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Orthopedic implant-associated infections cause serious complications primarily attributed to bacterial biofilm formation and are often characterized by increased antibiotic resistance and diminished treatment response. Yet, no methods currently exist to identify biofilms intraoperatively-surgeons rely solely on their eyes and hands and cannot detect or differentiate infected tissue to determine the location and extent of contamination. As the first step in addressing this unmet clinical need, here, we develop an optical coherence tomography (OCT)-based imaging method capable of detection and quantification of one of the most dangerous orthopedic biofilms formed by methicillin-resistant (MRSA). Growing biofilms on orthopedic hardware, we identify MRSA distinct optical signature through histogram-based multi-parametric texture analysis of OCT images and support the findings with bioluminescence imaging and scanning electron microscopy. Under identical experimental conditions, we identify an optical signature of ( ) biofilms and use it to distinguish and quantify both species within MRSA- biofilms. The developed OCT-based methodology was successfully tested for (1) MRSA colonies delineation, (2) detection of metal hardware (an important feature for clinical translation where the metal surface of most orthopedic hardware is not flat), (3) automated quantification of biofilm thickness and roughness, and (4) identification of pores and, therefore, ability to evaluate the role of porosity-one of the critical biological metrics in relation to biofilm maturity and response to treatment. For the first time, we demonstrated complex pore structures of thick ( ) MRSA biofilms with an unprecedented level of detail. The proposed rapid noninvasive detection/quantification of MRSA biofilms on metal surfaces and delineation of their complex network of pores opens new venues for label-free MRSA detection in preclinical models of trauma surgery, expansion to other bacterial strains, and further clinical translation.

Occult hemorrhages after trauma can be present insidiously, and if not detected early enough can result in patient death. This study evaluated a hemorrhage model on 18 human subjects, comparing the performance of traditional vital signs to multiple off-the-shelf non-invasive biomarkers. A validated lower body negative pressure (LBNP) model was used to induce progression towards hypovolemic cardiovascular instability. Traditional vital signs included mean arterial pressure (MAP), electrocardiography (ECG), plethysmography (Pleth), and the test systems utilized electrical impedance via commercial electrical impedance tomography (EIT) and multifrequency electrical impedance spectroscopy (EIS) devices. Absolute and relative metrics were used to evaluate the performance in addition to machine learning-based modeling. Relative EIT-based metrics measured on the thorax outperformed vital sign metrics (MAP, ECG, and Pleth) achieving an area-under-the-curve (AUC) of 0.99 (CI 0.95–1.00, 100% sensitivity, 87.5% specificity) at the smallest LBNP change (0–15 mmHg). The best vital sign metric (MAP) at this LBNP change yielded an AUC of 0.6 (CI 0.38–0.79, 100% sensitivity, 25% specificity). Out-of-sample predictive performance from machine learning models were strong, especially when combining signals from multiple technologies simultaneously. EIT, alone or in machine learning-based combination, appears promising as a technology for early detection of progression toward hemodynamic instability.

In orthopedic trauma surgery, spatially structured biofilm ecosystems of bacteria that colonize orthopedic devices account for up to 65% of all healthcare infections, including tens of millions of people affected in the United States. These biofilm infections typically show increased resistance to antibiotics due to their structure and composition, which contributes significantly to treatment failure. Anti-biofilm approaches are needed together with clinically usable microscopic-resolution imaging techniques for treatment efficacy assessment. Antimicrobial photodynamic therapy (aPDT) has been recently proposed to combat clinically relevant biofilms (chronic wound infections, dental biofilms, etc.) using photosensitizers excited with visible light to generate reactive oxygen species that can kill bacteria residing within pathogenic biofilms. We aim to assess the efficacy of this treatment for eradication of biofilms typically present on surfaces of orthopedic devices (e.g., intramedullary nails and osseointegrated prosthetic implants). In the first phase reported here, we test aPDT by growing biofilms of and bacteria (two of the seven most common pathogens found in orthopedic trauma patients) inside soft lithography-fabricated microfluidic devices. We treat these biofilms with 5-aminolevulinic acid (5-ALA)-based aPDT, evaluate treatment efficacy with optical coherence tomography, and compare with regular clinical antibiotic treatment outcomes. The antibacterial efficiency of 5-ALA-based aPDT showed nonlinear dependence on the photosensitizer concentration and the light power density, with low parameters ( light dose, 5-ALA concentration) being significantly more effective than antibiotic-treated groups ( ), reaching 99.98% of bacteria killed at light dose and 5-ALA concentration setting. Performed experiments enable the translation of this portable treatment/imaging platform to the second phase of the study: aPDT treatment response assessment of biofilms grown on orthopedic hardware.

