Linking candidate causal autoimmune variants to T cell networks using genetic and epigenetic screens in primary human T cells.

Genetic variants associated with autoimmune diseases are highly enriched within putative cis -regulatory regions of CD4 + T cells, suggesting that they alter disease risk via changes in gene regulation. However, very few genetic variants have been shown to affect T cell gene expression or function. We tested >18,000 autoimmune disease-associated variants for allele-specific expression using massively parallel reporter assays in primary human CD4 + T cells. The 545 expression-modulating variants (emVars) identified greatly enrich for likely causal variants. We provide evidence that many emVars are mediated by common upstream regulatory conduits, and that putative target genes of primary T cell emVars are highly enriched within a lymphocyte activation network. Using bulk and single-cell CRISPR-interference screens, we confirm that emVar-containing T cell cis -regulatory elements modulate both known and novel target genes that regulate T cell proliferation, providing plausible mechanisms by which these variants alter autoimmune disease risk.

Three-dimensional super-resolution passive cavitation mapping in laser lithotripsy.

Kidney stone disease is a major public health issue. By breaking stones with repeated laser irradiation, laser lithotripsy (LL) has become the main treatment for kidney stone disease. Laser-induced cavitation is closely associated with the stone damage in LL. Monitoring the cavitation activities during LL is thus crucial to optimizing the stone damage and maximizing LL efficiency. In this study, we have developed three-dimensional super-resolution passive cavitation mapping (3D-SRPCM), in which the cavitation bubble positions can be localized with an accuracy of 40 µm, which is 1/10th of the acoustic diffraction limit. Moreover, the 3D-SRPCM reconstruction speed has been improved by 300 times by adopting a GPU-based sparse-matrix beamforming approach. Using 3D-SRPCM, we studied LL-induced cavitation activities on BegoStones, both in free space of water and confined space of a kidney phantom. The dose-dependence analysis provided by 3D-SRPCM revealed that accumulated impact pressure on the stone surface has the highest correlation with the stone damage. By providing high-resolution cavitation mapping during LL treatment, we expect that 3D-SRPCM may become a powerful tool to improve the clinical LL efficiency and patient outcome.

Identification of functional enhancer variants associated with type I diabetes in CD4+ T cells.

Type I diabetes is an autoimmune disease mediated by T-cell destruction of ß cells in pancreatic islets. Currently, there is no known cure, and treatment consists of daily insulin injections. Genome-wide association studies and twin studies have indicated a strong genetic heritability for type I diabetes and implicated several genes. As most strongly associated variants are noncoding, there is still a lack of identification of functional and, therefore, likely causal variants. Given that many of these genetic variants reside in enhancer elements, we have tested 121 CD4+ T-cell enhancer variants associated with T1D. We found four to be functional through massively parallel reporter assays. Three of the enhancer variants weaken activity, while the fourth strengthens activity. We link these to their cognate genes using 3D genome architecture or eQTL data and validate them using CRISPR editing. Validated target genes include CLEC16A and SOCS1. While these genes have been previously implicated in type 1 diabetes and other autoimmune diseases, we show that enhancers controlling their expression harbor functional variants. These variants, therefore, may act as causal type 1 diabetic variants.

Bubble oscillations at low frequency ultrasound for biological applications.

Bubbles oscillating in the presence of ultrasound is commonly employed in biomedical applications for drug delivery, ultrasound enhanced thrombolysis, and the transport and manipulation of cells. This is possible because bubbles tend to interact with the ultrasound to undergo periodic shape changes known as shape-mode oscillation, concomitant with the generation of liquid agitation or streaming. This phenomenon is examined both experimentally and theoretically on a single bubble at a frequency of (45 ± 1) kHz. Effects of ultrasonic frequency and power on the flowfield were explored. Experiments revealed different trends in the development of liquid streaming velocities at different acoustic forcing conditions (5.53, 6.80 and 7.02 Vpp), with lowest (0.5 mm/s) and highest (1.1 mm/s) values of time-averaged mean streaming velocity occurring at 6.80 Vpp and 7.02 Vpp, respectively. Simulations captured the simultaneous evolution of bubble-shapes that helped create flow vortices in the liquid surrounding the bubble. These vortices collectively responsible in generating signature patterns in the liquid for a dominant shape-mode of the bubble, and could also generate localised shear stresses for practical application. The velocity and pressure profiles in the liquid around the bubble confirmed the connection of the applied and reflected soundwaves in driving this phenomenon.

