Chromatin regulation at the frontier of synthetic biology

As synthetic biology approaches are extended to diverse applications throughout medicine, biotechnology and basic biological research, there is an increasing need to engineer yeast, plant and mammalian cells. Eukaryotic genomes are regulated by the diverse biochemical and biophysical states of chromatin, which brings distinct challenges, as well as opportunities, over applications in bacteria. Recent synthetic approaches, including 'epigenome editing', have allowed the direct and functional dissection of many aspects of physiological chromatin regulation. These studies lay the foundation for biomedical and biotechnological engineering applications that could take advantage of the unique combinatorial and spatiotemporal layers of chromatin regulation to create synthetic systems of unprecedented sophistication.National Institute of General Medical Sciences (U.S.) (Ruth L. Kirschstein Postdoctoral Fellowship )United States. Defense Advanced Research Projects AgencyNational Institutes of Health (U.S.) (R24 Grant)Wyss Institute for Biologically Inspired EngineeringHoward Hughes Medical Institut

Path finding strategies in scale-free networks

We numerically investigate the scale-free network model of Barab{\'a}si and Albert [A. L. Barab{\'a}si and R. Albert, Science {\bf 286}, 509 (1999)] through the use of various path finding strategies. In real networks, global network information is not accessible to each vertex, and the actual path connecting two vertices can sometimes be much longer than the shortest one. A generalized diameter depending on the actual path finding strategy is introduced, and a simple strategy, which utilizes only local information on the connectivity, is suggested and shown to yield small-world behavior: the diameter $D$ of the network increases logarithmically with the network size $N$, the same as is found with global strategy. If paths are sought at random, $D \sim N^{0.5}$ is found.Comment: 4 pages, final for

Uso de espátula e dissector para otimização da ceratoplastia lamelar anterior profunda (DALK)

Objective: We describe a novel spatula and dissector to facilitate the big-bubble technique in deep anterior lamellar keratoplasty (DALK). Methods: A 29-year-old man who was diagnosed with bilateral keratoconus underwent deep anterior lamellar keratoplasty (DALK). After 350μm partial thickness incision of the recipient cornea, the Bonfadini dissector was inserted at the deepest point in the peripheral incision and could be advanced to the center of the cornea safely because of its semi-sharp tip. After achieving the big-bubble (BB) separation of Descemet membrane (DM) from the overlying stroma, the anterior stromal disc was removed. Viscoelastic material was placed on the stromal bed to prevent uncontrolled collapse and perforation of DM during the paracentesis blade incision into the BB. We could detect the safe opening of the BB using the Bonfadini dissector by the leakage of air bubbles into the viscoelastic material. After injecting viscoelastic material into the BB space, we inserted the Bonfadini spatula into the bigbubble safely because of its curved profile and blunt edges. The groove along the length of the Bonfadini spatula enables safe and efficient incision or the residual stromal tissue using the pointed end of a sharp blade while protecting the underlying DM. After removal of posterior stroma, the donor button was sutured with 16 interrupted 10-0 nylon sutures. Results: This technique and the use of the Bonfadini spatula and dissector facilitate exposure of Descemet membrane. Conclusion: The smooth Bonfadini DALK spatula and dissector facilitate safe and efficient completion of DALK surgery.Objetivo: Descrevemos o uso de novos instrumentais cirúrgicos para facilitar a técnica de big-bubble na ceratoplastia lamelar anterior profunda (DALK). Métodos: Paciente masculino, 29 anos, foi diagnosticado com ceratocone bilateral e submetido à ceratoplastia lamelar anterior profunda (DALK). Após incisão da córnea receptora numa profundidade de 350μm de espessura parcial, o dissector Bonfadini foi inserido no ponto mais profundo da incisão periférica e pode avançar para o centro da córnea com segurança devido à sua ponta semiafiada. Depois de realizar a big-bubble (BB) e atingir a separação da Membrana de Descemet (MD) do estroma sobrejacente, o disco corneano de estroma anterior foi removido. Um viscoelástico foi colocado sobre o leito do estroma remanescente para impedir o colapso não-controlado e perfuração da MD durante a incisão na BB com lâmina de paracentese. Verificamos segurança no rompimento do estroma remanescente com o auxílio do dissector Bonfadini, para liberação da bolha de ar da BB através do viscoelástico. Depois de injetar o viscoelástico no espaço da BB, inserimos a espátula Bonfadini neste espaço, o que demonstrou-se seguro devido ao formato curvo e das bordas arredondadas do instrumental. A chanfradura ao longo do comprimento da espátula Bonfadini permite a incisão pela ponta de uma lâmina afiada, protegendo assim a MD subjacente. Após a remoção do estroma posterior, o botão doador foi suturado com 16 pontos interrompidos de fio nylon 10.0. Resultados: Esta técnica e o uso da espátula Bonfadini e dissector facilitam a exposição de membrana de Descemet. Conclusão: A superfície lisa da espátula Bonfadini e dissector, facilitam a realização segura e eficiente da ceratoplastia lamelar anterior profunda (DALK).Johns Hopkins School of Medicine Wilmer Eye Institute Cornea & Anterior Segment ServiceRio de Janeiro Eye BankUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaCatholic University of Korea College of MedicineUNIFESP, EPMSciEL

Outcomes of Fusions From the Cervical Spine to the Pelvis.

