The present treatment of childhood T-cell leukemias involves the systemic administration of prokary-otic L-asparaginase (ASNase), which depletes plasma Asparagine (Asn) and inhibits protein synthesis. The mechanism of therapeutic action of ASNase is poorly understood, as are the etiologies of the side-effects incurred by treatment. Protein expression from genes bearing Asn homopolymeric coding regions (N-hCR) may be particularly susceptible to Asn level fluctuation. In mammals, N-hCR are rare, short and conserved. In humans, misfunctions of genes encoding N-hCR are associated with a cluster of disorders that mimic ASNase therapy side-effects which include impaired glycemic control, dislipidemia, pancreatitis, compromised vascular integrity, and neurological dysfunction. This paper proposes that dysregulation of Asn homeostasis, potentially even by ASNase produced by the microbiome, may contribute to several clinically important syndromes by altering expression of N-hCR bearing genes. By altering amino acid abundance and modulating ribosome translocation rates at codon repeats, the microbiomic environment may contribute to genome decoding and to shaping the proteome. We suggest that impaired translation at poly Asn codons elevates diabetes risk and severity. IRS2, encoding an insulin signal transducer, is the gene at the top of the list in Despite similar Asn codon usage, ~4%/gene, from plants to humans , mammalsin vitro (in vivo (Asparaginase (ASNase) is a component of highly effective chemotherapeutic regimens used to treat pediatric acute lymphoblastic leukemia (ALL) ,5 and soin vitro  and in v. In the abstract, reduced rates of translation anywhere along an mRNA would result directly in a reduced overall rate of target protein synthesis and, depending on protein halflife, result indirectly in decreased steady state levels of such proteins. High rates of translation may even increase the halflife of an mRNA , and eight percent with vasculature and hematopoesis. Of the remaing ~14%, many can be classified as involved with chromatin modification, DNA maintainance and repair, RNA transcription and processing or protein synthesis and turnover, some have Leucine rich repeats that can serve as pattern recognition elements. Some genes fall into multiple categories, e.g. epilepsy , aspectsepilepsy , Alzheimepilepsy –73, retiepilepsy , hippocaepilepsy , long teepilepsy , ataxia epilepsy , cardiacepilepsy , kidney epilepsy , renal depilepsy , breast epilepsy ,82, rhabepilepsy  and, in topoesis ,85. A lidiabetes .CXCR2, (EPQ18419), which has a 60N-hCR. Other genes of interest from mouse, that differ from human in N-hCR length, include MDR1 and CFTR , and TNFRSF16/BEX3A/NGFRAP1 (implicated in diabetes)  . For exaiabetes)  as well s)   . The genMPK/SIX5 ,112, GCLX5  subset of children and its mouse homolog regulates energy homeostasis and body weight (cf. KCNH43N-hCR and KCNH83N-hCR). Pancreatitis and diabetes are associated, respectively, with CFTR4N-hCR and SLC26A93N-hCR, the products of which physically and functionally interact. CFTR is an ion channel, closely related, by membership in the superfamily of ATP-binding cassette proteins, to the multidrug resistance transporter (MDR1) , very similar in location to Asn416 in the N-hCR of CFTR. Such polymorphisms in MDR1 have been proposed  between the membrane spanning domain (MSD) and the nucleotide binding domain (NBD) could, by analogy to the key MDR1 Gly412 substitution, alter translation rate at its Asn 415 to 418 region, under conditions of low Asn, to result in generation of CFTR protein folding variants (SLC26A93N-hCR)  –153. Somproposed  to affecproposed . The N-hvariants  with alt93N-hCR) –158, Saltibility , and timtibility .TRPM64NhCR and TRPM73N-hCR,4NhCR. Allelic variation of the former has been associated with elevated risk of diabetes, osteoporosis, asthma, and heart and vascular diseases  but also CFTR (Ile507 and ΔF508) (SCARB1 (Ala350)  and ischemic stroke  receptor that participates in lipid metabolism and flux of cholesterylesters   and SCAR(Ala350) . Rs5888,c stroke –171. Traylesters  into e.gylesters  and thusylesters . SCARB1 farction  and renafarction ,177 actifarction , and caufarction ; it indifarction , mitigatfarction , and afffarction  and macufarction . By inflfarction –188. A sfarction ; this suF53N-hCR104t (encoding coagulation Factor V) have been linked to coronary artery disease  associated with ischemic stroke  in diabetics  , age of onset of amyotrophic lateral sclerosis (ALS) (TTLL4 and LAMA3) (TMEM106B) (TTLL5) (TTLL5 and PHACTR1) (LRP4 and NCOA1) (TBC1D3 and TBC1D3F) (GRAIL/RNF128/isf2)   ,217, breCAM5/CEA , CYP19A1romatase , IRS2, C CLEC10A , LRP6 and TBC1D5 ], spinald LAMA3) , dementiMEM106B)  retinal  (TTLL5) , large aPHACTR1)  decreased NCOA1) , ovarianTBC1D3F)  T cell a28/isf2) –230, ast28/isf2) –233 and  -B, -C)  suggestiDZIP1, MAML2, BOC and CDON, and may present attractive targets for drug discovery via small molecules that accelerate turnover of specific proteins encoded by N-hCR bearing-genes, synergistically magnifying the impact of ASNase by altering the replacement rate and perhaps by establishing lowered steady state levels of the targeted protein. There is already a precedent for synergism of prednisolone with ASNase, which occurs by an as yet unknown mechanism. The halflife of WNT signalling complexes and the contribution of DSV to turnover of WNT coreceptors FZD and LRP6 has recently been characterized   ,244 comeisorders . Since SANK3, IRS2, SNCAIP, XIRP2, PPP1R9A and CACNA1-C. Low plasma Asn, via the 17 N-hCR-bearing-genes listed in SNCAIP; dental caries and peridontal disease as a diabetes comorbidity through TMEM178B or ANKRD17 in children (cf. LRP1B and periodontitis in adults) (LRP1B are age at menarche (cf. glaucoma), conditional erectile dysfunction in African American men, heart rate and multiple cancers. Deafness , heroin addiction vulnerability in African Americans (XIRP2 (PHACTR1 (cf. LRP6) and PPP1R9A  ; erythropoesis and quality control of mitochondria by BNIP3L; nucleic acid processing by COIL, PAPD5, THRAP3, MEX3B and C1orf86/FAAP20; and diabetes by THRAP3 (cf. CHRM3), PTPRD and IRS2.Adverse neurological outcomes have also been associated with N-hCR-bearing-genes children ,253; (cf adults) . Also afmenarche , APOE anmenarche , protectmenarche  as well Deafness ,259 is americans  and hears  ; bone dePEG10 and BNIP3L, have transcripts with long N-hCR that are encompassed within their initial two dozen codons. Both BNIP3L and PEG10 are apoptosis-related genes that are candidates for mediation of the cell death that has been observed to follow depletion of Asn either in cell culture  . Temperature sensitive mutants of Asn tRNA synthetase undergo cell cycle arrest in early S phase at the nonpermissive temperature, a phenomenon that has been posited to be consistent with the existence a protein required for cell cycle progression that is highly sensitive to the level of charged Asn-tRNA (cf. COIL above).The discussion above has focused on adverse events elicited by ASNase therapy, not the induction of tumor remission. Two N-hCR-bearing-genes,  culture  or in pend RYR2) , JAK2, K2, KCNA3 , UBE2Q2    were performed with defined templates containing Asn codons at predetermined sites. T7 RNA polymerase was used to generate transcripts that were either devoid of Asn codons or contained one, two, five or 23 Asn codons between the N- and C-terminal segments of a bipartite hybrid protein composed of two human genes with no Asn codons. The N-terminal portion was derived from TCL1A, and the C-terminal portion was derived from CKS2. The central, intragenic N-hCR was, on occasion, substituted by the programmed ribosomal frameshifting (PRF) region from PEG10 which contains an Asn (AAC) codon at the frameshifting site. The resulting in vitro transcripts were translated in rabbit reticulocyte cell free lysates with isotopically labelled 35S-methionine and the products were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography. This template gave extremely clean IVT results without the partial products seen with other templates such as PEG10 or gaussia luciferase. It was determined, with some appropriate control experiments, that there were quantities of ASNase that could be added to the translation mix to create different ratios of partial to full length products which could reflect relative degrees of pausing at the different poly Asn regions of length zero, one, two, five and 23 codons. Free Asn could subsequently be added back to the depleted reaction mix to 'chase', to a first approximation, the short 'TCL1A' proteins into longer, hybrid, 'TCL1A/CKS2' proteins. Conditions were also established in which the relative efficiency of frameshifting at the Asn codon of the PRF site of PEG10 was affected by exogenous ASNase added to the in vitro translation reaction, but this result was far less compelling than the effect of ASNase on translocation at N-hCR.ASNase in bsiella) , is encolower Km . Studiesynthesis . To deteynthesis ,274 in vSalmonella ASNase, of in vitro translation reactions  in adults compared to children underscores the difference between the physiology of children and adults.TBC1D33N-hCR gene of hominids, and the expansion, and perhaps positive selection in humans, of eight members of this N-hCR bearing-gene family (cf. TBC1D5 with Rab7 (TBC1D3 is involved in pinocytosis with ARF6 (cf. 285). These genes could also potentially regulate insulin or amino acid release from vesicular or lysosomal storage , or in human, mouse, rat, lizard, ~frog and chicken (BNIP3L), indicating that the two isoacceptor tRNAs may indeed be differentially regulated.Most of the poly Asn codon runs reported here consist of the two isoaccepter