Quantum Defect Sensitization via Phase-Changing Supercharged Antibody Fragments.

Quantum defects in single-walled carbon nanotubes promote exciton localization, which enables potential applications in biodevices and quantum light sources. However, the effects of local electric fields on the emissive energy states of quantum defects and how they can be controlled are unexplored. Here, we investigate quantum defect sensitization by engineering an intrinsically disordered protein to undergo a phase change at a quantum defect site. We designed a supercharged single-chain antibody fragment (scFv) to enable a full ligand-induced folding transition from an intrinsically disordered state to a compact folded state in the presence of a cytokine. The supercharged scFv was conjugated to a quantum defect to induce a substantial local electric change upon ligand binding. Employing the detection of a proinflammatory biomarker, interleukin-6, as a representative model system, supercharged scFv-coupled quantum defects exhibited robust fluorescence wavelength shifts concomitant with the protein folding transition. Quantum chemical simulations suggest that the quantum defects amplify the optical response to the localization of charges produced upon the antigen-induced folding of the proteins, which is difficult to achieve in unmodified nanotubes. These findings portend new approaches to modulate quantum defect emission for biomarker sensing and protein biophysics and to engineer proteins to modulate binding signal transduction.

A pan-cancer agent for screening, resection and wound monitoring via NIR and SWIR imaging.

Fluorescence guided surgery (FGS) facilitates real time tumor delineation and is being rapidly established clinically. FGS efficacy is tied to the utilized dye and provided tumor contrast over healthy tissue. Apoptosis, a cancer hallmark, is a desirable target for tumor delineation. Here, we preclinically in vitro and in vivo, validate an apoptosis sensitive commercial carbocyanine dye (CJ215), with absorption and emission spectra suitable for near infrared (NIR, 650-900nm) and shortwave infrared (SWIR, 900-1700nm) fluorescence imaging (NIRFI, SWIRFI). High contrast SWIRFI for solid tumor delineation is demonstrated in multiple murine and human models including breast, prostate, colon, fibrosarcoma and intraperitoneal colorectal metastasis. Organ necropsy and imaging highlighted renal clearance of CJ215. SWIRFI and CJ215 delineated all tumors under ambient lighting with a tumor-to-muscle ratio up to 100 and tumor-to-liver ratio up to 18, from 24 to 168 h post intravenous injection with minimal uptake in healthy organs. Additionally, SWIRFI and CJ215 achieved non-contact quantifiable wound monitoring through commercial bandages. CJ215 provides tumor screening, guided resection, and wound healing assessment compatible with existing and emerging clinical solutions.

Uterine washings as a novel method for early detection of ovarian cancer: Trials and tribulations.

Given the tubal origin of high-grade serous ovarian cancer (HGSC), we sought to investigate intrauterine lavage (IUL) as a novel method of biomarker detection. IUL and serum samples were collected from patients with HGSC or benign pathology. Although CA-125 and HE4 concentrations were significantly higher in IUL samples compared to serum, they were similar between IUL samples from patients with HGSC vs benign conditions. In contrast, CA-125 and HE4 serum concentrations differed between HGSC and benign pathology (P =.002 for both). IUL and tumor samples from patients with HGSC were subjected to targeted panel sequencing and droplet digital PCR (ddPCR). Tumor mutations were found in 75 % of matched IUL samples. Serum CA-125 and HE4 biomarker levels allowed for better differentiation of HGSC and benign pathology compared to IUL samples. We believe using IUL for early detection of HGSC requires optimization, and current strategies should focus on prevention until early detection strategies improve.

Astrocyte ensheathment of calyx-forming axons of the auditory brainstem precedes accelerated expression of myelin genes and myelination by oligodendrocytes.

