R-Loop Accumulation in Spliceosome Mutant Leukemias Confers Sensitivity to PARP1 Inhibition by Triggering Transcription-Replication Conflicts.

RNA splicing factor (SF) gene mutations are commonly observed in patients with myeloid malignancies. Here we showed that SRSF2- and U2AF1-mutant leukemias are preferentially sensitive to PARP inhibitors (PARPi), despite being proficient in homologous recombination repair. Instead, SF-mutant leukemias exhibited R-loop accumulation that elicited an R-loop-associated PARP1 response, rendering cells dependent on PARP1 activity for survival. Consequently, PARPi induced DNA damage and cell death in SF-mutant leukemias in an R-loop-dependent manner. PARPi further increased aberrant R-loop levels, causing higher transcription-replication collisions and triggering ATR activation in SF-mutant leukemias. Ultimately, PARPi-induced DNA damage and cell death in SF-mutant leukemias could be enhanced by ATR inhibition. Finally, the level of PARP1 activity at R-loops correlated with PARPi sensitivity, suggesting that R-loop-associated PARP1 activity could be predictive of PARPi sensitivity in patients harboring SF gene mutations. This study highlights the potential of targeting different R-loop response pathways caused by spliceosome gene mutations as a therapeutic strategy for treating cancer. SIGNIFICANCE: Spliceosome-mutant leukemias accumulate R-loops and require PARP1 to resolve transcription-replication conflicts and genomic instability, providing rationale to repurpose FDA-approved PARP inhibitors for patients carrying spliceosome gene mutations.

Stepwise differentiation of follicular helper T cells reveals distinct developmental and functional states.

Follicular helper T (Tfh) cells are essential for the formation of high affinity antibodies after vaccination or infection. Although the signals responsible for initiating Tfh differentiation from naïve T cells have been studied, the signals controlling sequential developmental stages culminating in optimal effector function are not well understood. Here we use fate mapping strategies for the cytokine IL-21 to uncover sequential developmental stages of Tfh differentiation including a progenitor-like stage, a fully developed effector stage and a post-effector Tfh stage that maintains transcriptional and epigenetic features without IL-21 production. We find that progression through these stages are controlled intrinsically by the transcription factor FoxP1 and extrinsically by follicular regulatory T cells. Through selective deletion of Tfh stages, we show that these cells control antibody dynamics during distinct stages of the germinal center reaction in response to a SARS-CoV-2 vaccine. Together, these studies demonstrate the sequential phases of Tfh development and how they promote humoral immunity.

Building block aspect ratio controls assembly, architecture, and mechanics of synthetic and natural protein networks.

Fibrous networks constructed from high aspect ratio protein building blocks are ubiquitous in nature. Despite this ubiquity, the functional advantage of such building blocks over globular proteins is not understood. To answer this question, we engineered hydrogel network building blocks with varying numbers of protein L domains to control the aspect ratio. The mechanical and structural properties of photochemically crosslinked protein L networks were then characterised using shear rheology and small angle neutron scattering. We show that aspect ratio is a crucial property that defines network architecture and mechanics, by shifting the formation from translationally diffusion dominated to rotationally diffusion dominated. Additionally, we demonstrate that a similar transition is observed in the model living system: fibrin blood clot networks. The functional advantages of this transition are increased mechanical strength and the rapid assembly of homogenous networks above a critical protein concentration, crucial for in vivo biological processes such as blood clotting. In addition, manipulating aspect ratio also provides a parameter in the design of future bio-mimetic and bio-inspired materials.

Transcriptionally Distinct B Cells Infiltrate Allografts After Kidney Transplantation.

