Control of Expression of Key Cell Cycle Enzymes Drives Cell Line-Specific Functions of CDK7 in Human PDAC Cells

Inhibition of the dual function cell cycle and transcription kinase CDK7 is known to affect the viability of cancer cells, but the mechanisms underlying cell line-specific growth control remain poorly understood. Here, we employed a previously developed, highly specific small molecule inhibitor that non-covalently blocks ATP binding to CDK7 (LDC4297) to study the mechanisms underlying cell line-specific growth using a panel of genetically heterogeneous human pancreatic tumor lines as model system. Although LDC4297 diminished both transcription rates and CDK T-loop phosphorylation in a comparable manner, some PDAC lines displayed significantly higher sensitivity than others. We focused our analyses on two well-responsive lines (Mia-Paca2 and Panc89) that, however, showed significant differences in their viability upon extended exposure to limiting LDC4297 concentrations. Biochemical and RNAseq analysis revealed striking differences in gene expression and cell cycle control. Especially the downregulation of a group of cell cycle control genes, among them CDK1/2 and CDC25A/C, correlated well to the observed viability differences in Panc89 versus Mia-Paca2 cells. A parallel downregulation of regulatory pathways supported the hypothesis of a feedforward programmatic effect of CDK7 inhibitors, eventually causing hypersensitivity of PDAC lines.

Fracturing Technology - Can We Finally Control Fracture Height?

For as long as we have been performing hydraulic fracturing, we have been trying to ensure that we stay out of undesirable horizons, potentially containing water and/or gas. The holy grail of hydraulic fracturing, an absolute control of created fracture height, has eluded the industry for more than 70 years. Of course, there have been many that have claimed solutions, but all the marketed approaches have at best merely created a delay to the inevitable growth and at worst been a snake-oil approach with little actual merit. Fundamentally, the applied techniques have attempted to delay or influence the underlying equations of net-pressure and stress variation; but having to ultimately honour them and by doing so then condemned themselves to limited success or outright failure. Fast forward to 2020, and a reassessment of the relative importance of height-growth constraint and what may have changed to help us achieve this. The development of unconventionals are focused on creating as much surface area as possible in micro/nano-Darcy environments, across almost any phase, but with typically poor line of sight to profit. However, the more valuable business of conventional oil and gas is working in thinner and thinner reservoirs with an often-deteriorating permeability, but with a significantly higher potential economic return. What unconventional has successfully delivered however, is a rapid deployment and acceleration in a range of completion technologies that were unavailable just a few years ago. We will demonstrate that these technologies potentially offer the capability of finally being able to control fracture height-growth. Consideration of a range of previously applied height-growth approaches will demonstrate how they attempted to fool or fudge height growth creation mechanisms. With this clarity, we can consider what advances in completion technology may offer in terms of delivering height growth control. We suggest that with the technology and approaches that are currently available today, that height-growth control is finally within reach. We will go on to describe a multi-well Pilot program, in deployment and execution in 2020/021 in Western Siberia; where billions of barrels remain to be recovered in thin oil-rim, low permeability sandstone reservoirs below gas or above water. A comprehensive assessment of the myriad of height-growth approaches that have been utilized over the last 70 years was performed, but in each case demonstrated the fallibility and limitations of each of these. However, rather than the interpretation that such control is not achievable, instead we will show a mathematically sound approach, along with field data and evidence that this is possible. The presentation will demonstrate that completion advances over the last 10 - 15 years make this approach a reality in the present day; and that broader field implementation is finally within reach.

Fracturing Height Growth Restriction Technique Successfully Extended into Horizontal Wells in Ostracod Formation

The Ostracod formation in the Awali brownfield is an extremely challenging layer to develop because the tight carbonate rock is interbedded with shaly streaks and because of the presence of a nearby water-bearing zone. Although the Ostracod formation has been in development since 1960, oil recovery has not yet reached 5% because past stimulation attempts experienced rapid production decline. The current project incorporated aggressive fracture design coupled with a unique height growth control (HGC) workflow, improving the development of Ostracod reserves. The HGC technology is a combination of an engineering workflow supported by geomechanical modeling and an advanced simulator of in-situ kinetics and materials transport to model the placement of a customized, impermeable mixture of particles that will restrict fracture growth. The optimized treatment design included injections of the HGC mixture prior to the main fracturing treatment. This injection was done with a nonviscous fluid to improve settling to create an artificial barrier. After the success of a trial campaign in vertical wells, the technique was adjusted for the horizontal wellbores. The high clay content within the Ostracod layers creates a significant challenge for successful stimulation. The high clay content prevents successful acid fracturing and leads to severe embedment with conventional proppant fracturing designs. We introduced a new approach to stimulate this formation with an aggressive tip-screenout design incorporating a large volume of 12/20-mesh proppant to obtain greater fracture width and conductivity, resulting in a significant and sustained oil production gain. The carefully designed HGC technique was efficient in avoiding fracture breakthrough into the nearby water zone, enabling treatments of up to 450,000 lbm to be successfully contained above a 20-ft-thick shaly barrier with small horizontal stress contrast. Independent measurements proved that the fracture height was successfully contained. This trial campaign in vertical wells proved that the combination of aggressive, large fracture designs with the HGC method could help unlock the Ostracod’s potential. Three horizontal wells were drilled and simulated, each with four stages of adjusted HGC technique to verify if this aggressive method was applicable to challenging sand admittance in case of transverse fractures. This rare implementation of HGC mixtures in horizontal wells showed operational success and proof of fracture containment based on pressure signatures and production monitoring. The applied HGC technique was modified with additional injections and improved by advanced modeling that only recently became available. These contributed to a significant increase of treatment volume, making the jobs placed in the Ostracod some of the world’s largest utilizing HGC techniques. The experience gained in this project can be of a paramount value to any project dealing with hydraulic fracturing near a water formation with insufficient or uncertain stress barriers.