Methicillin-resistant (MRSA) biofilm infections present a critical challenge in orthopedic trauma surgery and are notoriously resistant to systemic antibiotic therapy. Noninvasive, quantitative imaging methods are urgently needed to assess biofilm burden and therapeutic efficacy, especially for emerging photodynamic therapy (PDT) strategies. We aim to establish a quantitative framework using a combined bioluminescence and optical coherence tomography (OCT) imaging approach to correlate bioluminescent signal with viable MRSA burden in both planktonic and biofilm states and to determine how biofilm density and structure influence this relationship. Bioluminescent MRSA (SAP231-luxCDABE) was cultured in planktonic and biofilm forms using growth models in 24-well plates and custom macrofluidic devices, respectively. Bacteria bioluminescence intensity (BLI), counted colony-forming units (CFU), and OCT-based biofilm thickness measurements were collected to construct linear regression models to evaluate how well BLI alone, or combined with biofilm density (CFU/volume), predicts bacterial counts across culture conditions. Bioluminescence strongly correlated with CFU in planktonic cultures ( ). In biofilms, BLI per CFU decreased with density, indicating metabolic downregulation, and BLI alone was less reliable ( ). Incorporating biofilm density (CFU/volume) improved prediction ( ). A joint model for both states showed excellent fit ( ), but the biofilm versus planktonic group remained a significant factor ( ), revealing systematic differences. This highlights the need for a mixed-model approach that segments subvolumes by morphological features to improve accurate, generalizable CFU estimation across both growth states. Bioluminescence alone underestimates bacterial burden in dense, metabolically suppressed MRSA biofilms. The combination of BLI with OCT-derived structural metrics enables accurate, nondestructive quantification of viable bacterial load. This approach provides a robust toolset for preclinical evaluation of antimicrobial therapies, particularly for optimizing PDT dosimetry and assessing biofilm response in translational infection models.

A dynamic contrast-enhanced (DCE) near-infrared (NIR) method to measure cerebral blood flow (CBF) in the neurocritical care unit (NCU) is described. A primary concern in managing patients with acquired brain injury (ABI) is onset of delayed ischemic injury (DII) caused by complications during the days to weeks following the initial insult, resulting in reduced CBF and impaired oxygen delivery. The development of a safe, portable, and quantitative DCE-NIR method for measuring CBF in NCU patients is addressed by focusing on four main areas: designing a clinically compatible instrument, developing an appropriate analytical framework, creating a relevant ABI animal model, and validating the method against CT perfusion. In Chapter 2, depth-resolved continuous-wave NIR recovered values of CBF in a juvenile pig show strong correlation with CT perfusion CBF during mild ischemia and hyperemia (r=0.84, p<0.001). In particular, subject-specific light propagation modeling reduces the variability caused by extracerebral layer contamination. In Chapter 3, time-resolved (TR) NIR improves the signal sensitivity to brain tissue, and a relative CBF index is be both sensitive and specific to flow changes in the brain. In particular, when compared with the change in CBF measured with CT perfusion during hypocapnia, the deconvolution-based index has an error of 0.8%, compared to 21.8% with the time-to-peak method. To enable measurement of absolute CBF, a method for characterizing the AIF is described in Chapter 4, and the theoretical basis for an advanced analytical framework—the kinetic deconvolution optical reconstruction (KDOR)—is provided in Chapter 5. Finally, a multichannel TR-NIR system is combined with KDOR to quantify CBF in an adult pig model of ischemia (Chapter 6). In this final study, measurements of CBF obtained with the DCE-NIR technique show strong agreement with CT perfusion measurements of CBF in mild and moderate ischemia (r=0.86, p<0.001). The principle conclusion of this thesis is that the DCE-NIR method, combining multidistance TR instrumentation with the KDOR analytical framework, can recover CBF values that are in strong agreement with CT perfusion values of CBF. Ultimately, bedside CBF measurements could improve clinical management of ABI by detecting delayed ischemia before permanent brain damage occurs.

Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. We demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, we also show that targeting individual genes with multiple sgRNAs per cell improves efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Last, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments. Single-cell CRISPR screens are readily multiplexed and scaled with an improved version of Perturb-seq.

