Improved resolution in fiber bundle inline holographic microscopy using multiple illumination sources.

Recent work has shown that high-quality inline holographic microscopy images can be captured through fiber imaging bundles. Speckle patterns arising from modal interference within the bundle cores can be minimized by use of a partially-coherent optical source such as an LED delivered via a multimode fiber. This allows numerical refocusing of holograms from samples at working distances of up to approximately 1 mm from the fiber bundle before the finite coherence begins to degrade the lateral resolution. However, at short working distances the lateral resolution is limited not by coherence, but by sampling effects due to core-to-core spacing in the bundle. In this article we demonstrate that multiple shifted holograms can be combined to improve the resolution by a factor of two. The shifted holograms can be rapidly acquired by sequentially firing LEDs, which are each coupled to their own, mutually offset, illumination fiber. Following a one-time calibration, resolution-enhanced images are created in real-time at an equivalent net frame rate of up to 7.5 Hz. The resolution improvement is demonstrated quantitatively using a resolution target and qualitatively using mounted biological slides. At longer working distances, beyond 0.6 mm, the improvement is reduced as resolution becomes limited by the source spatial and temporal coherence.

Real-timing processing of fiber bundle endomicroscopy images in Python using PyFibreBundle.

Fiber imaging bundles allow the transfer of optical images from place-to-place along narrow and flexible conduits. Traditionally used extensively in medical endoscopy, bundles are now finding new applications in endoscopic microscopy and other emerging techniques. PyFibreBundle is an open-source Python package for fast processing of images acquired through imaging bundles. This includes detection and removal of the fiber core pattern by filtering or interpolation, and application of background and flat-field corrections. It also allows images to be stitched together to create mosaics and resolution to be improved by combining multiple shifted images. This paper describes the technical implementation of PyFibreBundle and provides example results from three endomicroscopy imaging systems: color transmission, monochrome transmission, and confocal fluorescence. This allows various processing options to be compared quantitatively and qualitatively, and benchmarking demonstrates that PyFibreBundle can achieve state-of-the-art performance in an open-source package. The paper demonstrates core removal by interpolation and mosaicing at over 100 fps, real-time multi-frame resolution enhancement and the first demonstration of real-time endomicroscopy image processing, including core removal, on a Raspberry Pi single board computer. This demonstrates that PyFibreBundle is potentially a valuable tool for the development of low-cost, high-performance fiber bundle imaging systems.

The role of anticipatory and reflexive compensatory muscle activation in catching errors under load uncertainty.

This experiment investigated the role of anticipatory and reflexive compensatory neuromotor control in catching errors occurring under load uncertainty. Participants performed 64 trials of a one-handed ball catching task using visually identical balls of four different weights without knowing the weight of the ball on each trial. Anticipatory and reflexive compensatory muscle activation were recorded in five muscles (anterior deltoid, biceps brachii, wrist flexors group, triceps brachii, lumbar erector spinae) using the EMG integral. In each muscle, the anticipatory and reflexive compensatory muscle activation were compared between successful catches and catching errors for the lightest ball and the heaviest ball. Anticipatory muscle activation was not implicated in errors made with the lightest ball. However, reflexive compensatory muscle activation in the anterior deltoid, biceps brachii, and wrist flexors were implicated in errors made with the lightest ball. Specifically, catching errors with the lightest ball were characterized by elevated reflexive compensatory muscle activation. In the case of the heaviest ball, both anticipatory (anterior deltoid, wrist flexors) and reflexive compensatory muscle activation (anterior deltoid, biceps brachii, wrist flexors) were implicated in catching errors. That is, catching errors with the heaviest ball were characterized by lower anticipatory and reflexive compensatory muscle activation. Results are considered in the context of the likely influence of limb compliance in catching under load uncertainty.

Eye Fixation Behaviors and Processing Time of People With Aphasia and Neurotypical Adults When Reading Narratives With and Without Text-to-Speech Support.