Early postnatal brain development involves complex interactions among maturing neurons and glial cells that drive tissue organization. We previously analyzed gene expression in tissue from the mouse medial nucleus of the trapezoid body (MNTB) during the first postnatal week to study changes that surround rapid growth of the large calyx of Held (CH) nerve terminal. Here, we present genes that show significant changes in gene expression level during the second postnatal week, a developmental timeframe that brackets the onset of airborne sound stimulation and the early stages of myelination. Gene Ontology analysis revealed that many of these genes are related to the myelination process. Further investigation of these genes using a previously published cell type-specific bulk RNA-Seq data set in cortex and our own single-cell RNA-Seq data set in the MNTB revealed enrichment of these genes in the oligodendrocyte lineage (OL) cells. Combining the postnatal day (P)6-P14 microarray gene expression data with the previously published P0-P6 data provided fine temporal resolution to investigate the initiation and subsequent waves of gene expression related to OL cell maturation and the process of myelination. Many genes showed increasing expression levels between P2 and P6 in patterns that reflect OL cell maturation. Correspondingly, the first myelin proteins were detected by P4. Using a complementary, developmental series of electron microscopy 3D image volumes, we analyzed the temporal progression of axon wrapping and myelination in the MNTB. By employing a combination of established ultrastructural criteria to classify reconstructed early postnatal glial cells in the 3D volumes, we demonstrated for the first time that astrocytes within the mouse MNTB extensively wrap the axons of the growing CH terminal prior to OL cell wrapping and compaction of myelin. Our data revealed significant expression of several myelin genes and enrichment of multiple genes associated with lipid metabolism in astrocytes, which may subserve axon wrapping in addition to myelin formation. The transition from axon wrapping by astrocytes to OL cells occurs rapidly between P4 and P9 and identifies a potential new role of astrocytes in priming calyceal axons for subsequent myelination.

Give them a fishing rod, if it is not urgent: The impact of help type on support for helpers' leadership.

Taking a follower's perspective on leadership and contributing to the new research stream on behaviors conducive to its emergence, we examined how distinct types of instrumental (task focused) helping-autonomy- versus dependency-helping-affected recipients' support for their helpers' leadership. Based on the literature on employees' needs for autonomy and mastery, combined with the empowering nature of autonomy-helping, we reasoned that autonomy- (vs. dependency-) helping typically signals greater benevolence toward recipients, enhancing their support for their helpers' leadership. Our findings were generalized across various samples (of U.S. and Israeli employees), manipulations, and research settings: simulations (Studies 1 and 2b), workplace role-play scenario (Study 2a), and recollections of helping events in the workplace (Study 3). We found that autonomy- (vs. dependency-) helping increased recipients' support for their helpers' leadership by heightening perceptions of helpers' benevolence-based (rather than ability-based) trustworthiness (Studies 1 and 3). We also showed time pressure to be a boundary condition under which the advantage of autonomy-helping disappeared (Studies 2a and 2b)-with dependency-helping then inducing comparable levels of perceived benevolence and thus similar support for the helper's potential leadership. Overall, we shed light on the development of informal leadership by uncovering how recipients interpret and respond to the two help types. Practically, this analysis opens the door to new ways for aspiring managers to enhance support for their leadership from potential followers, available even to those unlikely to be appointed to formal leadership positions. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Fragment-based drug nanoaggregation reveals drivers of self-assembly.

Drug nanoaggregates are particles that can deleteriously cause false positive results during drug screening efforts, but alternatively, they may be used to improve pharmacokinetics when developed for drug delivery purposes. The structural features of molecules that drive nanoaggregate formation remain elusive, however, and the prediction of intracellular aggregation and rational design of nanoaggregate-based carriers are still challenging. We investigate nanoaggregate self-assembly mechanisms using small molecule fragments to identify the critical molecular forces that contribute to self-assembly. We find that aromatic groups and hydrogen bond acceptors/donors are essential for nanoaggregate formation, suggesting that both π-π stacking and hydrogen bonding are drivers of nanoaggregation. We apply structure-assembly-relationship analysis to the drug sorafenib and discover that nanoaggregate formation can be predicted entirely using drug fragment substructures. We also find that drug nanoaggregates are stabilized in an amorphous core-shell structure. These findings demonstrate that rational design can address intracellular aggregation and pharmacologic/delivery challenges in conventional and fragment-based drug development processes.

Nanoreporter Identifies Lysosomal Storage Disease Lipid Accumulation Intracranially.

