Biology subjects

Caldwell, C. C.

Publications and source records attributed to Caldwell, C. C..

8 recordsLinked to original sources

bioRxiv2026

Mechanical characterization of human centromeres

Centromeres must withstand high pulling forces in metaphase, yet allow rapid and complete chromosome segregation in anaphase. Using optical tweezers measurements on isolated human metaphase chromosomes, we find that centromeres are stiffer at low forces and more frequently exhibit pronounced softening compared to whole chromosomes, while displaying a similar stiffening response at high forces. We capture these mechanical properties by establishing a softened hierarchical worm-like chain model. Cohesin cleavage alters the mechanical properties of centromeres but does not lead to chromosome segregation in vitro. Topoisomerase II is recruited to centromeric regions under significant tension, likely to resolve the persistent centromeric DNA entanglements that provide additional sister chromatid cohesion. These findings advance our understanding of the mechanical resilience and regulation of centromeres during mitosis.

biophysics↗

bioRxiv2026

Dynamic CTCF bridges drive genome organization

Human CCCTC-binding factor (CTCF) is a crucial factor in genome organization, regulating chromatin looping and gene expression. The loop extrusion model (LEM) designates cohesin as the sole active player and limits CTCF to a passive barrier, though emerging evidence suggests a more dynamic role. Using complementary single-molecule techniques, combining dual- and quad-trap optical tweezers, fluorescence microscopy, and atomic force microscopy, we show CTCF forms stable, dynamic DNA bridges. We demonstrate CTCF binds with high affinity and cooperativity, undergoes 1D diffusion, and stiffens DNA. Strikingly, CTCF stabilizes DNA loops without cohesin and forms bridges that slide at low force yet resist rupture at forces exceeding dsDNA stability. These findings suggest a modified LEM in which CTCF directly forms dynamic, force-resistant bridges to stabilize cohesin-established loops, revealing an active role in genome organization.

biophysics↗

bioRxiv2026

Base-pair scale dynamics of a repair helicase on DNA lesions reveal varied damage-sensing mechanisms

Nucleotide excision repair (NER) is a cellular pathway that removes DNA lesions caused by ultraviolet light and various mutagens. A critical component of the NER machinery is XPD helicase, which unwinds the duplex around the damage, allowing its excision and repair. XPD has also been increasingly implicated in sensing and verifying DNA damage. However, the detailed mechanisms by which XPD responds to DNA damage have to-date remained unclear. Here, we use optical tweezers to perform real-time, high-precision measurements of single molecules of XPD as they encounter a variety of well-defined DNA modifications, including a cyclobutane pyrimidine dimer (CPD), a natural substrate for NER. The observed XPD dynamics reveal different behaviors depending on the damage type and the relative orientations of the DNA fork, damage, and helicase. Most notably, XPD displays an almost complete inability to unwind past a CPD on the translocated strand, and instead exhibits a short pause around the lesion--though not a stall--followed by retreat. Combining the base pair-scale XPD kinetics with structural analyses, we identify two regions of XPD sensitive to DNA modifications.