BACKGROUND: Researchers have used eye-tracking technology to investigate eye movements in neurotypical adults (NAs) when reading. The technology can provide comparable information about people with aphasia (PWA). Eye fixations occurring when PWA do and do not have access to text-to-speech (TTS) technology are of interest because the support improves reading comprehension and decreases processing time for at least some PWA. AIMS: This study's purpose was to examine forward, regressive, and off-track eye fixations when PWA and NAs read narratives in read-only (RO) and TTS conditions. A secondary aim was to examine the influence of eye fixations on processing time. METHOD AND PROCEDURE: A Tobii Dynavox Pro Spectrum eye tracker recorded eye movements of nine PWA and nine NAs while reading narratives in two conditions. Movements of interest were forward fixations; within-word, within-sentence, and previous-sentence regressive fixations; and off-track fixations. OUTCOMES AND RESULTS: PWA exhibited significantly more forward and regressive fixations in the RO than TTS condition, whereas NAs showed opposite behaviors. NAs had significantly more off-track fixations in the TTS than RO condition, whereas PWA exhibited no difference across conditions. PWA took significantly longer to process content in the RO condition, whereas NAs took longer in the TTS condition. CONCLUSIONS: PWA and NAs differ in important ways when processing texts with and without TTS support. Examining eye-tracking data provides a means of gaining insight into the decoding and reading comprehension challenges of PWA and helps elucidate how assistive technology can mediate these challenges.

A tumor-restricted glycoform of podocalyxin is a highly selective marker of immunologically cold high-grade serous ovarian carcinoma.

Introduction: Targeted-immunotherapies such as antibody-drug conjugates (ADC), chimeric antigen receptor (CAR) T cells or bispecific T-cell engagers (eg, BiTE®) all aim to improve cancer treatment by directly targeting cancer cells while sparing healthy tissues. Success of these therapies requires tumor antigens that are abundantly expressed and, ideally, tumor specific. The CD34-related stem cell sialomucin, podocalyxin (PODXL), is a promising target as it is overexpressed on a variety of tumor types and its expression is consistently linked to poor prognosis. However, PODXL is also expressed in healthy tissues including kidney podocytes and endothelia. To circumvent this potential pitfall, we developed an antibody, named PODO447, that selectively targets a tumor-associated glycoform of PODXL. This tumor glycoepitope is expressed by 65% of high-grade serous ovarian carcinoma (HGSOC) tumors. Methods: In this study we characterize these PODO447-expressing tumors as a distinct subset of HGSOC using four different patient cohorts that include pre-chemotherapy, post-neoadjuvant chemotherapy (NACT) and relapsing tumors as well as tumors from various peritoneal locations. Results: We find that the PODO447 epitope expression is similar across tumor locations and negligibly impacted by chemotherapy. Invariably, tumors with high levels of the PODO447 epitope lack infiltrating CD8+ T cells and CD20+ B cells/plasma cells, an immune phenotype consistently associated with poor outcome. Discussion: We conclude that the PODO447 glycoepitope is an excellent biomarker of immune "cold" tumors and a candidate for the development of targeted-therapies for these hard-to-treat cancers.

Antibody-Drug Conjugates Targeting Tumor-Specific Mucin Glycoepitopes.

Finding the ideal epitope to target is a key element for the development of an antibody-drug conjugate (ADC). To maximize drug delivery to tumor cells and reduce side effects, this epitope should be specific to cancer cells and spare all normal tissue. During cancer progression, glycosylation pathways are frequently altered leading to the generation of new glycosylation patterns selective to cancer cells. Mucins are highly glycosylated proteins frequently expressed on tumors and, thus, ideal presenters of altered glycoepitopes. In this review, we describe three different types of glycoepitopes that are recognized by monoclonal antibodies (mAb) and, therefore, serve as ideal scaffolds for ADC; glycan-only, glycopeptide and shielded-peptide glycoepitopes. We review pre-clinical and clinical results obtained with ADCs targeting glycoepitopes expressed on MUC1 or podocalyxin (Podxl) and two mAbs targeting glycoepitopes expressed on MUC16 or MUC5AC as potential candidates for ADC development. Finally, we discuss current limits in using glycoepitope-targeting ADCs to treat cancer and propose methods to improve their efficacy and specificity.

Prognostic peripheral blood biomarkers at ICU admission predict COVID-19 clinical outcomes.