Dysregulated lipid metabolism contributes to neurodegenerative pathologies and neurological decline in lysosomal storage disorders as well as more common neurodegenerative diseases. Niemann-Pick type A (NPA) is a fatal neurodegenerative lysosomal storage disease characterized by abnormal sphingomyelin accumulation in the endolysosomal lumen. The ability to monitor abnormalities in lipid homeostasis intracranially could improve basic investigations and the development of effective treatment strategies. We investigated the carbon nanotube-based detection of intracranial lipid content. We found that the near-infrared emission of a carbon nanotube-based lipid sensor responds to lipid accumulation in neuronal and in vivo models of NPA. The nanosensor detected lipid accumulation intracranially in an acid sphingomyelinase knockout mouse via noninvasive near-infrared spectroscopy. This work indicates a tool to improve drug development processes in NPA, other lysosomal storage diseases, and neurodegenerative diseases.

Ambient Light Resistant Shortwave Infrared Fluorescence Imaging for Preclinical Tumor Delineation via the pH Low-Insertion Peptide Conjugated to Indocyanine Green.

Shortwave infrared (900-1,700 nm) fluorescence imaging (SWIRFI) has shown significant advantages over visible (400-650 nm) and near-infrared (700-900 nm) fluorescence imaging (reduced autofluorescence, improved contrast, tissue resolution, and depth sensitivity). However, there is a major lag in the clinical translation of preclinical SWIRFI systems and targeted SWIRFI probes. Methods: We preclinically show that the pH low-insertion peptide conjugated to indocyanine green (pHLIP ICG), currently in clinical trials, is an excellent candidate for cancer-targeted SWIRFI. Results: pHLIP ICG SWIRFI achieved picomolar sensitivity (0.4 nM) with binary and unambiguous tumor screening and resection up to 96 h after injection in an orthotopic breast cancer mouse model. SWIRFI tumor screening and resection had ambient light resistance (possible without gating or filtering) with outstanding signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) values at exposures from 10 to 0.1 ms. These SNR and CNR values were also found for the extended emission of pHLIP ICG in vivo (>1,100 nm, 300 ms). Conclusion: SWIRFI sensitivity and ambient light resistance enabled continued tracer clearance tracking with unparalleled SNR and CNR values at video rates for tumor delineation (achieving a tumor-to-muscle ratio above 20). In total, we provide a direct precedent for the democratic translation of an ambient light resistant SWIRFI and pHLIP ICG ecosystem, which can instantly improve tumor resection.

Design of Polarity-Dependent Immunosensors Based on the Structural Analysis of Engineered Antibodies.

"Reagentless" immunosensors are emerging to address the challenge of practical and sensitive detection of important biomarkers in real biological samples without the need for multistep assays and user intervention, with applications ranging from research tools to point-of-care diagnostics. Selective target binding to an affinity reagent is detected and reported in one step without the need for washing or additional reporters. In this study, we used a structure-guided approach to identify a mutation site in an antibody fragment for the polarity-dependent fluorophore, Anap, such that upon binding of the protein target cardiac troponin I, the Anap-labeled antibody would produce a detectable and dose-dependent shift in emission wavelength. We observed a significant emission wavelength shift of the Anap-labeled anti-cTnI mutant, with a blue shift of up to 37 nm, upon binding to the cTnI protein. Key differences in the resulting emission spectra between target peptides in comparison to whole proteins were also found; however, the affinity and binding characteristics remained unaffected when compared to the wild-type antibody. We also highlighted the potential flexibility of the approach by incorporating a near-infrared dye, IRDye800CW, into the same mutation site, which also resulted in a dose-dependent wavelength shift upon target incubation. These reagents can be used in experiments and devices to create simpler and more efficient biosensors across a range of research, medical laboratory, and point-of-care platforms.

Nanosensor-based monitoring of autophagy-associated lysosomal acidification in vivo.

Autophagy is a cellular process with important functions that drive neurodegenerative diseases and cancers. Lysosomal hyperacidification is a hallmark of autophagy. Lysosomal pH is currently measured by fluorescent probes in cell culture, but existing methods do not allow for quantitative, transient or in vivo measurements. In the present study, we developed near-infrared optical nanosensors using organic color centers (covalent sp3 defects on carbon nanotubes) to measure autophagy-mediated endolysosomal hyperacidification in live cells and in vivo. The nanosensors localize to the lysosomes, where the emission band shifts in response to local pH, enabling spatial, dynamic and quantitative mapping of subtle changes in lysosomal pH. Using the sensor, we observed cellular and intratumoral hyperacidification on administration of mTORC1 and V-ATPase modulators, revealing that lysosomal acidification mirrors the dynamics of S6K dephosphorylation and LC3B lipidation while diverging from p62 degradation. This sensor enables the transient and in vivo monitoring of the autophagy-lysosomal pathway.

