Nanobubble-actuated ultrasound neuromodulation for selectively shaping behavior in mice.

Ultrasound is an acoustic wave which can noninvasively penetrate the skull to deep brain regions, enabling neuromodulation. However, conventional ultrasound's spatial resolution is diffraction-limited and low-precision. Here, we report acoustic nanobubble-mediated ultrasound stimulation capable of localizing ultrasound's effects to only the desired brain region in male mice. By varying the delivery site of nanobubbles, ultrasound could activate specific regions of the mouse motor cortex, evoking EMG signaling and limb movement, and could also, separately, activate one of two nearby deep brain regions to elicit distinct behaviors (freezing or rotation). Sonicated neurons displayed reversible, low-latency calcium responses and increased c-Fos expression in the sub-millimeter-scale region with nanobubbles present. Ultrasound stimulation of the relevant region also modified depression-like behavior in a mouse model. We also provide evidence of a role for mechanosensitive ion channels. Altogether, our treatment scheme allows spatially-targetable, repeatable and temporally-precise activation of deep brain circuits for neuromodulation without needing genetic modification.

Gene Doping Control Analysis of Human Erythropoietin Transgene in Equine Plasma by PCR-Liquid Chromatography High-Resolution Tandem Mass Spectrometry.

Gene doping involves the misuse of genetic materials to alter an athlete's performance, which is banned at all times in both human and equine sports. Quantitative polymerase chain reaction (qPCR) assays have been used to control the misuse of transgenes in equine sports. Our laboratory recently developed and implemented duplex as well as multiplex qPCR assays for transgenes detection. To further advance gene doping control, we have developed for the first time a sensitive and definitive PCR-liquid chromatography high-resolution tandem mass spectrometry (PCR-LC-HRMS/MS) method for transgene detection with an estimated limit of detection of below 100 copies/mL for the human erythropoietin (hEPO) transgene in equine plasma. The method involved magnetic-glass-particle-based extraction of DNA from equine plasma prior to PCR amplification with 2'-deoxyuridine 5'-triphosphate (dUTP) followed by treatments with uracil DNA glycosylase and hot piperidine for selective cleavage to give small oligonucleotide fragments. The resulting DNA fragments were then analyzed by LC-HRMS/MS. The applicability of this method has been demonstrated by the successful detection of hEPO transgene in a blood sample collected from a gelding (castrated male horse) that had been administered the transgene. This novel approach not only serves as a complementary method for transgene detection but also paves the way for developing a generic PCR-LC-HRMS/MS method for the detection of multiple transgenes.

Partly linear single-index cure models with a nonparametric incidence link function.

In cancer studies, it is commonplace that a fraction of patients participating in the study are cured, such that not all of them will experience a recurrence, or death due to cancer. Also, it is plausible that some covariates, such as the treatment assigned to the patients or demographic characteristics, could affect both the patients' survival rates and cure/incidence rates. A common approach to accommodate these features in survival analysis is to consider a mixture cure survival model with the incidence rate modeled by a logistic regression model and latency part modeled by the Cox proportional hazards model. These modeling assumptions, though typical, restrict the structure of covariate effects on both the incidence and latency components. As a plausible recourse to attain flexibility, we study a class of semiparametric mixture cure models in this article, which incorporates two single-index functions for modeling the two regression components. A hybrid nonparametric maximum likelihood estimation method is proposed, where the cumulative baseline hazard function for uncured subjects is estimated nonparametrically, and the two single-index functions are estimated via Bernstein polynomials. Parameter estimation is carried out via a curated expectation-maximization algorithm. We also conducted a large-scale simulation study to assess the finite-sample performance of the estimator. The proposed methodology is illustrated via application to two cancer datasets.

The effects of Chinese proprietary medicine and vaccination on patients with COVID-19: a retrospective study in Macao.

