Layer-by-Layer Nanoparticle Assembly for Biomedicine: Mechanisms, Technologies, and Advancement via Acoustofluidics.

The deposition of thin films plays a crucial role in surface engineering, tailoring structural modifications, and functionalization across diverse applications. Layer-by-layer self-assembly, a prominent thin-film deposition method, has witnessed substantial growth since its mid-20th-century inception, driven by the discovery of eligible materials and innovative assembly technologies. Of these materials, micro- and nanoscopic substrates have received far less interest than their macroscopic counterparts; however, this is changing. The catalogue of eligible materials, including nanoparticles, quantum dots, polymers, proteins, cells and liposomes, along with some well-established layer-by-layer technologies, have combined to unlock impactful applications in biomedicine, as well as other areas like food fortification, and water remediation. To access these fields, several well-established technologies have been used, including tangential flow filtration, fluidized bed, atomization, electrophoretic assembly, and dielectrophoresis. Despite the invention of these technologies, the field of particle layer-by-layer still requires further technological development to achieve a high-yield, automatable, and industrially ready process, a requirement for the diverse, reactionary field of biomedicine and high-throughput pharmaceutical industry. This review provides a background on layer-by-layer, focusing on how its constituent building blocks and bonding mechanisms enable unmatched versatility. The discussion then extends to established and recent technologies employed for coating micro- and nanoscopic matter, evaluating their drawbacks and advantages, and highlighting promising areas in microfluidic approaches, where one distinctly auspicious technology emerges, acoustofluidics. The review also explores the potential and demonstrated application of acoustofluidics in layer-by-layer technology, as well as analyzing existing acoustofluidic technologies beyond LbL coating in areas such as cell trapping, cell sorting, and multidimensional particle manipulation. Finally, the review concludes with future perspectives on layer-by-layer nanoparticle coating and the potential impact of integrating acoustofluidic methods.

Alcohol use is associated with intracranial hemorrhage in older emergency department head trauma patients.

Objectives: Falls are common in adults aged 65 years and older and are the leading cause of traumatic brain injuries in this age group. Alcohol use may increase the risk of falls as well as the severity of resultant injuries. The aim of this study was to examine the association between self-reported alcohol use and the prevalence of intracranial hemorrhage (ICH) in this patient group. Methods: This was a secondary analysis of the Geriatric Head Trauma Short Term Outcomes Project (GREAT STOP), a study of older adults with blunt head trauma from a fall. We determined the characteristics of every fall event, including patient demographics and medical history, and clinical signs and symptoms related to head trauma. Self-reported alcohol use was categorized as none, occasionally, weekly, or daily. We defined ICH as any acute ICH detected by computed tomography scan. We evaluated the association between alcohol use frequency and ICH, adjusted for patient factors and head injury risk factors. Results: Of 3128 study participants, 18.2% (n = 567) reported alcohol use: 10.3% with occasional use, 1.9% with weekly use, and 6.0% with daily use. ICH was more common in patients who used alcohol (20.5%, 22.0%, and 25.1% for occasional, weekly, and daily alcohol users, respectively, vs. 12.0% for non-users, p < 0.001). The frequency of alcohol use was independently associated with ICH, adjusted for patient and head injury risk factors. The adjusted odds ratios (with 95% confidence intervals) for occasional, weekly, and daily alcohol users increased from 2.0 (1.5‒2.8) to 2.1 (1.1‒4.1) and 2.5 (1.7‒3.6), respectively, and showed the characteristics of dose‒response effect. Conclusions: Alcohol use in older adult emergency department patients with head trauma is relatively common. Self-reported alcohol use appears to be associated with a higher risk of ICH in a dose-dependent fashion. Fall prevention strategies may need to consider alcohol mitigation as a modifiable risk factor.

The anxiety care continuum and its association with viral suppression among persons with HIV.