Micro-engineering and nano-engineering approaches to investigate tumour ecosystems.

The interactions among tumour cells, the tumour microenvironment (TME) and non-tumour tissues are of interest to many cancer researchers. Micro-engineering approaches and nanotechnologies are under extensive exploration for modelling these interactions and measuring them in situ and in vivo to investigate therapeutic vulnerabilities in cancer and extend a systemic view of tumour ecosystems. Here we highlight the greatest opportunities for improving the understanding of tumour ecosystems using microfluidic devices, bioprinting or organ-on-a-chip approaches. We also discuss the potential of nanosensors that can transmit information from within the TME or elsewhere in the body to address scientific and clinical questions about changes in chemical gradients, enzymatic activities, metabolic and immune profiles of the TME and circulating analytes. This Review aims to connect the cancer biology and engineering communities, presenting biomedical technologies that may expand the methodologies of the former, while inspiring the latter to develop approaches for interrogating cancer ecosystems.

Hematoxylin Nuclear Stain Reports Oxidative Stress via Near-Infrared Emission.

Hematoxylin & eosin (H&E) is the gold standard histological stain used for medical diagnosis and has been used for over a century. Herein, we examined the near-infrared II (NIR-II) fluorescence of this stain. We observed significant NIR-II emission from the hematoxylin component of the H&E stain. We found that the emission intensity, using the common aluminum(III) hematoxylin mordant, could be modulated by the availability of endogenous iron(III), and this emission intensity increased at higher oxidative stress. Our mechanistic investigations found that hematoxylin emission reported the nuclear translocation of the iron via the protein ferritin. In human tumor tissue samples, oxidative stress biomarkers correlated with hematoxylin NIR-II emission intensity. Emission response of the stain was also observed in human Alzheimer's disease brain tissue regions affected by disease progression, suggesting that ferritin nuclear translocation is preserved in these regions as an oxidative stress response. These findings indicate that NIR-II emission from the H&E stain provides a new source of redox information in tissues with implications for biomedical research and clinical practice.

P-selectin-targeted nanocarriers induce active crossing of the blood-brain barrier via caveolin-1-dependent transcytosis.

Medulloblastoma is the most common malignant paediatric brain tumour, with ~30% mediated by Sonic hedgehog signalling. Vismodegib-mediated inhibition of the Sonic hedgehog effector Smoothened inhibits tumour growth but causes growth plate fusion at effective doses. Here, we report a nanotherapeutic approach targeting endothelial tumour vasculature to enhance blood-brain barrier crossing. We use fucoidan-based nanocarriers targeting endothelial P-selectin to induce caveolin-1-dependent transcytosis and thus nanocarrier transport into the brain tumour microenvironment in a selective and active manner, the efficiency of which is increased by radiation treatment. In a Sonic hedgehog medulloblastoma animal model, fucoidan-based nanoparticles encapsulating vismodegib exhibit a striking efficacy and marked reduced bone toxicity and drug exposure to healthy brain tissue. Overall, these findings demonstrate a potent strategy for targeted intracranial pharmacodelivery that overcomes the restrictive blood-brain barrier to achieve enhanced tumour-selective penetration and has therapeutic implications for diseases within the central nervous system.

NDUFA4L2 reduces mitochondrial respiration resulting in defective lysosomal trafficking in clear cell renal cell carcinoma.

