Advances in Chromatin and Chromosome Research: Perspectives from Multiple Fields.

Nucleosomes package genomic DNA into chromatin. By regulating DNA access for transcription, replication, DNA repair, and epigenetic modification, chromatin forms the nexus of most nuclear processes. In addition, dynamic organization of chromatin underlies both regulation of gene expression and evolution of chromosomes into individualized sister objects, which can segregate cleanly to different daughter cells at anaphase. This collaborative review shines a spotlight on technologies that will be crucial to interrogate key questions in chromatin and chromosome biology including state-of-the-art microscopy techniques, tools to physically manipulate chromatin, single-cell methods to measure chromatin accessibility, computational imaging with neural networks and analytical tools to interpret chromatin structure and dynamics. In addition, this review provides perspectives on how these tools can be applied to specific research fields such as genome stability and developmental biology and to test concepts such as phase separation of chromatin.

Application of Machine Learning for Calibrating Gas Sensors for Methane Emissions Monitoring.

Methane leaks are a significant component of greenhouse gas emissions and a global problem for the oil and gas industry. Emissions occur from a wide variety of sites with no discernable patterns, requiring methodologies to frequently monitor these releases throughout the entire production chain. To cost-effectively monitor widely dispersed well pads, we developed a methane point instrument to be deployed at facilities and connected to a cloud-based interpretation platform that provides real-time continuous monitoring in all weather conditions. The methane sensor is calibrated with machine learning methods of Gaussian process regression and the results are compared with artificial neural networks. A machine learning approach incorporates environmental effects into the sensor response and achieves the accuracies required for methane emissions monitoring with a small number of parameters. The sensors achieve an accuracy of 1 part per million methane (ppm) and can detect leaks at rates of less than 0.6 kg/h.

Instabilities and patterns in a submerged jelling jet.

When a thin stream of aqueous sodium alginate is extruded into a reacting calcium chloride bath, it polymerizes into a soft elastic tube that spontaneously forms helical coils due to the ambient fluid drag. We quantify the onset of this drag-induced instability and its nonlinear evolution using experiments, and explain the results using a combination of scaling, theory and simulations. By co-extruding a second (internal) liquid within the aqueous sodium alginate jet and varying the diameter of the jet and the rates of the co-extrusion of the two liquids, we show that we can tune the local composition of the composite filament and the nature of the ensuing instabilities to create soft filaments of variable relative buoyancy, shape and mechanical properties. Altogether, by harnessing the fundamental varicose (jetting) and sinuous (buckling) instabilities associated with the extrusion of a submerged jelling filament, we show that it is possible to print complex three-dimensional filamentous structures in an ambient fluid.

Self-Excited Motions of Volatile Drops on Swellable Sheets.

When a volatile droplet is deposited on a floating swellable sheet, it becomes asymmetric, lobed and mobile. We describe and quantify this phenomena that involves nonequilibrium swelling, evaporation and motion, working together to realize a self-excitable spatially extended oscillator. Solvent penetration causes the film to swell locally and eventually buckle, changing its shape and the drop responds by moving. Simultaneously, solvent evaporation from the swollen film causes it to regain its shape once the droplet has moved away. The process repeats and leads to complex pulsatile spinning and/or sliding movements. We use a one-dimensional experiment to highlight the slow swelling of and evaporation from the film and the fast motion of the drop, a characteristic of excitable systems. Finally, we provide a phase diagram for droplet excitability as a function of drop size and film thickness and scaling laws for the motion of the droplet.

Impact of extended infusional mesna prophylaxis on the incidence of BK viruria and hemorrhagic cystitis following post-transplantation cyclophosphamide and CTLA4Ig-based haploidentical transplantation.