codons AAT and AAC used in about equal frequency with a slight bias towards homopolymeric runs of AAC. In the gene ructures , RNA seqructures ]. InterePPP1R9A5Nx2-hCR, unique among the 17 genes of FAAP20 can form a highly structured α helix upon ubiquitin binding; this domain is interrupted by a 5N-hCR in certain variant isoforms. The 2N-hCR of TP53 is similarly located: adjacent to a pair of transactivation domains (TADs) that gain structure upon ligand binding   ]. The 4N member)  that may member) . A simil member) ,306, as  member) , a regio member) ,309. The member)  perhaps se model . TXNIP ose model ,312 and se model . Alteredse model . Deletiofunction ,314. Regfunction , but seefunction . hCR mayfunction  where prfunction . An altefunction .At least five different human tRNA synthetases can serve as autoantigens in inflammatory responses . Human tcf. L-hCR length polymorphisms and diabetic nephropathy in CNDP1   (RYR2 and CHRM3. RYR2 is involved with heart disease (CHRM33N-hCR is associated with response to an antidiabetic drug in African Americans (cf. CHRM24N-hCR associated with metabolic syndrome)  cf.  as well echanism , to mBEXm  . Asn lev disease  and asso disease  and ALL  disease , CHRM33Nmericans  (cf. CHRyndrome) . Another) : IL9R (ARID5B (cf. KCNA3) (IL9R shares a common γ subunit with other interleukin receptors) ]. ARID5B encodes part of a histone lysine demethylase complex : IL9R  and ARID. KCNA3) . IL9R shceptors)  IL9R has complex  and is n complex ,364–369  complex , low bir complex , diastol complex  rheumato complex , respons complex  (an anti complex , lipid b complex  and trig complex , as well complex . The contigation .We propose that the impaired translation which has been described above be termed the 'translational N-hamper effect' because there is nothing intrinsically impaired about a protein polymerization reaction in which one of the required components, activated Asn tRNA, is ratelimiting for the translocation reaction on the template mRNA. The verb of choice for slowed translocation could just as well have been cumbered movement instead of hampered movement. If the argument was first made for Gln, the Q-cumber effect could have encompassed this hypothetical phenomenon.The 'translational N-hamper effect' is a mechanism whereby protein expression is modulated by coupling fluctuations in appropriate aminoacylated-tRNA availability to ribosome translocation rates at corresponding hCR. Thus, ribosome movement could pause at hCR which would serve as punctuation marks to allow relative intracellular amino acid pool sizes to influence mRNA decoding and protein synthesis. Amino acid level fluctuation could potentially affect: mRNA halflife and accessibility to regulatory complexes, ribosome frameshifting efficiency, initiation rate and formation of stable translation complexes, and elongation rate and vestibule residence time to affect steady state levels of these proteins and of higher order structures in which they participate.Our model holds that Asn level reductions, such as those accompanying the administration of ASNase, cause impaired translation of N-hCR-bearing-genes to precipitate metabolic, vascular, immunological and neurological disorders and contends that this could result in insulin desensitization, impaired insulin release and, ultimately, diabetes. Thus the microbiome, by endogenously generating ASNase, could cotranslationally regulate a constellation of N-hCR-bearing-genes to initiate complex disease pathologies.


The effects of the stiffness of the microenvironment on the molecular response of 3D colony organization, at the maximum level of mammographic density (MD), are investigated. Phenotypic profiling reveals that 3D colony formation is heterogeneous and increased stiffness of the microenvironment, within the range of the MD, correlates with the increased frequency of aberrant 3D colony formation. Further integrative analysis of the genome-wide transcriptome and phenotypic profiling hypothesizes overexpression of ERBB2 in the premalignant MCF10A cell lines at a stiffness value that corresponds to the collagen component at high mammographic density. Subsequently, ERBB2 overexpression has been validated in the same cell line. Similar experiments with a more genetically stable cell line of 184A1 also revealed an increased frequency of aberrant colony formation with the increased stiffness; however, 184A1 did not demonstrate overexpression of ERBB2 at the same stiffness value of the high MD. These results suggest that stiffness exacerbates premalignant cell line of MCF10A. The extracellular matrix (ECM) plays important roles including regulating structure and function of the cell and cellular organization, and in achieving homeostasis361011in vivo models. Colony organization of the 3D cell culture models is an important endpoint for phenotypic profiling, which can also be mapped to the genomic aberrations. Previous studies have shown that colony organization for HMEC can be round and lumen forming, non-lumen forming, flat, or flat and invasive25This paper has two overarching goals. First, it advances the current understanding of the role of increased stiffness on colony organization and its molecular endpoints, within the range of mammographic density (MD), through detailed confocal microscopy and molecular profiling. MD represents the density of connective and epithelial tissue in breast17192022suggests that spatial organization of collagen has a preference for a phenotype with more fibrous in patients of high MD, as shown in establishes the baseline stiffness of ~800 and ~1800 Pa for the collagen regions, and ~500 and ~1100 Pa for epithelial in low and high MD, respectively, as shown in In order to establish the baseline stiffness for this study, AFM was used to characterize the matrix stiffness of breast tissue histology sections from patients with low and high MD who have gone through reduction mammoplasty. AFM provides an accurate spatial profiling of the stiffness of the microenvironment for each patch in a histology section. For each histology section, 3 patches were selected in triplicates  for collagen, epithelial, and fat regions. Each patch is 80-by-80 microns, and within each patch 9 grid points were randomly selected and nanoindented. The results are shown in In short, spatial organization of collagen appears less rough2930In order to mimic the stiffness of the microenvironment in low- and high-MD of breast tissues, agarose/Matrigel-based hydrogel is used to modulate the stiffness of the microenvironment in culture. Several strategies for modulating stiffness of the 3D of HMEC, using collagen and silk hydrogels, have been proposed in the past3537e.g., DAPI), β-catenin, and β4-integrin, and were imaged with confocal microscopy as shown in At low stiffness, colony organization is homogeneous and remains polarized.e.g., hollow spherical architecture) is coupled with the loss of polarity, as shown in At high stiffness, colony organization is heterogeneous, and in some cases, the loss of organization , heterogeneity analysis, and correlative analysis with the genome-wide molecular data for hypothesis generation. BioSig3Dviz., nuclear shape and geometry, colony size, number of cells in a colony, flatness of colony, and a proxy for lumen formation. These indices are then analyzed for their mean and distribution properties, using BioSig3D.Colony organization, imaged with phase contrast microscopy, can partially aid in classifying genomic aberrations26e.g., zero mean with variance of one) independent of the experimental factor, and then visualized with a heatmap, with an example shown in   partitions phenotypic signatures into two basic subgroups,  modulates colony organization in terms of flatness, size, and distances between adjacent nuclei in the same manner that genomically aberrant cell lines self-organize themselves24correlates with the increased colony size and number of cells per colony, independent of the increased nuclear size. Increased nuclear size, as a function of stiffness, has been reported previously in 2D cultures4041Computed indices are normalized , then the colony sizes are increasing independent of the nuclear size. Third, the frequency of aberrant flat colonies is increasing. Fourth, the distances between adjacent cells, within a colony, are also increasing. These observations are concomitant with aberrant colony organizations, per previous literature46Heterogeneity is usually computed in terms of entropy43In order to verify that heterogeneity is persistent over an extended culture period, an experiment was designed to quantify colony formations on days 9 and 21. The result is shown in e.g., ECM constituent, structural constituent of the cytoskeleton), with a p-value of 0.017. There were no other meaningful enrichments. Enrichment of structural activities is expected as cellular morphology and colony organization are altered as a result of increased stiffness. Furthermore, transcripts were evaluated for their dependencies with breast cancer biomarkers, and in particular ERBB2:TUBA1A, involved in G2/M transition, has been shown to be a part of a network that is associated with ERBB2 positive tumors in clinical samplesAnnexin A2 (ANXA2) has been found to be inversely correlated with ERBB2 expression in clinical samples and breast cancer cell linesTAGLN is activated by ERBB2 signaling by way of the Ingenuity Pathway Analysis (IPA).Network analysis partitioned these genes into 4 distinct functional categories, with Networks shown in Upstream analysis revealed a list of regulators, including ERBB2, with a p-value of 0.016 as shown in Utilizing IPA core analysis for the top 133 differentially expressed transcripts, two sets of analysis were performed:To