In clear cell renal cell carcinoma (ccRCC), activation of hypoxic signaling induces NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2) expression. Over 90% of ccRCCs exhibit overexpression of NDUFA4L2, which we previously showed contributes to ccRCC proliferation and survival. The function of NDUFA4L2 in ccRCC has not been fully elucidated. NDUFA4L2 was reported to reduce mitochondrial respiration via mitochondrial complex I inhibition. We found that NDUFA4L2 expression in human ccRCC cells increases the extracellular acidification rate, indicative of elevated glycolysis. Conversely, NDUFA4L2 expression in non-cancerous kidney epithelial cells decreases oxygen consumption rate while increasing extracellular acidification rate, suggesting that a Warburg-like effect is induced by NDUFA4L2 alone. We performed mass-spectrometry (MS)-based proteomics of NDUFA4L2 associated complexes. Comparing RCC4-P (parental) ccRCC cells with RCC4 in which NDUFA4L2 is knocked out by CRISPR-Cas9 (RCC4-KO-643), we identified 3,215 proteins enriched in the NDUFA4L2 immunoprecipitates. Among the top-ranking pathways were "Metabolic Reprogramming in Cancer" and "Glycolysis Activation in Cancer (Warburg Effect)." We also show that NDUFA4L2 enhances mitochondrial fragmentation, interacts with lysosomes, and increases mitochondrial-lysosomal associations, as assessed by high-resolution fluorescence microscopy and live cell imaging. We identified 161 lysosomal proteins, including Niemann-Pick Disease Type C Intracellular Cholesterol Transporters 1 and 2 (NPC1, NPC2), that are associated with NDUFA4L2 in RCC4-P cells. RCC4-P cells have larger and decreased numbers of lysosomes relative to RCC4 NDUFA4L2 knockout cells. These findings suggest that NDUFA4L2 regulates mitochondrial-lysosomal associations and potentially lysosomal size and abundance. Consequently, NDUFA4L2 may regulate not only mitochondrial, but also lysosomal functions in ccRCC.

Characteristics, ICU Interventions, and Clinical Outcomes of Patients With Palliative Care Triggers in a Mixed Community-Based Intensive Care Unit.

Objective: Integration of palliative care initiatives in the intensive care unit (ICU) benefit patients and improve outcomes. Palliative care triggers (PCTs) is a screening tool that aides in stratifying patients who would benefit most from an early palliative care approach. There is no consensus on PCT selection or best timing for implementation. We evaluated the clinical characteristics, ICU and palliative care interventions, and clinical outcomes of critically ill patients with PCT in a community-based mixed ICU. Methods: This retrospective study was conducted in a 44-bed adult, mixed ICU in a 407-bed community-based teaching hospital in Florida. Eleven PCTs were used as a screening tool during multidisciplinary rounds (MDRs). Patients were analyzed based on presence or absence of PCT as well as having met high (>2) versus low (<2) PCT. Data collected included patient demographics, ICU resource utilization and clinical outcomes. We considered a two-sided P value of less than .05 to indicate statistical significance with a 95% confidence interval. Results: Of 388 ICU patients, 189 (48.7%) met at least 1 PCT and 199 (51.3%) did not. The trigger group had higher Acute Physiology and Chronic Evaluation (APACHE) and Sequential Organ Failure Assessment (SOFA) scores within 24 h of ICU admission. The most common PCTs identified were ICU length of stay greater than 7 days or readmission to ICU, terminal prognosis and assisting family in transitioning goals of care. There were statistically significant differences in ICU resource utilization, palliative care interventions, and overall worse clinical outcomes in the trigger-detected group. Similar findings were seen in the cohort with high PCT (>2). Conclusions: Our study supports the implementation of a tailored 11-item palliative care screening tool to effectively identify ICU patients with high ICU and palliative care interventions and worse clinical outcomes.

Extraskeletal Ewing sarcoma of the sciatic nerve.

Extraskeletal Ewing sarcoma (EES) is a rare tumor diagnosed in children or young adults and is even more unusual in individuals over 30 years of age. Due to its rare occurrence and low index of suspicion, this tumor can pose diagnostic and therapeutic challenges. We present a case of a 60-year-old male with EES of the sciatic nerve, an unexpected entity given the patient's age, tumor type, and tumor location. This can mimic a nerve sheath tumor on imaging.

Revival of psychology in former Czechoslovakia and the contemporary Czech Republic after the fall of Totalitarian communist regimes.

After the general societal and political change in November 1989 in Czechoslovakia, the subject "History of Psychology" became the stable component of curriculum of studying psychology at the Department of Psychology of Faculty of Arts of Charles University in Prague. The author of this paper has taught "History of Psychology" in Czech since 1998 for more than 20 years all students of psychology and he is teaching this subject the students of ERASMUS+ program from whole Europe, studying at Charles University in Prague, now. Indivisible part of the curriculum is represented by the history of Czechoslovak and Czech psychology. In References, the most important publications in the field of history of Czechoslovak and Czech psychology are presented.

