Depicting Primate-Like Granular Dorsolateral Prefrontal Cortex in the Chinese Tree Shrew.

It remains unknown whether the Chinese tree shrew, regarded as the closest sister of primate, has evolved a dorsolateral prefrontal cortex (dlPFC) comparable with primates that is characterized by a fourth layer (L4) enriched with granular cells and reciprocal connections with the mediodorsal nucleus (MD). Here, we reported that following AAV-hSyn-EGFP expression in the MD neurons, the fluorescence micro-optical sectioning tomography revealed their projection trajectories and targeted brain areas, such as the hippocampus, the corpus striatum, and the dlPFC. Cre-dependent transsynaptic viral tracing identified the MD projection terminals that targeted the L4 of the dlPFC, in which the presence of granular cells was confirmed via cytoarchitectural studies by using the Nissl, Golgi, and vGlut2 stainings. Additionally, the L5/6 of the dlPFC projected back to the MD. These results suggest that the tree shrew has evolved a primate-like dlPFC which can serve as an alternative for studying cognition-related functions of the dlPFC.

Refractory COD removal from bio-treated paper wastewater using powdered activated coke adsorption technology with ozonation regeneration: Performance and molecular insights.

The present study employed powdered activated coke (PAC) for the adsorptive removal of refractory COD from the bio-treated paper wastewater (BTPW). The adsorption reached equilibrium after 3 h, resulting in a decrease in the COD concentration from 98.9 mg L-1 in BTPW to 42.6 mg L-1 when utilizing a PAC dosage of 5 g L-1. The dominant fractions of dissolved organic matter in BTPW were hydrophilic acids (HIA), hydrophilic neutrals (HIN), and hydrophobic acids (HOA), accounting for 48.8%, 34.2%, and 17.0% of the total dissolved organic carbon, respectively. Three fractions were all predominantly composed of humic/fulvic acid-like substances, while the HOA fraction exhibited highest susceptibility to adsorption by PAC, followed by the HIA and HIN fractions. FT-ICR MS data revealed PAC preferentially adsorbed the unsaturated and oxygen-rich substances containing more carboxyl groups. Additionally, the spent PAC was regenerated through ozonation and subsequently utilized in the adsorption cycles. The regeneration was successfully conducted under an ozone concentration of 1 mg L-1 for a duration of 10 min, and the regeneration efficiency remained about 87.0% even after undergoing five-cycle of adsorption-regeneration. The findings of this study demonstrate that PAC adsorption is a viable and efficacious treatment technology for efficiently removing refractory COD from BTPW.

Influence of an adjacent tunnel connecting zone shading shed on drivers' eye movement characteristics.

A tunnel shading shed is crucial in improving driving safety as a type of traffic facility to ease the transition of light environments. To study the effect of installation of a shading shed on the visual characteristics of drivers in the connecting zone of the adjacent tunnels, a total of 32 drivers were gathered to perform a real vehicle experiment. The study zone of the adjacent tunnels was divided into three sections: upstream tunnel exit; connecting zone; and downstream tunnel threshold zone. Fixation duration, saccade duration and saccade frequency were selected as research indexes. The results suggest that installation of a shading shed in the connecting zone significantly reduced the fixation (saccade) duration in the upstream tunnel exit and downstream tunnel threshold zones, with a significantly higher saccade frequency. In addition, fixation is better improved at the downstream tunnel entrance, and saccade is better enhanced at the upstream tunnel exit.

Cellular and circuit architecture of the lateral septum for reward processing.