BACKGROUND: Following allogeneic kidney transplantation, a substantial proportion of graft loss is attributed to the formation of donor-specific antibodies and antibody-mediated rejection. B cells infiltrate kidney grafts during antibody-mediated rejection; however, the origins, repertoires, and functions of these intrarenal B cells remain elusive. METHODS: Here, we use murine allogeneic kidney transplant models to study the origins, transcriptional programming and B cell receptor repertoire of intragraft B cells, and in vitro stimulation assays to evaluate the ability of intragraft B cells to promote CD4+ T cell expansion. RESULTS: B cells infiltrate kidney grafts in settings of allogeneic, but not syngeneic, transplantation. Intragraft B cells have characteristics of activation but are transcriptionally distinct from germinal center B cells and resemble innate-like B cells. B cell receptor sequencing demonstrates that the majority of intragraft B cells do not originate from lymph node germinal center B cells and are largely germline. Class-switched intragraft B cells are rare but can be donor-specific and produce IgG capable of binding to the kidney allograft. Lastly, intrarenal B cells are capable of stimulating naive T cells but have an altered ability to promote T follicular helper cell expansion. CONCLUSIONS: Together, these data demonstrate that intrarenal B cells during transplant rejection are transcriptionally distinct from lymph node B cells.

Follicular T cells optimize the germinal center response to SARS-CoV-2 protein vaccination in mice.

Follicular helper T (Tfh) cells promote, whereas follicular regulatory T (Tfr) cells restrain, germinal center (GC) reactions. However, the precise roles of these cells in the complex GC reaction remain poorly understood. Here, we perturb Tfh or Tfr cells after SARS-CoV-2 spike protein vaccination in mice. We find that Tfh cells promote the frequency and somatic hypermutation (SHM) of Spike-specific GC B cells and regulate clonal diversity. Tfr cells similarly control SHM and clonal diversity in the GC but do so by limiting clonal competition. In addition, deletion of Tfh or Tfr cells during primary vaccination results in changes in SHM after vaccine boosting. Aged mice, which have altered Tfh and Tfr cells, have lower GC responses, presenting a bimodal distribution of SHM. Together, these data demonstrate that GC responses to SARS-CoV-2 spike protein vaccines require a fine balance of positive and negative follicular T cell help to optimize humoral immunity.

Tfh-Mediated and Tfr-Suppressed Antigen-Driven IgG and IgE Assays.

T Follicular helper (Tfh) cells stimulate, whereas T follicular regulatory (Tfr) cells inhibit, effector B cell responses. Although new tools have been developed to assess the functional roles of Tfh and Tfr cells in vivo, methods to assess mechanisms have been limited. One such limitation has been the ability of in vitro functional assays to recapitulate robust germinal center-like responses. Although previous in vitro Tfh-mediated and Tfr-suppressed assays to assess antibody regulation have been developed, these classically have relied on polyclonal stimulation. To understand Tfh and Tfr cell functionality, more robust assays that utilize specific antigen are needed. Here we describe an in vitro approach for sensitively and quantitatively assessing the capacity of Tfh and Tfr cells to regulate B cell responses in an antigen-driven system. These assays allow the study of Tfh and Tfr cells in specific disease contexts, such as IgG production after vaccination or IgE responses during allergic airway disease.

Intermediate Structural Hierarchy in Biological Networks Modulates the Fractal Dimension and Force Distribution of Percolating Clusters.

Globular protein hydrogels are an emerging class of materials with the potential for rational design, and a generalized understanding of how their network properties emerge from the structure and dynamics of the building block is a key challenge. Here we computationally investigate the effect of intermediate (polymeric) nanoscale structure on the formation of protein hydrogels. We show that changes in both the cross-link topology and flexibility of the polymeric building block lead to changes in the force transmission around the system and provide insight into the dynamic network formation processes. The preassembled intermediate structure provides a novel structural coordinate for the hierarchical modulation of macroscopic network properties, as well as furthering our understanding of the general dynamics of network formation.

Synthesis and Propulsion of Magnetic Dimers under Orthogonally Applied Electric and Magnetic Fields.