Drug-Eluting Rubber Bands for Tissue Ligation.

Rubber band ligation is a commonly used method for the removal of tissue abnormalities. Most often, rubber band ligation is performed to remove internal hemorrhoids unresponsive to first line treatments to avoid surgery. While the procedure is considered safe, patients experience mild to significant pain and discomfort until the tissue sloughs off. As patients often require multiple bandings and sessions, reducing these side effects can have a considerable effect on patient adherence and quality of life. To reduce pain and discomfort, we developed drug-eluting rubber bands for ligation procedures. We investigated the potential for a band to elute anesthetics and drug combinations to durably manage pain for a period of up to 5 days while exhibiting similar mechanical properties to conventional rubber bands. We show that the rubber bands retain their mechanical properties despite significant drug loading. Lidocaine, released from the bands, successfully altered the calcium dynamics of cardiomyocytes in vitro and modulated heart rate in zebrafish embryos, while the bands exhibited lower cytotoxicity than conventional bands. Ex vivo studies demonstrated substantial local drug release in enteric tissues. These latex-free bands exhibited sufficient mechanical and drug-eluting properties to serve both ligation and local analgesic functions, potentially enabling pain reduction for multiple indications.

The IFNγ-PDL1 Pathway Enhances CD8T-DCT Interaction to Promote Hypertension.

BACKGROUND: Renal T cells contribute importantly to hypertension, but the underlying mechanism is incompletely understood. We reported that CD8Ts directly stimulate distal convoluted tubule cells (DCTs) to increase NCC (sodium chloride co-transporter) expression and salt reabsorption. However, the mechanistic basis of this pathogenic pathway that promotes hypertension remains to be elucidated. METHODS: We used mouse models of DOCA+salt (DOCA) treatment and adoptive transfer of CD8+ T cells (CD8T) from hypertensive animals to normotensive animals in in vivo studies. Co-culture of mouse DCTs and CD8Ts was used as in vitro model to test the effect of CD8T activation in promoting NCC-mediated sodium retention and to identify critical molecular players contributing to the CD8T-DCT interaction. Interferon (IFNγ)-KO mice and mice receiving renal tubule-specific knockdown of PDL1 were used to verify in vitro findings. Blood pressure was continuously monitored via radio-biotelemetry, and kidney samples were saved at experimental end points for analysis. RESULTS: We identified critical molecular players and demonstrated their roles in augmenting the CD8T-DCT interaction leading to salt-sensitive hypertension. We found that activated CD8Ts exhibit enhanced interaction with DCTs via IFN-γ-induced upregulation of MHC-I and PDL1 in DCTs, thereby stimulating higher expression of NCC in DCTs to cause excessive salt retention and progressive elevation of blood pressure. Eliminating IFN-γ or renal tubule-specific knockdown of PDL1 prevented T cell homing into the kidney, thereby attenuating hypertension in 2 different mouse models. CONCLUSIONS: Our results identified the role of activated CD8Ts in contributing to increased sodium retention in DCTS through the IFNγ-PDL1 pathway. These findings provide a new mechanism for T cell involvement in the pathogenesis of hypertension and reveal novel therapeutic targets.

Optical Nanosensor for Intracellular and Intracranial Detection of Amyloid-Beta.

Amyloid-beta (Aß) deposition occurs in the early stages of Alzheimer's disease (AD), but the early detection of Aß is a persistent challenge. Herein, we engineered a near-infrared optical nanosensor capable of detecting Aß intracellularly in live cells and intracranially in vivo. The sensor is composed of single-walled carbon nanotubes functionalized with Aß wherein Aß-Aß interactions drive the response. We found that the Aß nanosensors selectively responded to Aß via solvatochromic modulation of the near-infrared emission of the nanotube. The sensor tracked Aß accumulation in live cells and, upon intracranial administration in a genetic model of AD, signaled distinct responses in aged mice. This technology enables the interrogation of molecular mechanisms underlying Aß neurotoxicity in the development of AD in living systems.