The COVID-19 pandemic continues to challenge the capacities of hospital ICUs which currently lack the ability to identify prospectively those patients who may require extended management. In this study of 90 ICU COVID-19 patients, we evaluated serum levels of four cytokines (IL-1ß, IL-6, IL-10 and TNFα) as well as standard clinical and laboratory measurements. On 42 of these patients (binned into Initial and Replication Cohorts), we further performed CyTOF-based deep immunophenotyping of peripheral blood mononuclear cells with a panel of 38 antibodies. All measurements and patient samples were taken at time of ICU admission and retrospectively linked to patient clinical outcomes through statistical approaches. These analyses resulted in the definition of a new measure of patient clinical outcome: patients who will recover after short ICU stays (< 6 days) and those who will subsequently die or recover after long ICU stays (≥6 days). Based on these clinical outcome categories, we identified blood prognostic biomarkers that, at time of ICU admission, prospectively distinguish, with 91% sensitivity and 91% specificity (positive likelihood ratio 10.1), patients in the two clinical outcome groups. This is achieved through a tiered evaluation of serum IL-10 and targeted immunophenotyping of monocyte subsets, specifically, CD11clow classical monocytes. Both immune biomarkers were consistently elevated ( ≥15 pg/ml and ≥2.7 x107/L for serum IL-10 and CD11clow classical monocytes, respectively) in those patients who will subsequently die or recover after long ICU stays. This highly sensitive and specific prognostic test could prove useful in guiding clinical resource allocation.

Platelet signaling at the nexus of innate immunity and rheumatoid arthritis.

Rheumatoid arthritis (RA) is a debilitating autoimmune disorder characterized by chronic inflammation of the synovial tissues and progressive destruction of bone and cartilage. The inflammatory response and subsequent tissue degradation are orchestrated by complex signaling networks between immune cells and their products in the blood, vascular endothelia and the connective tissue cells residing in the joints. Platelets are recognized as immune-competent cells with an important role in chronic inflammatory diseases such as RA. Here we review the specific aspects of platelet function relevant to arthritic disease, including current knowledge of the molecular crosstalk between platelets and other innate immune cells that modulate RA pathogenesis.

Platelet factor 4 (CXCL4/PF4) upregulates matrix metalloproteinase-2 (MMP-2) in gingival fibroblasts.

Periodontitis is a chronic inflammatory disease characterized by the release of matrix metalloproteinases (MMPs) from resident connective tissue cells in tooth-supporting tissues (periodontium). Platelet activation, and the attendant release of pro-inflammatory chemokines such as platelet factor 4 (CXCL4/PF4), are associated with periodontitis although the associated biochemical pathways remain undefined. Here we report that recombinant PF4 is internalized by cultured human gingival fibroblasts (hGFs), resulting in significant (p < 0.05) upregulation in both the production and release of MMP-2 (gelatinase A). This finding was corroborated by elevated circulating levels of MMP-2 (p < 0.05) in PF4-overexpressing transgenic mice, relative to controls. We also determined that PF4 induces the phosphorylation of NF-κB; notably, the suppression of NF-κB signaling by the inhibitor BAY 11-7082 abrogated PF4-induced MMP-2 upregulation. Moreover, the inhibition of surface glycosaminoglycans (GAGs) blocked both PF4 binding and NF-κB phosphorylation. Partial blockade of PF4 binding to the cells was achieved by treatment with either chondroitinase ABC or heparinase III, suggesting that both chondroitin sulfate and heparan sulfate mediate PF4 signaling. These results identify a novel pathway in which PF4 upregulates MMP-2 release from fibroblasts in an NF-κB- and GAG-dependent manner, and further our comprehension of the role of platelet signaling in periodontal tissue homeostasis.

"Just one word, plastic!": Controversies and caveats in innate lymphoid cell plasticity.

