Multimodal Optothermal Manipulations along Various Surfaces.

Optical tweezers have provided tremendous opportunities for fundamental studies and applications in the life sciences, chemistry, and physics by offering contact-free manipulation of small objects. However, it requires sophisticated real-time imaging and feedback systems for conventional optical tweezers to achieve controlled motion of micro/nanoparticles along textured surfaces, which are required for such applications as high-resolution near-field characterizations of cell membranes with nanoparticles as probes. In addition, most optical tweezers systems are limited to single manipulation modes, restricting their broader applications. Herein, we develop an optothermal platform that enables the multimodal manipulation of micro/nanoparticles along various surfaces. Specifically, we achieve the manipulation of micro/nanoparticles through the synergy between the optical and thermal forces, which arise due to the temperature gradient self-generated by the particles absorbing the light. With a simple control of the laser beam, we achieve five switchable working modes [i.e., tweezing, rotating, rolling (toward), rolling (away), and shooting] for the versatile manipulation of both synthesized particles and biological cells along various substrates. More interestingly, we realize the manipulation of micro/nanoparticles on rough surfaces of live worms and their embryos for localized control of biological functions. By enabling the three-dimensional control of micro/nano-objects along various surfaces, including topologically uneven biological tissues, our multimodal optothermal platform will become a powerful tool in life sciences, nanotechnology, and colloidal sciences.

A fused biometrics information graph convolutional neural network for effective classification of patellofemoral pain syndrome.

Patellofemoral pain syndrome (PFPS) is a common, yet misunderstood, knee pathology. Early accurate diagnosis can help avoid the deterioration of the disease. However, the existing intelligent auxiliary diagnosis methods of PFPS mainly focused on the biosignal of individuals but neglected the common biometrics of patients. In this paper, we propose a PFPS classification method based on the fused biometrics information Graph Convolution Neural Networks (FBI-GCN) which focuses on both the biosignal information of individuals and the common characteristics of patients. The method first constructs a graph which uses each subject as a node and fuses the biometrics information (demographics and gait biosignal) of different subjects as edges. Then, the graph and node information [biosignal information, including the joint kinematics and surface electromyography (sEMG)] are used as the inputs to the GCN for diagnosis and classification of PFPS. The method is tested on a public dataset which contain walking and running data from 26 PFPS patients and 15 pain-free controls. The results suggest that our method can classify PFPS and pain-free with higher accuracy (mean accuracy = 0.8531 ± 0.047) than other methods with the biosignal information of individuals as input (mean accuracy = 0.813 ± 0.048). After optimal selection of input variables, the highest classification accuracy (mean accuracy = 0.9245 ± 0.034) can be obtained, and a high accuracy can still be obtained with a 40% reduction in test variables (mean accuracy = 0.8802 ± 0.035). Accordingly, the method effectively reflects the association between subjects, provides a simple and effective aid for physicians to diagnose PFPS, and gives new ideas for studying and validating risk factors related to PFPS.

A particle size threshold governs diffusion and segregation of PAR-3 during cell polarization.

The actomyosin cortex regulates the localization and function of proteins at the plasma membrane. Here, we study how membrane binding, cortical movements, and diffusion determine membrane protein distribution. In Caenorhabditis elegans zygotes, actomyosin flows transport PAR polarity proteins to establish the anterior-posterior axis. Oligomerization of a key scaffold protein, PAR-3, is required for polarization. PAR-3 oligomers are a heterogeneous population of many different sizes, and it remains unclear how oligomer size affects PAR-3 segregation. To address this question, we engineered PAR-3 to defined sizes. We report that PAR-3 trimers are necessary and sufficient for PAR-3 function during polarization and later embryo development. Quantitative analysis of PAR-3 diffusion shows that a threshold size of three subunits allows PAR-3 clusters to stably bind the membrane, where they are corralled and transported by the actomyosin cortex. Our study provides a quantitative model for size-dependent protein transportation of peripheral membrane proteins by cortical flow.

Non-invasive chimeric HaloTag labeling to study clustering and diffusion of membrane proteins.

As live imaging plays an increasingly critical role in cell biology research, the desire to label and track individual protein molecules in vivo has been growing. To address this, in this protocol we describe steps for sparse labeling using two different HaloTag ligand dyes in C. elegans. This labeling approach is simple, is non-invasive, and preserves the view of the bulk protein population. We further describe how to carry out single-particle tracking experiments and extract information about particle diffusion behavior. For complete details on the use and execution of this protocol, please refer to Chang and Dickinson (2022).1.

Phosphoprotein Gene of Wild-Type Rabies Virus Plays a Role in Limiting Viral Pathogenicity and Lowering the Enhancement of BBB Permeability.

Enhancement of blood-brain barrier (BBB) permeability is necessary for clearing virus in the central nervous system (CNS). It has been reported that only laboratory-attenuated rabies virus (RABV) induces inflammatory response to lead BBB transient breakdown rather than wild-type (wt) strains. As a component of ribonucleoprotein (RNP), phosphoprotein (P) of RABV plays a key role in viral replication and pathogenicity. To our knowledge, the function of RABV P gene during RABV invasion was unclear so far. In order to determine the role of RABV P gene during RABV infection, we evaluated the BBB permeability in vivo after infection with wt RABV strain (GD-SH-01), a lab-attenuated RABV strain (HEP-Flury), and a chimeric RABV strain (rHEP-SH-P) whose P gene cloned from GD-SH-01 was expressed in the genomic backbone of HEP-Flury. We found that rHEP-SH-P caused less enhancement of BBB permeability and was less pathogenic to adult mice than GD-SH-01 and HEP-Flury. In an effort to investigate the mechanism, we found that the replication of rHEP-SH-P has been limited due to the suppressed P protein expression and induced less response to maintain BBB integrity. Our data indicated that the P gene of wt RABV was a potential determinant in hampering viral replication in vivo, which kept BBB integrity. These findings provided an important foundation for understanding the viral invasion and development of novel vaccine.

