3 Mb and 2181-2189, respectively. Besides, the presence of various mobile genetic elements, antimicrobial resistance genes and biofilm related genes suggested their vital roles in virulence; further, adaptation to the host immune system as well as host pathogen interaction. Multi locus sequence analysis based on RIRDC scheme showed the presence of ST122 in all the three strains. wgMLST based phylogenic analysis suggested that HS causing Indian virulent field strains differed geographically and showed diversity from existing HS vaccine strain P52. The phylogenetic tree revealed that North Indian strains share high similarity with strains of Pakistan than South Indian Strain. Notably, a high divergence of SNPs between the HS causing circulating virulent strains of India and current HS vaccine strain P52 suggested an imminent need for relook in to HS vaccination strategy for livestock in India.

Development of recombinant NS1-NS3 antigen based indirect ELISA for detection of bluetongue antibodies in sheep.

Bluetongue is an insect borne (Culicoides) viral disease of small ruminants. The virus blankets the globe with a wide serotypic variation, numbered from 1 to 28. In India 21 different serotypes have been reported to be circulating across the various agro-climatic zones of the country. Non-structural proteins (NSPs) of bluetongue virus have always remained ideal target for differentiation of infected from vaccinated animals. The current study is an extrapolation of our previous work where a novel fusion construct comprising of bluetongue viral segment NS1 and NS3 was successfully cloned, expressed, purified with an efficient strategy for its suitable implementation as a diagnostic antigen. In this study, the applicability of the fusion construct has been further evaluated and optimised for field applicability. The fusion construct used in an ELISA platform projected a relative diagnostic sensitivity and specificity of 98.1% and 95.5% respectively against a pre-established test panel. The rNS1-NS3 ELISA showed substantially good agreement with the commercial BTV antibody detection kit. Finally, the study brings together the diagnostic capability of two NSPs, which can be a handy tool for sero-surveillance of bluetongue.

Variability in in vitro biofilm production and antimicrobial sensitivity pattern among Pasteurella multocida strains.

Biofilm production, hitherto an uncharacterized feature among circulating Pasteurella multocida strains, was studied along with the antibiotic susceptibility pattern. On the basis of biofilm formation ability, all the strains were categorized into four groups under six different culture conditions: strong biofilm-forming (22%), moderate (19%), weak (51%), and non-adherent (7%). Strains from serogroups A and B formed significant biofilms in at least one culture condition whereas strains from serogroup D were unable to form biofilms. All strains were found to be susceptible to tetracycline. In addition, the correlation between diverse factors (host, capsule type, clinical condition and the tadD gene) as well as antimicrobial susceptibility in biofilm production were analyzed by Joint distribution models, and showed that enrofloxacin and azithromycin resistant strains were positively correlated with strong biofilm production.

Comparative genetic diversity analysis based on virulence and repetitive genes profiling of circulating Pasteurella multocida isolates from animal hosts.

Virulence associated and/or housekeeping/repetitive genes either in single or multiple copies are being extensively targeted for bacterial pathogen detection and differentiation in epidemiological studies. In the present study, isolation of Pasteurella multocida from different animals and their genetic profiling based on the capsular types, virulence and repetitive elements (ERIC/REP) were carried out. A total 345 clinical samples from apparently healthy and diseased (pneumonic, septicaemia) animals (sheep, goat, pig, cattle, buffalo and rabbits) from different geographical regions of Karnataka, Uttar Pradesh, Mizoram and Assam states of India were screened. A total of 32% of the samples were found positive, of which 41 P. multocida isolates recovered. Virulence profiling of isolates indicated that omp87, ompA, ptfA, sodA, sodC, nanB, fur and exbB were present in 100% of isolates. Whereas, prevalence of other genes were; nanH (90%), ompH (71%), pfhA (63%), plpB (80%), hsf-1 (12%), hsf-2 (37%), pmHAS (78%), toxA (73%), hgbA (37%), hgbB (81%), tbpA (78%) and fimA (98%), among isolates. There was no influence of host or place on prevalence of virulence genes when assessed by fitting a Hierarchial Bayesian ordinal regression model. There was correlation (positive and negative) between broad groups of virulence genes. Both repetitive gene profiles (ERIC and REP) generated multiple amplicons (~200 to ~4000 bp). Cluster analysis with ERIC profiles revealed 5 clusters and 3 non- typable isolates with higher discriminatory power (D = 0.7991) than the REP-PCR profiles (D = 00.734) which revealed 4 clusters and 6 non- typable isolates. The results showed that a considerable level of genetic diversity exists among circulating P. multocida isolates despite belonging to the same geographical origin. The genetic diversity or clustering based on either virulence or repetitive elements among isolates could be largely driven by multiple factors acting together which lead to manifestations of particular disease symptoms.

