Microglial heterogeneity in the ischemic stroke mouse brain of both sexes.

BACKGROUND: Ischemic stroke elicits a complex and sustained immune response in the brain. Immunomodulatory treatments have long held promise for improving stroke outcomes, yet none have succeeded in the clinical setting. This lack of success is largely due to our incomplete understanding of how immune cells respond to stroke. The objective of the current study was to dissect the effect of permanent stroke on microglia, the resident immune cells within the brain parenchyma. METHODS: A permanent middle cerebral artery occlusion (pMCAO) model was used to induce ischemic stroke in young male and female mice. Microglia were sorted from fluorescence reporter mice after pMCAO or sham surgery and then subjected to single-cell RNA sequencing analysis. Various methods, including flow cytometry, RNA in situ hybridization, immunohistochemistry, whole-brain imaging, and bone marrow transplantation, were also employed to dissect the microglial response to stroke. Stroke outcomes were evaluated by infarct size and behavioral tests. RESULTS: First, we showed the morphologic and spatial changes in microglia after stroke. We then performed single-cell RNA sequencing analysis on microglia isolated from sham and stroke mice of both sexes. The data indicate no major sexual dimorphism in the microglial response to permanent stroke. Notably, we identified seven potential stroke-associated microglial clusters, including four major clusters characterized by a disease-associated microglia-like signature, a highly proliferative state, a macrophage-like profile, and an interferon (IFN) response signature, respectively. Importantly, we provided evidence that the macrophage-like cluster may represent the long-sought stroke-induced microglia subpopulation with increased CD45 expression. Lastly, given that the IFN-responsive subset constitutes the most prominent microglial population in the stroke brain, we used fludarabine to pharmacologically target STAT1 signaling and found that fludarabine treatment improved long-term stroke outcome. CONCLUSIONS: Our findings shed new light on microglia heterogeneity in stroke pathology and underscore the potential of targeting specific microglial populations for effective stroke therapies.

Isolating Immune Cells from Mouse Brain and Skull.

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

Characterizing the complete mitogenome of Odontothrips phaseoli (Thysanoptera: Thripidae) and its mitochondrial phylogeny.

Described originally from Heilongjiang, China, Odontothrips phaseoli is a potential pest of threatening bean plant in northern China. The complete mitochondrial genome of O. phaseoli was sequenced and assembled, with a total length of 15,540 bp. Within this genome, 37 genes have been identified: 13 PCGs, 22 tRNAs, two rRNAs, and two putative control regions. Most PCGs terminate with TAA, while four genes (atp8, nad1, nad2 and nad4) use an incomplete 'T' and nad6 employs TAG as the stop codon. Compared to the mitogenome of the ancestral insect, O. phaseoli displays significant gene rearrangement. However, it retains three conserved gene blocks in common with its related species, Megalurothrips usitatus, both of which belong to the Megalurothrips genus-group. The phylogenetic tree, constructed based on the entire mitogenome dataset of all thrips species available in NCBI, shows that the two species cluster closely together. This alignment might underscore the close link between gene arrangements and the phylogeny relationships.

New synonymy among gall thrips of the Asian genus Mesothrips, with revision of species from China (Thysanoptera, Haplothripini).

Historical, nomenclatural, technical, and biological problems associated with the 42 species of Mesothrips are discussed. Type specimens have been re-examined of 14 of the 25 species that were described prior to 1930 and remain known only from imperfectly slide-mounted specimens. As a result, seven new synonyms are recognised. From China, six species of Mesothrips have been listed, but the records of M.alluaudi and M.manii are rejected, and three new species are described: M.jianfengisp. nov., M.longistylussp. nov., and M.verniciasp. nov. These three species are divergent from other members of Mesothrips in lacking a prominent fore tarsal tooth and may indicate a possible generic relationship to the flower-living species in the Asian genus Dolichothrips. An illustrated key is provided to the seven Mesothrips species now known from China.

Therapeutic Potential of Targeting the PERK Signaling Pathway in Ischemic Stroke.

Many pathologic states can lead to the accumulation of unfolded/misfolded proteins in cells. This causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR), which encompasses three main adaptive branches. One of these UPR branches is mediated by protein kinase RNA-like ER kinase (PERK), an ER stress sensor. The primary consequence of PERK activation is the suppression of global protein synthesis, which reduces ER workload and facilitates the recovery of ER function. Ischemic stroke induces ER stress and activates the UPR. Studies have demonstrated the involvement of the PERK pathway in stroke pathophysiology; however, its role in stroke outcomes requires further clarification. Importantly, considering mounting evidence that supports the therapeutic potential of the PERK pathway in aging-related cognitive decline and neurodegenerative diseases, this pathway may represent a promising therapeutic target in stroke. Therefore, in this review, our aim is to discuss the current understanding of PERK in ischemic stroke, and to summarize pharmacologic tools for translational stroke research that targets PERK and its associated pathways.