Myostatin increases human trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 Signaling

Placental insufficiency disorders are major obstetric complications that share a common phenomenon of poor placental trophoblast cell invasion and remodeling of uterine tissues. Myostatin is a transforming growth factor (TGF)-β superfamily member well-known for its important role in muscle growth control. Myostatin is also produced in the placenta and has been shown to regulate some trophoblast functions. However, its roles in placental development are still poorly understood. In this study, we tested the hypothesis that myostatin increases trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling. Primary and immortalized (HTR8/SVneo) trophoblast cells were used as study models. Matrigel-coated transwell invasion assays were used to study the effects of recombinant human myostatin on trophoblast cell invasion. RT-qPCR and Western blot were used to measure myostatin effects on N-cadherin mRNA and protein levels, respectively. Small inhibitor molecules as well as siRNA-mediated knockdown were used to block myostatin receptor and downstream signaling, respectively. Data were analyzed either by unpaired Student T test or one-way ANOVA followed by Newman Keuls test for multiple group comparisons. Myostatin significantly increased primary and HTR8/SVneo trophoblast cell invasion. Moreover, myostatin upregulated N-cadherin mRNA and protein levels in a time dependent manner in both study models. These effects were blocked by inhibition of TGF-β type I receptors as well as siRNA-mediated knockdown of SMAD2/3 combined or common SMAD4. Importantly, myostatin-induced trophoblast cell invasion was abolished by knockdown of N-cadherin, SMAD2/3 or SMAD4. Myostatin may increase human trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling.

The Ceramide Synthase Subunit Lac1 Regulates Cell Growth and Size in Fission Yeast

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.

mGBP2 engages Galectin-9 and Cytoskeleton-associated Protein 4 for immunity against Toxoplasma gondii

Guanylate binding proteins (GBPs) are large interferon-inducible GTPases, executing essential host defense activities against Toxoplasma gondii, an invasive intracellular apicomplexan protozoan parasite of global importance. T. gondii establishes a parasitophorous vacuole (PV) which shields the parasite from the host's intracellular defense mechanisms. Murine GBPs (mGBPs) recognize T. gondii PVs and assemble into supramolecular mGBP homo- and heterocomplexes that are required for the disruption of the membrane of PVs eventually resulting in the cell-autonomous immune control of vacuole-resident pathogens. We have previously shown that mGBP2 plays an important role in T. gondii immune control. Here, in order to unravel mGBP2 functions, we report Galectin-9 (Gal9) and cytoskeleton-associated protein 4 (Ckap4) as critical mGBP2 interaction partners engaged for immunity to T. gondii. Interestingly, Gal9 and Ckap4 also accumulate and colocalize with mGBP2 at the T. gondii PV. Furthermore, we could prove the requirement of Gal9 and Ckap4 for growth control of T. gondii by CRISPR/Cas9 mediated gene editing. These discoveries clearly indicate that mGBP2 engages Gal9 and Ckap4 and that Gal9 and Ckap4 are critical factors for the mGBP2 coordinated cell autonomous host defense mechanism against T. gondii.

A Combination of Cabozantinib and Radiation Does Not Lead to an Improved Growth Control of Tumors in a Preclinical 4T1 Breast Cancer Model

The tyrosine kinase inhibitor Cabozantinib has been applied in clinical studies in combination with radiotherapy. We investigated the effect of such combination on triple-negative 4T1 cells as a metastatic breast cancer model in vitro and in vivo upon inoculation in BALB/c mice. In vitro assays indicated a potential for improved effects using the combination. Both Cabozantinib (2.5 µM) and 10 Gy of 250 kV x-rays were able to cease the growth of 4T1 cells as revealed by growth curves. In a clonogenic survival assay, the effect of Cabozantinib added on the effects of irradiation and the effectiveness of inhibiting the clonogenic survival was found to be 2 (RBE10). Additionally, cell death measurements of apoptosis plus necrosis revealed a synergistic effect when combining irradiation with Cabozantinib. Surprisingly, however, in vivo tumor growth kinetics showed no additional effect in growth control when irradiation was used together with Cabozantinib. Since both ionizing radiation and Cabozantinib are acknowledged to feature immunogenic effects, we additionally investigated the effect of the treatments on lung metastases. No difference to the control groups was found here, neither for irradiation nor Cabozantinib alone nor in combination. Yet, upon analysis of the mice’ livers, CD11b-positive cells, indicating immune suppressive myeloid derived suppressor cells were found diminished following treatment with Cabozantinib. In conclusion, despite promising in vitro controls of the combination of Cabozantinib and irradiation, tumor growth control was not increased by the combination, which was true also for the occurrence of lung metastases.

