The Improvement of Fluorescence In Situ Hybridization Technique Based on Explorations of Symbionts in Cicadas.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is widely used for the identification of microbes in complex samples, but it suffers from some limitations resulting in the weak or even absence of fluorescence signals of microbe(s), which may lead to the underestimation or misunderstanding of a microbial community. Herein, we explored symbionts in the bacteriomes and fat bodies of cicadas using modified FISH, aiming to improve this technique. We initially revealed that the probes of Candidatus Sulcia muelleri (Sulcia) and the yeast-like fungal symbiont (YLS) are suitable for detection of these symbionts in all cicadas and some other species of Auchenorrhyncha, whereas the probe of Candidatus Hodgkinia cicadicola (Hodgkinia) is only suitable for detection of Hodgkinia in a few cicada species. The fluorescence signal of Sulcia, Hodgkinia and YLS exhibited weak intensity without the addition of unlabeled oligonucleotides (helpers) and heat shock in some cicadas; however, it can be significantly improved by the addition of both helpers and heat shock. Results of this study suggest that heat shock denaturing rRNA and proteins of related microbe(s) together with helpers binding to the adjacent region of the probe's target sites prevent the re-establishment of the native secondary structure of rRNA; therefore, suitable probe(s) can more easily access to the probe's target sites of rRNA. Our results provide new information for the significant improvement of hybridization signal intensities of microbes in the FISH experiment, making it possible to achieve a more precise understanding of the microbial distribution, community and density in complex samples.

Human monoclonal antibodies against Staphylococcus aureus A protein identified by high-throughput single-cell sequencing of phase I clinical volunteers' B cells.

Methicillin-resistant Staphylococcus aureus, poses a significant threat through infections in both community and hospital settings. To address this challenge, we conducted a phase I clinical trial study involving a recombinant Staphylococcus aureus vaccine. Utilizing peripheral blood lymphocytes from 64 subjects, we isolated antigen-specific memory B cells for subsequent single-cell sequencing. Among the 676 identified antigen-binding IgG1+ clones, we selected the top 10 antibody strains for construction within expression vectors. Successful expression and purification of these monoclonal antibodies led to the discovery of a highly expressed human antibody, designated as IgG-6. This antibody specifically targets the pentameric form of the Staphylococcus aureus protein A (SpA5). In vivo assessments revealed that IgG-6 provided prophylactic protection against MRSA252 infection. This study underscores the potential of human antibodies as an innovative strategy against Staphylococcus aureus infections, offering a promising avenue for further research and clinical development.

Chronic stress hinders sensory axon regeneration via impairing mitochondrial cristae and OXPHOS.

Spinal cord injury (SCI) often leads to physical limitations, persistent pain, and major lifestyle shifts, enhancing the likelihood of prolonged psychological stress and associated disorders such as anxiety and depression. The mechanisms linking stress with regeneration remain elusive, despite understanding the detrimental impact of chronic stress on SCI recovery. In this study, we investigated the effect of chronic stress on primary sensory axon regeneration using a preconditioning lesions mouse model. Our data revealed that chronic stress-induced mitochondrial cristae loss and a decrease in oxidative phosphorylation (OXPHOS) within primary sensory neurons, impeding central axon regrowth. Corticosterone, a stress hormone, emerged as a pivotal player in this process, affecting satellite glial cells by reducing Kir4.1 expression. This led to increased neuronal hyperactivity and reactive oxygen species levels, which, in turn, deformed mitochondrial cristae and impaired OXPHOS, crucial for axonal regeneration. Our study underscores the need to manage psychological stress in patients with SCI for effective sensory-motor rehabilitation.

A Study on Symbiotic Systems of Cicadas Provides New Insights into Distribution of Microbial Symbionts and Improves Fluorescence In Situ Hybridization Technique.

Nutritional symbionts of sap-sucking auchenorrhynchan insects of Hemiptera are usually confined to the bacteriomes and/or fat bodies. Knowledge is limited about the distribution of microbial symbionts in other organs. We investigated the distribution of obligate symbionts in the salivary glands, gut tissues, reproductive organs, bacteriomes, and fat bodies of two cicada species, Karenia caelatata and Tanna sp., using integrated methods, including a modified fluorescence in situ hybridization (FISH) technique, which can greatly enhance the FISH signal intensity of related symbionts. We revealed that Candidatus Sulcia muelleri (Sulcia) and a yeast-like fungal symbiont (YLS) were harbored in the bacteriomes and fat bodies, respectively. Both of Sulcia and YLS can be transmitted to the offspring via ovaries, forming a "symbiont ball" in each egg. Neither Sulcia nor YLS were harbored in the salivary glands, gut tissues and testes. Phylogenetic trees of both Sulcia and cicadas confirm that K. caelatata is a member of the tribe Dundubiini, and the tribe Leptopsaltriini that comprises Ta. sp. is not monophyletic. YLS of K. caelatata is embedded inside the lineage of YLS of Dundubiini, whereas YLS of Ta. sp. is closely related to the clade comprising both cicada-parasitizing fungi Ophiocordyceps and YLS of Mogannia conica and Meimuna mongolica, suggesting an evolutionary replacement of YLS in Ta. sp. from an Ophiocordyceps fungus to another Ophiocordyceps fungus. Our results provide new insights into the symbiosis between Cicadidae and related symbionts. Modification through the addition of helpers and heat shock greatly enhanced the FISH signal intensity of YLS, which may provide guidelines for enhancement of the hybridization signal intensity of other symbiont(s) in the FISH experiments.

Training and asymmetrical transfer effects of working memory and inhibitory control in primary school children.

Working memory (WM) and inhibitory control (IC) are two fundamental and supportive components of executive function (EF) that are critical for school-age children. However, the direct comparison of the training and transfer effects of WM and IC training in school-age children still needs to be improved. This study adopted a "pre-, post-, and delayed posttest" design to compare the training, near-transfer, and far-transfer effects of WM and IC in school-age children. A total of 60 children aged 8 to 10 years were randomly assigned to the WM training group, IC training group, or control group. Children in the WM and IC training groups completed 12 sessions of multiple adaptive training tasks tapping different subcomponents of WM (visual-spatial and verbal WM) and IC (interference control and response inhibition) separately. In the pretraining, posttraining, and 6-month follow-up stages, we used WM and IC tasks to evaluate training and near-transfer effects and used analogical reasoning tasks to evaluate far-transfer effects. Results showed significant training effects on visual-spatial and verbal WM, near-transfer effects on response inhibition, and far-transfer effects on analogical reasoning for WM training in the posttraining stage. The improvements in verbal WM and analogical reasoning were maintained for 6 months, whereas for IC training only the training effects on response inhibition and the far-transfer effects on analogical reasoning were observed in the posttraining stage and only the training effects on response inhibition were maintained for 6 months. Results suggested positive training and asymmetrical transfer effects of WM and IC training, which provide new evidence for the effectiveness of WM and IC training in school-age children.