Innate lymphoid cells (ILCs) are frontline immune effectors involved in the early stages of host defense and maintenance of tissue homeostasis, particularly at mucosal surfaces such as the intestine, lung, and skin. Canonical ILCs are described as tissue-resident cells that populate peripheral tissues early in life and respond appropriately based on environmental exposure and their anatomical niche and tissue microenvironment. Intriguingly, there are accumulating reports of ILC "plasticity" that note the existence of non-canonical ILCs that exhibit distinct patterns of master transcription factor expression and cytokine production profiles in response to tissue inflammation. Yet this concept of ILC-plasticity is controversial due to several confounding caveats that include, among others, the independent large-scale recruitment of new ILC subsets from distal sites and the local, in situ, differentiation of uncommitted resident precursors. Nevertheless, the ability of ILCs to acquire unique characteristics and adapt to local environmental cues is an attractive paradigm because it would enable the rapid adaptation of innate responses to a wider array of pathogens even in the absence of pre-existing 'prototypical' ILC responder subsets. Despite the impressive recent progress in understanding ILC biology, the true contribution of ILC plasticity to tissue homeostasis and disease and how it is regulated remains obscure. Here, we detail current methodologies used to study ILC plasticity in mice and review the mechanisms that drive and regulate functional ILC plasticity in response to polarizing signals in their microenvironment and different cytokine milieus. Finally, we discuss the physiological relevance of ILC plasticity and its implications for potential therapeutics and treatments.

Comprehension, Processing Time, and Modality Preferences When People With Aphasia and Neurotypical Healthy Adults Read Books: A Pilot Study.

BACKGROUND: Many people with aphasia (PWA) want to read books. Text-to-speech (TTS) technology sometimes provides comprehension and processing time benefits when PWA read short, multisentence passages. Currently, no research examines the effect of TTS support when PWA read books. AIMS: This study's primary purpose was to examine comprehension accuracy and total processing time of PWA and neurotypical healthy adults (NHAs) when reading book sections in read-only versus TTS-supported conditions. A secondary aim was to examine condition preference and perceived degree of understanding by people in both participant groups. METHOD AND PROCEDURE: Ten PWA and 10 NHAs alternated between read-only and TTS-supported conditions to read a book. Participants answered comprehension questions and provided feedback about their reading experience, condition preference, and desire to use TTS technology for future book reading. Outcomes and Result: Overall, PWA exhibited less accurate comprehension and slower processing times compared to NHAs in both conditions. No significant comprehension accuracy difference occurred between conditions for either group. However, four PWA exhibited a 10% or greater increase in comprehension accuracy when receiving TTS support. A significant processing time difference occurred with PWA processing text faster with TTS support, whereas NHAs did not demonstrate processing time differences. Most PWA preferred the TTS condition and expressed a desire to use TTS technology in the future. Most NHAs expressed the opposite preference. CONCLUSIONS: TTS support during book reading promotes faster processing without compromising comprehension for PWA. Clinicians should discuss with PWA the relative importance of comprehension accuracy, processing time, and comfort with technology when determining whether using TTS support during book reading is desirable.

Gonadal hormones and sex chromosome complement differentially contribute to ethanol intake, preference, and relapse-like behaviour in four core genotypes mice.

Alcohol use and high-risk alcohol drinking behaviours among women are rapidly rising. In rodent models, females typically consume more ethanol (EtOH) than males. Here, we used the four core genotypes (FCG) mouse model to investigate the influence of gonadal hormones and sex chromosome complement on EtOH drinking behaviours. FCG mice were given access to escalating concentrations of EtOH in a two-bottle, 24-h continuous access drinking paradigm to assess consumption and preference. Relapse-like behaviour was measured by assessing escalated intake following repeated cycles of deprivation and re-exposure. Twenty-four-hour EtOH consumption was greater in mice with ovaries (Sry-), relative to those with testes, and in mice with the XX chromosome complement, relative to those with XY sex chromosomes. EtOH preference was higher in XX versus XY mice. For both consumption and preference, the influences of the Sry gene and sex chromosomes were concentration dependent. Escalated intake following repeated cycles of deprivation and re-exposure emerged only in XX mice (vs. XY). Mice with ovaries (Sry- FCG mice and C57BL/6J females) were also found to consume more water than mice with testes. These results demonstrate that aspects of EtOH drinking behaviour may be independently regulated by sex hormones and chromosomes and inform our understanding of the neurobiological mechanisms which contribute to EtOH dependence in male and female mice. Future investigation of the contribution of sex chromosomes to EtOH drinking behaviours is warranted. We used the FCG mouse model to investigate the influence of gonadal hormones and sex chromosome complement on EtOH drinking behaviours, including the alcohol deprivation effect. Escalated intake following repeated cycles of deprivation and re-exposure emerged only in XX mice (vs. XY). These results demonstrate that aspects of EtOH drinking behaviour may be independently regulated by sex hormones and chromosomes.