Leaf-feeding species of the genus Liothrips from China (Thysanoptera, Phlaeothripinae).

The 250 species of the second largest genus of Thysanoptera, Liothrips, are known as feeding mainly on green leaves, with many inducing galls or associated with galls. In China, 33 species are recognized including L. brevis sp. n., L. elongatus sp. n., L. longistylus sp. n., L. motuoensis sp. n., L. piceae sp. n., L. populi sp. n. and L. tibetanus sp. n., also seven species are recorded from this country for the first time. Four Hans Liothrips species are considered as new synonymies of L. vaneeckei that might be widespread in the Holarctic region. Three species are newly combined as Liothrips aporosae comb.n., Teuchothrips fuscus comb.n. and T. turkestanicus comb.n. The illustrated identification key to Chinese Liothrips species excludes L. hsuae but includes L. mirabilis due to its potential as a pest of Piper plants are growing throughout Southern China. Biology, structural variation, and generic relationships are also discussed.

The extraordinary rearrangement of mitochondrial genome of the wheat pest, Aptinothrips stylifer and the mitochondrial phylogeny of Thripidae (Thysanoptera).

The mitochondrial gene order in Thysanoptera is notably distinct and highly rearranged, with each species exhibiting its own unique arrangement. To elucidate the relationship between gene rearrangements and phylogeny, the complete mitochondrial genome (mitogenome) of the wheat pest, Aptinothrips stylifer, was sequenced and assembled, spanning a total length of 16,033 bp. Compared with the ancestral arthropod mitogenome, significant rearrangement differences were evident in A. stylifer, whereas the gene order between A. stylifer and Anaphothrips obscurus was similar. Phylogenetic trees were reconstructed based on all 13 protein-coding gene sequences using Bayesian inference and maximum-likelihood methods, both yielding similar topological structures. Notably, A. stylifer was robustly clustered with A. obscurus, affirming its classification within Anaphothrips genus group. This exemplifies the potential correlation between gene rearrangements and phylogeny in the Thripidae family. Additionally, the mitogenome of A. stylifer exhibited several atypical features, including: (1) Three putative control regions (CRs) in close proximity, with CR2 and CR3 displaying partial similarity, and CR1 differing in base composition; (2) Two transfer RNAs (tRNAs), trnS1 and trnV, lacking the DHU arm; (3) Two ribosomal RNA (rRNA) genes inverted and positioned distant from each other; (4) Negative AT and GC skew (AT skew = -0.001, GC skew = -0.077); (5) One transposition (nad6), one inverse transposition (trnQ), four inversions (trnF, trnH, trnC, and gene block nad1-trnL1-rrnL-trnV-rrnS), and four tandem duplication random loss events; and (6) Two protein-coding genes, nad2 and atp8, terminated with an incomplete stop codon "T".

Novel ratio-expressions of genes enables estimation of wound age in contused skeletal muscle.

Given that combination with multiple biomarkers may well raise the predictive value of wound age, it appears critically essential to identify new features under the limited cost. For this purpose, the present study explored whether the gene expression ratios provide unique time information as an additional indicator for wound age estimation not requiring the detection of new biomarkers and allowing full use of the available data. The expression levels of four wound-healing genes (Arid5a, Ier3, Stom, and Lcp1) were detected by real-time polymerase chain reaction, and a total of six expression ratios were calculated among these four genes. The results showed that the expression levels of four genes and six ratios of expression changed time-dependent during wound repair. The six expression ratios provided additional temporal information, distinct from the four genes analyzed separately by principal component analysis. The overall performance metrics for cross-validation and external validation of four typical prediction models were improved when six ratios of expression were added as additional input variables. Overall, expression ratios among genes provide temporal information and have excellent potential as predictive markers for wound age estimation. Combining the expression levels of genes with ratio-expression of genes may allow for more accurate estimates of the time of injury.

Taxonomic review of the Oriental genus Phylladothrips Priesner (Thysanoptera, Phlaeothripidae).

Species of the Oriental subtropical and tropical genus Phylladothrips of fungus-feeding thrips exhibit some diagnostic character states, usually with abdominal tergite VIII bearing two pairs of wing-retaining setae and male tergite IX setae S2 about as long as S1. These species are quite small, and the maxillary stylets unusually broad for Phlaeothripinae. Phylladothripstrisetaesp. nov. from Xizang, China and P.selangorsp. nov. from Selangor, Malaysia are described, and P.fasciae is newly recorded from China. All 11 species in this genus are revised with an illustrated key.

Sexual dimorphism of feeding stylets in some Thysanoptera - Phlaeothripinae, with description of two new species of Haplothrips from China.