An efficient production of hybrid recombinant protein comprising non-structural proteins (NS 1 & NS 3) of bluetongue virus in prokaryotic expression system.

Strategic design and suitable purification techniques are of paramount importance in the production of recombinant proteins, if intended for use in a diagnostic assay. However, there is no single protocol that can be universally adopted for obtaining proteins in requisite quality and quantity across various platforms. In this study, we have targeted proteins of bluetongue virus (BTV), which is the causative agent of an arthropod-borne infectious disease in ruminants. Traditionally, serological diagnosis of the disease has rested upon either virus neutralization test or on an ELISA test that employed a recombinant structural (VP1, VP7) protein. Among the non-structural (NS) proteins of BTV, NS1 and NS3, are preferred candidate antigens in development of immuno-diagnostics as these provide the option for identifying recent/ongoing infection. However, the difficulty in production/purification of recombinant full length NS proteins of BTV in sufficient quantity and quality in various expression systems, due to inherent structural complexities, have restricted their wider applicability as immunodiagnostic reagents. To circumvent the difficulties associated with production/purification, we developed a novel NS1 and NS3 fusion gene (∼1302 bp) encoding for NS1 N-terminus (1M to G252 aa) and NS3 protein containing the N- and C-termini with a deletion of two hydrophobic domains along with intervening variable central domain (118A to A182 aa) of bluetongue virus 23. This construct was cloned, over-expressed and efficiently purified by single step affinity chromatography under unique denaturing/renaturing condition. The purified fusion protein was found suitable for detection of antibodies against BTV in an indirect ELISA (iELISA).

Mutational analysis of foot and mouth disease virus nonstructural polyprotein 3AB-coding region to design a negative marker virus.

Inactivated purified whole virus vaccines are used for control of foot and mouth disease (FMD). ELISAs detecting antibodies to the nonstructural proteins (NSP), a marker of infection, are primarily used to differentiate FMD virus (FMDV) infected from vaccinated animals (DIVA). However, such DIVA assays have a limitation to their specificity since residual NSPs present in the relatively impure vaccines are suspected to induce an NSP-antibody response in the repeatedly vaccinated animals. Epitope-deleted negative marker vaccine strategy seems to have an advantage over the conventional vaccines in identifying the infected animals with accuracy. NSP 3AB contains an abundance of immunodominant B-cell epitopes of diagnostic importance. This study addresses the feasibility of producing 3AB-truncated FMDV mutant as a potential negative marker vaccine candidate. An infectious cDNA clone of FMDV serotype Asia 1 strain was used to engineer an array of deletion mutations in the established antigenic domain of 3AB. The maximum length of deletion tolerated by the virus was found to be restricted to amino acid residues 87-144 in the C-terminal half of 3A protein along with deletion of the first two copies of 3B peptide. The 3AB-truncated marker virus (Asia 1 IND 491/1997Δ3A87-1443B1,2+FLAG) demonstrated infectivity titres comparable to that of the parental virus in BHK-21 (log10 7.42 TCID50/ml) and LFBK-αVß6 (log10 8.30 TCID50/ml) cell monolayer culture. The protein fragment corresponding to the viable deletion in the 3AB region was expressed in a prokaryotic system to standardize a companion assay (3A87-1533B1,2 I-ELISA) for the negative marker virus which showed reasonably high diagnostic sensitivity (96.9%) and specificity (100% for naïve and 97.1% for uninfected vaccinated samples). The marker virus and its companion ELISA designed in this study provide a basis to devise a marker vaccine strategy for FMD control.

Production of recombinant non-structural protein-3 hydrophobic domain deletion (NS3ΔHD) protein of bluetongue virus from prokaryotic expression system as an efficient diagnostic reagent.

Serological diagnostics for bluetongue (BT), which is an infectious, non-contagious and arthropod-borne virus disease of ruminants, are primarily dependent on availability of high quality native or recombinant antigen(s) based on either structural/non-structural proteins in sufficient quantity. Non-structural proteins (NS1-NS4) of BT virus are presumed candidate antigens in development of DIVA diagnostics. In the present study, NS3 fusion gene encoding for NS3 protein containing the N- and C-termini with a deletion of two hydrophobic domains (118A to S141 aa and 162S to A182 aa) and intervening variable central domain (142D to K161 aa) of bluetongue virus 23 was constructed, cloned and over-expressed using prokaryotic expression system. The recombinant NS3ΔHD fusion protein (∼38 kDa) including hexa-histidine tag on its both termini was found to be non-cytotoxic to recombinant Escherichia coli cells and purified by affinity chromatography. The purified rNS3ΔHD fusion protein was found to efficiently detect BTV-NS3 specific antibodies in indirect-ELISA format with diagnostic sensitivity (DSn = 94.4%) and specificity (DSp = 93.9%). The study indicated the potential utility of rNS3ΔHD fusion protein as candidate diagnostic reagent in developing an indirect-ELISA for sero-surveillance of animals for BTV antibodies under DIVA strategy, wherever monovalent/polyvalent killed BT vaccine formulations devoid of NS proteins are being practiced for immunization.