Anisotropic particles have been widely used to make micro/nanomotors that convert chemical, ultrasonic, electrical, or magnetic energy into mechanical energy. The moving directions of most colloidal motors are, however, difficult to control. For example, asymmetric dimers with two lobes of different sizes, ζ-potential, or chemical composition have shown rich propulsion behaviors under alternating current (AC) electric fields due to unbalanced electrohydrodynamic flow. While they always propel in a direction perpendicular to the applied electric field, their moving directions along the substrate are hard to control, limiting their applications for cargo delivery. Inspired by two separate engine and steering wheel systems in automobiles, we use orthogonally applied AC electric field and direct current (DC) magnetic field to control the dimer's speed and direction independently. To this end, we first synthesize magnetic dimers by coating dopamine-functionalized nanoparticles on geometrically asymmetric polystyrene dimers. We further characterize their static and dynamic susceptibilities by measuring the hysteresis diagram and rotation speed experimentally and comparing them with theoretical predictions. The synthesized dimers align their long axes quickly with a planar DC magnetic field, allowing us to control the particles' orientation accurately. The propulsion speed of the dimers, on the other hand, is tunable by an AC electric field applied perpendicularly to the substrate. As a result, we can direct the particle's motion with predesigned trajectories of complex shapes. Our bulk-synthesis approach has the potential to make other types of magnetically anisotropic particles. And the combination of electric and magnetic fields will help pave the way for the assembly of magnetically anisotropic particles into complex structures.

Control of Nanoscale In Situ Protein Unfolding Defines Network Architecture and Mechanics of Protein Hydrogels.

Hierarchical assemblies of proteins exhibit a wide-range of material properties that are exploited both in nature and by artificially by humankind. However, little is understood about the importance of protein unfolding on the network assembly, severely limiting opportunities to utilize this nanoscale transition in the development of biomimetic and bioinspired materials. Here we control the force lability of a single protein building block, bovine serum albumin (BSA), and demonstrate that protein unfolding plays a critical role in defining the architecture and mechanics of a photochemically cross-linked native protein network. The internal nanoscale structure of BSA contains "molecular reinforcement" in the form of 17 covalent disulphide "nanostaples", preventing force-induced unfolding. Upon addition of reducing agents, these nanostaples are broken rendering the protein force labile. Employing a combination of circular dichroism (CD) spectroscopy, small-angle scattering (SAS), rheology, and modeling, we show that stapled protein forms reasonably homogeneous networks of cross-linked fractal-like clusters connected by an intercluster region of folded protein. Conversely, in situ protein unfolding results in more heterogeneous networks of denser fractal-like clusters connected by an intercluster region populated by unfolded protein. In addition, gelation-induced protein unfolding and cross-linking in the intercluster region changes the hydrogel mechanics, as measured by a 3-fold enhancement of the storage modulus, an increase in both the loss ratio and energy dissipation, and markedly different relaxation behavior. By controlling the protein's ability to unfold through nanoscale (un)stapling, we demonstrate the importance of in situ unfolding in defining both network architecture and mechanics, providing insight into fundamental hierarchical mechanics and a route to tune biomaterials for future applications.

PK11195 Protects From Cell Death Only When Applied During Reperfusion: Succinate-Mediated Mechanism of Action.