OBJECTIVE: It is unclear how often anxiety is diagnosed and treated and whether anxiety treatment is associated with improved viral suppression in persons with HIV. In this study, we characterized the anxiety care continuum and its association with viral suppression in a large urban HIV clinic in the United States. DESIGN: Observational cohort study. METHODS: We described the anxiety care continuum by combining data on self-reported anxiety symptoms, engagement in mental health care, clinical diagnoses and prescriptions from 1,967 persons receiving HIV care and treatment in Baltimore, Maryland, from 2014-23. We examined cross-sectional associations with viral suppression. All analyses were stratified by sex and race/ethnicity; a secondary analysis adjusted for age, years in care, and depressive symptoms. RESULTS: Nearly 1 in 5 patients reported mild-severe symptoms of anxiety but were not currently receiving mental health care or pharmacologic treatment for anxiety; 6% of patients reported anxiety symptoms but were receiving treatment, and 7% had been treated for anxiety that was currently in remission. The prevalence of viral suppression ranged from 87-89% across the anxiety care continuum except among patients with untreated moderate-severe anxiety, only 81% of whom were virally suppressed (95% CI: 80, 83). In adjusted models, untreated moderate-severe anxiety remained associated with viral non-suppression across demographic groups. CONCLUSION: We observed a robust association between untreated anxiety and viral non-suppression in a large urban cohort of persons with HIV. Screening for anxiety may identify patients with unmet mental health care needs who face barriers to maintaining viral suppression.

Patellofemoral Disorders in Soccer Players.

Patellofemoral disorders are common in the world of soccer and impact players across all levels and ages of the sport. Patellofemoral disorders encompass a spectrum of conditions, from anterior knee pain to patellar instability, and are often influenced by complex biomechanical factors and anatomic variations that can predispose to these conditions. In recent years, there has been a growing emphasis on injury prevention strategies and data-driven approaches, championed by organizations like the Union of European Football Associations and individual professional clubs. Conservative management remains the initial approach for many players, including physical therapy and supportive devices. However, surgical intervention, particularly in cases of recurrent patellar dislocations, is often necessary. The understanding of patellofemoral biomechanics in soccer continues to evolve and offers opportunities for more effective injury prevention and tailored treatment strategies. Despite the challenges, a comprehensive approach to patellofemoral disorders in soccer is essential to preserve player health, enhance performance, and sustain the sport's vitality.

Toward once-monthly insulin therapy via synergy in two pharmacokinetic protractors: Fc-conjugation and fatty acid acylation.

Pharmacokinetic properties and duration of therapeutic action of a pharmaceutical agent can be significantly extended through the combination of two distinct strategies aimed at increasing plasma half-life: fatty acid acylation and Fc-conjugation. Using insulin as a case study, we demonstrate that a doubly protracted insulin analog produces a substantial prolongation of pharmacodynamic effect to lower blood glucose in STZ-treated mice when compared to the Fc-only counterparts. This enhancement is further corroborated by direct pharmacokinetic measurements in rat and dog models, demonstrating the potential for once-monthly insulin therapy. The results suggest that this approach might have broad application across a diverse spectrum of peptide- and protein-based therapeutics.

Sex Differences in the Risk of Stroke Associated With Traditional and Non-Traditional Factors in a US Cohort of People With HIV Infection.

BACKGROUND AND OBJECTIVES: Although stroke risk associated with HIV may be greater for women than men, little is known about whether the impact of different factors on cerebrovascular risk varies by sex in people with HIV (PWH) and contributes to stroke risk disparities in this population. The primary objective of this study was to examine whether sex modifies the effect of demographics, cardiometabolic factors, health-related behaviors, and HIV-specific variables on stroke risk in PWH from the Centers for AIDS Research Network of Integrated Clinical Systems (CNICS) cohort. METHODS: In this observational cohort study, we analyzed data from clinical encounters for PWH followed at 5 CNICS sites from approximately 2005 to 2020. All potential stroke events were adjudicated by neurologists. Patient-reported outcomes collected at clinic visits, including substance use and depression, were also available. We used Cox proportional hazards models to determine whether sex modified the association of predictors of interest with incident stroke. RESULTS: Among 13,573 PWH (19% female sex at birth, mean age 44 years, mean follow-up 5.6 years), female sex was associated with a higher risk of stroke only among individuals aged 50 years or younger (hazard ratio [HR] 2.01 at age 40 [1.25-3.21] vs HR 0.60 at age 60 [0.34-1.06]; p = 0.001 for the interaction). Younger female participants who developed a stroke were more likely to have treated hypertension, a higher cardiovascular risk score, and detectable HIV than younger male participants whereas these factors were comparable by sex among older participants who developed a stroke. Sex modified the effect of detectable HIV (HR 4.66 for female participants [2.48-8.74] vs HR 1.30 for male participants [0.83-2.03]; p = 0.001 for the interaction), methamphetamine use (HR 4.78 for female participants [1.47-15.56] vs HR 1.19 for male participants [0.62-2.29]; p = 0.04 for the interaction), and treated hypertension (HR 3.44 for female participants [1.74-6.81] vs HR 1.66 for male participants [1.14-2.41]; p = 0.06 for the interaction) on stroke risk. DISCUSSION: Younger female participants with HIV were at elevated cerebrovascular risk compared with younger male participants. Several risk factors had a greater adverse effect on stroke risk in female participants than in male participants, including HIV viremia, methamphetamine use, and treated hypertension. These findings underscore the importance of a personalized approach to predict and prevent cerebrovascular risk among PWH.