investigate changes in gene expression as a result of increased stiffness, genome-wide profiling was performed, in parallel, with phenotypic profiling, i.e., exact same culture conditions. Expression data were analyzed using several complementary methods.Finally, genome-wide expression data were correlated with computed phenotypic indices such as colony size and flatness. In the case of colony size, correlation coefficients of 0.9 or better (in both directions), and with the p-value of 0.02 or better, were filtered for further enrichment analysis. ERBB2 was identified as an upstream regulator, with a p-value of 0.006. Similar analysis with colony flatness revealed PPARG as an upstream regulator. These analyses hypothesize ERBB2 as a potential endpoint for validation, even though differential gene expression analysis does not show ERBB2 as having a strong p-value.e.g., in SKBR3 cell lines) and in approximately 20% of the patients . Because genome-wide transcriptome analysis has also enriched ERBB2 signaling, samples have been stained with ERBB2 antibodies in each of the treatment groups. Results indicate that ERBB2 is overexpressed at high stiffness values of the microenvironment that correspond to high MD. A representative example is shown in 50Overexpression of ERBB2 is associated with aberrant colony formation in culture , whereas tumor tissue is quite stiff and of the order of 4000 Pa. Stiffness of the microenvironment has been measured in normal and tumor regions of mammary glands in miceHaving reduced the technical variations, 3D colony profiling revealed that aberrant colony formation is gradually increased as a function of the stiffness of the microenvironment. These observations are consistent for MCF10A and 184A1. Quantitative profiling was performed using mean-based analysis and subtyping, to investigate global changes as well as heterogeneity in colony formation. In short, stiffness modulates aberrant colony formation. Parallel cultures for genome-wide transcriptome profiling hypothesized ERBB2 as a potential regulator in MCF10A, which was subsequently validated in 3D cultures at astiffness value corresponding to the high MD. However, ERBB2 was not overexpressed in (i) a genomically stable cell line of 184A1, and (ii) in 2D cultures of MCF10A at the high stiffness value of the microenvironment. Therefore, overexpression of ERBB2 is strictly a 3D effect and is exacerbated by the pre-malignant state of the MCF10A cell line.In summary, it is concluded that stiffness, within the range of MD, plays a major role in promoting aberrant colony organization with clinically relevant upregulation of ERBB2 in MCF10A. Because women with high MD have an increased risk factor to develop breast cancer, it is necessary to investigate the overexpression of ERBB2 at high stiffness further. A second experiment with a genomically stable 184A1 HMEC revealed that ERBB2 is not overexpressed with the increased stiffness. Although both MCF10A and 184A1 are immortalized premalignant cell lines, MCF10A has probably lost more barriers toward transformation, and uniquely expresses ERRB2 under high stiffness of the microenvironment. In this particular case, stiffness exacerbates the premalignant phenotype.e.g., collagen, epithelial, fat) were identified and marked in the H&E stained images, and subsequently matched in adjacent non-stained sections, for profiling with atomic force microscopy (AFM).Normal breast histology sections from low and high MD (BIRADS score 1 and 4) were collected from patients who had undergone reduction mammoplasty at UCSF. For each BIRADS score, tissue sections from 3 different patients were collected, and, for each patient, 4 non-stained and 2 H&E stained sections were acquired at 10 micron thickness. Tissue sections were collected and transported in frozen conditions. Landmarks associated with distinct regions of microanatomy  hydrogel, which is modulated between 480 Pa to 4470 Pa. The details of the PA hydrogel preparation is outlined in Lin The 3D cell culture model utilizes embedded protocol as opposed to the “ontop” method−1 of spring constant), was acquired from Novascan Tech. The AFM tip was brought down, with maximum strain set at 20% to indent the samples. Next, the Young’s Modulus was calculated. Tissue slides (3 frozen and 1 HE stained) from 3 patients for each category of mammographic density were used for the AFM profiling. H&E stained slide was used to select three regions per tissue compartment , which adds up to 9 regions per tissue section and 54 regions for the entire sample size. For each region, specified on the H&E stained slide, the corresponding region on the frozen samples was marked. Frozen samples were then thawed, and the size of each region was set at 80 μm × 80 μm. Each region was divided into 9 sub-regions for individual spot test. 3 spot measurements , in each sub-region, were performed. These data were then plotted as the mean ± standard deviation (SD) in AFM was used to quantify the local stiffness within the histology sections as well as engineering matrices for modulating local tension. Briefly, AFM indentation experiments were carried out with a MFP-3D AFM . A special AFM tip, fitted with a borosilicate glass sphere  supplemented with 5% horse serum (Life Science Technology), 10 ng/mL FGF (Life Science Technology), 50 ng/mL chlora toxin (Sigma), 200 μg/mL insulin (Sigma), 100 ng/mL hydroxylcortisone (Sigma), and 1% penicillin-streptomycin (Life Science Technology). 184A1 cells were cultured in M87A medium, which was provided by Martha Stampfer’s lab at the Lawrence Berkeley National Lab.Two HMEC were cultured sequentially following an initial pilot project, validation, and hypothesis generation. These were MCF10A and 184A1. The MCF10A model system was obtained from ATCC at passage >50, and was then subcultured (P5-P7) for the proposed studies. The 184A1 cells were obtained from the Martha Stampfer labe.g., ERBB2, β-catenin, β4-integrin) (Abcam) were diluted in blocking solution (1:500), incubated for 1 h, and followed by 3 PBS washes. Goat anti rabbit secondary antibody conjugated with Alexa 488 and Alexa 565 (Life Science Technology) was diluted in the blocking solution (1:250), conjugated to primary antibody for 1 h, and followed by 3 PBS washes. Subsequently, nuclei were stained with 4′-6-diamidino-2-phenylindole (50 ng/mL) .Cells in the hydrogel were fixed at room temperature in 10% formalin for 30 min. After 3 times of PBS washes, cells were permeabilized using a 0.1% Triton X-100 solution for 5 min. Hydrogels were washed 3 times with PBS, blocked using a 1% bovine serum albumin (BSA) solution (Sigma) supplemented with 1% goat IgG (Jackson ImmunoResearch), and the blocking solution was maintained for 1 h. In some cases, samples were stained for a specific molecular endpoint, but in all cases, DAPI nuclear stain was applied for profiling colony organization. Monocolonal rabbit anti human primary antibodies (Stained samples were imaged using a Zeiss LSM 710 system equipped with a Zeiss Apochromat 40X/1.1 (0.8 mm WD) water immersion objective lens. The excitation filters were set at 405 and 561 nm, and the emission filters were set to receive signals between 420–480 and 597–700 nm, respectively. The laser intensity was set at 1%. A twin-gate main beam splitter with two wheels, each wheel having 10 filter positions (and thus 100 possible combinations), was used to separate excitation and emission beams. The pinhole was set at “1”. The digital gain was adjusted to approximately ¾ of the maximum gain, which kept the dynamic range of the pixels between 500–2000 (12 bits). The 3D stack function of the Zen software was used to collect raw 3D information for each colony, and was concurrent with “Z correction” of the pixel intensity for thick samples. The voxel size was set to 0.25 μm × 0.25 μm × 1 μm, the image files were saved as lsm files, and uploaded to the BioSig3D imaging bioinformatics systemTotal RNA was extracted using a Qiagen RNAeasy micro kit, following the in-kit protocol. The RNA quality was checked to assure that RNA integrity number (RIN) was over 7, out of maximum 10. Total RNA extractions were sent to the UCLA UNGC core facility for profiling, where UCLA performed their own quality control check independently. Subsequently, UCLA amplified the total RNA by Ambion amplification and profiled gene expression on Illumina human ht-12 chip in triplicates. The chips were scanned, gene expression data were normalized, and p-values for the quality of each gene was computed, and a spreadsheet was delivered for integrative analysis.e.g., colony flatness) represent colony shape attributes globally. Graphical features  represent local attributes of the colony organization. These analyses were performed by BioSig3D, which was also developed by our Laboratory. This is publicly available at biosig.lbl.gov, and a special version of it will be released as a virtual machine with the data that have been collected for this manuscript. BioSig3D provides an end-to-end solution for designing high-content screening assays, robust algorithms for quantitative analysis of multicellular systems and their organization, visualization of raw and processed 3D volumetric data, and integration of bioinformatics analysis. As a result, colony organization can be represented as a heat map or similarity matrices that correspond to colony heterogeneity. In this paper, BioSig3D has been utilized to characterize heterogeneity in colony formation for cell lines. For each experimental condition, 40 3D datasets were uploaded for analysis.3D Colony organizations were computed by segmenting nuclear regions, from 3D images, and by computing a series of geometric and graphical features per colony. Geometrical features .