Study designRetrospective cohort study.ObjectiveDetermine the indications, complications, and clinical outcomes in patients requiring fusions from the cervical spine to the pelvis. Several investigators have examined fusions from the thoracic spine to the sacrum, but no similar study has been performed for cervical-to-pelvis fusions.MethodsPatients from 2003 to 2014 with an upper instrumented vertebrae (UIV) in the cervical spine (any level) and a lower instrumented vertebrae (LIV) in the sacrum or pelvis were included in the study. Those with infectious or acute trauma-related deformities were excluded. Patient demographics, medical history, diagnosis, operative procedure, and health-related quality of life measures were analyzed. Student's t test, Kruskal-Wallis test, and χ2 test were used as appropriate; significance was set at P < .05 for all tests.ResultsFifty-five patients met inclusion criteria for the study. Average follow-up was 2.8 years. Proximal junctional kyphosis was the most common indication for cervical-to-pelvis fusions (36%). The most common UIV was C2 (29%) followed by C7 (24%). There was an average 31° correction in maximum kyphosis and a 3.3 cm improvement in sagittal vertical axis. In adults, the rate of complication was 71.4%, with a major complication rate of 39.3% and reoperation rate of 53.6%. There was significant improvement in the Scoliosis Research Society (SRS-22r) score (3.0 to 3.5; P < .01).ConclusionProximal junctional kyphosis is the most common indication for patients requiring fusion to the cervical spine. Adult patients incur a significant risk of major complications and reoperations. However, significant improvement in SRS-22r outcomes are noted in these patients

Subnormal Cytokine Profile in the Tear Fluid of Keratoconus Patients

Keratoconus, historically viewed as a non-inflammatory disease, is an ectatic corneal disorder associated with progressive thinning of the corneal stroma. Recently, a few inflammatory mediators have been reported to be elevated in the tear fluid of keratoconus patients. Consequently, we investigated a wide range of inflammation regulating cytokines in the tears and sera of keratoconus and control subjects. Interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, interferon (IFN)-γ, chemokine C-C motif ligand 5 (CCL5) and tumor necrosis factor (TNF)-α were tested in tear samples and sera of keratoconus and control individuals by multiplex immuno-bead assays. Selected cytokines were further tested by standard ELISA on pooled tear samples. All cytokines in the sera were generally low, with no significant changes between keratoconus and control subjects. However, in tear fluids, clear differences were detected between the two groups. These differences include increased IL-6, and decreased IL-12, TNF-α, IFN-γ, IL-4, IL-13 and CCL5 in keratoconus compared to control tear fluids. The decreases in IL-12, TNF-α and CCL5 were statistically significant, while the IL-13 decrease was statistically significant in the severe keratoconus group only. IL-17 could not be detected by multiplex immuno-bead assay, but showed an increase in keratoconus by conventional ELISA on a limited number of pooled tear samples. Our findings confirm increased IL-6, but dispute earlier reports of increased TNF-α, and suggest a cytokine imbalance in keratoconus disrupting corneal homeostasis. Moreover, an increase in IL-17 suggests tissue degenerative processes at work, contributing to the thinning and weakening of the corneal connective tissue in keratoconus

Growing Scale-Free Networks with Tunable Clustering

We extend the standard scale-free network model to include a ``triad formation step''. We analyze the geometric properties of networks generated by this algorithm both analytically and by numerical calculations, and find that our model possesses the same characteristics as the standard scale-free networks like the power-law degree distribution and the small average geodesic length, but with the high-clustering at the same time. In our model, the clustering coefficient is also shown to be tunable simply by changing a control parameter - the average number of triad formation trials per time step.Comment: Accepted for publication in Phys. Rev.