Integrated renal and sympathetic mechanisms underlying the development of sex- and age-dependent hypertension and the salt sensitivity of blood pressure.

Aging is a non-modifiable understudied risk factor for hypertension. We hypothesized that sympathetically mediated activation of renal sodium reabsorption drives age-dependent hypertension and the salt sensitivity of blood pressure (BP). Using 3-, 8-, and 16-month-old male and female Sprague-Dawley rats as a model of normal aging, we assessed BP, indices of sympathetic tone, and the physiological responses to acute and chronic sodium challenge including sodium chloride cotransporter (NCC) regulation. The effects of renal nerve ablation and NCC antagonism were assessed in hypertensive male rats. We observed sex-dependent impaired renal sodium handling (24 h sodium balance (meq), male 3-month 0.36 ± 0.1 vs. 16-month 0.84 ± 0.2; sodium load excreted during 5% bodyweight isotonic saline volume expansion (%) male 3-month 77 ± 5 vs. 16-month 22 ± 8), hypertension (MAP (mmHg) male 3-month 123 ± 4 vs. 16-month 148 ± 6), and the salt sensitivity of BP in aged male, but not female, rats. Attenuated sympathoinhibitory afferent renal nerve (ARN) responses contributed to increased sympathetic tone and hypertension in male rats. Increased sympathetic tone contributes to renal sodium retention, in part through increased NCC activity via a dysfunctional with-no-lysine kinase-(WNK) STE20/SPS1-related proline/alanine-rich kinase signaling pathway, to drive hypertension and the salt sensitivity of BP in aged male rats. NCC antagonism and renal nerve ablation, which reduced WNK dysfunction and decreased NCC activity, attenuated age-dependent hypertension in male Sprague-Dawley rats. The contribution of an impaired sympathoinhibitory ARN reflex to sex- and age-dependent hypertension in an NCC-dependent manner, via an impaired WNK1/WNK4 dynamic, suggests this pathway as a mechanism-based target for the treatment of age-dependent hypertension.

The Interaction of a Gemini Surfactant with a DNA Quadruplex.

DNA secondary structures are stabilized by mono- and divalent cations. To examine the stability of the DNA quadruplex formed from (TTAGGG)4, its interaction with a dicationic Gemini surfactant in standard phosphate buffer was investigated. The Gemini surfactant begins to form micelles in buffer at a cmc (critical micelle concentration) of 1.5 mM. In this study, solutions of DNA were prepared in buffer with surfactant concentrations ranging from 0.0 to 3.0 mM, i.e., above and below the cmc of the surfactant. In all samples of DNA and surfactant, a precipitate formed. The fraction of DNA precipitated depends upon both the initial DNA concentration and the initial concentration of the surfactant. In those samples where the DNA did not totally precipitate, the residual DNA assumed a quadruplex conformation. It was determined that two surfactant molecules per DNA phosphate are needed to completely precipitate all of the DNA in a particular sample. An estimated apparent K sp for the DNA:surfactant complex was determined.

High-Spin State of a Ferrocene Electron Donor Revealed by Optical and X-ray Transient Absorption Spectroscopy.