IMPLEMENTASI PROGRAM KELUARGA BERENCANA DI PUSKESMAS SIMPANG BARU KOTA PEKANBARU TAHUN 2020

Keluarga Berencana (KB) adalah upaya mengatur kelahiran anak, jarak usia ideal melahirkan, dan mengatur kehamilan melalui promosi perlindungan dan bantuan sesuai hak reproduksi untuk mewujudkan keluarga berkualitas serta mewujudkan norma keluarga kecil bahagia dan sejahtera yang menjadi dasar bagi terwujudnya masyarakat yang sejahtera melalui pengendalian kelahiran dan pengendalian pertumbuhan. Permasalahan yang ditemukan di Puskesmas Simpang Baru untuk Program Keluarga Berencana ialah masih kurang memadai nya sumber daya manusia, sarana prasarana serta penyuluhan yang masih kurang merata. Tujuan penelitian adalah untuk mengetahui pelaksanaan Program Keluarga Berencana di Puskesmas Simpang Baru tahun 2020. Jenis Penelitian ini adalah penelitian kualitatif dan metode penelitian deskriftif. Subjek Penelitian yaitu Kepala Puskesmas, Pemengang Program KIA/KB, Bidan, Akseptor KB. Pengumpulan Data dilakukan dengan wawancara mendalam berikatan dengan program Keluarga Berencana di Puskesmas,penelusuran dokumen, dan observasi. Hasil Penilitian ini menunjukan Masih kurangnya Sumber Daya Manusia di Puskesmas serta Sumber Daya Manusia di dalam Program Keluarga Berencana lalu masih belum tersedianya sarana prasarana seperti meja litotomi dan meja steril lalu masih kurang merata penyuluhan yang dilakukan oleh pihak Puskesmas yang dapat menghambat pelaksanaan Progran didalam Puskesmas . Saran dari penelitian ini adalah perlu nya penambahan Tenaga Kesehatan serta penambahan sarana prasarana di dan penyuluhan yang secara merata. Family Planning is the effort to manage the child birth, the space of delivery ideal age, and manage the pregnancy through protection promotion and assistance in accordance with the reproduction right to achieve quality family and to create happily small and prosperous family which becomes the basic of wealthy community through the birth control and growth control. The problem found at Communith Health Center Simpang Baru for its Family Planning Program still has less adequate human resources, less facilities, and less socialized of Family Planning Program. The aims of this study are to know the implementation of Family Planning Program at Communith Health Center (Puskesmas) Simpang Baru in 2020. The type of this research was qualitative research and descriptive research method. The research subject was the head of Communith Health Center (Puskesmas), the holder of KIA/KB Program, and Acceptor of KB. Data collection was conducted through deep interview related to Family Program Planning at Communith Health Center (Puskesmas), document search, and observation. The results of this research show that the human resources at Communith Health Center (Puskesmas) are still less adequate, and the facilities such as table for lithotomy and sterile table are not provided, and less evenly distributed socialization of Family Planning conducted by Communith Health Center (Puskesmas) party which can inhibit the implementation of the program at the Communith Health Center (Puskesmas) itself.

Meta-Analysis: The Influence of Knowledge, Education, and Husband's Support on The Selection of Long Acting Contraception Methods

The population is expected to increase by 8,5 billion persons in 2030 and 10,9 billion persons in 2100, population is growing at a rate of around 1,1% per year. One of population growth control by birth control with contraception. The most effective contraception is long-acting contraception, however, on IDHS (Indonesian Health Demographic Survey) only 13% of currently married women use long-acting contraception. Many factors affect contraceptive use among married women, such as knowledge, education, and husband’s support. The purpose of this study was to determine the effect of knowledge, education, and husband's support on the selection of MKJP. This study used meta-analysis with the search engines by Google scholar, PUBMED, science direct, and ProQuest. The study was selected using PRISMA and it was evaluated by AMSTAR. Data synthesis was conducted by STATA 16.0. The results of this study obtained by knowledge [OR = 0,99; 95% Cl : 0,90-1,08, p = 0,000; I2 = 74,8%], education [OR = 0,84; 95% Cl : 0,77-0,92, p = 0,000; I2 = 86,3%], and husband’s [OR = 0,94; 95% Cl : 0,69-1,20, p = 0,000; I2 = 81,5%]. This means that knowledge, education, and husband’s support have an impact on the use of long-acting contraception.