Utilizing Three Years of Epidemiological Data from Medical Missions in Cambodia to Shape the Mobile Medical Clinic Formulary

Objective: The purpose of this project was to gather epidemiological data on common diseases and medications dispensed during medical mission teams to Cambodia to shape the mobile medical clinic formulary. Methods: Data for patients seen during week-long, mobile, medical clinics was collected in Cambodia during Septembers 2012 to 2014. Patient’s gender, age, weight, blood pressure, glucose, pertinent laboratory values, diagnoses, and medications dispensed were collected. Blood pressure and glucose were measured in patients 18 years and above. Data collected onto paper intake forms were transferred onto spreadsheets without patient identifying information and analyzed for aggregate means, common diseases, and most dispensed medications. This project received institutional review board approval. Results: A total of 1,015 patients were seen over three years. Women made up 61.4% and the mean age was 41.8 years. The most common diagnosis was gastrointestinal disorders (22.9%), which included gastroesophageal reflux disease and intestinal parasites. Next, 20.1% of patients had hypertension (BP\u3e140/90), 18.0% had presbyopia, 15.4% had back and joint pain, followed by 8.8% with headache, including migraines. Approximately 8.4% of patients had hyperglycemia (RPG \u3e140 mg/dl). Top five medications dispensed were acetaminophen, omeprazole, multivitamin, ibuprofen and metformin. For hypertension, amlodipine and lisinopril were dispensed. Conclusion: Cambodia lacks systematic public health collection of epidemiological data for prevalence of diseases. Hence, investigators collected and analyzed information from week-long mobile medical clinics over three years. Proton-pump inhibitors and H. pylori lab tests are recommended for gastrointestinal disorders. Acetaminophen and ibuprofen are recommended for pain. Angiotensin-converting-enzyme inhibitors and dihydropyridine calcium channel blockers are recommended over diuretics since patients are already dehydrated. Metformin is recommended for diabetes. Vitamins and supplements are recommended for malnourished patients. Hemoglobin machine and urine test strips are suggested. This information should help future teams decide what medications and laboratory tests are most beneficial on medical teams in Cambodia

Integrated stress response and decreased ECM in cultured stromal cells from keratoconus corneas

Purpose: Keratoconus (KC) is a multifactorial disease where progressive thinning and weakening of the cornea leads to loss of visual acuity. Although the underlying etiology is poorly understood, a major endpoint is a dysfunctional stromal connective tissue matrix. Using multiple individual KC corneas, we determined that matrix production by keratocytes is severely impeded due to an altered stress response program. Methods: KC and donor (DN) stromal keratocytes were cultured in low glucose serum-free medium containing insulin, selenium and transferrin. Fibronectin, collagens and proteins related to their chaperone, processing and export, matrix metalloproteinase, and stress response related proteins were investigated by immunoblotting, immunocytochemistry, hydroxyproline quantification, and gelatin zymography. Multiplexed mass spectrometry was used for global proteomic profiling of 5 individual DN and KC cell culture. Transcription of selected proteins was assayed by qPCR. Results: DN and KC cells showed comparable survival and growth. However, immunoblotting of selected ECM proteins and global proteomics showed decreased fibronectin, collagens, PCOLCE, ADAMTS2, BMP1, HSP47, other structural and cytoskeletal proteins in KC. Phosphorylated (p) eIF2α, a translation regulator and its target, ATF4 were increased in KC cultured cells and corneal sections. Conclusions: The profound decrease in structural proteins in cultured KC cells and increase in the p-eIF2α, and ATF4, suggest a stress related blockade in structural proteins not immediately needed for cell survival. Therefore, this cell culture system reveals an intrinsic aggravated stress response with consequent decrease in ECM proteins as potential pathogenic underpinnings in KC

Ultrasound-evoked immediate early gene expression in the brainstem of the Chinese torrent frog, Odorrana tormota

The concave-eared torrent frog, Odorrana tormota, has evolved the extraordinary ability to communicate ultrasonically (i.e., using frequencies>20kHz), and electrophysiological experiments have demonstrated that neurons in the frog’s midbrain (torus semicircularis) respond to frequencies up to 34kHz. However, at this time, it is unclear which region(s) of the torus and what other brainstem nuclei are involved in the detection of ultrasound. To gain insight into the anatomical substrate of ultrasound detection, we mapped expression of the activity-dependent gene, egr-1, in the brain in response to a full-spectrum mating call, a filtered, ultrasound-only call, and no sound. We found that the ultrasound-only call elicited egr-1 expression in the superior olivary and principal nucleus of the torus semicircularis. In sampled areas of the principal nucleus, the ultrasound-only call tended to evoke higher egr-1 expression than the full-spectrum call and, in the center of the nucleus, induced significantly higher egr-1 levels than the no-sound control. In the superior olivary nucleus, the full-spectrum and ultrasound-only calls evoked similar levels of expression that were significantly greater than the control, and egr-1 induction in the laminar nucleus showed no evidence of acoustic modulation. These data suggest that the sampled areas of the principal nucleus are among the regions sensitive to ultrasound in this species