Ferrocene is one of the most common electron donors, and mapping its ligand-field excited states is critical to designing donor-acceptor (D-A) molecules with long-lived charge transfer states. Although 3(d-d) states are commonly invoked in the photophysics of ferrocene complexes, mention of the high-spin 5(d-d) state is scarce. Here, we provide clear evidence of 5(d-d) formation in a bimetallic D-A molecule, ferrocenyl cobaltocenium hexafluorophosphate ([FcCc]PF6). Femtosecond optical transient absorption (OTA) spectroscopy reveals two distinct electronic excited states with 30 and 500 ps lifetimes. Using a combination of ultraviolet, visible, near-infrared, and short-wave infrared probe pulses, we capture the spectral features of these states over an ultrabroadband range spanning 320 to 2200 nm. Time-dependent density functional theory (DFT) calculations of the lowest triplet and quintet states, both primarily Fe(II) (d-d) in character, qualitatively agree with the experimental OTA spectra, allowing us to assign the 30 ps state as the 3(d-d) state and the 500 ps state as the high-spin 5(d-d) state. To confirm the ferrocene-centered high-spin character of the 500 ps state, we performed X-ray transient absorption (XTA) spectroscopy at the Fe and Co K edges. The Fe K-edge XTA spectrum at 150 ps shows a red shift of the absorption edge that is consistent with an Fe(II) high-spin state, as supported by ab initio calculations. The transient signal detected at the Co K-edge is 50× weaker, confirming the ferrocene-centered character of the excited state. Fitting of the transient extended X-ray absorption fine structure region yields an Fe-C bond length increase of 0.25 ± 0.1 Å in the excited state, as expected for the high-spin state based on DFT. Altogether, these results demonstrate that the high-spin state of ferrocene should be considered when designing donor-acceptor assemblies for photocatalysis and photovoltaics.

Identification of Proteomic Biomarkers of Acetaminophen-Induced Hepatotoxicity Using Stable Isotope Labeling.

Quantitative proteomics approaches based on stable isotopic labeling and mass spectrometry have been widely applied to disease research, drug target discovery, biomarker identification, and systems biology. One of the notable stable isotopic labeling approaches is trypsin-catalyzed 18O/16O labeling, which has its own advantages of low sample consumption, simple labeling procedure, cost-effectiveness, and absence of chemical reactions that potentially generate by-products. In this chapter, a protocol for 18O/16O labeling-based quantitative proteomics approach is described with an application to the identification of proteomic biomarkers of acetaminophen (APAP)-induced hepatotoxicity in rats. The protocol involves first the extraction of proteins from liver tissues of control and APAP-treated rats and digestion into peptides by trypsin. After cleaning of the peptides by solid-phase extraction, equal amounts of peptides from the APAP treatment and the control groups are then subject to trypsin-catalyzed 18O/16O labeling. The labeled peptides are combined and fractionated by off-line strong cation exchange liquid chromatography (SCXLC), and each fraction is then analyzed by nanoflow reversed-phase LC coupled online with tandem mass spectrometry (RPLC-MS/MS) for identification and quantification of differential protein expression between APAP-treated rats and controls. The protocol is applicable to quantitative proteomic analysis for a variety of biological samples.

Presynaptic sensor and silencer of peptidergic transmission reveal neuropeptides as primary transmitters in pontine fear circuit.

Neurons produce and release neuropeptides to communicate with one another. Despite their importance in brain function, circuit-based mechanisms of peptidergic transmission are poorly understood, primarily due to the lack of tools for monitoring and manipulating neuropeptide release in vivo. Here, we report the development of two genetically encoded tools for investigating peptidergic transmission in behaving mice: a genetically encoded large dense core vesicle (LDCV) sensor that detects presynaptic neuropeptide release and a genetically encoded silencer that specifically degrades neuropeptides inside LDCVs. Using these tools, we show that neuropeptides, not glutamate, encode the unconditioned stimulus in the parabrachial-to-amygdalar threat pathway during Pavlovian threat learning. We also show that neuropeptides play important roles in encoding positive valence and suppressing conditioned threat response in the amygdala-to-parabrachial endogenous opioidergic circuit. These results show that our sensor and silencer for presynaptic peptidergic transmission are reliable tools to investigate neuropeptidergic systems in awake, behaving animals.