Human RSV is one of the most prevalent viral pathogens of early childhood for which no vaccine is available. Herein we provide an analysis of RSV epitope data to examine its application to vaccine design and development. Our objective was to provide an overview of antigenic coverage, identify critical antibody and T cell determinants, and then analyze the cumulative RSV epitope data from the standpoint of functional responses using a combinational approach to characterize antigenic structure and epitope location. A review of the cumulative data revealed, not surprisingly, that the vast majority of epitopes have been defined for the two major surface antigens, F and G. Antibody and T cell determinants have been reported from multiple hosts, including those from human subjects following natural infection, however human data represent a minority of the data. A structural analysis of the major surface antigen, F, showed that the majority of epitopes defined for functional antibodies  were either shown to bind pre-F or to be accessible in both pre- and post-F forms. This finding may have has implications for on-going vaccine design and development. These interpretations are in agreement with previous work and can be applied in the larger context of functional epitopes on the F protein. It is our hope that this work will provide the basis for further RSV-specific epitope discovery and investigation into the nature of antigen conformation in immunogenicity. Despite the fact that viral pathogenesis and immunity to RSV are well characterized in humans and in animal models of disease –4, human+ T cell-driven Th2 cytokine response  to comprehensively analyze all antibody/B cell and T cell epitopes described to date for the F protein. We used a combination of in silico prediction tools housed at the IEDB and other computational methods to characterize the nature and structural features of these epitopes on available 3D structures of the F protein and then compared these results to known  functional/protective sites. We found that the majority of epitopes defined for functional antibodies was either shown to bind pre-F or to be solvent accessible in both pre- and post-F forms. Finally, we observed an alignment of functional B and T cell epitopes mapped along the length of the F protein, which might suggest a relationship between the location of epitopes and protein functional sites. These findings are in agreement with previous works [Our aim was to use the data and tools housed in the Immune Epitope Database (IEDB) and Analysis resource [us works –20 and cwww.iedb.org]. For more complex queries the advanced search interface  was utilized. Results were downloaded in Excel format for detailed analysis. Excel spreadsheets containing all B and T cell assay data were used to identify epitope sequence and source, assay type, immunization details, antibody isotype, effector cells (CD4/CD8), host, etc. RSV-specific queries were conducted using the Antigen organism finder, using the input ‘Pneumovirus.’ In the IEDB, this genus currently includes human RSV , bovine RSV (BRSV) and Ovine RSV (ORSV). The latter strains are used as animal models of natural infection and the viruses are antigenically homologous. Antigen-specific queries were performed using Antigen Finder or through analysis of above spreadsheets for response-association. Unless otherwise indicated, all reported data herein represent positive epitopes and/or assays only.All queries were performed using the Immune Epitope Database and Analysis Resource (IEDB) home page search interface  showing epitope-specific killing of target cells, as well as in vitro cytokine production , wherein peripheral blood mononuclear cells (PBMCs) or lymphocytes are stimulated with or respond to the epitope as assay antigen.The IEDB defines an epitope the unique molecular structures  experimentally shown to react with a B cell or T cell (no predictions). This includes peptides less than or equal to 50 amino acids in size and non-peptidic structures less than or equal to 5000 Daltons. These structures must be experimentally tested for binding to an adaptive immune receptor (T cell receptor (TCR), antibody or B cell receptor (BCR), or major histocompatibility complex (MHC)) or the receptor must be known and stated to be epitope specific in order to be included in the IEDB. Positive epitopes are defined as those residues or structures shown to bind in at least one positive assay; negative data (residues shown to be non-binding) are also captured in the IEDB. With respect to assay type, B cell/antibody assays considered herein to define ‘functional epitopes’  include,in vitro neutralization in a standard microneutralization assay. Epitopes captured to date were also defined by crystallographic analysis of antigen-antibody complexes  .Also of note with respect to the source of the data presented herein, discontinuous epitopes  were most frequently defined using virus escape mutants whereby the alteration or substitution of critical residues conferred escape from www.rcsb.org/pdb] structures for the pre-F  and post-F  conformations were used. Separate chains comprising the trimeric structure of the F protein were re-worked manually into a single chain, which was used as input for the B cell epitope prediction tools, ElliPro , and a higher score indicates that a larger fraction of individuals responded. The square root is a correction factor, approximating one standard deviation for the number of responding donors. This gives a higher score to epitopes studied with larger sample sizes. . Thus, thttp://tools.immuneepitope.org/esm/userMappingFrontP.jsp or through the IEDB Analysis Tools menu. This tool uses input from the user (epitope residues or epitope ID) to map individual or a group of epitopes onto the three dimensional structure of the source protein provided by entering a SWISS-PROT, GenBank ID or FASTA. JAVA version 7 is required for this application.The Homology Mapping tool can be accessed at Pneumovirus. As of January 2015, the IEDB contained 140 references describing 672 epitopes derived from this genus, including human RSV (567), bovine RSV (86), murine pneumonia virus (19) and ovine RSV (1) [number in parentheses means total epitopes for each virus]. These data include antibody/B cell (243) and T cell (323) epitopes, as well as epitopes naturally-eluted from human MHC-peptide complexes and those tested in MHC binding assays (160). Of the B cell epitopes, linear epitopes (208) vastly outnumbered discontinuous (35). For T cell epitopes, the majority have been described for class II/CD4+ (206) compared to class I/CD8+ (97). Thus the majority of reported data were from human RSV, representing a fairly broad range of immune reactivities, including all those within the broad categories of assay types: binding assays , functional assays  and in vivo challenge assays. Of the total T and B cell epitopes reported to date, 214 epitopes (32%) have been defined in humans. Non-human animal models used in the assays identifying RSV epitopes included mice (307), cows (88), rabbits (39), guinea pigs (2), rhesus monkeys (2) and Cynomolgus monkeys (1).To determine the breadth of RSV-specific data available in the IEDB, we performed a broad query to enumerate all data reported from viruses within the genus Epitopes have been reported for all ten RSV protein antigens: fusion (F) glycoprotein, attachment (G) glycoprotein, RNA-dependent RNA polymerase (L), nucleoprotein (NP), matrix (M), matrix 2 (M2), non-structural protein 1 (NS1), non-structural protein 2 (NS2), phosphoprotein (P) and the small hydrophobic protein (SH). However, the degree of coverage varied greatly among individual antigens. Not surprisingly, the major surface glycoproteins, F and G, represented the majority of identified epitopes , followed by L (12%), NP (5%), M2 (4%), M (3%), NS2 (2%), P (1%), SH (1%) and NS1 (1%). The predominance of epitopes derived from F and G was not unexpected, as these antigens are well-documented as the major targets of immune response to RSV infection . In partin vivo protection assays and those defining in vitro correlates of protection, virus neutralization/fusion-inhibition (described in the Methods). From here forward, we will refer to this specific subset of data as ‘functional epitopes.’ Many epitope mapping studies are carried out by testing overlapping 15-20-mer peptides spanning the entire length of an antigen. While this is a standard approach for epitope mapping, the interaction it defines is less frequently indicative of a biologically relevant activity. Assays that defined binding alone have therefore have been excluded.To characterize in depth the B cell/antibody data for the F and G proteins, we focused on the subset of data describing functional assays, including only data related to in vivo protection, respectively. Data for linear epitopes include epitopes identified in assays using both polyclonal sera (PC) and monoclonal antibodies (mAbs). Discontinuous epitopes were defined for both human and murine mAbs, and include well-known mAb D25  and post-F  [PDB: 3RThus to quantify and compare the change in accessibility from pre- to post-F, we calculated residues relative solvent accessibility (RSA) for each structure. The ability to map or identify B cell epitopes has been historically difficult, due in large part to the conformation-dependent nature of these determinants, which imposes numerous technical challenges related to the analysis of proteins in their native, 3-dimentional state. Also likely at issue, is the more practical problem of limited access to samples from human donors. Consequently, there has been increased interested in supplementing the current experimental methods with other analyses and bioinformatics tools. The idea being that these additional analytic tools may help decrease time and effort by identifying specific regions or sites of the greatest potential for activity that could then be tested empirically in the lab.Using the tools at our disposal, we next sought to explore the relationship (if any) between solvent exposure , evolutionary conservation scores and epitope prediction outcomes to determine if these measurements, when used in combination, could