Modeling the Cellular Level of Natural Sensing with the Functional Basis for the Design of Biomimetic Sensor Technology

After surveying biology for natural sensing solutions six main types of extraneous sensing were identified across the biological kingdoms. Natural sensing happens at the cellular level with receptor cells that respond to photo, chemo, eletro, mechano, thermo and magnetoreceptor-type stimuli. At the highest level, all natural sensing systems have the same reaction sequence to stimuli: perception, transduction, and response. This research is exploring methods for knowledge transfer between the biological and engineering domains. With the use of the Functional Basis, a well-defined modeling language, the ingenuity of natural sensing can be captured through functional models and crossed over into the engineering domain, for design or inspiration. Furthermore, a morph-matrix that lists each component in the model can easily compare and contrast the biological and engineering design components, effectively bridging the two design domains. The six main types of receptor families were modeled for the Animalia and Plantae Kingdoms, from the highest to the 4th sub-level, with emphasis on the transduction sequence. To make the biological sensing models accessible to design engineers they were placed in the Missouri University of Science & Technology Design Repository as artifacts. The models can then be utilized for concept generation and biomimetic design through searching the design repository by functional characteristics. An example of a biomimetic navigation product based on the principle of electric fish is provided to illustrate the utilization of the natural sensing models, morph-matrices and design repository

Revised genetic requirements for the decatenation G2 checkpoint: The role of ATM

The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in CHEK2, but no appreciable phosphorylation of Ser139 on H2AX. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage

'Working out’ identity: distance runners and the management of disrupted identity

This article contributes fresh perspectives to the empirical literature on the sociology of the body, and of leisure and identity, by analysing the impact of long-term injury on the identities of two amateur but serious middle/long-distance runners. Employing a symbolic interactionist framework,and utilising data derived from a collaborative autoethnographic project, it explores the role of ‘identity work’ in providing continuity of identity during the liminality of long-term injury and rehabilitation, which poses a fundamental challenge to athletic identity. Specifically, the analysis applies Snow and Anderson’s (1995) and Perinbanayagam’s (2000) theoretical conceptualisations in order to examine the various forms of identity work undertaken by the injured participants, along the dimensions of materialistic, associative and vocabularic identifications. Such identity work was found to be crucial in sustaining a credible sporting identity in the face of disruption to the running self, and in generating momentum towards the goal of restitution to full running fitness and reengagement with a cherished form of leisure. KEYWORDS: identity work, symbolic interactionism, distance running, disrupted identit

Loss of the Metalloprotease ADAM9 Leads to Cone-Rod Dystrophy in Humans and Retinal Degeneration in Mice

Cone-rod dystrophy (CRD) is an inherited progressive retinal dystrophy affecting the function of cone and rod photoreceptors. By autozygosity mapping, we identified null mutations in the ADAM metallopeptidase domain 9 (ADAM9) gene in four consanguineous families with recessively inherited early-onset CRD. We also found reduced photoreceptor responses in Adam9 knockout mice, previously reported to be asymptomatic. In 12-month-old knockout mice, photoreceptors appear normal, but the apical processes of the retinal pigment epithelium (RPE) cells are disorganized and contact between photoreceptor outer segments (POSs) and the RPE apical surface is compromised. In 20-month-old mice, there is clear evidence of progressive retinal degeneration with disorganized POS and thinning of the outer nuclear layer (ONL) in addition to the anomaly at the POS-RPE junction. RPE basal deposits and macrophages were also apparent in older mice. These findings therefore not only identify ADAM9 as a CRD gene but also identify a form of pathology wherein retinal disease first manifests at the POS-RPE junction

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Multiple Novel Functions of Henipavirus O-glycans: The First O-glycan Functions Identified in the Paramyxovirus Family.

O-linked glycosylation is a ubiquitous protein modification in organisms belonging to several kingdoms. Both microbial and host protein glycans are used by many pathogens for host invasion and immune evasion, yet little is known about the roles of O-glycans in viral pathogenesis. Reportedly, there is no single function attributed to O-glycans for the significant paramyxovirus family. The paramyxovirus family includes many important pathogens, such as measles, mumps, parainfluenza, metapneumo- and the deadly Henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviral cell entry requires the coordinated actions of two viral membrane glycoproteins: the attachment (HN/H/G) and fusion (F) glycoproteins. O-glycan sites in HeV G were recently identified, facilitating use of the attachment protein of this deadly paramyxovirus as a model to study O-glycan functions. We mutated the identified HeV G O-glycosylation sites and found mutants with altered cell-cell fusion, G conformation, G/F association, viral entry in a pseudotyped viral system, and, quite unexpectedly, pseudotyped viral F protein incorporation and processing phenotypes. These are all important functions of viral glycoproteins. These phenotypes were broadly conserved for equivalent NiV mutants. Thus our results identify multiple novel and pathologically important functions of paramyxoviral O-glycans, paving the way to study O-glycan functions in other paramyxoviruses and enveloped viruses