Hemorrhagic cystitis (HC) has been reported with increased frequency following post-transplantation cyclophosphamide (PTCy)-based haploidentical hematopoietic cell transplantation (HCT) along with a strong association with BK viruria. We prospectively evaluated the incidence of BK viruria and HC in 115 patients (median age 20 years, 2-65) undergoing PTCy-based haploidentical HCT with (n = 71) or without (n = 44) CTLA4Ig. HC prophylaxis consisted of a continuous infusion of mesna 30 min prior and 48 h post-PTCy. The overall incidence of BK viruria was 65.7%. None with BK viruria < 104 copies/ml developed clinical symptoms (n = 65). The incidence of BK viruria ≥ 104 copies/ml was 7.1% (n = 8) and 75% developed HC. The incidence of HC was 5.4% at a median of 30 days. Both BK viruria ≥ 104 copies/ml and HC were strongly associated with acute GVHD (p < 0.001). A higher NRM was observed in those with BK viruria ≥ 104 copies/ml, related to GVHD and its complications (41.7% vs 12.6%, p = 0.04). The incidences of acute GVHD, vis-à-vis, overall BK viruria, BK viruria ≥ 104 copies/ml, and HC, tended to be lower in patients receiving CTLA4Ig. Thus, extended infusional mesna, coupled with significant reduction in alloreactivity along with possible preservation of antiviral immunity associated with the use of CTLA4Ig, was probably responsible for a much lower incidence of BK viruria and resultant HC than reported previously following PTCy-based haploidentical HCT.

CTLA4Ig Primed Donor Lymphocyte Infusion: A Novel Approach to Immunotherapy after Haploidentical Transplantation for Advanced Leukemia.

CTLA4Ig attenuates T cell activation by co-stimulation blockade, but natural killer (NK) cells are not only resistant to CTLA4Ig, they also may demonstrate better antileukemia effect in the presence of CTLA4Ig. To explore this phenomenon we used sequential CTLA4Ig primed donor lymphocyte infusion (DLI) after post-transplant cyclophosphamide-based haploidentical transplantation. Thirty patients (CTLA4Ig-DLI group) with advanced leukemia received CTLA4Ig on day -1 and subsequently on days +7, +21, and +35, followed 12hours later by DLI of 1 to 10 × 106 CD3+ T cells/kg containing .1 to 3.27 × 106/kg CD56+ NK cells, with low dose cyclosporine for 60days. The incidences of acute graft-versus-host disease (GVHD), chronic GVHD and nonrelapse mortality (NRM) were 6.7%, 21%, and 4.5 %, respectively, with disease progression of 23.3% and overall survival of 79% at 18 months. Patients without disease progression had a significant early surge in CD56dimCD16+NK cells with lower NKG2A expression. CTLA4Ig primed DLI was associated with an upregulation of CD86 in mature NK cells that was not witnessed with CTLA4Ig administration alone. Thus, CTLA4Ig primed DLI resulted in early proliferation of mature NK cells with cytotoxic potential enabling early institution of adoptive immunotherapy to mitigate the risk of relapse in advanced leukemia with reduced GVHD and NRM.

Impact of Preemptive Granulocyte Infusions During Febrile Neutropenia in Patients Colonized with Carbapenem-Resistant Gram-Negative Bacteria Undergoing Haploidentical Transplantation.

We prospectively studied the impact of preemptive granulocyte infusions (pGIs) in 69 patients colonized with carbapenem-resistant gram-negative bacteria (CRGNB) undergoing haploidentical hematopoietic cell transplantation (HCT) compared with a previous cohort of 33 patients who received only antimicrobials directed toward CRGNB at the onset of neutropenic fever (non-pGI group). All patients developed neutropenic fever at a median of day +8 (range, -4 to +12) after transplantation. Engraftment kinetics were similar for both groups. The median number of GIs was 2 (range, 1 to 7), and the median dose of granulocytes infused was 5 × 1010 granulocytes per infusion (range, 1 to 30). The overall incidence of CRGNB bloodstream infections (BSIs) was 21.2% in non-pGI group (7/33) and 17.5% (12/69) in the pGI group (P = .8). However, the CRGNB-related mortality among those with BSI was 100% (7/7) in the non-pGI group versus 16.6% (2/12) in the pGI group (P = .001). The day 100 (4.4% versus 24.4%, P = .002) and 2-year nonrelapse mortality (7.5% versus 35.6%, P = .0001) were significantly reduced in the pGI group. The overall survival at 2 years was 75.6% in the pGI group versus 21.2% in the non-pGI group (P = .0001). Colonization and subsequent BSI with CRGNB are associated with a high incidence of mortality in patients undergoing HCT. pGI reduced early mortality associated with CRGNB in colonized patients undergoing post-transplant cyclophosphamide-based haploidentical HCT.

