ROCK signalling induced gene expression changes in mouse pancreatic ductal adenocarcinoma cells

The RhoA and RhoC GTPases act via the ROCK1 and ROCK2 kinases to promote actomyosin contraction, resulting in directly induced changes in cytoskeleton structures and altered gene transcription via several possible indirect routes. Elevated activation of the Rho/ROCK pathway has been reported in several diseases and pathological conditions, including disorders of the central nervous system, cardiovascular dysfunctions and cancer. To determine how increased ROCK signalling affected gene expression in pancreatic ductal adenocarcinoma (PDAC) cells, we transduced mouse PDAC cell lines with retroviral constructs encoding fusion proteins that enable conditional activation of ROCK1 or ROCK2, and subsequently performed RNA sequencing (RNA-Seq) using the Illumina NextSeq 500 platform. We describe how gene expression datasets were generated and validated by comparing data obtained by RNA-Seq with RT-qPCR results. Activation of ROCK1 or ROCK2 signalling induced significant changes in gene expression that could be used to determine how actomyosin contractility influences gene transcription in pancreatic cancer

Letter from A. F. Rath to Eugene Burdick Regarding Beaded Moccasins, February 20, 1941

This letter dated February 20, 1941, from A. F. Rath to Eugene Burdick, requests information about beaded moccasins that Burdick may have available for sale. Rath specifically requests a list of items and prices. See also: Letter from Eugene Burdick to A. F. Rath Regarding Beaded Moccasins, February 25, 1941https://commons.und.edu/burdick-papers/1066/thumbnail.jp

Targeting ROCK activity to disrupt and prime pancreatic cancer for chemotherapy

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease; the identification of novel targets and development of effective treatment strategies are urgently needed to improve patient outcomes. Remodeling of the pancreatic stroma occurs during PDAC development, which drives disease progression and impairs responses to therapy. The actomyosin regulatory ROCK1 and ROCK2 kinases govern cell motility and contractility, and have been suggested to be potential targets for cancer therapy, particularly to reduce the metastatic spread of tumor cells. However, ROCK inhibitors are not currently used for cancer patient treatment, largely due to the overwhelming challenge faced in the development of anti-metastatic drugs, and a lack of clarity as to the cancer types most likely to benefit from ROCK inhibitor therapy. In 2 recent publications, we discovered that ROCK1 and ROCK2 expression were increased in PDAC, and that increased ROCK activity was associated with reduced survival and PDAC progression by enabling extracellular matrix (ECM) remodeling and invasive growth of pancreatic cancer cells. We also used intravital imaging to optimize ROCK inhibition using the pharmacological ROCK inhibitor fasudil (HA-1077), and demonstrated that short-term ROCK targeting, or ‘priming’, improved chemotherapy efficacy, disrupted cancer cell collective movement, and impaired metastasis. This body of work strongly indicates that the use of ROCK inhibitors in pancreatic cancer therapy as ‘priming’ agents warrants further consideration, and provides insights as to how transient mechanical manipulation, or fine-tuning the ECM, rather than chronic stromal ablation might be beneficial for improving chemotherapeutic efficacy in the treatment of this deadly disease

A one-step procedure to probe the viscoelastic properties of cells by Atomic Force Microscopy

The increasingly recognised importance of viscoelastic properties of cells in pathological conditions requires rapid development of advanced cell microrheology technologies. Here, we present a novel Atomic Force Microscopy (AFM)-microrheology (AFM2) method for measuring the viscoelastic properties in living cells, over a wide range of continuous frequencies (0.005 Hz ~ 200 Hz), from a simple stress-relaxation nanoindentation. Experimental data were directly analysed without the need for pre-conceived viscoelastic models. We show the method had an excellent agreement with conventional oscillatory bulk-rheology measurements in gels, opening a new avenue for viscoelastic characterisation of soft matter using minute quantity of materials (or cells). Using this capability, we investigate the viscoelastic responses of cells in association with cancer cell invasive activity modulated by two important molecular regulators (i.e. mutation of the p53 gene and Rho kinase activity). The analysis of elastic (G′(ω)) and viscous (G″(ω)) moduli of living cells has led to the discovery of a characteristic transitions of the loss tangent (G″(ω)/G′(ω)) in the low frequency range (0.005 Hz ~ 0.1 Hz) that is indicative of the capability for cell restructuring of F-actin network. Our method is ready to be implemented in conventional AFMs, providing a simple yet powerful tool for measuring the viscoelastic properties of living cells