Eosinophils Decrease Pulmonary Metastatic Mammary Tumor Growth.

Metastatic breast cancer is challenging to effectively treat, highlighting the need for an improved understanding of host factors that influence metastatic tumor cell colonization and growth in distant tissues. The lungs are a common site of breast cancer metastasis and are host to a population of tissue-resident eosinophils. Eosinophils are granulocytic innate immune cells known for their prominent roles in allergy and Th2 immunity. Though their presence in solid tumors and metastases have been reported for decades, the influence of eosinophils on metastatic tumor growth in the lungs is unclear. We used transgenic mouse models characterized by elevated pulmonary eosinophils (IL5Tg mice) and eosinophil-deficiency (ΔdblGATA mice), as well as antibody-mediated depletion of eosinophils, to study the role of eosinophils in EO771 mammary tumor growth in the lungs. We found that IL5Tg mice exhibit reduced pulmonary metastatic colonization and decreased metastatic tumor burden compared to wild-type (WT) mice or eosinophil-deficient mice. Eosinophils co-cultured with tumor cells ex vivo produced peroxidase activity and induced tumor cell death, indicating that eosinophils are capable of releasing eosinophil peroxidase (EPX) and killing EO771 tumor cells. We found that lung eosinophils expressed phenotypic markers of activation during EO771 tumor growth in the lungs, and that metastatic growth was accelerated in eosinophil-deficient mice and in WT mice after immunological depletion of eosinophils. Our results highlight an important role for eosinophils in restricting mammary tumor cell growth in the lungs and support further work to determine whether strategies to trigger local eosinophil degranulation may decrease pulmonary metastatic growth.

Targeting a Tumor-Specific Epitope on Podocalyxin Increases Survival in Human Tumor Preclinical Models.

Podocalyxin (Podxl) is a CD34-related cell surface sialomucin that is normally highly expressed by adult vascular endothelia and kidney podocytes where it plays a key role in blocking adhesion. Importantly, it is also frequently upregulated on a wide array of human tumors and its expression often correlates with poor prognosis. We previously showed that, in xenograft studies, Podxl plays a key role in metastatic disease by making tumor initiating cells more mobile and invasive. Recently, we developed a novel antibody, PODO447, which shows exquisite specificity for a tumor-restricted glycoform of Podxl but does not react with Podxl expressed by normal adult tissue. Here we utilized an array of glycosylation defective cell lines to further define the PODO447 reactive epitope and reveal it as an O-linked core 1 glycan presented in the context of the Podxl peptide backbone. Further, we show that when coupled to monomethyl auristatin E (MMAE) toxic payload, PODO447 functions as a highly specific and effective antibody drug conjugate (ADC) in killing ovarian, pancreatic, glioblastoma and leukemia cell lines in vitro. Finally, we demonstrate PODO447-ADCs are highly effective in targeting human pancreatic and ovarian tumors in xenografted NSG and Nude mouse models. These data reveal PODO447-ADCs as exquisitely tumor-specific and highly efficacious immunotherapeutic reagents for the targeting of human tumors. Thus, PODO447 exhibits the appropriate characteristics for further development as a targeted clinical immunotherapy.

Endoscopic en-face optical coherence tomography and fluorescence imaging using correlation-based probe tracking.

Forward-viewing endoscopic optical coherence tomography (OCT) provides 3D imaging in vivo, and can be combined with widefield fluorescence imaging by use of a double-clad fiber. However, it is technically challenging to build a high-performance miniaturized 2D scanning system with a large field-of-view. In this paper we demonstrate how a 1D scanning probe, which produces cross-sectional OCT images (B-scans) and 1D fluorescence T-scans, can be transformed into a 2D scanning probe by manual scanning along the second axis. OCT volumes are assembled from the B-scans using speckle decorrelation measurements to estimate the out-of-plane motion along the manual scan direction. Motion within the plane of the B-scans is corrected using image registration by normalized cross correlation. En-face OCT slices and fluorescence images, corrected for probe motion in 3D, can be displayed in real-time during the scan. For a B-scan frame rate of 250 Hz, and an OCT lateral resolution of approximately 20 µ m , the approach can handle out-of-plane motion at speeds of up to 4 mm/s.