Rotavirus infection following post-transplantation cyclophosphamide based haploidentical hematopoietic cell transplantation in children is associated with hemophagocytic syndrome and high mortality.

We evaluated the incidences and consequences of rotavirus induced diarrhea in a cohort of 115 patients undergoing T-cell replete haploidentical transplantation. Four out of 115 patients developed rotavirus-induced diarrhea between 47 and 147 days. The incidence of rotavirus infection was 9.7% in children compared to none in adults (P = .01). This was 25.3% in those with GVHD compared to 1.2% in those without GVHD (P = .001). Rotavirus infection was followed by post-transplantation hemophagocytic syndrome (PTHPS) at a median of 4 days (range, 3-10 days) in all four patients. Three patients succumbed to the complications related to PTHPS. Only one patient, who is long-term survivor, was able to eliminate this virus after 2 weeks. Children undergoing T-replete haploidentical hematopoietic cell transplantation who develop GVHD are at a higher risk of community-acquired rotavirus infection which was strongly associated with PTHPS with poor outcome.

Higher CD45RA+ Regulatory T Cells in the Graft Improves Outcome in Younger Patients Undergoing T Cell-Replete Haploidentical Transplantation: Where Donor Age Matters.

To understand the phenomenon of early alloreactivity (EA) in younger children undergoing post-transplantation cyclophosphamide (PTCy)-based haploidentical transplantation, we studied the graft composition and the immune reconstitution in 32 consecutive patients (aged 2 to 25 years) undergoing PTCy and T cell costimulation blockade based peripheral blood stem cell transplantation with emphasis on CD45RA+ subset of regulatory T cells (Tregs). All but 1 engrafted, and 14 patients experienced EA (acute graft-versus-host disease grades II to IV, n = 8; and post-transplantation hemophagocytic syndrome, n = 6) with a cumulative incidence of 43.7%; 42% developed mild chronic graft-versus-host disease. The overall survival was 70.2% with a nonrelapse mortality of 16.8% at a median of 19 months. Age < 10 years, donor age > 45 years, and poor recovery of Tregs correlated with EA. Not Tregs but higher CD45RA+ Tregs in the graft was associated with less EA (11.7% versus 32.5%, P = .0001). Higher donor age correlated with a lower CD45RA+ Tregs in the graft (P = .01). However, only higher CD45RA+ Treg percentage in the graft favorably impacted EA as well as nonrelapse mortality and overall survival. Our study demonstrates a critical role for CD45RA+ Tregs in determining EA and outcome after PTCy-based haploidentical peripheral blood stem cell transplantation, and the age-related physiologic decline in this population might be responsible for adverse impact of donor age.

Elastowetting of Soft Hydrogel Spheres.

When a soft hydrogel sphere is placed on a rigid hydrophilic substrate, it undergoes arrested spreading by forming an axisymmetric foot near the contact line, while conserving its global spherical shape. In contrast, liquid water (that constitutes greater than 90% of the hydrogel's volume) spreads into a thin film on the same surface. We study systematically this elastowetting of gel spheres on substrates of different surface energies and find that their contact angle increases as the work of adhesion between the gel and the substrate decreases, as one would observe for drops of pure water-albeit being larger than in the latter case. This difference in the contact angles of gel and water appears to be due to the elastic shear stresses that develop in the gel and oppose its spreading. Indeed, by increasing the elastic modulus of the gel spheres, we find that their contact angle also increases. In addition, the length of the contact foot increases with the work of adhesion and sphere size, while it decreases when the elastic modulus of the gel is increased. We discuss those experimental results in light of a minimal analysis based on energy minimization, volume conservation, and scaling arguments.

Wetting and phase separation in soft adhesion.