Accuracy of predicting milk yield from alternative milk recording schemes

peer-reviewedThe effect of reducing the frequency of official milk recording and the number of recorded samples per test-day on the accuracy of predicting daily yield and cumulative 305-day yield was investigated. A control data set consisting of 58 210 primiparous cows with milk test-day records every 4 weeks was used to investigate the influence of reduced milk recording frequencies. The accuracy of prediction of daily yield with one milk sample per test-day was investigated using 41 874 testday records from 683 cows. Results show that five or more test-day records taken at 8-weekly intervals (A8) predicted 305-day yield with a high level of accuracy. Correlations between 305-day yield predicted from 4-weekly recording intervals (A4) and from 8-weekly intervals were 0.99, 0.98 and 0.98 for milk, fat and protein, respectively. The mean error in estimating 305-day yield from the A8 scheme was 6.8 kg (s.d. 191 kg) for milk yield, 0.3 kg (s.d. 10 kg) for fat yield, and −0.3 kg (s.d. 7 kg) for protein yield, compared with the A4 scheme. Milk yield and composition taken during either morning (AM) or evening (PM) milking predicted 24-h yield with a high degree of accuracy. Alternating between AM and PM sampling every 4 weeks predicted 305-day yield with a higher degree of accuracy than either all AM or all PM sampling. Alternate AM-PM recording every 4 weeks and AM + PM recording every 8 weeks produced very similar accuracies in predicting 305-day yield compared with the official AM + PM recording every 4 weeks

Tissue-selective expression of a conditionally-active ROCK2-estrogen receptor fusion protein

The serine/threonine kinases ROCK1 and ROCK2 are central mediators of actomyosin contractile force generation that act downstream of the RhoA small GTP-binding protein. As a result, they have key roles in regulating cell morphology and proliferation, and have been implicated in numerous pathological conditions and diseases including hypertension and cancer. Here we describe the generation of a gene-targeted mouse line that enables CRE-inducible expression of a conditionally-active fusion between the ROCK2 kinase domain and the hormone-binding domain of a mutated estrogen receptor (ROCK2:ER). This two-stage system of regulation allows for tissue-selective expression of the ROCK2:ER fusion protein, which then requires administration of estrogen analogues such as tamoxifen or 4-hydroxytamoxifen to elicit kinase activity. This conditional gain-of-function system was validated in multiple tissues by crossing with mice expressing CRE recombinase under the transcriptional control of cytokeratin14 (K14), murine mammary tumor virus (MMTV) or cytochrome P450 Cyp1A1 (Ah) promoters, driving appropriate expression in the epidermis, mammary or intestinal epithelia respectively. Given the interest in ROCK signaling in normal physiology and disease, this mouse line will facilitate research into the consequences of ROCK activation that could be used to complement conditional knockout models

A heuristic solution method for node routing based solid waste collection problems

This paper considers a real world waste collection problem in which glass, metal, plastics, or paper is brought to certain waste collection points by the citizens of a certain region. The collection of this waste from the collection points is therefore a node routing problem. The waste is delivered to special sites, so called intermediate facilities (IF), that are typically not identical with the vehicle depot. Since most waste collection points need not be visited every day, a planning period of several days has to be considered. In this context three related planning problems are considered. First, the periodic vehicle routing problem with intermediate facilities (PVRP-IF) is considered and an exact problem formulation is proposed. A set of benchmark instances is developed and an efficient hybrid solution method based on variable neighborhood search and dynamic programming is presented. Second, in a real world application the PVRP-IF is modified by permitting the return of partly loaded vehicles to the depots and by considering capacity limits at the IF. An average improvement of 25% in the routing cost is obtained compared to the current solution. Finally, a different but related problem, the so called multi-depot vehicle routing problem with inter-depot routes (MDVRPI) is considered. In this problem class just a single day is considered and the depots can act as an intermediate facility only at the end of a tour. For this problem several instances and benchmark solutions are available. It is shown that the algorithm outperforms all previously published metaheuristics for this problem class and finds the best solutions for all available benchmark instances

Characterization of Staphylococcus Aureus Isolated from the Nasal Cavity Flora of Nursing Majors