Subphase Exchange Cell for Studying Fluid-Fluid Interfaces with Optical Microscopy.

A subphase exchange cell was designed to observe fluid-fluid interfaces with a conventional optical microscope while simultaneously changing the subphase chemistry. Materials including phospholipids, asphaltenes, and nanoparticles at fluid-fluid interfaces exhibit unique morphological changes as a function of the bulk-phase chemistry. These changes can affect their interfacial material properties and, ultimately, the emergent bulk material properties of the films, foams, and emulsions produced from such interfacial systems. In this work, we combine experiments, computational fluid dynamics simulations, and modeling to establish the operating parameters for a subphase exchange cell of this type to reach a desired concentration. We used the experimental setup to investigate changes to a graphene film during a common wet-etching transfer process. Observations reveal that capillary interactions can induce defects and deformations in the graphene film during the wet-etching process, an important finding that must be considered for any wet-etching transfer technique for 2D materials. More generally, conventional optical microscopy was shown to be able to image the dynamics of interfacial systems during a bulk-phase chemistry change. Potential applications for this equipment and technique include observing morphological dynamics of phospholipid film structure with subphase salinity, asphaltene film structure with subphase pH, and particle film synthesis with subphase chemistry.

Exploring optimal settings for safe and effective thulium fibre laser lithotripsy in a kidney model.

OBJECTIVES: To explore the optimal laser settings and treatment strategies for thulium fibre laser (TFL) lithotripsy, namely, those with the highest treatment efficiency, lowest thermal injury risk, and shortest procedure time. MATERIALS AND METHODS: An in vitro kidney model was used to assess the efficacy of TFL lithotripsy in the upper calyx. Stone ablation experiments were performed on BegoStone phantoms at different combinations of pulse energy (EP ) and frequency (F) to determine the optimal settings. Temperature changes and thermal injury risks were monitored using embedded thermocouples. Experiments were also performed on calcium oxalate monohydrate (COM) stones to validate the optimal settings. RESULTS: High EP /low F settings demonstrated superior treatment efficiency compared to low EP /high F settings using the same power. Specifically, 0.8 J/12 Hz was the optimal setting, resulting in a twofold increase in treatment efficiency, a 39% reduction in energy expenditure per unit of ablated stone mass, a 35% reduction in residual fragments, and a 36% reduction in total procedure time compared to the 0.2 J/50 Hz setting for COM stones. Thermal injury risk assessment indicated that 10 W power settings with high EP /low F combinations remained below the threshold for tissue injury, while higher power settings (>10 W) consistently exceeded the safety threshold. CONCLUSIONS: Our findings suggest that high EP /low F settings, such as 0.8 J/12 Hz, are optimal for TFL lithotripsy in the treatment of COM stones. These settings demonstrated significantly improved treatment efficiency with reduced residual fragments compared to conventional settings while keeping the thermal dose below the injury threshold. This study highlights the importance of using the high EP /low F combination with low power settings, which maximizes treatment efficiency and minimizes potential thermal injury. Further studies are warranted to determine the optimal settings for TFL for treating kidney stones with different compositions.

In vitro investigation of stone ablation efficiency, char formation, spark generation, and damage mechanism produced by thulium fiber laser.