The lateral septum (LS) is composed of heterogeneous cell types that are important for various motivated behaviors. However, the transcriptional profiles, spatial arrangement, function, and connectivity of these cell types have not been systematically studied. Using single-nucleus RNA sequencing, we delineated diverse genetically defined cell types in the LS that play distinct roles in reward processing. Notably, we found that estrogen receptor 1 (Esr1)-expressing neurons in the ventral LS (LSEsr1) are key drivers of reward seeking via projections to the ventral tegmental area, and these neurons play an essential role in methamphetamine (METH) reward and METH-seeking behavior. Extended exposure to METH increases the excitability of LSEsr1 neurons by upregulating hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, thereby contributing to METH-induced locomotor sensitization. These insights not only elucidate the intricate molecular, circuit, and functional architecture of the septal region in reward processing but also reveal a neural pathway critical for METH reward and behavioral sensitization.

Oncolytic Activity of Sindbis Virus with the Help of GM-CSF in Hepatocellular Carcinoma.

Hepatocellular carcinoma is a refractory tumor with poor prognosis and high mortality. Many oncolytic viruses are currently being investigated for the treatment of hepatocellular carcinoma. Based on previous studies, we constructed a recombinant GM-CSF-carrying Sindbis virus, named SINV-GM-CSF, which contains a mutation (G to S) at amino acid 285 in the nsp1 protein of the viral vector. The potential of this mutated vector for liver cancer therapy was verified at the cellular level and in vivo, respectively, and the changes in the tumor microenvironment after treatment were also described. The results showed that the Sindbis virus could effectively infect hepatocellular carcinoma cell lines and induce cell death. Furthermore, the addition of GM-CSF enhanced the tumor-killing effect of the Sindbis virus and increased the number of immune cells in the intra-tumor microenvironment during the treatment. In particular, SINV-GM-CSF was able to efficiently kill tumors in a mouse tumor model of hepatocellular carcinoma by regulating the elevation of M1-type macrophages (which have a tumor-resistant ability) and the decrease in M2-type macrophages (which have a tumor-promoting capacity). Overall, SINV-GM-CSF is an attractive vector platform with clinical potential for use as a safe and effective oncolytic virus.

Dendrite-Free All-Solid-State Lithium Metal Batteries by In Situ Phase Transformation of the Soft Carbon-Li3N Interface Layer.

The accelerated formation of lithium dendrites has considerably impeded the advancement and practical deployment of all-solid-state lithium metal batteries (ASSLMBs). In this study, a soft carbon (SC)-Li3N interface layer was developed with both ionic and electronic conductivity, for which the in situ lithiation reaction not only lithiated SC into LiC6 with good electronic/ionic conductivity but also successfully transformed the mixed-phase Li3N into pure-phase ß-Li3N with a high ionic conductivity/ion diffusion coefficient and stability to lithium metal. The mixed conductive interface layer facilitates fast Li+ transport at the interface and induces the homogeneous deposition of lithium metal inside it. This effectively inhibits the formation of lithium dendrites and greatly improves the performance of the ASSLMB. The ASSLMB assembled with the SC-Li3N interface layer exhibits high areal capacity (15 mA h cm-2), high current density (7.5 mA cm-2), and long cycle life (6000 cycles). These results indicate that this interface layer has great potential for practical applications in high-energy-density ASSLMBs.

Mapping the Behavioral Signatures of Shank3b Mice in Both Sexes.

Autism spectrum disorders (ASD) are characterized by social and repetitive abnormalities. Although the ASD mouse model with Shank3b mutations is widely used in ASD research, the behavioral phenotype of this model has not been fully elucidated. Here, a 3D-motion capture system and linear discriminant analysis were used to comprehensively record and analyze the behavioral patterns of male and female Shank3b mutant mice. It was found that both sexes replicated the core and accompanied symptoms of ASD, with significant sex differences. Further, Shank3b heterozygous knockout mice exhibited distinct autistic behaviors, that were significantly different from those those observed in the wild type and homozygous knockout groups. Our findings provide evidence for the inclusion of both sexes and experimental approaches to efficiently characterize heterozygous transgenic models, which are more clinically relevant in autistic studies.

Engineered AAV13 variants with enhanced transduction and confined spread.