Antigenic evidence of bluetongue virus from small ruminant population of two different geographical regions of Odisha, India.

AIM: The aim of the present study was to carry out antigenic detection of bluetongue virus (BTV) among the small ruminant population of two different geographical regions of Odisha (coastal and central) using recombinant VP7 (r-VP-7) based sandwich enzyme-linked immunosorbent assay (s-ELISA). MATERIALS AND METHODS: Blood samples (n=274) were collected from two different geographical pockets of Odisha, which covered mostly the coastal and central regions. Of the total samples under study 185 were from goat and 89 were from sheep. The blood samples were tested for the presence of BTV antigen by r-VP7 based s-ELISA. RESULTS: r-VP-7 s-ELISA detected BTV antigen in 52.43% and 44.94% of the goat and sheep population under study, respectively. This study highlights the antigenic persistence of BTV in the state for the 1(st) time. CONCLUSION: This high antigenic presence in both sheep and goat population suggests an alarming BTV infection in field conditions which warrants more systematic study directed toward isolation and characterization studies as well as the implementation of control strategy for BT in Odisha.

A coiled-coil motif in non-structural protein 3 (NS3) of bluetongue virus forms an oligomer.

Bluetongue, an arthropod-borne non-contagious hemorrhagic disease of small ruminants, is caused by bluetongue virus (BTV). Several structural and non-structural proteins encoded by BTV have been associated with virulence mechanisms. In the present study, the NS3 protein sequences of bluetongue viral serotypes were analyzed for the presence of heptad regions and oligomer formation. Bioinformatic analysis of NS3 sequences of all 26 BTV serotypes revealed the presence of at least three coiled-coil motifs (CCMs). A conserved α-helical heptad sequence was identified at 14-26 aa (CCM-I), 185-198aa (CCM-II), and 94-116 aa (CCM-III). Among these, CCM-I occurs close to the N-terminus of NS3 and was presumed to be involved in oligomerization. Furthermore, the N-terminus of NS3 (1M-R117 aa) was over-expressed as a recombinant fusion protein in a prokaryotic expression system. Biochemical characterization of recombinant NS3Nt protein revealed that it forms SDS-resistant dimers and high-order oligomers (hexamer and/or octamer) under reducing or non-reducing conditions. Coiled-coil motifs are believed to be critical for NS protein oligomerization and have potential roles in the formation of viroporin ring/pore either with six/eight subunits and this is the first study toward characterization of CCMs in NS3 of bluetongue virus.

Recombinant transferrin binding protein A (rTbpA) fragments of Pasteurella multocida serogroup B:2 provide variable protection following homologous challenge in mouse model.

Transferrin binding protein A (TbpA), an iron acquisition surface protein that also acts as virulence factor, is widely distributed among strains of Pasteurella multocida. In the present study, a total of seven clones of TbpA fragments (39D to F777; 39D to Q697; 188V to F777; 188V to Q697; 39D to P377; 188V to P377 and 39D to F187) belonging to P. multocida B:2 were constructed, over-expressed and purified as recombinant fusion proteins from Escherichia coli using affinity chromatography. Immunization of mice with rTbpA fragments resulted in a significant (p < 0.05) rise in antigen specific serum total IgG and subtypes (IgG1 and IgG2a) tires. All immunized mice challenged with 8 LD50 of P. multocida B:2 resulted in a variable protective efficacy up to 50%. The study indicated the potential possibilities to incorporate full length TbpA in subunit vaccine formulation composed of synergistic subunit antigens against haemorrhagic septicaemia (HS) in cattle and buffalo.

Homogeneity of VacJ outer membrane lipoproteins among Pasteurella multocida strains and heterogeneity among members of Pasteurellaceae.