Sexual dimorphism in feeding stylets is recorded among some Phlaeothripinae that have maxillary stylets long and close together in females but wider apart in males. These atypical long feeding stylets have been found in two new species of Haplothrips from China, both taken on Artemisia from Plateau zone. Two species are described and illustrated: H.helanshanensissp. nov. from Helanshan Mountain and H.longistylussp. nov. from Tibet. There is no evidence of any association between feeding behaviors and feeding stylet orientation.

Review of the genus Gigantothrips Zimmermann from China and Southeast Asia (Thysanoptera, Phlaeothripidae, Phlaeothripinae).

Gigantothrips is a genus of leaf-feeding species from the Old World tropics that is distinguished from Gynaikothrips and Leeuwenia by the large number of tergal wing-retaining setae. Eight species are recognized from China and Southeast Asia including G.tibetanussp. nov. from Tibet and G.yunnanensissp. nov. from Yunnan, both taken on the leaves of Ficus trees. An illustrated identification key to these eight species is provided here.

The genus Liothrips (Thysanoptera, Phlaeothripidae) in Australia.

Eight species of Liothrips are recognised from Australia, including L. burwelli sp.n., L. chionanthes sp.n. and L. timonii sp.n., also three species shared with southeast Asia. Evidence is presented that L. vaneeckei, the Lily Bulb Thrips, is widespread in eastern Australia presumably on native plants, and that it has been on this continent for many years. The illustrated identification key includes a ninth species, L. urichi, as a potential introduction to Australia for biocontrol purposes. L. brevifemur Girault is considered a nomen dubium known only from fragments of the unique holotype. Two species are newly transferred from Liothrips as Teuchothrips soror (Hood) comb. nov. and Kellyia tenuis (Hood) comb. nov. Generic relationships are discussed, particularly with one new species that is intermediate in structure between Liothrips and Gynaikothrips.

Exploring postmortem succession of rat intestinal microbiome for PMI based on machine learning algorithms and potential use for humans.

The microbial communities may undergo a meaningful successional change during the progress of decay and decomposition that could aid in determining the post-mortem interval (PMI). However, there are still challenges to applying microbiome-based evidence in law enforcement practice. In this study, we attempted to investigate the principles governing microbial community succession during decomposition of rat and human corpse, and explore their potential use for PMI of human cadavers. A controlled experiment was conducted to characterize temporal changes in microbial communities associated with rat corpses as they decomposed for 30 days. Obvious differences of microbial community structures were observed among different stages of decomposition, especially between decomposition of 0-7d and 9-30d. Thus, a two-layer model for PMI prediction was developed based on the succession of bacteria by combining classification and regression models using machine learning algorithms. Our results achieved 90.48% accuracy for discriminating groups of PMI 0-7d and 9-30d, and yielded a mean absolute error of 0.580d within 7d decomposition and 3.165d within 9-30d decomposition. Furthermore, samples from human cadavers were collected to gain the common succession of microbial community between rats and humans. Based on the 44 shared genera of rats and humans, a two-layer model of PMI was rebuilt to be applied for PMI prediction of human cadavers. Accurate estimates indicated a reproducible succession of gut microbes across rats and humans. Together these results suggest that microbial succession was predictable and can be developed into a forensic tool for estimating PMI.

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Sustained overexpression of spliced X-box-binding protein-1 in neurons leads to spontaneous seizures and sudden death in mice.

The underlying etiologies of seizures are highly heterogeneous and remain incompletely understood. While studying the unfolded protein response (UPR) pathways in the brain, we unexpectedly discovered that transgenic mice (XBP1s-TG) expressing spliced X-box-binding protein-1 (Xbp1s), a key effector of UPR signaling, in forebrain excitatory neurons, rapidly develop neurologic deficits, most notably recurrent spontaneous seizures. This seizure phenotype begins around 8 days after Xbp1s transgene expression is induced in XBP1s-TG mice, and by approximately 14 days post induction, the seizures evolve into status epilepticus with nearly continuous seizure activity followed by sudden death. Animal death is likely due to severe seizures because the anticonvulsant valproic acid could significantly prolong the lives of XBP1s-TG mice. Mechanistically, our gene profiling analysis indicates that compared to control mice, XBP1s-TG mice exhibit 591 differentially regulated genes (mostly upregulated) in the brain, including several GABAA receptor genes that are notably downregulated. Finally, whole-cell patch clamp analysis reveals a significant reduction in both spontaneous and tonic GABAergic inhibitory responses in Xbp1s-expressing neurons. Taken together, our findings unravel a link between XBP1s signaling and seizure occurrence.

Novel Prediction Method Applied to Wound Age Estimation: Developing a Stacking Ensemble Model to Improve Predictive Performance Based on Multi-mRNA.