Mild traumatic brain injury (TBI) is associated with persistent sleep-wake dysfunction, including insomnia and circadian rhythm disruption, which can exacerbate functional outcomes including mood, pain, and quality of life. Present therapies to treat sleep-wake disturbances in those with TBI (e.g., cognitive behavioral therapy for insomnia) are limited by marginal efficacy, poor patient acceptability, and/or high patient/provider burden. Thus, this study aimed to assess the feasibility and preliminary efficacy of morning bright light therapy, to improve sleep in Veterans with TBI (NCT03578003). Thirty-three Veterans with history of TBI were prospectively enrolled in a single-arm, open-label intervention using a lightbox (~10,000 lux at the eye) for 60-minutes every morning for 4-weeks. Pre- and post-intervention outcomes included questionnaires related to sleep, mood, TBI, post-traumatic stress disorder (PTSD), and pain; wrist actigraphy as a proxy for objective sleep; and blood-based biomarkers related to TBI/sleep. The protocol was rated favorably by ~75% of participants, with adherence to the lightbox and actigraphy being ~87% and 97%, respectively. Post-intervention improvements were observed in self-reported symptoms related to insomnia, mood, and pain; actigraphy-derived measures of sleep; and blood-based biomarkers related to peripheral inflammatory balance. The severity of comorbid PTSD was a significant positive predictor of response to treatment. Morning bright light therapy is a feasible and acceptable intervention that shows preliminary efficacy to treat disrupted sleep in Veterans with TBI. A full-scale randomized, placebo-controlled study with longitudinal follow-up is warranted to assess the efficacy of morning bright light therapy to improve sleep, biomarkers, and other TBI related symptoms.

Feline hyperthyroidism (FHT) is a debilitating disease affecting > 10% of elderly cats. It is generally characterised by chronic elevation of thyroid hormone in the absence of circulating TSH. Understanding of the molecular pathogenesis of FHT is currently limited. However, FHT shares clinical and histopathological similarities with human toxic multinodular goitre, which has been associated with activating mutations in TSH receptor (TSHR) and G s α encoding genes. Using RNA-seq transcriptomic analysis of thyroid tissue from hyperthyroid and euthyroid cats, we identified differentially expressed genes and dysregulated pathways in FHT, many of which are downstream of TSHR. In addition, we detected missense variants in thyroid RNA-seq reads that alter the structure of both TSHR and G s α. All FHT-associated mutations were absent in germline sequence from paired blood samples. Only a small number of hyperthyroid cats demonstrated TSHR variation, however all thyroids from advanced cases of FHT carried at least one missense variant affecting G s α. The activating nature of the acquired G s α mutations was demonstrated by increased cAMP production in vitro. These data indicate that constitutive activation of signalling downstream of TSHR is central to the TSH-independent production of thyroid hormone in FHT, offering a novel therapeutic target pathway in this common disease.

The Kepler mission was designed to determine the frequency of Earth-sized planets in and near the habitable zone of Sun-like stars. The habitable zone is the region where planetary temperatures are suitable for water to exist on a planet's surface. During the first 6 weeks of observations, Kepler monitored 156,000 stars, and five new exoplanets with sizes between 0.37 and 1.6 Jupiter radii and orbital periods from 3.2 to 4.9 days were discovered. The density of the Neptune-sized Kepler-4b is similar to that of Neptune and GJ 436b, even though the irradiation level is 800,000 times higher. Kepler-7b is one of the lowest-density planets (approximately 0.17 gram per cubic centimeter) yet detected. Kepler-5b, -6b, and -8b confirm the existence of planets with densities lower than those predicted for gas giant planets.

A new class of ultra-long-duration γ-ray bursts (GRBs) has recently been suggested, with durations in excess of 10,000 seconds, and now a supernova (SN 2011kl) has been found to be associated with the ultra-long-duration GRB 111209A, allowing a physical understanding of the nature of ultra-long-duration GRBs. A γ-ray burst/supernova pairing This paper reports that supernova SN 2011kl was associated with the ultra-long-duration γ-ray burst GRB 111209A at z = 0.677, adding support to the recent suggestion of a new class of ultra-long-duration (longer than 10,000 seconds) γ-ray bursts. SN 2011kl was more luminous than type Ic supernovae associated with long γ-ray bursts and its spectrum is distinctly different, with a low metal-line opacity. Its properties are best represented by a model in which extra energy is injected by a strongly magnetized neutron star (a magnetar), a type of object that has also been proposed as the explanation for super-luminous supernovae. A new class of ultra-long-duration (more than 10,000 seconds) γ-ray bursts has recently been suggested 1 , 2 , 3 . They may originate in the explosion of stars with much larger radii than those producing normal long-duration γ-ray bursts 3 , 4 or in the tidal disruption of a star 3 . No clear supernova has yet been associated with an ultra-long-duration γ-ray burst. Here we report that a supernova (SN 2011kl) was associated with the ultra-long-duration γ-ray burst GRB 111209A, at a redshift z of 0.677. This supernova is more than three times more luminous than type Ic supernovae associated with long-duration γ-ray bursts 5 , 6 , 7 , and its spectrum is distinctly different. The slope of the continuum resembles those of super-luminous supernovae 8 , 9 , but extends further down into the rest-frame ultraviolet implying a low metal content. The light curve evolves much more rapidly than those of super-luminous supernovae. This combination of high luminosity and low metal-line opacity cannot be reconciled with typical type Ic supernovae, but can be reproduced by a model where extra energy is injected by a strongly magnetized neutron star (a magnetar), which has also been proposed as the explanation for super-luminous supernovae 10 .