To investigate stone ablation characteristics of thulium fiber laser (TFL), BegoStone phantoms were spot-treated in water at various fiber tip-to-stone standoff distances (SDs, 0.5 ~ 2 mm) over a broad range of pulse energy (Ep, 0.2 ~ 2 J), frequency (F, 5 ~ 150 Hz), and power (P, 10 ~ 30 W) settings. In general, the ablation speed (mm3/s) in BegoStone decreased with SD and increased with Ep, reaching a peak around 0.8 ~ 1.0 J. Additional experiments with calcium phosphate (CaP), uric acid (UA), and calcium oxalate monohydrate (COM) stones were conducted under two distinctly different settings: 0.2 J/100 Hz and 0.8 J/12 Hz. The concomitant bubble dynamics, spark generation and pressure transients were analyzed. Higher ablation speeds were consistently produced at 0.8 J/12 Hz than at 0.2 J/100 Hz, with CaP stones most difficult yet COM and UA stones easier to ablate. Charring was mostly observed in CaP stones at 0.2 J/100 Hz, accompanied by strong spark-generation, explosive combustion, and diminished pressure transients, but not at 0.8 J/12 Hz. By treating stones in parallel fiber orientation and leveraging the proximity effect of a ureteroscope, the contribution of bubble collapse to stone ablation was found to be substantial (16% ~ 59%) at 0.8 J/12 Hz, but not at 0.2 J/100 Hz. Overall, TFL ablation efficiency is significantly better at high Ep/low F setting, attributable to increased cavitation damage with less char formation.

Dissimilar cavitation dynamics and damage patterns produced by parallel fiber alignment to the stone surface in holmium:yttrium aluminum garnet laser lithotripsy.

Recent studies indicate that cavitation may play a vital role in laser lithotripsy. However, the underlying bubble dynamics and associated damage mechanisms are largely unknown. In this study, we use ultra-high-speed shadowgraph imaging, hydrophone measurements, three-dimensional passive cavitation mapping (3D-PCM), and phantom test to investigate the transient dynamics of vapor bubbles induced by a holmium:yttrium aluminum garnet laser and their correlation with solid damage. We vary the standoff distance (SD) between the fiber tip and solid boundary under parallel fiber alignment and observe several distinctive features in bubble dynamics. First, long pulsed laser irradiation and solid boundary interaction create an elongated "pear-shaped" bubble that collapses asymmetrically and forms multiple jets in sequence. Second, unlike nanosecond laser-induced cavitation bubbles, jet impact on solid boundary generates negligible pressure transients and causes no direct damage. A non-circular toroidal bubble forms, particularly following the primary and secondary bubble collapses at SD = 1.0 and 3.0 mm, respectively. We observe three intensified bubble collapses with strong shock wave emissions: the intensified bubble collapse by shock wave, the ensuing reflected shock wave from the solid boundary, and self-intensified collapse of an inverted "triangle-shaped" or "horseshoe-shaped" bubble. Third, high-speed shadowgraph imaging and 3D-PCM confirm that the shock origins from the distinctive bubble collapse form either two discrete spots or a "smiling-face" shape. The spatial collapse pattern is consistent with the similar BegoStone surface damage, suggesting that the shockwave emissions during the intensified asymmetric collapse of the pear-shaped bubble are decisive for the solid damage.

Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts.

The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.

Technical, economic and environmental feasibility of resource recovery technologies from wastewater.

An enormous amount of wastewater is generated across the world from different industrial or municipal sectors. Traditional wastewater treatment plants (WWTP) have primarily focused on the treatment of wastewater rather than the recovery of valuable resources. A shift from a linear to a circular economy may offer a unique platform for recovering valuable resources including energy, nutrients, and high-value goods from wastewater. However, transitioning from conventional frameworks to sustainable WWT systems remains a significant challenge. Thus, this review paper focuses on the avenues of resource recovery from WWTPs, by evaluating the potential for nutrients, water, and energy recovery from different types of wastewaters and sewage sludge. It discusses in detail a variety of available and advanced technologies for resource recovery. Further, the feasibility of these technologies from a sustainable standpoint is discussed, covering the technical, economic, and environmental facets.

In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal γ-globin in ß-YAC mice.