BACKGROUND: COVID-19 is continuing to ravage globally and has resulted in a huge health and financial burden. Chinese proprietary medicines, such as Lianhua Qingwen (LHQW) and Huoxiang Zhengqi (HXZQ) capsules, have been recommended for non-high-risk patients with COVID-19 in China. Based on this, we described the baseline information, using status of LHQW and HXZQ capsules and inoculation history of quarantined patients in the second half of 2022 in Macao. Additionally, we analyzed the underlying association among medicines administration, vaccination and COVID-19 indices, in order to explore novel clues for the regular control and prevention of local epidemic situation in the future. METHODS: A total of 976 patients in Macao quarantine hotels from June to August 2022 were included in the present study, of which, 857 subjects were followed-up for prognosis evaluation. During quarantine, the baseline demographic information, including sex, age, BMI, occupation and personal habits were collected. Additionally, the inoculation history, medicine employment status and cycle threshold (Ct) values were also reported. We interviewed the patients for collection of their symptoms at the beginning and end of quarantine, as well as prognostic ones. Basic statistical description of baseline information, vaccination history and medication were displayed. Chi-squared test or with continuous correction test was employed for comparison of dichotomous data between two or multiple groups. Binary logistic regression was applied to reveal the correlation between potential risk factors and Ct values or prognosis symptoms. We also used Cox regression model to identify the effect of different types of vaccine products on Ct value altering rate. RESULTS: Patients who were female (52.0%), engaged in service industry (31.8%), from Macao native (65.8%), never took physical exercises (33.6%) and preferred irritated diet (59.5%) enjoyed more dominant proportions. Over 80% of participants were inoculated and 74.6% of them chose inactivated COVID-19 vaccine produced by China National Biotech Group (CNBG). Participants used LHQW capsules accounted for 92.1% and the duration of medicating lasted for one to two weeks. All of the reported symptoms were significantly ameliorated after quarantine and the duration of quarantine was concentrated on 21 days. People with different age, sex, occupation and region had different choices of HXZQ administration and vaccination. Additionally, middle dose (4-5 boxes) of LHQW capsules exhibited evidently negative association with positive Ct values (adjusted, - 0.037 ± 0.19, p = 0.04). Two doses of CNBG and one dose of mRNA vaccine had obvious protective effect on reducing Ct positive rate (p = 0.041). Meanwhile, symptoms after quarantine were significantly positive correlated with those in prognosis (adjusted, 1.38 ± 0.18, p < 0.0001). CONCLUSION: Our study found that the administration of LHQW capsules was beneficial for Ct value turning negative, meanwhile, certain mixed inoculation may be the promoting factor to reduce the positive rate of Ct value. These findings provide data basis for the Chinese proprietary medicine treatment and mixed vaccination applying for prevention and control of local COVID-19 epidemic in the future.

Coping style and mental health amid the first wave of the COVID-19 pandemic: a culture-moderated meta-analysis of 44 nations.

In the first wave of the COVID-19 pandemic, the rapid transmission of a novel virus and the unprecedented disease-mitigation measures have elicited considerable stress in many countries worldwide. Coping with pandemic stress may be differentially related to psychological symptoms across countries characterised by distinct cultural values. This study aimed to: (a) synthesise the literature by investigating the associations between some major types of coping style and psychological symptoms, and (b) investigate the moderating effects of culture on these associations. We performed a three-level random-effects meta-analysis, which included 151 independent samples from 44 countries across eight world regions (n = 137,088, 66% women, Mage = 36.08). For both problem-focused and avoidant coping styles, their hypothesised associations with psychological symptoms were robust across the countries (anxiety: rs = -.11 and .31; depression: rs = -.19 and .33; ps < .0001). For both emotion-focused and social support seeking styles, their associations with psychological symptoms were moderated by two Hofstede's cultural dimensions: uncertainty avoidance (intolerance of ambiguity) and masculinity (concern for achievement and success). The hypothesised negative coping style-symptom associations were found only in the countries with lower levels of uncertainty avoidance or masculinity, but opposite patterns of findings were found in those with higher levels of either of these two cultural dimensions.

Screening and confirmation of recombinant human follistatin in equine plasma for doping control purposes.