In the classic theory of solid adhesion, surface energy drives deformation to increase contact area whereas bulk elasticity opposes it. Recently, solid surface stress has been shown also to play an important role in opposing deformation of soft materials. This suggests that the contact line in soft adhesion should mimic that of a liquid droplet, with a contact angle determined by surface tensions. Consistent with this hypothesis, we observe a contact angle of a soft silicone substrate on rigid silica spheres that depends on the surface functionalization but not the sphere size. However, to satisfy this wetting condition without a divergent elastic stress, the gel phase separates from its solvent near the contact line. This creates a four-phase contact zone with two additional contact lines hidden below the surface of the substrate. Whereas the geometries of these contact lines are independent of the size of the sphere, the volume of the phase-separated region is not, but rather depends on the indentation volume. These results indicate that theories of adhesion of soft gels need to account for both the compressibility of the gel network and a nonzero surface stress between the gel and its solvent.

CD56-enriched donor cell infusion after post-transplantation cyclophosphamide for haploidentical transplantation of advanced myeloid malignancies is associated with prompt reconstitution of mature natural killer cells and regulatory T cells with reduced incidence of acute graft versus host disease: A pilot study.

We conducted a pilot study on the feasibility of CD56-enriched donor cell infusion after post-transplantation cyclophosphamide (PTCy) for 10 patients with advanced myeloid malignancies undergoing haploidentical peripheral blood stem cell transplantation with cyclosporine alone as graft-versus-host disease (GVHD) prophylaxis and compared the outcome and immune reconstitution with a control group of 20 patients undergoing the same without CD56-enriched donor cell infusion. An early and rapid surge of mature NK cells as well as CD4+ T cells and regulatory T cells (Tregs) was noted compared with the control group. KIR of donor phenotype reconstituted as early as day 30 with expression of CD56dimCD16+NKG2A-KIR+ phenotype. None experienced viral or fungal infections, and non-relapse mortality was 10% only. The incidence of grade 2-4 acute GVHD was 50% in the control group with none in the CD56 group (P = 0.01). Only two had de novo chronic GVHD in each group. Relapse occurred in five patients in CD56 group with a median follow-up of 12 months, similar to the control group. Our preliminary data show that CD56+ donor cell infusion after PTCy and short-course cyclosporine is feasible with prompt engraftment, rapid reconstitution of CD4+T cells, Tregs and NK cells and reduced incidence of acute GVHD.

Haploidentical Peripheral Blood Stem Cell Transplantation with Post-Transplantation Cyclophosphamide in Children with Advanced Acute Leukemia with Fludarabine-, Busulfan-, and Melphalan-Based Conditioning.

Post-transplantation cyclophosphamide (PTCY) therapy has made haploidentical transplantation a global reality in adults, but the literature is largely silent on the feasibility of this approach in children. We conducted a prospective study of 20 patients (median age, 12 years; range, 2-20 years) with advanced acute leukemia to evaluate the feasibility of PTCY-based haploidentical peripheral blood stem cell (PBSC) transplantation in children. The conditioning regimen comprised fludarabine, i.v. busulfan, and melphalan (Flu-Bu-Mel). PTCY on days +3 and +4 was followed by mycophenolate mofetil for 14-21 days and cyclosporine for 60 days. Thirteen patients (65%) had refractory or relapsed myelogenous leukemia, and the remainder had high-risk lymphoblastic leukemia. Prompt engraftment was noted at a median of 14 days, with full donor chimerism by day +28. The cumulative incidence of acute and chronic graft-versus-host disease was 35% and 5%, respectively. Nonrelapse mortality at 1 year was 20%. The incidence of disease progression was 25.7%. The actuarial overall survival at 2 years was 64.3% (95% confidence interval, 53.4%-75.2%). Our data suggest that Flu-Bu-Mel-based conditioning followed by PTCY-based haploidentical PBSC transplantation with reduced duration of immunosuppression is feasible in pediatric patients with advanced leukemia.

Improved Outcome of Refractory/Relapsed Acute Myeloid Leukemia after Post-Transplantation Cyclophosphamide-Based Haploidentical Transplantation with Myeloablative Conditioning and Early Prophylactic Granulocyte Colony-Stimulating Factor-Mobilized Donor Lymphocyte Infusions.