Effect of non-contact boxing training on the frequency and timing of anticipatory postural adjustments in healthy adults.

BACKGROUND: The experiment tested the effect of non-contact boxing training on the frequency and timing of anticipatory postural adjustments (APAs) resulting from self-induced postural perturbations in healthy adults. METHODS: The 8-week non-contact boxing intervention study involved 33 healthy adults between 18 and 27 years of age who had no boxing experience (control group = 17 participants, boxing group = 16 participants). Pretests and post-tests utilized rapid bilateral arm raising as a focal movement to elicit APAs. EMG in the anterior deltoid, thoracic and lumbar erector spinae, semitendinosus, and soleus muscles was recorded. The boxing group completed twenty 90-min non-contact boxing training sessions over 8 weeks, whereas the control group kept physical activity consistent during the intervention period. RESULTS: Non-contact boxing training caused APAs to become more frequent during the focal movement, in comparison to the control group, in the soleus and in the semitendinosus after an outlier was removed. Non-contact boxing training caused earlier APA onset during the focal movement, in comparison to the control group, in the lumbar erector spinae after an outlier was removed. CONCLUSIONS: Non-contact boxing training had a modest positive effect on the frequency and timing of APAs resulting from self-induced postural perturbations in healthy adults.

Effect of a single cold stress exposure on the reproductive behavior of male crickets.

Cold stress is an important abiotic factor that can impact insect physiology, behavior, and overall fitness. Upon exposure to cold temperature, many insects enter a reversible state of immobility called chill coma. If the cold stress is brief and mild enough, insects can recover and regain full mobility upon return to warmer temperatures. However, the long-term impact of sublethal cold stress on insect behavior has been understudied. Here, sexually naïve adult male Acheta domesticus crickets were exposed to a single 0 °C cold stress for 6 h. One week later, the ability of these males to mate with a female was examined. For mating trials, a cold stressed male cricket was paired with a non-cold stressed, control female. Control pairs were comprised of a non-cold stressed control male and control female. Cold exposed males were less successful at mating than control males because most did not carry a spermatophore at the time of their mating trials. However, when these cold stressed males were allowed 1 h of chemosensory contact with a female, most produced a spermatophore. Males that produced spermatophores were given the opportunity to mate once with a female, and stressed males that successfully mated sired as many offspring as did control males. However, our results support that a single cold stress exposure can negatively impact the reproductive fitness of male crickets since it reduced their capacity to carry spermatophores and, as a consequence, to attract females.

Butyrate Shapes Immune Cell Fate and Function in Allergic Asthma.

The microbiome plays a fundamental role in how the immune system develops and how inflammatory responses are shaped and regulated. The "gut-lung axis" is a relatively new term that highlights a crucial biological crosstalk between the intestinal microbiome and lung. A growing body of literature suggests that dysbiosis, perturbation of the gut microbiome, is a driving force behind the development, and severity of allergic asthma. Animal models have given researchers new insights into how gut microbe-derived components and metabolites, such as short-chain fatty acids (SCFAs), influence the development of asthma. While the full understanding of how SCFAs influence allergic airway disease remains obscure, a recurring theme of epigenetic regulation of gene expression in several immune cell compartments is emerging. This review will address our current understanding of how SCFAs, and specifically butyrate, orchestrates cell behavior, and epigenetic changes and will provide a detailed overview of the effects of these modifications on immune cells in the context of allergic airway disease.

Inline holographic microscopy through fiber imaging bundles.

Fiber imaging bundles are widely used as thin, passive image conduits for miniaturized and endoscopic microscopy, particularly for confocal fluorescence imaging. Holographic microscopy through fiber bundles is more challenging; phase conjugation approaches are complex and require extensive calibration. This paper describes how simple inline holographic microscopy can be performed through an imaging bundle using a partially coherent illumination source from a multimode fiber. The sample is imaged in transmission, with the intensity hologram sampled by the bundle and transmitted to a remote camera. The hologram can then be numerically refocused for volumetric imaging, achieving a resolution of approximately 6 µm over a depth range of 1 mm. The scheme does not require any complex prior calibration and hence is insensitive to bending.