Aim: Reperfusion after myocardial ischemia causes cellular injury, in part due to changes in mitochondrial Ca2+ handling, oxidative stress, and myocyte energetics. We have previously shown that the 18-kDa translocator protein of the outer mitochondrial membrane (TSPO) can modulate Ca2+ handling. Here, we aim to evaluate the role of the TSPO in ischemia/reperfusion (I/R) injury. Methods: Rabbit ventricular myocytes underwent simulated acute ischemia (20 min) and reperfusion (at 15 min, 1 h, and 3 h) in the absence and presence of 50 µM PK11195, a TSPO inhibitor. Cell death was measured by lactate dehydrogenase (LDH) assay, while changes in mitochondrial Ca2+, membrane potential (ΔΨm), and reactive oxygen species (ROS) generation were monitored using confocal microscopy in combination with fluorescent indicators. Substrate utilization was measured with Biolog mitochondrial plates. Results: Cell death was increased by ~200% following I/R compared to control untreated ventricular myocytes. Incubation with 50 µM PK11195 during both ischemia and reperfusion did not reduce cell death but increased mitochondrial Ca2+ uptake and ROS generation. However, application of 50 µM PK11195 only at the onset and during reperfusion effectively protected against cell death. The large-scale oscillations in ΔΨm observed after ~1 h of reperfusion were significantly delayed by 1 µM cyclosporin A and almost completely prevented by 50 µM PK11195 applied during 3 h of reperfusion. After an initial increase, mitochondrial Ca2+, measured with Myticam, rapidly declined during 3 h of reperfusion after the initial transient increase. This decline was prevented by application of PK11195 at the onset and during reperfusion. PK11195 prevented a significant increase in succinate utilization following I/R and succinate-induced forward-mode ROS generation. Treatment with PK11195 was also associated with a significant increase in glutamate and a decrease in leucine utilization. Conclusion: PK11195 administered specifically at the moment of reperfusion limited ROS-induced ROS release and cell death, likely in part, by a shift from succinate to glutamate utilization. These data demonstrate a unique mechanism to limit cardiac injury after I/R.

Impaired host response and the presence of Acinetobacter baumannii in the serum microbiome of type-II diabetic patients.

Type II diabetes (T2D) affects over 10% of the US population and is a growing disease worldwide that manifests with numerous comorbidities and defects in inflammation. This dysbiotic host response allows for infection of the host by numerous microorganisms. In the course of T2D disease, individuals can develop chronic infections including foot ulcers and periodontitis, which lead to further complications and opportunistic infections in multiple body sites. In this study, we investigated the serum of healthy subjects and patients with T2D with (T2DP) or without periodontitis for both microbiome signatures in addition to cytokine profiles. Surprisingly, we detected the presence of Acinetobacter baumanii in the serum of 23% individuals with T2D/T2DP tested. In T2DP, IL-1ß, TNF-α, MCP-1, IL-6, IL-8, and IFN-γ were significantly elevated in ABC-positive subjects. As an emerging pathogen, A. baumanii infection represents a risk for impaired inflammation and the development of comorbidities in subjects with T2D.

Follicular T cells mediate donor-specific antibody and rejection after solid organ transplantation.

Following solid organ transplantation, a substantial proportion of chronic allograft loss is attributed to the formation of donor-specific antibodies (DSAs) and antibody-mediated rejection (AbMR). The frequency and phenotype of T follicular helper (Tfh) and T follicular regulatory (Tfr) cells is altered in the setting of kidney transplantation, particularly in patients who develop AbMR. However, the roles of Tfh and Tfr cells in AbMR after solid organ transplantation is unclear. We developed mouse models to inducibly and potently perturb Tfh and Tfr cells to assess the roles of these cells in the development of DSA and AbMR. We found that Tfh cells are required for both de novo DSA responses as well as augmentation of DSA following presensitization. Using orthotopic allogeneic kidney transplantation models, we found that deletion of Tfh cells at the time of transplantation resulted in less severe transplant rejection. Furthermore, using inducible Tfr cell deletion strategies we found that Tfr cells inhibit de novo DSA formation but only have a minor role in controlling kidney transplant rejection. These studies demonstrate that Tfh cells promote, whereas Tfr cells inhibit, DSA to control rejection after kidney transplantation. Therefore, targeting these cells represent a new therapeutic strategy to prevent and treat AbMR.

Modeling the Joint Effect of Leader and Follower Authenticity on Work and Non-Work Outcomes.

Recent theoretical and empirical work has developed the concept of authenticity, both as it relates to leader authenticity and as it relates to authenticity in followers. The present study examines perceptions of employee authenticity and perceived leader authenticity and highlights the utility of jointly examining the congruence between the two to predict individual-level outcomes. The present study utilized Qualtrics Panels to recruit three waves of survey participants. A total of 420 participants responded to all three time points. Polynomial regression, response surface modeling, and a block variable approach with indirect effects were used to test our hypotheses. Results supported the effect of authenticity congruence on individual-level work-related outcomes. Leader-member exchange was found to be a mediating variable between leader-follower authenticity congruence and outcomes in the work and non-work domains.