Factors Influencing Neuromuscular Blockade Reversal Choice in the United States Before and During the COVID-19 Pandemic: Retrospective Longitudinal Analysis.

BACKGROUND: Neuromuscular blockade (NMB) agents are a critical component of balanced anesthesia. NMB reversal methods can include spontaneous reversal, sugammadex, or neostigmine and the choice of reversal strategy can depend on various factors. Unanticipated changes to clinical practice emerged due to the COVID-19 pandemic, and a better understanding of how NMB reversal trends were affected by the pandemic may help provide insight into how providers view the tradeoffs in the choice of NMB reversal agents. OBJECTIVE: We aim to analyze NMB reversal agent use patterns for US adult inpatient surgeries before and after the COVID-19 outbreak to determine whether pandemic-related practice changes affected use trends. METHODS: A retrospective longitudinal analysis of a large all-payer national electronic US health care database (PINC AI Healthcare Database) was conducted to identify the use patterns of NMB reversal during early, middle, and late COVID-19 (EC, MC, and LC, respectively) time periods. Factors associated with NMB reversal choices in inpatient surgeries were assessed before and after the COVID-19 pandemic reached the United States. Multivariate logistic regression assessed the impact of the pandemic on NMB reversal, accounting for patient, clinical, procedural, and site characteristics. A counterfactual framework was used to understand if patient characteristics affected how COVID-19-era patients would have been treated before the pandemic. RESULTS: More than 3.2 million inpatients experiencing over 3.6 million surgical procedures across 931 sites that met all inclusion criteria were identified between March 1, 2017, and December 31, 2021. NMB reversal trends showed a steady increase in reversal with sugammadex over time, with the trend from January 2018 onwards being linear with time (R2>0.99). Multivariate analysis showed that the post-COVID-19 time periods had a small but statistically significant effect on the trend, as measured by the interaction terms of the COVID-19 time periods and the time trend in NMB reversal. A slight increase in the likelihood of sugammadex reversal was observed during EC relative to the pre-COVID-19 trend (odds ratio [OR] 1.008, 95% CI 1.003-1.014; P=.003), followed by negation of that increase during MC (OR 0.992, 95% CI 0.987-0.997; P<.001), and no significant interaction identified during LC (OR 1.001, 95% CI 0.996-1.005; P=.81). Conversely, active reversal (using either sugammadex or neostigmine) did not show a significant association relative to spontaneous reversal, or a change in trend, during EC or MC (P>.05), though a slight decrease in the active reversal trend was observed during LC (OR 0.987, 95% CI 0.983-0.992; P<.001). CONCLUSIONS: We observed a steady increase in NMB active reversal overall, and specifically with sugammadex compared to neostigmine, during periods before and after the COVID-19 outbreak. Small, transitory alterations in the NMB reversal trends were observed during the height of the COVID-19 pandemic, though these alterations were independent of the underlying NMB reversal time trends.

Intrinsic PARG inhibitor sensitivity is mimicked by TIMELESS haploinsufficiency and rescued by nucleoside supplementation.

A subset of cancer cells are intrinsically sensitive to inhibitors targeting PARG, the poly(ADP-ribose) glycohydrolase that degrades PAR chains. Sensitivity is accompanied by persistent DNA replication stress, and can be induced by inhibition of TIMELESS, a replisome accelerator. However, the nature of the vulnerability responsible for intrinsic sensitivity remains undetermined. To understand PARG activity dependency, we analysed Timeless model systems and intrinsically sensitive ovarian cancer cells. We show that nucleoside supplementation rescues all phenotypes associated with PARG inhibitor sensitivity, including replisome speed and fork stalling, S-phase completion and mitotic entry, proliferation dynamics and clonogenic potential. Importantly nucleoside supplementation restores PARG inhibitor resistance despite the continued presence of PAR chains, indicating that sensitivity does not correlate with PAR levels. In addition, we show that inhibition of thymidylate synthase, an enzyme required for dNTP homeostasis, induces PARG-dependency. Together, these observations suggest that PARG inhibitor sensitivity reflects an inability to control replisome speed and/or maintain helicase-polymerase coupling in response to nucleotide imbalances.