biophysics↗

bioRxiv2024

Determining potential immunomodulatory drug efficacy in sepsis using ELISpot

PurposeThis study evaluated the ability of ELISpot to identify immuno-modulatory drug therapies for their potential efficacy in patients with sepsis. MethodsELISpot was performed using diluted whole blood from 61 septic patients and 48 healthy matched controls. Innate and adaptive immunity were evaluated by ex vivo stimulated production of TNF- and IFN-{gamma} respectively. Potential drug efficacy was determined by the drugs effects to increase or decrease the number of cytokine-producing cells and amount of cytokine produced per cell as determined by spot size and intensity. The corticosteroid dexamethasone was evaluated for its ability to down modulate TNF- and IFN-{gamma} production. The TLR7/8 agonist resiquimod (R848) and T-cell stimulants IL-7 and anti-PD-1 mAb were tested for their ability to enhance immune responses in sepsis. ResultsSpontaneous production of TNF- and IFN-{gamma} varied among healthy subjects and septic patients. LPS or resiquimod stimulation increased total TNF- production in septic patients by 1,648% and 1,929% respectively. Conversely, dexamethasone diminished the responses to LPS or resiquimod by 71% and 61% respectively. IL-7, but not anti-PD-1 mAb markedly increased IFN-{gamma} production in both healthy subjects (127%) and septic patients (79%). Dexamethasone also reduced anti-CD3/CD28 mAb stimulated IFN-{gamma} production by 54%; while IL-7 ameliorated dexamethasone-induced suppression. IL-7 significantly enhanced lymphocyte function in over 90% of septic patients. ConclusionELISpot can reveal host immune response patterns and the effects of drugs to selectively down- or up-regulate patient immunity. Furthermore, the ability of ELISpot to detect the effect of specific immuno-modulatory drugs to independently regulate the innate and adaptive host response could enable precision-based immune drug therapies in sepsis.

immunology↗

bioRxiv2024

Development and optimization of a diluted whole blood ELISpot assay to test immune function

BackgroundSepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in Sepsis ( SPIES) is a prospective, multicenter observational study testing the utility of ELISpot as a functional bioassay specifically measuring cytokine-producing cells after stimulation to identify the immunosuppressed endotype, predict clinical outcomes in septic patients, and test potential immune stimulants for clinical development. Most ELISpot protocols call for the isolation of PBMC prior to their inclusion in the assay. In contrast, we developed a diluted whole blood (DWB) ELISpot protocol that has been validated across multiple laboratories. MethodsHeparinized whole blood was collected from healthy donors and septic patients and tested under different stimulation conditions to evaluate the impact of blood dilution, stimulant concentration, blood storage, and length of stimulation on ex vivo IFN{gamma} and TNF production as measured by ELISpot. ResultsWe demonstrate a dynamic range of whole blood dilutions that give a robust ex vivo cytokine response to stimuli. Additionally, a wide range of stimulant concentrations can be utilized to induce cytokine production. Further modifications demonstrate anticoagulated whole blood can be stored up to 24 hours at room temperature without losing significant functionality. Finally, we show ex vivo stimulation can be as brief as 4 hours allowing for a substantial decrease in processing time. ConclusionsThe data demonstrate the feasibility of using ELISpot to measure the functional capacity of cells within DWB under a variety of stimulation conditions to inform clinicians on the extent of immune dysregulation in septic patients.

immunology↗

bioRxiv2023

Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair

Nucleotide excision repair (NER) promotes genomic integrity by correcting bulky DNA adducts damage caused by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the DNA adduct. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP) was incorporated at Arg-153 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent human XPA protein (hXPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate hXPACy3 and detail experimental conditions required to stably maintain the protein during biochemical and biophysical studies.

biochemistry↗

bioRxiv2021

KERA: Analysis Tool for Multi-Process, Multi-State Single-Molecule Data

Molecular machines within cells dynamically assemble, disassemble, and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy (CoSMoS), in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy (TIRFM) approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell-time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERAs utility by analyzing conformational dynamics of two DNA binding proteins: RPA and XPD helicase.

biophysics↗

bioRxiv2018

Dynamics and Selective Remodeling of the DNA Binding Domains of RPA

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-strand DNA (ssDNA) intermediates, activating the DNA damage response, and handing off ssDNA to appropriate downstream players. Six DNA binding domains (DBDs) in RPA promote high affinity binding to ssDNA, but also allow RPA displacement by lower affinity proteins. We have made fluorescent versions of RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA, while RPA as a whole remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. This demonstrates how RPA interacting proteins, with lower ssDNA binding affinity, can access the occluded ssDNA and remodel individual DBDs to replace RPA.\n\nOne Sentence SummaryThe choreography of binding and rearrangement of the individual domains of RPA during homologous recombination is revealed.

biochemistry↗