Base editors are capable of installing precise genomic alterations without creating double-strand DNA breaks. In this study, we targeted critical motifs regulating γ-globin reactivation with base editors delivered via HDAd5/35++ vectors. Through optimized design, we successfully produced a panel of cytidine and adenine base editor (ABE) vectors targeting the erythroid BCL11A enhancer or recreating naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations in the HBG1/2 promoter. All 5 tested vectors efficiently installed target base conversion and led to γ-globin reactivation in human erythroid progenitor cells. We observed ~23% γ-globin protein production over ß-globin, when using an ABE vector (HDAd-ABE-sgHBG-2) specific to the -113A>G HPFH mutation. In a ß-YAC mouse model, in vivo hematopoietic progenitor/stem cell (HSPC) transduction with HDAd-ABE-sgHBG-2 followed by in vivo selection resulted in >40% γ-globin+ erythrocytes in the peripheral blood. This result corresponded to 21% γ-globin production over human ß-globin. The average -113A>G conversion in total bone marrow cells was 20%. No alterations in hematological parameters, erythropoiesis, and bone marrow cellular composition were observed after treatment. No detectable editing was found at top-scoring, off-target genomic sites. Bone marrow lineage-negative cells from primary mice were capable of reconstituting secondary transplant-recipient mice with stable γ-globin expression. Importantly, the advantage of base editing over CRISPR/Cas9 was reflected by the markedly lower rates of intergenic HBG1/2 deletion and the absence of detectable toxicity in human CD34+ cells. Our observations suggest that HDAd-vectorized base editors represent a promising strategy for precise in vivo genome engineering for the treatment of ß-hemoglobinopathies.

Ab initio Description of Bond Breaking in Large Electric Fields.

Strong (10^{10} V/m) electric fields capable of inducing atomic bond breaking represent a powerful tool for surface chemistry. However, their exact effects are difficult to predict due to a lack of suitable tools to probe their associated atomic-scale mechanisms. Here we introduce a generalized dipole correction for charged repeated-slab models that controls the electric field on both sides of the slab, thereby enabling direct theoretical treatment of field-induced bond-breaking events. As a prototype application, we consider field evaporation from a kinked W surface. We reveal two qualitatively different desorption mechanisms that can be selected by the magnitude of the applied field.

Candidate silencer elements for the human and mouse genomes.

The study of gene regulation is dominated by a focus on the control of gene activation or increase in the level of expression. Just as critical is the process of gene repression or silencing. Chromatin signatures have identified enhancers, however, genome-wide identification of silencers by computational or experimental approaches are lacking. Here, we first define uncharacterized cis-regulatory elements likely containing silencers and find that 41.5% of ~7500 tested elements show silencer activity using massively parallel reporter assay (MPRA). We trained a support vector machine classifier based on MPRA data to predict candidate silencers in over 100 human and mouse cell or tissue types. The predicted candidate silencers exhibit characteristics expected of silencers. Leveraging promoter-capture HiC data, we find that over 50% of silencers are interacting with gene promoters having very low to no expression. Our results suggest a general strategy for genome-wide identification and characterization of silencer elements.

Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing.

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

Targeted Integration and High-Level Transgene Expression in AAVS1 Transgenic Mice after In Vivo HSC Transduction with HDAd5/35++ Vectors.

Our goal is the development of in vivo hematopoietic stem cell (HSC) transduction technology with targeted integration. To achieve this, we modified helper-dependent HDAd5/35++ vectors to express a CRISPR/Cas9 specific to the "safe harbor" adeno-associated virus integration site 1 (AAVS1) locus and to provide a donor template for targeted integration through homology-dependent repair. We tested the HDAd-CRISPR + HDAd-donor vector system in AAVS1 transgenic mice using a standard ex vivo HSC gene therapy approach as well as a new in vivo HSC transduction approach that involves HSC mobilization and intravenous HDAd5/35++ injections. In both settings, the majority of treated mice had transgenes (GFP or human γ-globin) integrated into the AAVS1 locus. On average, >60% of peripheral blood cells expressed the transgene after in vivo selection with low-dose O6BG/bis-chloroethylnitrosourea (BCNU). Ex vivo and in vivo HSC transduction and selection studies with HDAd-CRISPR + HDAd-globin-donor resulted in stable γ-globin expression at levels that were significantly higher (>20% γ-globin of adult mouse globin) than those achieved in previous studies with a SB100x-transposase-based HDAd5/35++ system that mediates random integration. The ability to achieve therapeutically relevant transgene expression levels after in vivo HSC transduction and selection and targeted integration make our HDAd5/35++-based vector system a new tool in HSC gene therapy.

