Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(ω). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.     

Improving the Accuracy and Efficiency of Respiratory Rate Measurements in Children Using Mobile Devices

The recommended method for measuring respiratory rate (RR) is counting breaths for 60 s using a timer. This method is not efficient in a busy clinical setting. There is an urgent need for a robust, low-cost method that can help front-line health care workers to measure RR quickly and accurately. Our aim was to develop a more efficient RR assessment method. RR was estimated by measuring the median time interval between breaths obtained from tapping on the touch screen of a mobile device. The estimation was continuously validated by measuring consistency (% deviation from the median) of each interval. Data from 30 subjects estimating RR from 10 standard videos with a mobile phone application were collected. A sensitivity analysis and an optimization experiment were performed to verify that a RR could be obtained in less than 60 s; that the accuracy improves when more taps are included into the calculation; and that accuracy improves when inconsistent taps are excluded. The sensitivity analysis showed that excluding inconsistent tapping and increasing the number of tap intervals improved the RR estimation. Efficiency (time to complete measurement) was significantly improved compared to traditional methods that require counting for 60 s. There was a trade-off between accuracy and efficiency. The most balanced optimization result provided a mean efficiency of 9.9 s and a normalized root mean square error of 5.6%, corresponding to 2.2 breaths/min at a respiratory rate of 40 breaths/min. The obtained 6-fold increase in mean efficiency combined with a clinically acceptable error makes this approach a viable solution for further clinical testing. The sensitivity analysis illustrating the trade-off between accuracy and efficiency will be a useful tool to define a target product profile for any novel RR estimation device.     

Social Isolation Shortens Telomeres in African Grey Parrots (Psittacus erithacus erithacus)

Telomeres, the caps of eukaryotic chromosomes, control chromosome stability and cellular senescence, but aging and exposure to chronic stress are suspected to cause attrition of telomere length. We investigated the effect of social isolation on telomere length in the highly social and intelligent African Grey parrot (Psittacus erithacus erithacus). Our study population consisted of single-housed (n = 26) and pair-housed (n = 19) captive individuals between 0.75 to 45 years of age. Relative telomere length of erythrocyte DNA was measured by quantitative real-time PCR. We found that telomere length declined with age (p<0.001), and socially isolated parrots had significantly shorter telomeres compared to pair-housed birds (p<0.001) – even among birds of similar ages. Our findings provide the first evidence that social isolation affects telomere length, which supports the hypothesis that telomeres provide a biomarker indicating exposure to chronic stress.     

Vitamin D Deficiency in a Multiethnic Healthy Control Cohort and Altered Immune Response in Vitamin D Deficient European-American Healthy Controls

Objective

In recent years, vitamin D has been shown to possess a wide range of immunomodulatory effects. Although there is extensive amount of research on vitamin D, we lack a comprehensive understanding of the prevalence of vitamin D deficiency or the mechanism by which vitamin D regulates the human immune system. This study examined the prevalence and correlates of vitamin D deficiency and the relationship between vitamin D and the immune system in healthy individuals.

Methods

Healthy individuals (n = 774) comprised of European-Americans (EA, n = 470), African–Americans (AA, n = 125), and Native Americans (NA, n = 179) were screened for 25-hydroxyvitamin D [25(OH)D] levels by ELISA. To identify the most noticeable effects of vitamin D on the immune system, 20 EA individuals with severely deficient (<11.3 ng/mL) and sufficient (>24.8 ng/mL) vitamin D levels were matched and selected for further analysis. Serum cytokine level measurement, immune cell phenotyping, and phosphoflow cytometry were performed.

Results

Vitamin D sufficiency was observed in 37.5% of the study cohort. By multivariate analysis, AA, NA, and females with a high body mass index (BMI, >30) demonstrate higher rates of vitamin D deficiency (p<0.05). Individuals with vitamin D deficiency had significantly higher levels of serum GM-CSF (p = 0.04), decreased circulating activated CD4⁺ (p = 0.04) and CD8⁺ T (p = 0.04) cell frequencies than individuals with sufficient vitamin D levels.

Conclusion

A large portion of healthy individuals have vitamin D deficiency. These individuals have altered T and B cell responses, indicating that the absence of sufficient vitamin D levels could result in undesirable cellular and molecular alterations ultimately contributing to immune dysregulation.     

Syk and Src Family Kinases Regulate C-type Lectin Receptor 2 (CLEC-2)-mediated Clustering of Podoplanin and Platelet Adhesion to Lymphatic Endothelial Cells

The interaction of C-type lectin receptor 2 (CLEC-2) on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signaling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signaling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the center of the platelet to form a single structure. Fluorescence lifetime imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilized Podoplanin using direct stochastic optical reconstruction microscopy. These findings provide mechanistic insight by which CLEC-2 signaling promotes adhesion to Podoplanin and regulation of Podoplanin signaling, thereby contributing to lymphatic vasculature development.     

Structural Basis for the De-N-acetylation of Poly-β-1,6-N-acetyl-d-glucosamine in Gram-positive Bacteria

Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In staphylococci, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the extracellular protein IcaB is required for biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of IcaB from Ammonifex degensii (IcaB_Ad) has been determined to 1.7 Å resolution. The structure of IcaB_Ad reveals a (β/α)₇ barrel common to the family four carbohydrate esterases (CE4s) with the canonical motifs circularly permuted. The metal dependence of IcaB_Ad is similar to most CE4s showing the maximum rates of de-N-acetylation with Ni²⁺, Co²⁺, and Zn²⁺. From docking studies with β-1,6-GlcNAc oligomers and structural comparison to PgaB from Escherichia coli, the Gram-negative homologue of IcaB, we identify Arg-45, Tyr-67, and Trp-180 as key residues for PNAG binding during catalysis. The absence of these residues in PgaB provides a rationale for the requirement of a C-terminal domain for efficient deacetylation of PNAG in Gram-negative species. Mutational analysis of conserved active site residues suggests that IcaB uses an altered catalytic mechanism in comparison to other characterized CE4 members. Furthermore, we identified a conserved surface-exposed hydrophobic loop found only in Gram-positive homologues of IcaB. Our data suggest that this loop is required for membrane association and likely anchors IcaB to the membrane during polysaccharide biosynthesis. The work presented herein will help guide the design of IcaB inhibitors to combat biofilm formation by staphylococci.     

Palmitoylation and p8-Mediated Human T-Cell Leukemia Virus Type 1 Transmission

The orf-I gene of human T-cell leukemia type 1 (HTLV-1) encodes p8 and p12 and has a conserved cysteine at position 39. p8 and p12 form disulfide-linked dimers, and only the monomeric forms of p8 and p12 are palmitoylated. Mutation of cysteine 39 to alanine (C39A) abrogated dimerization and palmitoylation of both proteins. However, the ability of p8 to localize to the cell surface and to increase cell adhesion and viral transmission was not affected by the C39A mutation.     

Design of 1-piperazinyl-4-arylphthalazines as potent Smoothened antagonists

The Hedgehog (Hh) signaling pathway regulates cell proliferation and differentiation in developing tissues, and abnormal activation of the Hh pathway has been linked to several tumor subsets. As a transducer of Hh signaling, the GPCR-like protein Smoothened (Smo) is a promising target for disruption of unregulated Hh signaling. A series of 1-amino-4-arylphthalazines was developed as potent and orally bioavailable inhibitors of Smo. A representative compound from this class demonstrated significant tumor volume reduction in a mouse medulloblastoma model.