Combined effects of matrix stiffness and obesity-associated signaling directs progressive phenotype in PANC-1 pancreatic cancer cells in vitro.

Obesity is a leading risk factor of pancreatic ductal adenocarcinoma (PDAC) that contributes to poor disease prognosis and outcomes. Retrospective studies have identified this link, but interactions surrounding obesity and PDAC are still unclear. Research has shifted to contributions of fibrosis (desmoplasia) on malignancy, which involves increased deposition of collagens and other extracellular matrix (ECM) molecules and increased ECM crosslinking, all of which contribute to increased tissue stiffening. However, fibrotic stiffening is underrepresented as a model feature in current PDAC models. Fibrosis is shared between PDAC and obesity, and can be leveraged for in vitro model design, as current animal obesity models of PDAC are limited in their ability to isolate individual components of fibrosis to study cell behavior. In the current study, methacrylated type I collagen (PhotoCol®) was photo-crosslinked to pathological stiffness levels to recapitulate fibrotic ECM stiffening. PANC-1 cells were encapsulated within PhotoCol®, and the tumor-tissue constructs were prepared to represent normal (healthy) (∼600 Pa) and pathological (∼2000 Pa) tissues. Separately, human mesenchymal stem cells were differentiated into adipocytes representing lean (2D differentiation) and obese fat tissue (3D collagen matrix differentiation), and conditioned media was applied to PANC-1 tumor-tissue constructs. Conditioned media from obese adipocytes showed increased vimentin expression, a hallmark of invasiveness and progression, that was not seen after exposure to media from lean adipocytes or control media. Characterization of the obese adipocyte secretome suggested that some PANC-1 differences may arise from increased interleukin-8 and -10 compared to lean adipocytes. Additionally, high matrix stiffness associated induced an amoeboid morphology in PANC-1 cells that was not present at low stiffness. Amoeboid morphology is an accessory to epithelial-to-mesenchymal transition and is used to navigate complex ECM environments. This plasticity has greater implications for treatment efficacy of metastatic cancers. Overall, this work 1) highlights the importance of investigating PDAC-obesity interactions to study the effects on disease progression and persistence, 2) establishes PhotoCol® as a matrix material that can be leveraged to study amoeboid morphology and invasion in PDAC, and 3) emphasizes the importance of integrating both biophysical and biochemical interactions associated within both pathologies for in vitro PDAC models.

Care ally-assisted massage for Veterans with chronic neck pain: TOMCATT results.

PURPOSE: Chronic neck pain (CNP) is prevalent and challenging to treat. Despite evidence of massage's effectiveness for CNP, multiple accessibility barriers exist. The Trial Outcomes for Massage: Care Ally-Assisted vs. Therapist Treated (TOMCATT) study examined a care ally-assisted massage (CA-M) approach compared to a waitlist control prior to a study design modification (WL-C0). METHODS: CA-M consisted of in-person training for veteran/care-ally dyads to learn a standardized 30-minue massage routine, instructional DVD, and printed treatment manual. Participants were to complete three care ally-assisted massage sessions weekly for 12-weeks. Outcomes collected at baseline, 1-, 3-, and 6-months included validated measures of neck pain severity and associated disability. Linear mixed-model approaches were used for analysis with 3-months as the primary outcome timepoint. RESULTS: Participants (N = 203) were 56.7 ± 14 years old, 75% White, 15% female, and 75% married/partnered. Among 102 CA-M participants, 45% did not attend the in-person training and subsequently withdrew from the study and were more likely to be younger (p = .016) and employed (p = .004). Compared to WL-C0, CA-M participants had statistically significant reductions in pain-related disability at 3-months (-3.4, 95%CI = [-5.8, -1.0]; p = .006) and 6-months (-4.6, 95%CI = [-7.0, -2.1]; p < .001) and pain severity at 3-months (-1.3, 95%CI = [-1.9, -0.8]; p < .001) and 6-months (-1.0, 95%CI = [-1.6, -0.4]; p = .007), respectively. CONCLUSION: In this analysis, CA-M led to greater reductions in CNP with disability and pain severity compared to WL-C0, despite treatment engagement and retention challenges. Future work is needed to determine how to better engage Veterans and their care-allies to attend CA-M training.