Three-dimensional genome architecture and emerging technologies: looping in disease.

Genome compaction is a universal feature of cells and has emerged as a global regulator of gene expression. Compaction is maintained by a multitude of architectural proteins, long non-coding RNAs (lncRNAs), and regulatory DNA. Each component comprises interlinked regulatory circuits that organize the genome in three-dimensional (3D) space to manage gene expression. In this review, we update the current state of 3D genome catalogues and focus on how recent technological advances in 3D genomics are leading to an enhanced understanding of disease mechanisms. We highlight the use of genome-wide chromatin conformation capture (Hi-C) coupled with oligonucleotide capture technology (capture Hi-C) to map interactions between gene promoters and distal regulatory elements such as enhancers that are enriched for disease variants from genome-wide association studies (GWASs). We discuss how aberrations in architectural units are associated with various pathological outcomes, and explore how recent advances in genome and epigenome editing show great promise for a systematic understanding of complex genetic disorders. Our growing understanding of 3D genome architecture-coupled with the ability to engineer changes in it-may create novel therapeutic opportunities.

Influence of Ionic Liquids on Thermodynamics of Small Molecule-DNA Interaction: The Binding of Ethidium Bromide to Calf Thymus DNA.

Ionic liquids (ILs) are salts with poor ionic coordination, resultantly remaining in liquid state below 100 °C and some may retain liquid state even at room temperature. ILs are known to provide a conducive environment for many biological enzymatic reactions, but their interaction with biomacromolecules are poorly understood. In the present study, we investigate the effect of various ionic liquids on DNA-small molecule interaction using calf thymus DNA (ctDNA)-ethidium bromide (EB) as a model system. The effect of various ionic liquids on these interactions is studied by an array of techniques such as circular dichroism (CD), UV melting, fluorescence exclusion and isothermal titration calorimetry. Interestingly, we observed that presence of IL increased the stability of ctDNA without altering its structure. The binding affinities Kbs for EB binding to ctDNA in the presence of 300 mM ILs are about half order of magnitude smaller than the Kbs in absence of ILs and correspond to a less favorable free energy. We noted that, when adjusted to corresponding buffer condition, the unfavorable shift in ΔG of ctDNA-EB interaction is attributed to decreased entropy in the case of ILs, whereas the same effect by NaCl was due to increased enthalpy.

The structure of Rv3717 reveals a novel amidase from Mycobacterium tuberculosis.

Bacterial N-acetylmuramoyl-L-alanine amidases are cell-wall hydrolases that hydrolyze the bond between N-acetylmuramic acid and L-alanine in cell-wall glycopeptides. Rv3717 of Mycobacterium tuberculosis has been identified as a unique autolysin that lacks a cell-wall-binding domain (CBD) and its structure has been determined to 1.7 Šresolution by the Pt-SAD phasing method. Rv3717 possesses an α/ß-fold and is a zinc-dependent hydrolase. The structure reveals a short flexible hairpin turn that partially occludes the active site and may be involved in autoregulation. This type of autoregulation of activity of PG hydrolases has been observed in Bartonella henselae amidase (AmiB) and may be a general mechanism used by some of the redundant amidases to regulate cell-wall hydrolase activity in bacteria. Rv3717 utilizes its net positive charge for substrate binding and exhibits activity towards a broad spectrum of substrate cell walls. The enzymatic activity of Rv3717 was confirmed by isolation and identification of its enzymatic products by LC/MS. These studies indicate that Rv3717, an N-acetylmuramoyl-L-alanine amidase from M. tuberculosis, represents a new family of lytic amidases that do not have a separate CBD and are regulated conformationally.

Integration host factor of Mycobacterium tuberculosis, mIHF, compacts DNA by a bending mechanism.

The bacterial chromosomal DNA is folded into a compact structure called as 'nucleoid' so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like "PAKK" motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF) for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of E. coli and mycobacterial IHF in DNA compaction.