be discriminating factors in identifying true epitopes. Because the IEDB captures both positive and negative data, residues shown to be uniquely negative (non-binding) can be used to analyze truly non-immunogenic residues or regions. Here, RSA values and evolutionary conservation scores were calculated for non-binding, negative residues and compared to those calculated for all positive epitopes considered thus far from the subset of 22 mAbs from . This waWe found that on the pre-F PDB structures, residues from all positive residues (known epitopes) had a tendency to be solvent exposed in comparison to those from non-binding, negative residues, given the average RSA values of 43% and 12%, respectively . By contrast, on post-F PDB structures there was no difference observed in RSA values between positive residues (epitopes) and negative residues, with the average values for positive epitopes of 29% and 27% . Thus while positive and negative residues are discriminated on the pre-F structure by calculated RSA values, on post-F, both positive and negative residues are equally solvent accessible, showing RSA values indicating intermediate or partial exposure.Next, we calculated residue conservancy scores for each PDB F structure and again made comparisons between known epitopes (positives) and negative residues. Here we found no significant difference between residue conservancy scores for positive and negative residues.Lastly, we compared epitope prediction outcomes between the pre- and post-F conformations using two published B cell prediction methods, ElliPro  and DiscThus the results of the combined analysis reveal a correspondence of RSA values and epitope prediction scores with positive and negative residues, but this was the case only when considering the pre-F structures. There was no observed tendency for the residues in functional epitopes to be more or less conserved than in reported non-binding, negative residues. No significant correlation was found between epitope prediction scores, RSA values and sequence conservation considered together for any structure (data not shown). All results for each of the analyzed structures are summarized in the + and CD8+ T cell subsets have been shown to play a critical role in protection from disease, and while involvement for each subset in disease enhancement has been implicated in certain animal models, in humans a balanced CD4+ Th1:Th2 cytokine response seems to promote development of neutralizing antibodies, while CD8+ cytolytic activity effects viral clearance from the lungs [in vitro IFNγ production (CD4/CD8), degranulation (perforin/granzyme B release), cytolytic activity (CTL), and in vivo protection. Unlike the case described for B cell/Ab epitopes above, we were not as limited in repertoire and could therefore select the human-specific subset of T cell data.Both CD4he lungs , 45. Her+ and CD8+) and/or cytotoxicity (CD8+ T cell) [Tables + epitopes associated with IFNγ production and cytotoxicity, whereas epitopes associated with CD4+ IFNγ were most often reported from F. These data highlight the potential utility of NP as a vaccine antigen, and also show that in addition to being a major target of protective antibody responses, F is also a target of CD4+ and CD8+ T cell responses. Interestingly and perhaps not surprisingly, many of the epitopes described for CD8+ T cells are derived from antigens active within the cytoplasm (e.g. RNA binding role) [Tables + T cell epitopes are derived from extracellular antigens active at the cell surface [More than 50 peptides, from F, G, M2, M, NP and NS1, have been reported in the context of IFNγ CD4) Tables –5. NP re) Tables  and 4, w Tables – surface .As a further analysis, a broad query was performed to identify all overlapping T and B cell epitopes that were recognized by children/infants and adults following (post) or during (acute) natural RSV infection . These ain vivo survival), making these potential reagents for use in evaluating candidate vaccine formulations for specific T cell response types.Finally, we were interested in reported tetramer data, which are useful tools for studying T cell responses in the research and vaccine evaluation settings. A total of 11 RSV-derived tetramers were described, including epitopes restricted by both HLA class I and class II alleles . All butHerein we have provided an analysis of RSV specific immune epitope data with the goal of examining this body of work for its application to vaccine design and development. To this end, we first provided an overview of all available data, showing overall coverage and identifying critical knowledge gaps. Then we further analyzed the cumulative RSV epitope data from the standpoint of functional responses, and used a combinational approach to characterize antigenic structure and epitope location. This work represents a comprehensive analysis of RSV epitope data that provides an update for previous work by Anderson et al. .Our initial assessment of the cumulative data revealed that the vast majority of epitope mapping has focused on just two antigens, F and G. While this is not surprising, it may be useful to pursue the expanded epitope mapping of this virus, not only to complete our understanding of these two critical proteins, but perhaps to also include additional antigens to more fully understand the immune response to all pertinent antigens having key roles in viral pathogenesis. Further, the balance of T cell versus antibody epitopes should be addressed, especially in light of increasing evidence from the literature regarding known correlates of protection, which suggests a role for both humoral and cellular responses in disease resolution. If it is found that combined antibody and T cell responses prove to be efficacious and safe, antigens such as NP might provide a potential target for inducing CMI, allowing the vaccine to generate the needed balance of both humoral and cellular responses. Indeed, our review of the T cell epitope data suggested there may be a need to incorporate multiple antigens (besides F) into candidate vaccines in order to optimize required response, but further empirical testing is warranted to make this determination.in vivo protection, etc.) and 3) future epitope discovery or mapping in the context of human disease, especially infants and young children would be beneficial to increase the characterization of immunity at the molecular level. Historically speaking, epitopes have not played a prominent role in RSV vaccine development; however, going forward, this information may help to more fully elucidate certain as yet poorly understood or partially characterized aspects of RSV immunobiology. While animal models of RSV have historically provided essential platforms through which we have gained valuable insights with respect to vaccine-enhanced disease and virus-induce airway hypersensitivity, and in which to test proof-of-principle for prophylactic and therapeutic agents [Somewhat unexpected was the low number of human data; only 32% of the epitopes described to date are derived from human hosts, and of these a mere 3 epitopes were defined in humans describing neutralizing or protective activity. Thus our assessment of the totality of RSV epitope data is 1) that additional mapping of all proteins is warranted, including F and G, 2) a greater breadth in T cell, as well as B cell/antibody epitope repertories is needed, especially in humans and specifically using functional assays . This is surprising given the ubiquitous nature of this pathogen, by comparison to influenza A virus as an example. To date, there are 379 assays defining more than 50 flu A neutralizing sites in humans captured from the peer-reviewed literature in the IEDB. Historically, we do know that vaccine-induced partial immunity against RSV has been a double-edge sword. If we can more fully characterize the nature of protection at the molecular level against RSV we may, for example, be better positioned to answer the question, ‘are the majority of the neutralizing epitopes present on pre-F, or are these critical epitopes located on both pre- and post-F?’ The answer may influence the chosen vaccine modality  and its complexity from a formulation standpoint.+ and CD8+ epitopes. Furthermore, many of these same overlapping epitopes residues are also recognized in the context of natural infection. However, we acknowledge that antibody and T cell responses represent functionally distinct processes and concede that the data are as yet insufficient to elucidate a true relationship. It may simply be that functionally important antigens are made in abundance and are therefore likely “seen” more frequently by B and T cells. We hope that ultimately it will be feasible to apply this layering approach using subsets of data selected for high stringency  to define epitopes/antigens that are “heavy hitters” from an immunological perspective.As part of our investigation into the nature of protective epitopes, we sought to determine to what extent, if any, functional antibody and T cell epitopes residues overlapped. We reasoned that such analysis was relevant to vaccine design and development since evidence to date demonstrates that the generation of a combined humoral and cellular response is optimal against RSV. Thus considering the antibody data together with the T cell data may be of value for vaccine development, especially if the aim is to develop, for example, a subunit vaccine  that targets both humoral and cellular immunity. Interestingly, we found that there was overlap between certain Ab and T cell sites . Moreover, some of these overlapping residues occur within important structural/functional features of the respective antigens. This was shown to be true for linear  and discontinuous antibody epitopes (mAbs), as well as both CD4Finally, we also gained some insights into B cell epitope prediction, which suggested that future B cell/antibody prediction tools may incorporate the identification of solvent accessibility scores, as well as of a protein’s features of structural significance. Indeed, there are now databases devoted to protein structure that may be useful in this regard  . Our datS1 Table(XLSX)Click here for additional data file.