(1) Background: Accurate diagnosis of wound age is crucial for investigating violent cases in forensic practice. However, effective biomarkers and forecast methods are lacking. (2) Methods: Samples were collected from rats divided randomly into control and contusion groups at 0, 4, 8, 12, 16, 20, and 24 h post-injury. The characteristics of concern were nine mRNA expression levels. Internal validation data were used to train different machine learning algorithms, namely random forest (RF), support vector machine (SVM), multilayer perceptron (MLP), gradient boosting (GB), and stochastic gradient descent (SGD), to predict wound age. These models were considered the base learners, which were then applied to developing 26 stacking ensemble models combining two, three, four, or five base learners. The best-performing stacking model and base learner were evaluated through external validation data. (3) Results: The best results were obtained using a stacking model of RF + SVM + MLP (accuracy = 92.85%, area under the receiver operating characteristic curve (AUROC) = 0.93, root-mean-square-error (RMSE) = 1.06 h). The wound age prediction performance of the stacking models was also confirmed for another independent dataset. (4) Conclusions: We illustrate that machine learning techniques, especially ensemble algorithms, have a high potential to be used to predict wound age. According to the results, the strategy can be applied to other types of forensic forecasts.

A novel method for determining postmortem interval based on the metabolomics of multiple organs combined with ensemble learning techniques.

Determining postmortem interval (PMI) is one of the most challenging and essential endeavors in forensic science. Developments in PMI estimation can take advantage of machine learning techniques. Currently, applying an algorithm to obtain information on multiple organs and conducting joint analysis to accurately estimate PMI are still in the early stages. This study aimed to establish a multi-organ stacking model that estimates PMI by analyzing differential compounds of four organs in rats. In a total of 140 rats, skeletal muscle, liver, lung, and kidney tissue samples were collected at each time point after death. Ultra-performance liquid chromatography coupled with high-resolution mass spectrometry was used to determine the compound profiles of the samples. The original data were preprocessed using multivariate statistical analysis to determine discriminant compounds. In addition, three interrelated and increasingly complex patterns (single organ optimal model, single organ stacking model, multi-organ stacking model) were established to estimate PMI. The accuracy and generalized area under the receiver operating characteristic curve of the multi-organ stacking model were the highest at 93% and 0.96, respectively. Only 1 of the 14 external validation samples was misclassified by the multi-organ stacking model. The results demonstrate that the application of the multi-organ combination to the stacking algorithm is a potential forensic tool for the accurate estimation of PMI.

The Oriental fungus-feeding genus Azaleothrips Ananthakrishnan, 1964 from China with one new species and four new records (Thysanoptera, Phlaeothripidae, Phlaeothripinae).

Azaleothrips, a genus of fungus-feeding Phlaeothripinae , is easily recognized by the complex sculpture on the body surface. It is species-rich in the Oriental region, with 10 species here recognized from China, including A.sphaericussp. nov. and four new records. An illustrated key to the species from China is provided.

Structural diversity among the leaf-feeding thrips of Australia in the genus Teuchothrips (Thysanoptera, Phlaeothripinae) with 20 new species.

An identification system, together with illustrated notes, is presented to 34 species of the genus Teuchothrips known from Australia, including the following 20 species newly described: T. agonis sp.n., T. aliceae sp.n., T. badu sp.n., T. bundjalong sp.n., T. dodonaea sp.n., T. gangurru sp.n., T. garrunggam sp.n., T. jarowair sp.n., T. jukun sp.n., T. kaurna sp.n., T. kokatha sp.n., T. larrakia sp.n., T. leptospermum sp.n., T. lutruwita sp.n., T. mareeba sp.n., T. miriwoong sp.n., T. monga sp.n., T. mooni sp.n., T. tolga sp.n., T. toowoomba sp.n. Two species from Philippines are removed from Teuchothrips as Liothrips capitulatus (Reyes) comb.n. and L. pedanus (Reyes) comb.n. The genus Teuchothrips is very species rich in Australia, particularly in the northern tropical zone, presumably in association with the richer flora of perennial shrubs in the warmer and more humid north.

Ant-mimicking thrips of the genus Compsothrips Reuter from China(Phlaeothripidae, Idolothripinae).

Species of Compsothrips are ant-mimics in body form and structure. In contrast to the predatory species of ant-mimicking Aeolothripidae, these species feed by ingesting fungal spores. Worldwide, there are 27 species listed in this genus, with three recorded here from China. Compsothrips tenebronus is here considered a new synonym of C. sinensis, and C. timur is newly recorded from China in Tibet. Furthermore, C. reticulates is redescribed, with details of the female given for the first time. An illustrated key is provided to the three species of Compsothrips from China.