Continuum mechanical parameterisation of cytoplasmic dynein from atomistic simulation.

Cytoplasmic dynein is responsible for intra-cellular transport in eukaryotic cells. Using Fluctuating Finite Element Analysis (FFEA), a novel algorithm that represents proteins as continuum viscoelastic solids subject to thermal noise, we are building computational tools to study the mechanics of these molecular machines. Here we present a methodology for obtaining the material parameters required to represent the flexibility of cytoplasmic dynein within FFEA from atomistic molecular dynamics (MD) simulations, and show that this continuum representation is sufficient to capture the principal dynamic properties of the motor.

Time-restricted feeding mice a high-fat diet induces a unique lipidomic profile.

Time-restricted feeding (TRF) can reduce adiposity and lessen the co-morbidities of obesity. Mice consuming obesogenic high-fat (HF) diets develop insulin resistance and hepatic steatosis, but have elevated indices of long-chain polyunsaturated fatty acids (LCPUFA) that may be beneficial. While TRF impacts lipid metabolism, scant data exist regarding the impact of TRF upon lipidomic composition of tissues. We (1) tested the hypothesis that TRF of a HF diet elevates LCPUFA indices while preventing insulin resistance and hepatic steatosis and (2) determined the impact of TRF upon the lipidome in plasma, liver, and adipose tissue. For 12 weeks, male, adult mice were fed a control diet ad libitum, a HF diet ad libitum (HF-AL), or a HF diet with TRF, 12 hours during the dark phase (HF-TRF). HF-TRF prevented insulin resistance and hepatic steatosis resulting from by HF-AL treatment. TRF-blocked plasma increases in LCPUFA induced by HF-AL treatment but elevated concentrations of triacylglycerols and non-esterified saturated fatty acids. Analysis of the hepatic lipidome demonstrated that TRF did not elevate LCPUFA while reducing steatosis. However, TRF created (1) a separate hepatic lipid signature for triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine species and (2) modified gene and protein expression consistent with reduced fatty acid synthesis and restoration of diurnal gene signaling. TRF increased the saturated fatty acid content in visceral adipose tissue. In summary, TRF of a HF diet alters the lipidomic profile of plasma, liver, and adipose tissue, creating a third distinct lipid metabolic state indicative of positive metabolic adaptations following HF intake.

Exploring the dynamics of flagellar dynein within the axoneme with Fluctuating Finite Element Analysis.

Flagellar dyneins are the molecular motors responsible for producing the propagating bending motions of cilia and flagella. They are located within a densely packed and highly organised super-macromolecular cytoskeletal structure known as the axoneme. Using the mesoscale simulation technique Fluctuating Finite Element Analysis (FFEA), which represents proteins as viscoelastic continuum objects subject to explicit thermal noise, we have quantified the constraints on the range of molecular conformations that can be explored by dynein-c within the crowded architecture of the axoneme. We subsequently assess the influence of crowding on the 3D exploration of microtubule-binding sites, and specifically on the axial step length. Our calculations combine experimental information on the shape, flexibility and environment of dynein-c from three distinct sources; negative stain electron microscopy, cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET). Our FFEA simulations show that the super-macromolecular organisation of multiple protein complexes into higher-order structures can have a significant influence on the effective flexibility of the individual molecular components, and may, therefore, play an important role in the physical mechanisms underlying their biological function.

Nano-encapsulated Escherichia coli Divisome Anchor ZipA, and in Complex with FtsZ.