During vascular injury, platelets adhere to exposed subendothelial proteins, such as collagen, on the blood vessel walls to trigger clot formation. Although the biochemical signalings of platelet-collagen interactions have been well characterized, little is known about the role microenvironmental biomechanical properties, such as vascular wall stiffness, may have on clot formation. To that end, we investigated how substrates of varying stiffness conjugated with the same concentration of Type I collagen affect platelet adhesion, spreading, and activation. Using collagen-conjugated polyacrylamide (PA) gels of different stiffnesses, we observed that platelets do in fact mechanotransduce the stiffness cues of collagen substrates, manifesting in increased platelet spreading on stiffer substrates. In addition, increasing substrate stiffness also increases phosphatidylserine exposure, a key aspect of platelet activation that initiates coagulation on the platelet surface. Mechanistically, these collagen substrate stiffness effects are mediated by extracellular calcium levels and actomyosin pathways driven by myosin light chain kinase but not Rho-associated protein kinase. Overall, our results improve our understanding of how the mechanics of different tissues and stroma affect clot formation, what role the increased vessel wall stiffness in atherosclerosis may directly have on thrombosis leading to heart attacks and strokes, and how age-related increased vessel wall stiffness affects hemostasis and thrombosis. IIbβ3 [, the release of granules to recruit other platelets [During a vascular injury, platelets are exposed to and adhere to subendothelial proteins such as collagen, initiating the process of clot formation. After initial adhesion, platelets then spread and activate, undergoing a myriad of changes including the activation of integrin αIIbβ3 , 2, the latelets  and phoslatelets , 6. BiocIn other cell types such as endothelial cells and fibroblasts, the microenvironmental mechanical cues, such as stiffness, of the underlying substrate alter the physiological processes of those cells via mechanotransduction –9. InterAs recent studies have documented that the stiffness of subendothelial collagen correlates with the degree of different conditions, such as atherosclerosis or increasing age, these studies are clinically relevant –15. To bTo that end and to decouple the mechanical cues from biological and biochemical factors that may be involved, we covalently conjugated collagen on polyacrylamide (PA) gels of varied stiffnesses and investigated how platelets biologically respond to these differences in substrate stiffness. We observed that on collagen-conjugated PA gels with stiffness of over 5 kPa, the average spreading area of platelets adhered onto those gels was significantly increased as compared to those adhered on collagen-conjugated PA gels softer than 5 kPa. No difference was detected in the number of adhered platelets on PA gels of different stiffnesses. Interestingly, PS exposure, a marker of platelet activation and “procoagulabilty”, increased with increasing PA stiffness. To investigate the underlying mechanisms of these phenomena, we then conducted studies using pharmacologic cytoskeletal inhibitors, differential calcium levels and inhibitors of ADP signaling and thromboxane A2 generation.12 mm glass slides were exposed to oxygen plasma for 1 minute and were silanized with 10% (3-aminopropyl)—trimethoxysilane (Sigma-Aldrich) in 95% ethanol for 45 minutes. Afterwards, the glass slides were treated with 0.5% glutaldehyde solution (Sigma-Aldrich) for 30 minutes. For gel fabrication, different ratios of acrylamide (Sigma-Aldrich) and bis-acrylamide (Sigma-Aldrich), crosslinked with 0.1% ammonium persulfate and tetramethylethrlenediamine (Sigma-Aldrich), were used to create different PA gels of varying stiffness according to standard protocols . The PA 5 platelets/ml and 1 mM of Ca2+ and 2 mM of Mg2+ were added to the suspension of platelets, which were incubated onto the collagen-conjugated PA gels for 1 hour at 37°C. 50 minutes into the incubation, a fluorescent membrane dye, CellMask Orange , was added to the solution. The PA gels were then washed with PBS 3 times and fixed for 10 minutes with 4% PFA. For experiments using pharmacologic cytoskeletal inhibitors, ML-7 , Y-27632 , or latrunculin A  was used to pre-treat washed platelets for 1hour before incubation on collagen-conjugated PA gels. To study the effect of ADP and thromboxane A2 signaling on platelet mechanosensing on collagen, washed platelets were also pre-treated with apyrase (0.05 U/mL), aspirin (1 mM) or clopidogrel (250 μM) for 1hour before incubation on collagen-conjugated PA gels.The protocol was approved by Emory University IRBs and written informed consent was received from all participants. To isolate platelets, 3 ml of blood was drawn into conical tubes with ACD anticoagulant and spun at 150 G’s for 15 minutes without a brake or acceleration. Platelet-rich plasma was then collected and centrifuged again at 900 G’s with 10% (v/v) ACD for 5 minutes. Platelet-poor plasma was then removed and the remaining platelet pellet was suspended in Tyrode’s buffer with 0.1% BSA. Platelets were then diluted to 5.5 x 10Adherent platelets on PA gels were imaged with epi-fluorescence microscopy (Nikon TE2000-U) using a 20x objective. The number of adherent platelets in each image was counted using ImageJ, and the numbers from three images were averaged for each gel. Adherent platelets were also imaged with Z stack using confocal microscopy (Zeiss LSM700). Similarly, three representative images were taken for each gel. The Z-stack images were then flattened, and the spreading area of each platelet was also measured using ImageJ. Spreading area of platelets in all three images was averaged for each gel.IIbβ3 activation, PAC-1-FITC (BD Science) was applied to adherent platelets for 5 minutes before fixation. Similarily, to visualize PS exposure on adhered platelets, Annexin V-Alexa Fluor 488  was incubated with adherent platelet for 5 minutes before fixation. Stained platelets were then imaged with Z stack using confocal microscopy. The same laser light intensity and gain settings were used for all images of either PAC-1-FITC or Annexin-V-Alexa Fluor 488 staining. The Z-stack images were flattened, and the average fluorescence intensity over the spreading area of each adherent platelet was measured with ImageJ.To visualize platelet activation as measured by integrin αThe data in the figures are presented with ± standard deviation bars. Using Minitab Statistical Analysis Software these groups were analyzed for differences using Student’s t-test with a p-value of less than 0.05 denoting statistical significance.2 adhered on the PA gels after 1 hour incubation  compared to control conditions (~30 μm2)  . Pre-tre~30 μm2) . This in2+ affects stiffness-mediated platelet spreading on collagen, platelets were incubated with different concentrations of calcium. Interestingly, a lower calcium concentration at 0.1 mM significantly increased platelet spreading on substrates of all stiffness compared to those at calcium concentration of 1 mM, while higher calcium concentrations (10 mM) decreased platelet spreading on substrates of all stiffnesses  on collagen.Adhesion of platelets on collagen can lead to ADP release and thromboxane A2 generation. To investigate the effect of ADP and thromboxane A2 on substrate stiffness-mediated platelet spreading on collagen, washed platelets were pretreated with apyrase, an ADP-degrading enzyme, and aspirin, a thromboxane A2 inhibitor. While apyrase and aspirin exposure increased platelet spreading on all the substrates regardless of the stiffness, the substrate stiffness- mediated platelet spreading was augmented as adherent platelets spread significantly more on both 5 and 50 kPa gels compared to that on 0.5 kPa gels . More in apyrase . Taken tIIbβ3 integrins for platelets aggregation and clot formation, and it can also leads to PS exposure and formation of procoagulant platelets [IIbβ3 integrins and exposed PS as biomarkers to determine how substrate stiffness mediates platelet activation. Since the antibody, PAC-1-FITC, can specifically bind to activated αIIbβ3 integrins, we quantified the fluorescence intensity to compare the platelet activation on substrate across all the stiffness  of adherent platelets. Strikingly, the PS exposure of adherent platelets was mediated by the substrate stiffness . On 0.5 Overall, our data show that platelets mechanosense the mechanical properties of the subendothelial collagen matrix of the vascular wall and exhibit differential degrees of spreading and activation on substrates of varying stiffness. To the best of our knowledge, this is first reported observation that platelets can mechanosense the underlying substrate stiffness when adhered to collagen. We used PA gels conjugated with the same density of rat type I collagen but varied in stiffness, ranging from 0.25 to 100 kPa, enabled decoupling of the biochemical from the mechanical cues of the subendothelium , 25, 26.Platelets bind to collagen through two transmembrane receptors, glycoprotein VI (GPVI) and integrin α2β1, 27–29, Mechanistically, we observed that the substrate stiffness-mediated effects of platelet mechanotransduction of collagen-conjugated PA gels are driven by actin polymerization and MLCK. ROCK, however, is not involved in this process, suggesting that platelet mechanotransduction on collagen substrates involves a specific actomyosin pathway.2+ also plays a role in this process: the lower the extracellular Ca2+ concentration, the more pronounced substrate stiffness-mediated platelet spreading is. This augmentation of the substrate-mediated effects at low Ca2+ levels may be due to the fact that the contractile actomyosin pathways are Ca2+-dependent [2+ levels may inhibit contractile pathways, which in turn may “un-inhibit” platelet spreading. We also observed that with higher concentration of Ca2+, more bleb formation appeared on adherent platelets on collagen. Adhesion to collagen has been reported to cause Ca2+ release and influx, and result in sustained high cytosolic Ca2+ level [2+-dependent protease, calpain [-/- mice exhibit enhanced spreading on both collagen and fibrinogen [2+ levels can cause faster or more calpain activity, which results in more bleb formation and reduces the stiffness-mediated effect on platelet spreading.Interestingly, we also observed that extracellular Caependent . Decreas2+ level . It also calpain , 24, whi calpain , 22. It brinogen . It is t2+ signaling of platelets adherent on collagen [2+, thus decreasing bleb formation and increasing substrate stiffness-mediated platelet spreading, which is consistent with a previous report [Adhesion of platelets on collagen can lead to ADP release and thromboxane A2 generation , which wcollagen –40, whiccollagen  and couls report .2+ concentrations. These experiments have direct clinical relevance as vessel wall stiffness is noted to increase with age and with atherosclerosis [Overall, our study demonstrates that platelets have the capability to sense the mechanical properties of underlying collagen substrates and mechanotransduce these cues to alter their physiology to directly affect clot formation as in the case of PS exposure . These cclerosis –15. Our


It was 1913, and one of those rare occasions when Ernest Starling disagreed with his fellow UCL Professor of Physiology and brother‐in‐law, William Bayliss, about a physiological issue. The question related to the admission of women to membership in The Physiological Society. Founded in 1876 as a dining club, the Society had never explicitly excluded women. But habit and practice had meant that women did not attend its meetings as members, although they had certainly contributed to some of its meetings, which from December 1880 included live scientific demonstrations and the reading of papers. The centrality of the dinner, which followed the Society's scientific proceedings and which routinely included live animal experiments at that time, is emphasized by Starling's view, when considering women as possible members, that ‘it would be improper to dine with ladies smelling of dog – the men smelling of dog that is’ Evans, . DespiteThe Journal of Physiology  or the Quarterly Journal of Experimental Physiology  or both, although little is yet known about their lives  also included two additional women authors, both American. One, Harriet Bills, was listed only as ‘assisting’ V. C. Vaughan (Fig. As noted above, Florence Buchanan was the author of the very first paper in the  Journal 878/9 als Journal /9 also ihan Fig. . Then a  Journal /9 also ihan Fig. .The Journal (Chick & Martin, The Journal, working with J. S. Edkins, who later became her brother‐in‐law (Edkins & Tweedy, The Journal in 1949. Like Winifred Cullis, she influenced generations of women students, being promoted to the headship of the Department of Physiology at Bedford College in 1929, although unlike Cullis she was denied the title of Professor.The lists in Tables The Journal, that there was never any discrimination against women authors. Indeed a substantial cohort of women authors has been revealed, and their publications in The Journal, and later the QJEP, contributed to several distinguished careers, many sadly now forgotten.Now, a century after the election of the first women to The Physiological Society, it seems strange to read of a time when it was difficult for them to be full members of a scientific society. It does seem clear, however, from an examination of the early volumes of None declared.