Recombinant human follistatin (rhFST) is a potential performance-enhancing agent owing to its stimulating effect on muscle growth. Administration of rhFST to athletes is prohibited in human sports by the World Anti-Doping Agency (WADA) and in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering published by the International Federation of Horseracing Authorities (IFHA). For effective control of the potential misuse of rhFST in flat racing, methods for screening and confirmatory analysis are required. This paper describes the development and validation of a complete solution for detecting rhFST and confirming its presence in plasma samples collected from racehorses. A high-throughput analysis of rhFST with a commercially available enzyme-linked immunosorbent assay (ELISA) was evaluated for the screening of equine plasma samples. Any suspicious finding would then be subjected to a confirmatory analysis using immunocapture, followed by nano-liquid chromatography/high-resolution tandem mass spectrometry (nanoLC-MS/HRMS). The confirmation of rhFST by nanoLC-MS/HRMS was achieved by comparing the retention times and relative abundances of three characteristic product-ions with those from the reference standard in accordance with the industry criteria published by the Association of Official Racing Chemists. The two methods achieved comparable limit of detection (~2.5-5 ng/mL) and limit of confirmation (2.5 ng/mL or below), as well as adequate specificity, precision and reproducibility. To our knowledge, this is the first report of the screening and confirmation methods for rhFST in equine samples.

Mendelian randomization with incomplete measurements on the exposure in the Hispanic Community Health Study/Study of Latinos.

Mendelian randomization has been widely used to assess the causal effect of a heritable exposure variable on an outcome of interest, using genetic variants as instrumental variables. In practice, data on the exposure variable can be incomplete due to high cost of measurement and technical limits of detection. In this paper, we propose a valid and efficient method to handle both unmeasured and undetectable values of the exposure variable in one-sample Mendelian randomization analysis with individual-level data. We estimate the causal effect of the exposure variable on the outcome using maximum likelihood estimation and develop an expectation maximization algorithm for the computation of the estimator. Simulation studies show that the proposed method performs well in making inference on the causal effect. We apply our method to the Hispanic Community Health Study/Study of Latinos, a community-based prospective cohort study, and estimate the causal effect of several metabolites on phenotypes of interest.

An enhanced label-free proteomics approach for deep-diving into equine plasma proteome, including the discovery of protein biomarkers for strenuous exercise.

Plasma proteins have been a valuable source of biomarkers for clinical uses and for monitoring of the illicit use of prohibited substances or practices in equine sports. We have previously reported the first use of label-free proteomics in profiling equine plasma proteome. This study aimed to refine the method by systematically evaluating various plasma fractionation methods and the use of narrower precursor mass ranges in data-independent acquisition (DIA) mass spectrometry (MS). Tandem fractionations of equine plasma with octanoic acid precipitation followed by solid-phase extraction (SPE) with C4 cartridges provided the largest increase in the number of new proteins identified. The use of two narrow precursor mass ranges of m/z 400-600 and 600-800 in DIA not only identified most proteins detectable by using a single mass range of m/z 350-1500 but also identified ~27% more proteins. The improved method was applied to analyse the plasma proteome of 'postrace' samples which, unlike other samples, had been collected from racehorses soon after racing. Multivariate data analysis has identified upregulation of 14 proteins and downregulation of six proteins in postrace plasma compared with the non-postrace plasma samples. Literature review of these proteins has provided evidence of exercise-induced haemolysis and changes in antioxidant enzyme activities, kinin system, insulin signalling and energy metabolism after strenuous exercise. The improved method has enabled a deeper profiling of the equine plasma proteome and identified the proteins associated with normal physiological changes after racing which are potential confounding factors in the development of a biomarker approach for doping control.

Comprehensive structural, infrared spectroscopic and kinetic investigations of the roles of the active-site arginine in bidirectional hydrogen activation by the [NiFe]-hydrogenase 'Hyd-2' from Escherichia coli.