Electron microscopy snapshots of single particles from single cells.

Cryo-electron microscopy (cryo-EM) has become an indispensable tool for structural studies of biological macromolecules. Two additional predominant methods are available for studying the architectures of multiprotein complexes: 1) single-particle analysis of purified samples and 2) tomography of whole cells or cell sections. The former can produce high-resolution structures but is limited to highly purified samples, whereas the latter can capture proteins in their native state but has a low signal-to-noise ratio and yields lower-resolution structures. Here, we present a simple, adaptable method combining microfluidic single-cell extraction with single-particle analysis by EM to characterize protein complexes from individual Caenorhabditis elegans embryos. Using this approach, we uncover 3D structures of ribosomes directly from single embryo extracts. Moreover, we investigated structural dynamics during development by counting the number of ribosomes per polysome in early and late embryos. This approach has significant potential applications for counting protein complexes and studying protein architectures from single cells in developmental, evolutionary, and disease contexts.

Brain Activation Patterns in Response to Conspecific and Heterospecific Social Acoustic Signals in Female Plainfin Midshipman Fish, Porichthys notatus.

While the peripheral auditory system of fish has been well studied, less is known about how the fish's brain and central auditory system process complex social acoustic signals. The plainfin midshipman fish, Porichthys notatus, has become a good species for investigating the neural basis of acoustic communication because the production and reception of acoustic signals is paramount for this species' reproductive success. Nesting males produce long-duration advertisement calls that females detect and localize among the noise in the intertidal zone to successfully find mates and spawn. How female midshipman are able to discriminate male advertisement calls from environmental noise and other acoustic stimuli is unknown. Using the immediate early gene product cFos as a marker for neural activity, we quantified neural activation of the ascending auditory pathway in female midshipman exposed to conspecific advertisement calls, heterospecific white seabass calls, or ambient environment noise. We hypothesized that auditory hindbrain nuclei would be activated by general acoustic stimuli (ambient noise and other biotic acoustic stimuli) whereas auditory neurons in the midbrain and forebrain would be selectively activated by conspecific advertisement calls. We show that neural activation in two regions of the auditory hindbrain, i.e., the rostral intermediate division of the descending octaval nucleus and the ventral division of the secondary octaval nucleus, did not differ via cFos immunoreactive (cFos-ir) activity when exposed to different acoustic stimuli. In contrast, female midshipman exposed to conspecific advertisement calls showed greater cFos-ir in the nucleus centralis of the midbrain torus semicircularis compared to fish exposed only to ambient noise. No difference in cFos-ir was observed in the torus semicircularis of animals exposed to conspecific versus heterospecific calls. However, cFos-ir was greater in two forebrain structures that receive auditory input, i.e., the central posterior nucleus of the thalamus and the anterior tuberal hypothalamus, when exposed to conspecific calls versus either ambient noise or heterospecific calls. Our results suggest that higher-order neurons in the female midshipman midbrain torus semicircularis, thalamic central posterior nucleus, and hypothalamic anterior tuberal nucleus may be necessary for the discrimination of complex social acoustic signals. Furthermore, neurons in the central posterior and anterior tuberal nuclei are differentially activated by exposure to conspecific versus other acoustic stimuli.

Protective Effect of Klotho against Ischemic Brain Injury Is Associated with Inhibition of RIG-I/NF-κB Signaling.

Aging is the greatest independent risk factor for the occurrence of stroke and poor outcomes, at least partially through progressive increases in oxidative stress and inflammation with advanced age. Klotho is an antiaging gene, the expression of which declines with age. Klotho may protect against neuronal oxidative damage that is induced by glutamate. The present study investigated the effects of Klotho overexpression and knockdown by an intracerebroventricular injection of a lentiviral vector that encoded murine Klotho (LV-KL) or rat Klotho short-hairpin RNA (LV-KL shRNA) on cerebral ischemia injury and the underlying anti-neuroinflammatory mechanism. The overexpression of Klotho induced by LV-KL significantly improved neurobehavioral deficits and increased the number of live neurons in the hippocampal CA1 and caudate putamen subregions 72 h after cerebral hypoperfusion that was induced by transient bilateral common carotid artery occlusion (2VO) in mice. The overexpression of Klotho significantly decreased the immunoreactivity of glial fibrillary acidic protein and ionized calcium binding adaptor molecule-1, the expression of retinoic-acid-inducible gene-I, the nuclear translocation of nuclear factor-κB, and the production of proinflammatory cytokines (tumor necrosis factor α and interleukin-6) in 2VO mice. The knockdown of Klotho mediated by LV-KL shRNA in the brain exacerbated neurological dysfunction and cerebral infarct after 22 h of reperfusion following 2 h middle cerebral artery occlusion in rats. These findings suggest that Klotho itself or enhancers of Klotho may compensate for its aging-related decline, thus providing a promising therapeutic approach for acute ischemic stroke during advanced age.