Approximately 30% of people have the bacterium Staphylococcus aureus (S. aureus) in their nasal passages. Within this group, approximately 1-2% are colonized with methicillin-resistant S. aureus (MRSA), although many do not display any symptoms.1 MRSA is an opportunistic pathogen that can potentially cause diseases such as pneumonia, skin infections and sepsis. MRSA infections are commonly grouped into two categories, hospital associated (HA) or community associated (CA) based on where the infection was acquired and the profile of antibiotic resistance. S. aureus and MRSA can spread between individuals through physical contact and presents a serious health hazard to patients if healthcare professionals are carriers. This research focuses on the detection and characterization of S. aureus strains found among a population of healthy nursing majors in multiple sections of a laboratory course at St. John Fisher College. Samples collected from the nasal passages of students were characterized using mannitol salt agar, Gram-staining procedures, blood agar, CHROMagar MRSA II and were subjected to antibiotic resistance testing. Based on the findings of this experiment, it is clear that S. aureus is present in healthy individuals. Results from the spring and fall trials demonstrated that S. aureus was present in 31% and 50% of the sample population respectively. Despite only one strain testing positive for MRSA, many other strains did exhibit antibiotic resistance similar to that of HA-MRSA. Our results reveal a vast array of S. aureus strains present in healthcare workers and support the argument that there needs to be increased awareness and policies to help prevent the transmission of infection to patients

Preliminary study of feasibility of an experiment looking for excited state double beta transitions in tin

An attempt to study the feasibility of a new experiment to search for double beta decay in $^{112}$Sn and $^{124}$Sn was carried out by using ultra-low background HPGe detector (244 cm$^{3}$) inside the Gran Sasso National Laboratory (LNGS) of the INFN (Italy). A small sample of natural Sn was examined for 2367.5 h. The radioactive contamination of the sample has been estimated. The data has also been considered to calculate the present sensitivity for the proposed search; half-life limits $\sim$ $10^{17} - 10^{18}$ years for $\beta^{+}$EC and EC-EC processes in $^{112}$Sn and $\sim$ $10^{18}$ years for $\beta^{-}\beta^{-}$ transition in $^{124}$Sn were measured. In the last section of the paper the enhancement of the sensitivity for a proposed experiment with larger mass to reach theoretically estimated values of half-lives is discussed.Comment: 20 pages, 11 figures, 4 tables, accepted for publication in NIMA (in press

Nuclear deformation and neutrinoless double-β\beta decay of 94,96^{94,96}Zr, 98,100^{98,100}Mo, 104^{104}Ru, 110^{110}Pd, 128,130^{128,130}Te and 150^{150}Nd nuclei in mass mechanism

The $(\beta ^{-}\beta ^{-})_{0\nu}$ decay of $^{94,96}$Zr, $^{98,100}$Mo, $^{104}$Ru, $^{110}$Pd, $^{128,130}$Te and $^{150}$Nd isotopes for the $0^{+}\to 0^{+}$ transition is studied in the Projected Hartree-Fock-Bogoliubov framework. In our earlier work, the reliability of HFB intrinsic wave functions participating in the $\beta ^{-}\beta ^{-}$ decay of the above mentioned nuclei has been established by obtaining an overall agreement between the theoretically calculated spectroscopic properties, namely yrast spectra, reduced $B(E2$:$0^{+}\to 2^{+})$ transition probabilities, quadrupole moments $Q(2^{+})$, gyromagnetic factors $g(2^{+})$ as well as half-lives $T_{1/2}^{2\nu}$ for the $0^{+}\to 0^{+}$ transition and the available experimental data. In the present work, we study the $(\beta ^{-}\beta ^{-})_{0\nu}$ decay for the $0^{+}\to 0^{+}$ transition in the mass mechanism and extract limits on effective mass of light as well as heavy neutrinos from the observed half-lives $T_{1/2}^{0\nu}(0^{+}\to 0^{+})$ using nuclear transition matrix elements calculated with the same set of wave functions. Further, the effect of deformation on the nuclear transition matrix elements required to study the $(\beta ^{-}\beta ^{-})_{0\nu}$ decay in the mass mechanism is investigated. It is noticed that the deformation effect on nuclear transition matrix elements is of approximately same magnitude in $(\beta ^{-}\beta ^{-})_{2\nu}$ and $(\beta ^{-}\beta ^{-})_{0\nu}$ decay.Comment: 15 pages, 1 figur