The diagnosis of hypochondroplasia (HCH) is hampered by absence of radiological criteria relevant to the age-dependent manifestations, particularly those in the neonatal period. This work deals with the radiological features in hypochondroplastic neonates with a FGFR3 mutation, including quantitative measurement that facilitates the definitive diagnosis. We propose the radiological criteria for HCH in the neonatal period.Subjects included six HCH neonates with FGFR3 mutations and 30 control subjects, in whom radiological examination was available as a neonate. The following findings were evaluated: 1) short ilia, 2) squared ilia, 3) short greater sciatic notch, 4) horizontal acetabula, 5) short femora, 6) stubby femora, 7) metaphyseal flaring, 8) lumboscaral interpediculate distance narrowing, and 9) oval radiolucency in the proximal femora.All measurement parameters for short ilia, short greater sciatic notch, horizontal acetabula, short femora and stubby femora  were statistically different between HCH and control, while the other parameters 2, 7, and 8 were not. Based on these results, we tentatively made the criteria and scoring system for the diagnosis of HCH. The major criteria that are given a score of 2 comprise parameters 1, 3, and 6, whose distribution was not overlapped between HCH and control. The minor criteria that are given a score of 1 point comprise parameters 4 and 5, and 9, because the parameter 4 was overlapped in distribution between HCH and control, the parameter 5 is a non-specific finding, and the parameter 9 is subjective in assessment. We presumed that a total score of 6 points or higher warrant a diagnosis of HCH.HCH was clearly distinguishable from normal infants assessing the skeletal findings of the ilia and proximal femora on neonatal roentgenograms. We compiled a set of diagnostic criteria for the early diagnosis of hypochondroplastic neonates.


To date, there has been no adequate biomechanical model that would allow a quantitative comparison in terms of stability/stiffness between a corpectomy with the posterior column preserved and a total spondylectomy with the posterior column sacrificed. The objective of this study was to perform a biomechanical comparison of 360° stabilizations for corpectomy and total spondylectomy, using the human thoracolumbar spine.Five human cadaveric thoracolumbar spines (T8-L2) were tested according to the following loading protocol: axial compression, flexion, extension, lateral bending to the right and left, and axial rotation to the right and left. This loading protocol was applied three times. Each specimen was tested intact, after corpectomy, and after total spondylectomy. The relative stiffness of each motion segment was determined for each test.There was no significant difference in stiffness after reconstruction of total spondylectomy versus corpectomy in our thoracolumbar model. Our construct consisted of an anterior cage and four-level pedicle screw instrumentation (two above and two below) and provided similar stiffness in both models. Despite the additional bone resection in a total spondylectomy versus corpectomy, the constructs did not differ biomechanically. Additionally, there was no significant difference in stiffness between the intact specimen and either reconstruction model.A classic corpectomy, which leaves the posterior column intact, is no better in terms of stability/stiffness than a total spondylectomy carried out using a shorter cage, followed by compression using posterior instrumentation. Spine surgeons have used combined anterior and posterior surgery for the treatment of patients with spinal fractures, tumors, and deformities. Corpectomy is generally followed by anterior reconstruction with a strut graft or a cage and posterior instrumentation with pedicle screws. This procedure is performed to provide sufficient mechanical stability. However, total spondylectomy  by means of a posterior approach is sometimes preferred, because pedicle screws and a posterior approach technique can provide secure fixation. Posterior total spondylectomy can be useful in cases where the anterior approach is considered difficult, such as in cases of severe adhesions due to previous anterior surgery, suboptimal pulmonary function, severe kyphosis, and trunk shortening –3.Classic corpectomy leaves the posterior column intact, thus, offering good biomechanical stability, since it provides support at both the front and back –6. On thTo date, there has been no adequate biomechanical model that would allow a quantitative comparison in terms of stability/stiffness between a corpectomy and a total spondylectomy. We decided to compare biomechanical stabilities using a thoracolumbar reconstructed spine (T9-L1), with anterior cage and multilevel posterior instrumentation after corpectomy (T11), and the same spine with a 10-mm shorter anterior cage and multilevel posterior instrumentation after total spondylectomy (T11).Thoracolumbar spines (T8-L2) were harvested from five fresh human cadavers . The specimens were examined grossly and radiographically to rule out malignancy or fractures that could have interfered with the results. The specimens were then frozen at −20 °C until the day before testing. After thawing to room temperature overnight, the surrounding soft tissues and muscles were dissected meticulously to preserve the osseous and ligamentous structures. The specimens were kept moist throughout the procedure.2, all of the vertebrae were classified as osteoporotic.The bone mineral density (BMD) of all specimens was measured in a water bath, using dual-energy X-ray absorptiometry . Using the World Health Organization definition of osteoporosis as BMD <0.8 g/cmThe biomechanical testing was carried out using a materials testing machine . The vertebral bodies of T8 and L2 were potted in 10-cm diameter polyvinyl chloride endcaps using dental cement . The specimen (T8-L2) in its two pots was then clamped on the testing machine in the upright position Fig. . Each spStep 1 (establishing the center of rotation): The center of rotation for flexion-extension and lateral bending was established as follows. A pure compressive load of 50 N was applied through a roller bearing to the top surface of the upper end-cap containing T8. With the roller bearing positioned, the load was applied, then repositioned and applied again, until no angular rotation in the coronal or sagittal planes could be detected when a compressive force was applied to the upper end-cap. This spot on the top surface of the end-cap was deemed to be the center of rotation and remained as the reference spot in subsequent testing on that specific specimen. A cyclic compression force was then applied as conditioning (40 ± 10 N at 0.5 Hz for 15 min) to remove excess fluid from the discs and to return the disc to its predeath height .Step 2 (compression): The specimen was then loaded in pure compression (with the load applied at the center of rotation) at a displacement rate of 0.25 cm/min. The load was applied up to a maximum of 200 N. A load-deformation curve was obtained. The specimen was then loaded two more times, and load-deformation curves were obtained each time. The three load-deformation curves were essentially identical, and only one was used to calculate the stiffness. Stiffness was calculated as the slope of the linear portion of the load-deformation curve .Step 3 (flexion): The test was repeated with a 100 N load applied 2 cm anterior to the center of rotation, to produce a maximum bending moment of 2 Nm  in flexion. The load was then released and applied twice more. The three load-deformation curves were essentially identical, and only one was used to calculate the stiffness.Step 4 (extension): The test was repeated with a 100 N load applied 2 cm posterior to the center of rotation, to send the specimen into extension. The load was then released and applied twice more. The three load-deformation curves were essentially identical, and only one was used to calculate the stiffness.Step 5 and 6 : The test was repeated with a 100 N load applied 2 cm to the right of the center of rotation, to send the specimen into right lateral bending. The load was then released and applied twice more. Three load-deformation curves were obtained for right lateral bending. The three right load-deformation curves were essentially identical, and only one was used to calculate the stiffness. The experiment was then repeated for left lateral bending, and the same criteria applied.Step 7 and 8 : To apply axial rotation, the specimen was first compressed to 100 N, and an axial torque was then applied in a clockwise motion (about the center of rotation) to a maximum of 10°. Three torque-angle deformation curves were obtained for right rotation. The three right torque-angle deformation curves were essentially identical, and only one was used to calculate the stiffness. The experiment was then repeated for left rotation, and the same criteria applied.After the test on the intact specimen had been carried out (as described above), pedicle screws were inserted at T9, T10, T12, and L1 ). A corpectomy at T11 was performed by incising and removing the T10-11 and T11-12 intervertebral discs and the cartilaginous endplates. The entire T11 vertebral body was removed after creating bilateral osteotomies through the T11 pedicles and resecting the anterior and posterior longitudinal ligaments at T11; the posterior elements were left intact. Vertebral body reconstruction was performed by implanting a Harms titanium mesh cage  between T10 and T12, depending on the corpectomy height Figs. . The diaAfter the test on the corpectomy model had been carried out, the rod on one side was removed to allow access for the removal of the posterior elements. A total spondylectomy at T11 was carried out by removing the posterior elements. A cage 10 mm shorter than the one used in the corpectomy model was inserted at T11. After the “short” cage was inserted into the anterior defect, the posterior instrumentation was adjusted, using a compressor and rod-screw junctions, to compress the inserted cage Figs.  and 3. WThus, each specimen was biomechanically tested three times: intact, as a corpectomy model, and as a total spondylectomy model. The order of assembling and testing the models was the same for all five specimens.P < 0.05. For statistical analysis, SAS version 9.2  was used.Results for stiffness are presented as the mean of