We carried out post-transplantation cyclophosphamide (PTCy)-based haploidentical peripheral blood stem cell transplantation in 51 patients with refractory/relapsed acute myeloid leukemia not in remission. The first 10 patients received nonmyeloablative conditioning followed by planned granulocyte colony-stimulating factor (G-CSF)-mobilized donor lymphocyte infusions (DLIs) on days 35, 60, and 90. No patient developed graft-versus-host disease (GVHD), but 90% had disease progression between 3 and 6 months. A subsequent 41 patients received myeloablative conditioning (MAC); the first 20 patients did not receive DLIs (MAC group) and the next 21 patients received G-CSF-mobilized DLIs (G-DLI) on days 21, 35, and 60 (MAC-DLI group). The incidence of disease progression and progression-free survival at 18 months were 66% and 25% in the MAC group compared with 21.4% and 61.9% in the MAC-DLI group (P = .01). Chronic GVHD but not acute GVHD was increased in the MAC-DLI group (41.2% versus 11%, P = .05). Natural killer cell alloreactive donor was associated with lower incidence of disease progression in the MAC but not in MAC-DLI group. The only factor favorably influencing disease progression and progression-free survival was administration of G-DLI after myeloablative conditioning. Our study shows that early administration of G-DLI is feasible after PTCy-based haploidentical hematopoietic stem cell transplantation for refractory/relapsed acute myeloid leukemia and might be associated with improved survival after MAC.

Haploidentical transplantation in children with unmanipulated peripheral blood stem cell graft: The need to look beyond post-transplantation cyclophosphamide in younger children.

Haploidentical transplantation with PTCY following marrow or PBSC graft has been associated with low incidence of GVHD in adults with similar data lacking in children. We report on the outcome of 25 patients <20 yr of age (median age 12 yr), undergoing a haploidentical PBSC transplantation for both malignant and non-malignant disorders. Engraftment was prompt and sustained. Cumulative incidences of acute GVHD and chronic GVHD were 40.3% and 16.7%, respectively. On subgroup analysis, it was evident that acute GVHD developed in 80% of patients <10 yr compared to only 13.3% in those between 10 and 20 yr [log-rank p = 0.001], despite similar graft composition with significantly higher NRM (60% vs. 0%; p = 0.001). The FFS was 63.5%; (79% in >10 yr and 40% in <10 yr, p = 0.01). Our data suggest that PTCY-based haploidentical PBSC transplantation is feasible in older children, but results in early and severe alloreactivity in younger children. These findings, despite being counterintuitive, could be explained by the variable metabolism of CY and oral mycophenolate in younger children indicating that PTCY-based approach as used in adults might not be adequate for younger children.

Attraction of mesoscale objects on the surface of a thin elastic film supported on a liquid.

We study the interaction of two parallel rigid cylinders on the surface of a thin elastic film supported on a pool of liquid. The excess energy of the surface due to the curvature of the stretched film induces attraction of the cylinders that can be quantified by the variation of their gravitational potential energies as they descend into the liquid while still floating on the film. Although the experimental results follow the trend predicted from the balance of the gravitational and elastic energies of the system, they are somewhat underestimated. The origin of this discrepancy is the hysteresis of adhesion between the cylinder and the elastic film that does not allow the conversion of the total available energy into gravitational potential energy, as some part of it is recovered in stretching the film behind the cylinders while they approach each other. A modification of the model accounting for the effects of adhesion hysteresis improves the agreement between theoretical and experimental results. The contribution of the adhesion hysteresis can be reduced considerably by introducing a thin hydrogel layer atop the elastic film that enhances the range of attraction of the cylinders (as well as rigid spheres) in a dramatic way. Morphological instabilities in the gel project corrugated paths to the motion of small spheres, thus leading to a large numbers of particles to aggregate along their defects. These observations suggest that a thin hydrogel layer supported on a deformable elastic film affords an effective model system to study elasticity and defects mediated interaction of particles on its surface.

Generation of Motion of Drops with Interfacial Contact.

A liquid drop moves on a solid surface if it is subjected to a gradient of wettability or temperature. However, the pinning defects on the surface manifested in terms of a wetting hysteresis, or first-order nonlinear friction, limit the motion in the sense that a critical size has to be exceeded for a drop to move. The effect of hysteresis can, however, be mitigated by an external vibration that can be either structured or stochastic, thereby creating a directed motion of the drop. Many of the well-known features of rectification, amplification, and switching that are generic to electronics can be engineered with such types of movements. A specific case of interest is the random coalescence of drops on a surface that gives rise to self-generated noise. This noise overcomes the pinning potential, thereby generating a random motion of the coalesced drops. Randomly moving coalesced drops themselves exhibit a directed diffusive flux when a boundary is present to eliminate them by absorption. With the presence of a bias, the coalesced drops execute a diffusive drift motion that can have useful applications in various water and thermal management technologies.