The active site of [NiFe]-hydrogenases contains a strictly-conserved pendant arginine, the guanidine head group of which is suspended immediately above the Ni and Fe atoms. Replacement of this arginine (R479) in hydrogenase-2 from E. coli results in an enzyme that is isolated with a very tightly-bound diatomic ligand attached end-on to the Ni and stabilised by hydrogen bonding to the Nζ atom of the pendant lysine and one of the three additional water molecules located in the active site of the variant. The diatomic ligand is bound under oxidising conditions and is removed only after a prolonged period of reduction with H2 and reduced methyl viologen. Once freed of the diatomic ligand, the R479K variant catalyses both H2 oxidation and evolution but with greatly decreased rates compared to the native enzyme. Key kinetic characteristics are revealed by protein film electrochemistry: most importantly, a very low activation energy for H2 oxidation that is not linked to an increased H/D isotope effect. Native electrocatalytic reversibility is retained. The results show that the sluggish kinetics observed for the lysine variant arise most obviously because the advantage of a more favourable low-energy pathway is massively offset by an extremely unfavourable activation entropy. Extensive efforts to establish the identity of the diatomic ligand, the tight binding of which is an unexpected further consequence of replacing the pendant arginine, prove inconclusive.

Survival analysis with a random change-point.

Contemporary works in change-point survival models mainly focus on an unknown universal change-point shared by the whole study population. However, in some situations, the change-point is plausibly individual-specific, such as when it corresponds to the telomere length or menopausal age. Also, maximum-likelihood-based inference for the fixed change-point parameter is notoriously complicated. The asymptotic distribution of the maximum-likelihood estimator is non-standard, and computationally intensive bootstrap techniques are commonly used to retrieve its sampling distribution. This article is motivated by a breast cancer study, where the disease-free survival time of the patients is postulated to be regulated by the menopausal age, which is unobserved. As menopausal age varies across patients, a fixed change-point survival model may be inadequate. Therefore, we propose a novel proportional hazards model with a random change-point. We develop a nonparametric maximum-likelihood estimation approach and devise a stable expectation-maximization algorithm to compute the estimators. Because the model is regular, we employ conventional likelihood theory for inference based on the asymptotic normality of the Euclidean parameter estimators, and the variance of the asymptotic distribution can be consistently estimated by a profile-likelihood approach. A simulation study demonstrates the satisfactory finite-sample performance of the proposed methods, which yield small bias and proper coverage probabilities. The methods are applied to the motivating breast cancer study.

Activation of N-Methyl-D-aspartate receptor contributed to the ultrasonic modulation of neurons in vitro.

Ultrasound stimulation is increasingly used to investigate brain function and treat brain diseases due to its high level of safety and precise spatiotemporal resolution. Therefore, it is crucial to understand the underlying mechanisms involved in ultrasound brain stimulation. In this study, we investigate the role of NMDA receptors in mediating the effects of ultrasound on primary hippocampal neurons in mice. Our results show that ultrasound alone can activate heterologous NMDA receptor subunits, including NR1A, NR2A, and NR2B, in 293T cells, as well as endogenous NMDA receptors in primary neurons. This activation leads to an influx of calcium and an increase in nuclear c-Fos expression in primary neurons that have not been pre-treated with an NMDA receptor inhibitor. In conclusion, our findings demonstrate that NMDA receptors contribute to neuronal activation by ultrasound stimulation in vitro, providing insight into the molecular mechanisms of ultrasound neuromodulation and a new mediator for the sonogenetics technique.

Popliteus Tendon Injury: A Rare Cause of Acute Locked Knee.

The acute locked knee is a common presentation of meniscal tears or other intra-articular injuries. However, a popliteus tendon tear, an uncommon cause of acute locked knee, is often overlooked as a possible diagnosis. Here, we present the case of a 29-year-old male who experienced an acute locked knee following a sports injury. An arthroscopic examination revealed an intrasubstance tear in the popliteus tendon and a complete anterior cruciate ligament tear, while the menisci remained intact. Due to the extension lag caused by the popliteus tendon tear, the anterior cruciate ligament reconstruction was postponed. The patient then underwent a course of physiotherapy before the anterior cruciate ligament reconstruction and eventually achieved full knee extension after six weeks. Further surgical intervention was then performed to address the ligament injury. Our case highlights the importance of considering a popliteus tendon tear as a possible cause of an acute locked knee. Proper diagnosis and management are crucial for achieving optimal outcomes for patients with an acute locked knee and concomitant ligamentous injury.