determinations, with error bars representing standard deviations. Means and standard deviations were computed for the five specimens, for each measure under each experimental condition. A repeated measures analysis of variance with the three models  was performed, followed by post hoc pairwise comparisons based on a Bonferroni correction. Post hoc comparisons were made for the intact spine versus each of the two models and for the corpectomy model versus the total spondylectomy model. The level of significance was None of the specimens fractured, and none of the pedicle screws or rods loosened during the experiments. Visual inspection revealed no major pedicle violations. There was one minor lateral perforation, with threads barely visible outside of the pedicle, but this did not compromise fixation.The mean ± standard deviation (SD) values of the stiffness values in axial compression, flexion, extension, lateral bending, and axial rotation are shown in Table No significant difference was observed between the two reconstruction models, or between the intact specimen and either of the two reconstruction models.Both reconstruction models provided greater stiffness than that shown by the intact specimen, but the differences were not significant. The total spondylectomy model provided greater stiffness than the corpectomy model, but the difference was not significant.The two reconstruction models provided greater stiffness than the intact specimen, but the differences were not significant. The total spondylectomy model provided greater stiffness than the corpectomy model, but the difference was not significant.The values of stiffness measured for right and left lateral bending were averaged for each specimen, since each of our specimens showed some slight scoliotic changes . Both of the reconstruction models provided greater stiffness than that shown by the intact specimen, but the differences were not significant. The corpectomy model provided greater stiffness than the total spondylectomy model, but the difference was not significant.The two reconstruction models provided greater stiffness than that shown by the intact specimen, but the differences were not significant. The corpectomy model provided greater stiffness than the total spondylectomy model, but the difference was not significant.The two reconstruction models provided greater stiffness than that shown by the intact specimen, but the differences were not significant. The corpectomy model provided greater stiffness than the total spondylectomy model, but the difference was not significant.Spinal pathologic conditions, such as fractures, tumors, and degenerative diseases, lead to spinal instability, deformity, and neural element compression –14. The Total spondylectomy  leads to complete loss of spinal continuity and stability. Therefore, subsequent secure and stable spinal reconstruction is absolutely required. Some authors reported that applied compression forces on the anterior strut graft or cage using posterior instrumentation improves stability , 20, 21.In order to evaluate spinal stability after a total spondylectomy, the stiffness of the reconstructed spine and the stress generated in the instrument used for the spinal reconstruction have been investigated by a finite element method and loading test using a cadaveric spine. Experimentation using a cadaveric spine is a direct means to analyze the behavior of the implant through the displacement response of instrumented segments to applied forces, compared to intact segments. However, experimental results are sensitive to limitations caused by testing procedures and variability among specimens. A finite element model presents a great advantage, because it enables the same procedure  and the same vertebral segment  to be used for different spinal implants and for each implant to analyze the influence of a different parameter. Therefore, a validated finite element model would constitute a powerful simulation tool, for the clinician as well as for the implant designer . NeverthPosterior pedicle screw segmental instrumentation could allow more rigid fixation with three columns. The length of the posterior fixation is a major determinant for rigidity of the construct. Long posterior fixations were significantly more stable compared to intact specimens. The functional relationship between length of posterior fixation and spinal stiffness following spondylectomy and subsequent reconstruction is emphasized by the decreased range of motion in the long posterior fixation group, when compared to short fixation and an intact specimen. In most of the biomechanical studies dealing with corpectomy or total spondylectomy models, an anterior cage combined with multilevel posterior fixation (two above and two below) was able to provide more stiffness than the intact specimens. On the other hand, there is evidence to suggest that eliminating mechanical loads on healing bone when using rigid fixation may result in negative bone remodeling and net bone loss. Thus, less rigid fixation that permits a certain degree of micromotion may accelerate the time to union. Akamaru et al. made a three-dimensional finite element model of the reconstructed spine (T10-L4) following total spondylectomy at T12. They concluded that a reconstruction method with multilevel posterior instrumentation (two above and two below) and no anterior fixation should be better at allowing stress for remodeling of the bone graft inside the titanium mesh cage . TherefoThere was no significant difference in stiffness after reconstruction of total spondylectomy versus corpectomy in this cadaveric model. Additionally, there was no significant difference in stiffness between the intact specimen and either of the two reconstruction models. Despite the additional bone resection performed when doing a total spondylectomy versus corpectomy, the constructs did not differ biomechanically. Some authors similarly refer to the importance of applied compression forces on the anterior strut graft to improve stability , 20, 21.Several limitations to the study should be considered. 1) The loads and torques chosen to test the specimens in various configurations were low. We chose low loads and torques since there were many testing steps, and we wanted to avoid damage to the specimens before the entire sequence had been completed. 2) Our three models were tested immediately after reconstruction, so conclusions cannot be drawn about the long-term stability in a spine subject to cyclic loads applied daily. 3) The relatively small sample size may also be a limitation, although the results are very clear (no significant differences). The vagaries of postmortem collection always present problems. Most of the spines available for collection and subsequent testing are usually from older cadavers and often have multiple massive osteophytes that render the spines unsuitable for the testing described above. Most of the thoracic spines made available to us did not meet our selection criteria. 4) Thoracolumbar specimens were used in the present study, and only a T11 spondylectomy model was evaluated. Corpectomy and total spondylectomy are performed not only in the lumbar spine but also in the proximal and midthoracic spine. The biomechanical environment of the thoracic spine is quite different from that of the lumbar spine because of the stabilizing effects of the costovertebral joints and rib cage. T11 at the thoracolumbar junction is exposed to forces similar to the lumbar spine. Therefore, reconstruction stability after total spondylectomy in the thoracic spine remains unknown. Further biomechanical studies examining the comparative multisegmental mechanics of total spondylectomy in the thoracic region are required for certainty. 5) The results of the present study do not allow for a recommendation regarding the length of constructs to be used clinically, when performing either total spondylectomy or corpectomy. Depending on the bone purchase achieved by the pedicle screws, as well as the overall sagittal alignment, additional pedicle screw fixation may be needed in clinical practice.The choice of management for spinal surgery depends on many factors, which surgeons must weigh before determining the ideal surgical approach. These in vitro biomechanical data provide helpful guidelines for planning surgical management.A classic corpectomy, which leaves the posterior column intact, is no better in terms of stability/stiffness than a total spondylectomy, when the total spondylectomy is carried out using a shorter cage followed by compression using posterior instrumentation.


Regarding the paper titled: ‘‘They think surgery is just a quick fix’’ by Karen Synne Groven.International Journal of Qualitative Studies on Health and Well-being, 11 July 2014. Citation: Int J Qualitative Stud Health Well-being 2014, 9: 24378 - http://dx.doi.org/10.3402/qhw.v9.24378Published in On page 2, second column second paragraph (“Both the risk of regaining weight …”), the sentence currently reads: “However, we also found that the women's changing habits were intimately intertwined with their changed and changing bodies, including side-effects and changes in their bodily capacity .”This sentence should read: “However, we also found that the women's changing habits were intimately intertwined with their changed and changing bodies, including side-effects and changes in their bodily capacity .”On page 11, first column first paragraph (“As for those women who emphasized …”), the sentence currently reads: “In line with previous findings, undergoing gastric bypass surgery can be considered a turning point in terms of physical activity and exercise .”This sentence should read: “In line with previous findings, undergoing gastric bypass surgery can be considered a turning point in terms of physical activity and exercise .”Health Care for Women International. DOI: 10.1080/07399332.2013.794465.”One reference was missing from the article. The references have now been supplemented with: “Groven, K. S., Råheim, M., & Engelsrud, G. (2013a). Changing bodies, changing habits: Women's experiences of intervall training following gastric bypass surgery. The reference in page 13 should read: “Groven, K. S., Råheim, M., & Engelsrud, G. (2013b). Disappearence and dys-appearance anew: Living with excess skin and intestinal changes following weight loss surgery. Medicine, Health Care and Philosophy. DOI: 10.1007/s11019-012-9397-5.”