Soft Lithography Using Nectar Droplets.

In spite of significant advances in replication technologies, methods to produce well-defined three-dimensional structures are still at its infancy. Such a limitation would be evident if we were to produce a large array of simple and, especially, compound convex lenses, also guaranteeing that their surfaces would be molecularly smooth. Here, we report a novel method to produce such structures by cloning the 3D shape of nectar drops, found widely in nature, using conventional soft lithography.The elementary process involves transfer of a thin patch of the sugar solution coated on a glass slide onto a hydrophobic substrate on which this patch evolves into a microdroplet. Upon the absorption of water vapor, such a microdroplet grows linearly with time, and its final size can be controlled by varying its exposure time to water vapor. At any stage of the evolution of the size of the drop, its shape can be cloned onto a soft elastomer by following the well-known methods of molding and cross-linking the same. A unique new science that emerges in our attempt to understand the transfer of the sugar patch and its evolution to a spherical drop is the elucidation of the mechanics underlying the contact of a deformable sphere against a solid support intervening a thin liquid film. A unique aspect of this work is to demonstrate that higher level structures can also be generated by transferring even smaller nucleation sites on the surface of the primary lenses and then allowing them to grow by absorption of water vapor. What results at the end is either a well-controlled distribution of smooth hemispherical lenses or compound structures that could have potential applications in the fundamental studies of contact mechanics, wettability, and even in optics.

Adhesion-induced instabilities and pattern formation in thin films of elastomers and gels.

A hydrostatically stressed soft elastic film circumvents the imposed constraint by undergoing a morphological instability, the wavelength of which is dictated by the minimization of the surface and the elastic strain energies of the film. While for a single film, the wavelength is entirely dependent on its thickness, a co-operative energy minimization dictates that the wavelength depends on both the elastic moduli and thicknesses of two contacting films. The wavelength can also depend on the material properties of a film if its surface tension has a pronounced effect in comparison to its elasticity. When such a confined film is subjected to a continually increasing normal displacement, the morphological patterns evolve into cracks, which, in turn, govern the adhesive fracture behavior of the interface. While, in general, the thickness provides the relevant length scale underlying the well-known Griffith-Kendall criterion of debonding of a rigid disc from a confined film, it is modified non-trivially by the elasto-capillary number for an ultra-soft film. Depending upon the degree of confinement and the spatial distribution of external stress, various analogs of the canonical instability patterns in liquid systems can also be reproduced with thin confined elastic films.

Elastocapillary interaction of particles on the surfaces of ultrasoft gels: a novel route to study self-assembly and soft lubrication.

We study the interaction of small hydrophobic particles on the surface of an ultrasoft elastic gel, in which a small amount of elasticity of the medium balances the weights of the particles. The excess energy of the surface of the deformed gel causes them to attract as is the case with the generic capillary interactions of particles on a liquid surface. The variation of the gravitational potential energies of the particles resulting from their descents in the gel coupled with the superposition principle of Nicolson allow a fair estimation of the distance dependent attractive energy of the particles. This energy follows a modified Bessel function of the second kind with a characteristic elastocapillary decay length that decreases with the elasticity of the medium. An interesting finding of this study is that the particles on the gel move toward each other as if the system possesses a negative diffusivity that is inversely proportional to friction. This study illustrates how the capillary interaction of particles is modified by the elasticity of the medium, which is expected to have important implications in the surface force driven self-assembly of particles. In particular, this study points out that the range and the strength of the capillary interaction can be tuned in by appropriate choices of the elasticity of the support and the interfacial tension of the surrounding medium. Manipulation of the particle interactions is exemplified in such fascinating mimicry of the biological processes as the tubulation and phagocytic engulfment and in the assembly of particles that can be used to study nucleation and clustering phenomena in well-controlled settings.