PENALIZED REGRESSION FOR MULTIPLE TYPES OF MANY FEATURES WITH MISSING DATA.

Recent technological advances have made it possible to measure multiple types of many features in biomedical studies. However, some data types or features may not be measured for all study subjects because of cost or other constraints. We use a latent variable model to characterize the relationships across and within data types and to infer missing values from observed data. We develop a penalized-likelihood approach for variable selection and parameter estimation and devise an efficient expectation-maximization algorithm to implement our approach. We establish the asymptotic properties of the proposed estimators when the number of features increases at a polynomial rate of the sample size. Finally, we demonstrate the usefulness of the proposed methods using extensive simulation studies and provide an application to a motivating multi-platform genomics study.

Modulation of deep neural circuits with sonogenetics.

Noninvasive control of neuronal activity in the deep brain can be illuminating for probing brain function and treating dysfunctions. Here, we present a sonogenetic approach for controlling distinct mouse behavior with circuit specificity and subsecond temporal resolution. Targeted neurons in subcortical regions were made to express a mutant large conductance mechanosensitive ion channel (MscL-G22S), enabling ultrasound to trigger activity in MscL-expressing neurons in the dorsal striatum and increase locomotion in freely moving mice. Ultrasound stimulation of MscL-expressing neurons in the ventral tegmental area could activate the mesolimbic pathway to trigger dopamine release in the nucleus accumbens and modulate appetitive conditioning. Moreover, sonogenetic stimulation of the subthalamic nuclei of Parkinson's disease model mice improved their motor coordination and mobile time. Neuronal responses to ultrasound pulse trains were rapid, reversible, and repeatable. We also confirmed that the MscL-G22S mutant is more effective to sensitize neurons to ultrasound compared to the wild-type MscL. Altogether, we lay out a sonogenetic approach which can selectively manipulate targeted cells to activate defined neural pathways, affect specific behaviors, and relieve symptoms of neurodegenerative disease.

The mechanosensitive ion channel Piezo1 contributes to ultrasound neuromodulation.

Transcranial low-intensity ultrasound is a promising neuromodulation modality, with the advantages of noninvasiveness, deep penetration, and high spatiotemporal accuracy. However, the underlying biological mechanism of ultrasonic neuromodulation remains unclear, hindering the development of efficacious treatments. Here, the well-known Piezo1 was studied through a conditional knockout mouse model as a major mediator for ultrasound neuromodulation ex vivo and in vivo. We showed that Piezo1 knockout (P1KO) in the right motor cortex of mice significantly reduced ultrasound-induced neuronal calcium responses, limb movement, and muscle electromyogram (EMG) responses. We also detected higher Piezo1 expression in the central amygdala (CEA), which was found to be more sensitive to ultrasound stimulation than the cortex was. Knocking out the Piezo1 in CEA neurons showed a significant reduction of response under ultrasound stimulation, while knocking out astrocytic Piezo1 showed no-obvious changes in neuronal responses. Additionally, we excluded an auditory confound by monitoring auditory cortical activation and using smooth waveform ultrasound with randomized parameters to stimulate P1KO ipsilateral and contralateral regions of the same brain and recording evoked movement in the corresponding limb. Thus, we demonstrate that Piezo1 is functionally expressed in different brain regions and that it is an important mediator of ultrasound neuromodulation in the brain, laying the ground for further mechanistic studies of ultrasound.

A multiplex qPCR assay for transgenes detection: A novel approach for gene doping control in horseracing using conventional laboratory setup.