Multiple Novel Functions of Henipavirus O-glycans: The First O-glycan Functions Identified in the Paramyxovirus Family

O-linked glycosylation is a ubiquitous protein modification in organisms belonging to several kingdoms. Both microbial and host protein glycans are used by many pathogens for host invasion and immune evasion, yet little is known about the roles of O-glycans in viral pathogenesis. Reportedly, there is no single function attributed to O-glycans for the significant paramyxovirus family. The paramyxovirus family includes many important pathogens, such as measles, mumps, parainfluenza, metapneumo- and the deadly Henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviral cell entry requires the coordinated actions of two viral membrane glycoproteins: the attachment (HN/H/G) and fusion (F) glycoproteins. O-glycan sites in HeV G were recently identified, facilitating use of the attachment protein of this deadly paramyxovirus as a model to study O-glycan functions. We mutated the identified HeV G O-glycosylation sites and found mutants with altered cell-cell fusion, G conformation, G/F association, viral entry in a pseudotyped viral system, and, quite unexpectedly, pseudotyped viral F protein incorporation and processing phenotypes. These are all important functions of viral glycoproteins. These phenotypes were broadly conserved for equivalent NiV mutants. Thus our results identify multiple novel and pathologically important functions of paramyxoviral O-glycans, paving the way to study O-glycan functions in other paramyxoviruses and enveloped viruses

Loss of O-glycans in HeV G affect HeV F incorporation and processing in pseudotyped virions, affecting viral entry.

<p>A) Western blot of HeV/VSV viral lysates. HeV G (top panel) and HeV F (bottom panel) were detected as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005445#ppat.1005445.g002" target="_blank">Fig 2</a>. B) F incorporation and F processing of the HeV mutant pseudotyped virions determined by densitometry. F incorporation values were normalized to those of wt HeV. F processing was measured by the ratio of F₁/ (F₀+F₁) for HeV pseudotyped virions. N = 3. Statistically significant differences, as determined by a Student’s t-test, are marked with an * (p<0.01). C) Viral entry of HeV/VSV virions at log dilutions of viral input. Values for the mutant virions are compared to wt HeV values. Virions expressing only HeV G (no HeV F) were used as a negative control. Averages and standard deviations are shown. N = 3.</p

Associations with F are altered by O-glycan loss.

<p>A) Cell lysates of cells transfected with wt F and either wt or mutant HeV G. HeV G (top) is blotted with rabbit anti-HA, and HeV F (bottom) is blotted with mouse anti-AU1. B) Pulldown of HeV G and co-IP of F for transfected cells. Antibody blotting was performed as in A). C) Cell lysates of cells transfected with wt F and wt or mutant NiV G. D) Co-IP of NiV transfected cells. E) Co-IP values of HeV and NiV O-glycan mutants, determined by densitometry. Image Lab software (Biorad) was used to measure the densitometry of Western blot bands. Co-IP values were determined by dividing either the F₀, F₁, or total F densitometry values for the co-IP bands by the co-IP G densitometry values to account for any differences in co-IP G pulldown. These values were then divided by F₀, F₁, or total F densitometry values for cell lysates to account for differences in F mutant expression. Values were then normalized to those of wt HNV G. Averages are shown. N = 3. Statistically significant differences, as determined by a Student’s t-test, are marked with an * (p<0.05).</p

O-glycans on the HNV G stalk modulate fusion.

<p>A) Schematic of the HNV G protein. The dashed lines denote the O-glycan rich region in which mutants were created, with the insert showing the amino acid sequence of that region. Amino acids determined by mass spectrometry to be O-glycosylated in HeV are in bold. CT = cytoplasmic tail, TM = transmembrane. B&D) Cell surface expression (CSE, white) fusion (black), and ephrinB2 binding (grey) levels of generated HeV G (B) or NiV G (D) mutants. All values were normalized to wt HeV or NiV G. Averages and standard deviations are shown. N = 5. Statistically significant differences, as determined by a Student’s t-test, are marked with an * (p< 0.05). C, E) Western blot analysis of cell lysates from HeV G (C) or NiV G (E) mutants.</p