Induction and characterization of Ph1 wheat mutants.

The cloning of genes for complex traits in polyploid plants that possess large genomes, such as hexaploid wheat, requires an efficient strategy. We present here one such strategy focusing on the homologous pairing suppressor (Ph1) locus of wheat. This locus has been shown to affect both premeiotic and meiotic processes, possibly suggesting a complex control. The strategy combined the identification of lines carrying specific deletions using multiplex PCR screening of fast-neutron irradiated wheat populations with the approach of physically mapping the region in the rice genome equivalent to the deletion to reveal its gene content. As a result, we have located the Ph1 factor controlling the euploid-like level of homologous chromosome pairing to the region between two loci (Xrgc846 and Xpsr150A). These loci are located within 400 kb of each other in the rice genome. By sequencing this region of the rice genome, it should now be possible to define the nature of this factor.

A simple PCR-based method for scoring the ph1b deletion in wheat.

The amplified fragment length polymorphism (AFLP) technique was used to isolate DNA sequences present in the euploid wheat Chinese Spring but not in the Chinese Spring ph1b mutant (which has a deletion of the Ph1 gene, a suppressor of homoeologous chromosome pairing). The polymorphic DNA fragments identified by AFLP were then cloned, sequenced, and used to design two primer pairs. These primers were used in a PCR-based assay to specifically amplify products from the Chinese Spring euploid but not from the ph1b mutant. This PCR assay can be carried out from extracted genomic DNA or directly from alkaline-treated wheat leaves, and the reaction products can be scored on a plus-minus basis, making the screening amenable to automation. The reliability of the assay was tested using a F1-derived doubled-haploid population of 55 lines which segregate for the ph1b deletion. This PCR-screening technique is less time and labour consuming, and more accurate and reliable, than cytologically based conventional methods.

Detailed comparative mapping of cereal chromosome regions corresponding to the Ph1 locus in wheat.

Detailed physical mapping of markers from rice chromosome 9, and from syntenous (at the genetic level) regions of other cereal genomes, has resulted in rice yeast artificial chromosome (YAC) contigs spanning parts of rice 9. This physical mapping, together with comparative genetic mapping, has demonstrated that synteny has been largely maintained between the genomes of several cereals at the level of contiged YACs. Markers located in one region of rice chromosome 9 encompassed by the YAC contigs have exhibited restriction fragment length polymorphism (RFLP) using deletion lines for the Ph1 locus. This has allowed demarcation of the region of rice chromosome 9 syntenous with the ph1b and ph1c deletions in wheat chromosome 5B. A group of probes located in wheat homoeologous group 5 and barley chromosome 5H, however, have synteny with rice chromosomes other than 9. This suggests that the usefulness of comparative trait analysis and of the rice genome as a tool to facilitate gene isolation will differ from one region to the next, and implies that the rice genome is more ancestral in structure than those of the Triticeae.

Are rice chromosomes components of a holocentric chromosome ancestor?

Comparative genomics reveals that cereal genomes are composed of similar genomic building blocks (linkage blocks). By stacking these blocks in a unique order, it is possible to construct a single ancestral 'chromosome' which can be cleaved to give the basic structure of the 56 different chromosomes found in wheat, rice, maize, sorghum, millet and sugarcane. The borders of linkage blocks are defined by cereal centromeric and telomeric sites. However, a number of studies have shown that telomeric heterochromatin has neocentromeric activity, implying that linkage blocks are in fact defined by centromeric-like sites with conserved sequences. The structure of the ancestral cereal genome thus resembles a holocentric chromosome, which is the chromosome structure shared by the closest relatives of the Gramineae, the Cypericeae and Juncaceae.

Centromeric sites and cereal chromosome evolution.

Comparative genome analysis enables the sites of centromeres, telomeres and nucleolar organiser regions to be aligned with borders that define the sets of linked genes conserved across the cereal genomes. This provides a basis for studying cereal genome evolution.

Pattern of axonal loss in longstanding papilledema due to idiopathic intracranial hypertension.