Illicit administration of transgene into horses is a form of gene doping that has been a key concern in equine sports. The large number of potential performance-enhancing transgenes has demanded a cost-effective and reliable detection method. Multiplex qPCR is a relevant technique, but the cross-talking between fluorophores and high background noise limits the method sensitivity and specificity. This study reports a simpler multiplexing approach by using the same fluorophore for four hydrolysis probes each targeting one of the four transgenes: human growth hormone, insulin-like growth factor 1, equine erythropoietin and interleukin-10. Any positive findings from this multiplex qPCR assay can then be confirmed by individual qPCR assays to identify potential transgene(s). This has effectively eliminated the cross-talking issue and allowed an improved signal-to-noise than conventional multiplex qPCR assay. It has also removed the limitation imposed by the available choice of fluorophores and optical channels of qPCR instruments on the number of transgenes that can be analysed in a multiplex qPCR assay. This novel multiplex qPCR has been successfully validated. The estimated limits of detection were ~1500-2500 copies/mL of blood, thus demonstrating comparable sensitivity with the corresponding duplex qPCR assays. Concurring results were obtained by analysing hundreds of official blood samples provided by racehorses with this multiplex qPCR assay and the accredited individual duplex qPCR assays. This novel multiplex qPCR assay for detecting multiple transgenes is a cost-effective screening method using a conventional laboratory setup and has opened up the potential to include the testing of additional transgenes in a single assay.

The mechanosensitive ion channel Piezo1 modulates the migration and immune response of microglia.

Microglia are the brain's resident immune cells, performing surveillance to promote homeostasis and healthy functioning. While microglial chemical signaling is well-studied, mechanical cues regulating their function are less well-understood. Here, we investigate the role of the mechanosensitive ion channel Piezo1 in microglia migration, pro-inflammatory cytokine production, and stiffness sensing. In Piezo1 knockout transgenic mice, we demonstrated the functional expression of Piezo1 in microglia and identified genes whose expression was consequently affected. Functional assays revealed that Piezo1 deficiency in microglia enhanced migration toward amyloid ß-protein, and decreased levels of pro-inflammatory cytokines produced upon stimulation by lipopolysaccharide, both in vitro and in vivo. The phenomenon could be mimicked or reversed chemically using a Piezo1-specific agonist or antagonist. Finally, we also showed that Piezo1 mediated the effect of substrate stiffness-induced migration and cytokine expression. Altogether, we show that Piezo1 is an important molecular mediator for microglia, its activation modulating microglial migration and immune responses.

Electric field manipulation of spin chirality and skyrmion dynamic.

The Dzyaloshinskii-Moriya interaction (DMI) is an antisymmetric exchange interaction that stabilizes spin chirality. One scientific and technological challenge is understanding and controlling the interaction between spin chirality and electric field. In this study, we investigate an unconventional electric field effect on interfacial DMI, skyrmion helicity, and skyrmion dynamics in a system with broken inversion symmetry. We design heterostructures with a 3d-5d atomic orbital interface to demonstrate the gate bias control of the DMI energy and thus transform the DMI between opposite chiralities. Furthermore, we use this voltage-controlled DMI (VCDMI) to manipulate the skyrmion spin texture. As a result, a type of intermediate skyrmion with a unique helicity is created, and its motion can be controlled and made to go straight. Our work shows the effective control of spin chirality, skyrmion helicity, and skyrmion dynamics by VCDMI. It promotes the emerging field of voltage-controlled chiral interactions and voltage-controlled skyrmionics.

Altered Mechanisms for Acid-Catalyzed RNA Cleavage and Isomerization Reactions Models.

RNA strand cleavage by 2'-O-transphosphorylation is catalyzed not only by numerous nucleolytic RNA enzymes (ribozymes) but also by hydroxide or hydronium ions. In experiments, both cleavage of the 5'-linked nucleoside and isomerization between 3',5'- and 2',5'-phosphodiesters occur under acidic conditions, while only the cleavage reaction is observed under basic conditions. An ab initio path-integral approach for simulating kinetic isotope effects is used to reveal the reaction mechanisms for RNA cleavage and isomerization reactions under acidic conditions. Moreover, the proposed mechanisms can also be combined through the experimental pH-rate profiles.