Conscious but not thinking-Mind-blanks during visuomotor tracking: An fMRI study of endogenous attention lapses.

Attention lapses (ALs) are complete lapses of responsiveness in which performance is briefly but completely disrupted and during which, as opposed to microsleeps, the eyes remain open. Although the phenomenon of ALs has been investigated by behavioural and physiological means, the underlying cause of an AL has largely remained elusive. This study aimed to investigate the underlying physiological substrates of behaviourally identified endogenous ALs during a continuous visuomotor task, primarily to answer the question: Were the ALs during this task due to extreme mind-wandering or mind-blanks? The data from two studies were combined, resulting in data from 40 healthy non-sleep-deprived subjects (20M/20F; mean age 27.1 years, 20-45). Only 17 of the 40 subjects were used in the analysis due to a need for a minimum of two ALs per subject. Subjects performed a random 2-D continuous visuomotor tracking task for 50 and 20 min in Studies 1 and 2, respectively. Tracking performance, eye-video, and functional magnetic resonance imaging (fMRI) were recorded simultaneously. A human expert visually inspected the tracking performance and eye-video recordings to identify and categorise lapses of responsiveness as microsleeps or ALs. Changes in neural activity during 85 ALs (17 subjects) relative to responsive tracking were estimated by whole-brain voxel-wise fMRI and by haemodynamic response (HR) analysis in regions of interest (ROIs) from seven key networks to reveal the neural signature of ALs. Changes in functional connectivity (FC) within and between the key ROIs were also estimated. Networks explored were the default mode network, dorsal attention network, frontoparietal network, sensorimotor network, salience network, visual network, and working memory network. Voxel-wise analysis revealed a significant increase in blood-oxygen-level-dependent activity in the overlapping dorsal anterior cingulate cortex and supplementary motor area region but no significant decreases in activity; the increased activity is considered to represent a recovery-of-responsiveness process following an AL. This increased activity was also seen in the HR of the corresponding ROI. Importantly, HR analysis revealed no trend of increased activity in the posterior cingulate of the default mode network, which has been repeatedly demonstrated to be a strong biomarker of mind-wandering. FC analysis showed decoupling of external attention, which supports the involuntary nature of ALs, in addition to the neural recovery processes. Other findings were a decrease in HR in the frontoparietal network before the onset of ALs, and a decrease in FC between default mode network and working memory network. These findings converge to our conclusion that the ALs observed during our task were involuntary mind-blanks. This is further supported behaviourally by the short duration of the ALs (mean 1.7 s), which is considered too brief to be instances of extreme mind-wandering. This is the first study to demonstrate that at least the majority of complete losses of responsiveness on a continuous visuomotor task are, if not due to microsleeps, due to involuntary mind-blanks.

Treatment of failed cervical total disc replacements in a series of 53 cases and description of a management strategy.

PURPOSE: To describe modes of failure of cervical TDR, their related treatment strategies, and to describe a management strategy for the treatment of failed cervical TDR. METHODS: This retrospective study was based on a consecutive series of 53 cervical TDR patients who underwent removal or revision surgery. Chart review was conducted to collect general descriptive data, reasons for TDR removal/revision, duration from index implantation to re-operation, and the subsequent procedure performed. RESULTS: Among 53 patients, 36 underwent TDR removal and fusion, 16 underwent TDR removal and replacement with another TDR, and one patient's TDR was revised by repositioning. The mean duration from index surgery to removal/revision was 40.1 months (range: 3 days-222 months). In all cases, removal/revision surgery was completed without complication. The most common reason for removal was severe osteolysis, often involving C. acnes infection, and was primarily associated with one implant type. TDR removal and fusion were performed for subsidence, device migration, treatment of symptoms arising from posterior anatomy (facet joints, etc.), approach-related complications and pain. TDR replacement was feasible for hypermobility, metal allergy, implant locked in kyphosis, and oversized implant use. In one case of TDR malpositioning, the device was successfully revised into appropriate position. CONCLUSION: After cervical TDR failure, replacing a TDR with another implant can be feasible. Reasons for revision or removal after cervical TDR surgery include biomechanical failure, implant migration, surgeon or technical error, or biological reasons. The type of failure can help the surgeon create a strategy to address these complications.