The E. coli membrane protein ZipA, binds to the tubulin homologue FtsZ, in the early stage of cell division. We isolated ZipA in a Styrene Maleic Acid lipid particle (SMALP) preserving its position and integrity with native E. coli membrane lipids. Direct binding of ZipA to FtsZ is demonstrated, including FtsZ fibre bundles decorated with ZipA. Using Cryo-Electron Microscopy, small-angle X-ray and neutron scattering, we determine the encapsulated-ZipA structure in isolation, and in complex with FtsZ to a resolution of 1.6 nm. Three regions can be identified from the structure which correspond to, SMALP encapsulated membrane and ZipA transmembrane helix, a separate short compact tether, and ZipA globular head which binds FtsZ. The complex extends 12 nm from the membrane in a compact structure, supported by mesoscale modelling techniques, measuring the movement and stiffness of the regions within ZipA provides molecular scale analysis and visualisation of the early divisome.

The hierarchical emergence of worm-like chain behaviour from globular domain polymer chains.

Biological organisms make use of hierarchically organised structures to modulate mechanical behaviour across multiple lengthscales, allowing microscopic objects to generate macroscopic effects. Within these structural hierarchies, the resultant physical behaviour of the entire system is determined not only by the intrinsic mechanical properties of constituent subunits, but also by their organisation in three-dimensional space. When these subunits are polyproteins, colloidal chains or other globular domain polymers, the Kratky-Porod model is often assumed for the individual subunits. Hence, it is implicitly asserted that the polymeric object has an intrinsic parameter, the persistence length, that defines its flexibility. However, the persistence lengths extracted from experiment vary, and are often relatively small. Through a series of simulations on polymer chains formed of globular subunits, we show that the persistence length itself is a hierarchical structural property, related not only to the intrinsic mechanical properties of the underlying monomeric subunits, but emerging due to the organisation of inhomogenous geometry along the polymer contour.

Simplified Mass Spectrometric Analysis of Ceramides using a Common Collision Energy.

Ceramides (CER) are biologically active sphingolipid precursors that are mechanistically linked to several pathogenic states including cancer, insulin resistance, and neurodegeneration. CER are commonly quantified through mass spectrometry-based methods founded upon a product ion scan (PIS) in positive mode to produce a characteristic m/z 264 ion. The ionization efficiency (IE) of CER species decreases with an increase in CER mass, thus quantitation of CER typically involves application of mass-dependent response factors (RF) for each CER species. In this work, we observed that the RF were systematically dependent on the number of fatty acid acyl carbons and the collision energy (CE) used to generate the m/z 264 ion. Using these complimentary trends, we determined an "isosbestic" CE where the RF for all CER species were equivalent, allowing for CER quantitation without postcollection correction factors. A comparison of this common CE/common RF method to the multiple RF method demonstrated good agreement between the two methods. Use of the common CE/common RF method will reduce data processing and reduce the use of multiple CER species standards.

Investigating Leader Role Congruity and Counterproductive Work Behavior.

The present study sought to examine the role of fulfilled vs. unfulfilled expectations in work-related and non-work domains. Specifically, we examined how congruence and incongruence between implicit leadership theories across multiple categories of leaders (typical, ideal, and effective leaders) and characteristics recognized on one's supervisor affect leader-member exchange (LMX), work-family conflict, and subsequent counter-productive work behavior. We tested our hypotheses using polynomial regression and response surface modeling. The results of this study showed that congruence between implicit leadership theories (ILT's) of typical, ideal, and effective leaders and supervisor recognition, as well as incongruity between ILT's of ideal leaders and characteristics recognized in one's supervisor, significantly impacts perceived LMX quality with supervisors. Incongruity between ILT's of typical and ideal leaders and supervisor recognition was also associated with higher ratings of work-family conflict (WFC). Finally, the results showed support for a significant indirect effect of congruence between ILTs of typical and ideal leaders and characteristics recognized in one's supervisor on CWB through WFC. These results add meaningful depth to literature on congruent implicit theories in two ways: (a) we add to existing knowledge of outcomes associated with congruence on ILT's, and, (b) we examine these associations using ILT's across multiple categories of leaders. Implications are also discussed.