There is both clinical and histopathologic evidence for peripheral visual field loss and optic nerve degeneration in longstanding papilledema due to idiopathic intracranial hypertension (IIH). The purpose of this study was to look at the extent and distribution of axonal dropout in secondary optic atrophy due to IIH. Both optic nerves from a 29-year-old man with a two year history of IIH were examined histologically and morphometrically. A high-contrast lipid (myelin) stain, paraphenylenediamine (PPD), and a semiautomated image analysis system were employed to resolve sufficiently the optic nerve fiber images for counts and for measurement. There were 80% and 90% losses of axons, respectively, in the right and left optic nerves consequent to IIH. The axonal loss in the peripheral area of each optic nerve was much more severe than that in inner sectors (= 0.001 for the right optic nerve and = 0.005 for the left). This pattern of axonal dropout is consistent with the preservation of good central visual acuity despite devastating optic nerve atrophy, and with the severe peripheral visual field loss noted in this patient.

Organization of retro-element and stem-loop repeat families in the genomes and nuclei of cereals.

Sequences homologous to the retro-element BIS-1 and the stem-loop repeat Hi-10 are present in the genomes of a number of cereal species. A detailed characterization of these elements indicated that they are non-randomly organized in the genomes of at least two of these species, namely barley and rye. In contrast to the BIS-1 retro-elements, the stem-loop repeats are also non-randomly organized into discrete domains in interphase nuclei from barley and rye. Features of the organization of these repeats along chromosomes and within interphase nuclei of rye, barley and rice are discussed.

Optic nerve morphometry following axonal degeneration from SAIDS in rhesus monkeys.

In order to further study AIDS (acquired immunodeficiency syndrome) related neuropathologic findings, specifically progressive diffuse leuko-encephalopathy (PDL) and optic neuropathy, ten macaque monkeys (Macaca mulatta) were infected with simian immunodeficiency virus (SIV), observed for varying periods, and then sacrificed and tissue prepared for analysis. A marked difference from human AIDS pathology was found: in all monkeys, there was significant peripheral axonal dropout, as opposed to relatively little dropout in the central areas of the nerves (t stat. = -5.9, p < .001). In those monkeys infected with SIV for over one year, the axonal loss in the periphery was also greater than in the central optic nerve (t stat. = -5.03, p < .001); monkeys infected with SIV for less than one year had slightly less peripheral than central loss (t stat. = -4.5, p = .001). When compared with humans, however, it was found that the overall loss of axons was less (15% in monkeys vs. up to 45% in humans). There was also a lack of discernible retinal pathology, such as cotton wool spots, in the monkey tissue.

Key features of cereal genome organization as revealed by the use of cytosine methylation-sensitive restriction endonucleases.

Unlike mammalian genomes, cereal (Gramineae) genomes exhibit little suppression of CpG dinucleotides. In cereal genomes, however, most of the numerous potential recognition sites for CpG methylation-sensitive restriction enzymes are methylated. Analysis of cereal genomic libraries and of regions flanking genes indicates that unmethylated NotI sites are useful landmarks for regions containing genes/single-copy sequences. Studies of a rye chromosome arm indicate that its pericentromeric region has a reduced density of unmethylated NotI (and MluI) sites and therefore of genes. Unmethylated MluI and NruI sites are distributed nonrandomly in the genomes of wheat, barley, and rice. Analysis of the genomic blocks defined by these sites in wheat and barley indicates that they are most likely to have arisen by amplification. These observations form the basis of a proposed model for the organization and evolution of the wheat, barley, and rice genomes.

Targeting deletion (homoeologous chromosome pairing locus) or addition line single copy sequences from cereal genomes.

We describe here a protocol for obtaining clones containing sequences present in low copy-number from genomic DNA where moderately and highly repeated sequences predominate. Specific chromosomal regions can be targeted by using deletion or addition line material. We have used this protocol to identify a sequence which has been deleted in both the tetraploid and hexaploid wheat mutants for the homoeologous chromosome pairing locus.

Watchful waiting of a fetus with zone III amniotic fluid values.

A case of Rh negative, sensitized patient was observed while her amniotic fluid values climbed through zone III of Liley's graph. The fetus was monitored weekly with ultrasound and delivered when it had a mature lung profile. It is not always necessary to proceed with immediate fetal therapy whenever amniotic fluid values are in zone III.