Outer membrane lipoproteins are widely distributed in Gram-negative bacteria which are involved in diverse mechanisms of physiology/pathogenesis. Various pathogenic bacterial strains belonging to the family-Pasteurellaceae have several surface exposed virulence factors including VacJ/VacJ-like lipoproteins. In the present study, vacJ gene encoding for VacJ outer membrane lipoprotein of different Pasteurella multocida strains (n = 10) were amplified, sequenced and compared with available VacJ/VacJ-like sequences (n = 45) of Pasteurellaceae members. Comparative multiple sequence analysis at amino acid level indicated absolute homogeneity of VacJ lipoprotein among different P. multocida strains. However, heterogeneity (18.0-89.9%) of VacJ lipoprotein was noticed among members of Pasteurellaceae. A predicted lipobox motif (L-3-[A/S/T/V]-2-[G/A]-1-C) was found to be conserved between 12-32aa residues at N-terminus among all VacJ sequences. Bioinformatic analysis indicated that VacJ is a chromosomal gene product exposed on the bacterial surface, possibly essential for either physiological or pathogenicity process of Pasteurellae and distributed widely among P. multocida serogroups. The study indicated potential possibilities of using absolutely conserved VacJ lipoprotein either as 'signature gene/protein' in developing diagnostic assay or as a recombinant subunit vaccine for P. multocida infections in livestock.

Covalent functionalization of dipole-modulating molecules on trilayer graphene: an avenue for graphene-interfaced molecular machines.

The molecular dipole moment plays a significant role in governing important phenomena like molecular interactions, molecular configuration, and charge transfer, which are important in several electronic, electrochemical, and optoelectronic systems. Here, the effect of the change in the dipole moment of a tethered molecule on the carrier properties of (functionalized) trilayer graphene--a stack of three layers of sp(2)-hybridized carbon atoms--is demonstrated. It is shown that, due to the high carrier confinement and large quantum capacitance, the trans-to-cis isomerisation of 'covalently attached' azobenzene molecules, with a change in dipole moment of 3D, leads to the generation of a high effective gating voltage. Consequently, 6 units of holes are produced per azobenzene molecule (hole density increases by 440 000 holes µm(-2)). Based on Raman and X-ray photoelectron spectroscopy data, a model is outlined for outer-layer, azobenzene-functionalized trilayer graphene with current modulation in the inner sp(2) matrix. Here, 0.097 V are applied by the isomerisation of the functionalized azobenzene. Further, the large measured quantum capacitance of 72.5 µF cm(-2) justifies the large Dirac point in the heavily doped system. The mechanism defining the effect of dipole modulation of covalently tethered molecules on graphene will enable future sensors and molecular-machine interfaces with graphene.

Nanotomy-based production of transferable and dispersible graphene nanostructures of controlled shape and size.

Because of the edge states and quantum confinement, the shape and size of graphene nanostructures dictate their electrical, optical, magnetic and chemical properties. The current synthesis methods for graphene nanostructures do not produce large quantities of graphene nanostructures that are easily transferable to different substrates/solvents, do not produce graphene nanostructures of different and controlled shapes, or do not allow control of GN dimensions over a wide range (up to 100 nm). Here we report the production of graphene nanostructures with predetermined shapes (square, rectangle, triangle and ribbon) and controlled dimensions. This is achieved by diamond-edge-induced nanotomy (nanoscale-cutting) of graphite into graphite nanoblocks, which are then exfoliated. Our results show that the edges of the produced graphene nanostructures are straight and relatively smooth with an I(D)/I(G) of 0.22-0.28 and roughness <1 nm. Further, thin films of GN-ribbons exhibit a bandgap evolution with width reduction (0, 10 and ~35 meV for 50, 25 and 15 nm, respectively).

Synthesis and characterization of amphiphilic reduced graphene oxide with epoxidized methyl oleate.

Amphiphilic reduced graphene oxide is obtained by oleo-functionalization with epoxidized methyl oleate (renewable feedstock) using a green process. The excellent diverse solvent-dispersivity of the oleo-reduced amphiphilic graphene and its reduction chemistry are confirmed in this study. Oleo-reduction of amphiphilic graphene is amenable to industrially viable processes to produce future graphene-based polymer composites and systems.

Impermeable graphenic encasement of bacteria.

Transmission electron microscopy (TEM) of hygroscopic, permeable, and electron-absorbing biological cells has been an important challenge due to the volumetric shrinkage, electrostatic charging, and structural degradation of cells under high vacuum and fixed electron beam.(1-3) Here we show that bacterial cells can be encased within a graphenic chamber to preserve their dimensional and topological characteristics under high vacuum (10(-5) Torr) and beam current (150 A/cm(2)). The strongly repelling π clouds in the interstitial sites of graphene's lattice(4) reduces the graphene-encased-cell's permeability(5) from 7.6-20 nm/s to 0 nm/s. The C-C bond flexibility(5,6) enables conformal encasement of cells. Additionally, graphene's high Young's modulus(6,7) retains cell's structural integrity under TEM conditions, while its high electrical(8) and thermal conductivity(9) significantly abates electrostatic charging. We envision that the graphenic encasement approach will facilitate real-time TEM imaging of fluidic samples and potentially biochemical activity.