Precise targeting of specific regions within the central nervous system (CNS) is crucial for both scientific research and gene therapy in the context of brain diseases. Adeno-associated virus 13 (AAV13) is known for its restricted diffusion range within the CNS, making it an ideal choice for precise labeling and administration within small brain regions. However, AAV13 mediates relatively low expression of target genes. Here, we introduced specifically engineered modifications to the AAV13 capsid protein to enhance its transduction efficiency. We first constructed AAV13-YF by mutating tyrosine to phenylalanine on the surface of the AAV13 capsid. We then inserted the 7m8 peptide, known to enhance cell transduction, into positions 587/588 and 585/586 of the AAV13 capsid, resulting in two distinct variants named AAV13-587-7m8 and AAV13-585-7m8, respectively. We found that AAV13-YF exhibited superior in vitro infectivity in HEK293T cells compared to AAV13, while AAV13-587-7m8 and AAV13-585-7m8 showed enhanced CNS infection capabilities in C57BL/6 mice, with AAV13-587-7m8 infection retaining a limited spread range. These modified AAV13 variants hold promising potential for applications in gene therapy and neuroscience research.

LncRNA TubAR complexes with TUBB4A and TUBA1A to promote microtubule assembly and maintain myelination.

A long-standing hypothesis proposes that certain RNA(s) must exhibit structural roles in microtubule assembly. Here, we identify a long noncoding RNA (TubAR) that is highly expressed in cerebellum and forms RNA-protein complex with TUBB4A and TUBA1A, two tubulins clinically linked to cerebellar and myelination defects. TubAR knockdown in mouse cerebellum causes loss of oligodendrocytes and Purkinje cells, demyelination, and decreased locomotor activity. Biochemically, we establish the roles of TubAR in promoting TUBB4A-TUBA1A heterodimer formation and microtubule assembly. Intriguingly, different from the hypomyelination-causing mutations, the non-hypomyelination-causing mutation TUBB4A-R2G confers gain-of-function for an RNA-independent interaction with TUBA1A. Experimental use of R2G/A mutations restores TUBB4A-TUBA1A heterodimer formation, and rescues the neuronal cell death phenotype caused by TubAR knockdown. Together, we uncover TubAR as the long-elusive structural RNA for microtubule assembly and demonstrate how TubAR mediates microtubule assembly specifically from αß-tubulin heterodimers, which is crucial for maintenance of cerebellar myelination and activity.

Brain-wide neuronal circuit connectome of human glioblastoma.

Glioblastoma (GBM), a universally fatal brain cancer, infiltrates the brain and can be synaptically innervated by neurons, which drives tumor progression 1-6 . Synaptic inputs onto GBM cells identified so far are largely short-range and glutamatergic 7-9 . The extent of integration of GBM cells into brain-wide neuronal circuitry is not well understood. Here we applied a rabies virus-mediated retrograde monosynaptic tracing approach 10-12 to systematically investigate circuit integration of human GBM organoids transplanted into adult mice. We found that GBM cells from multiple patients rapidly integrated into brain-wide neuronal circuits and exhibited diverse local and long-range connectivity. Beyond glutamatergic inputs, we identified a variety of neuromodulatory inputs across the brain, including cholinergic inputs from the basal forebrain. Acute acetylcholine stimulation induced sustained calcium oscillations and long-lasting transcriptional reprogramming of GBM cells into a more invasive state via the metabotropic CHRM3 receptor. CHRM3 downregulation suppressed GBM cell invasion, proliferation, and survival in vitro and in vivo. Together, these results reveal the capacity of human GBM cells to rapidly and robustly integrate into anatomically and molecularly diverse neuronal circuitry in the adult brain and support a model wherein rapid synapse formation onto GBM cells and transient activation of upstream neurons may lead to a long-lasting increase in fitness to promote tumor infiltration and progression.

fMRI, LFP, and anatomical evidence for hierarchical nociceptive routing pathway between somatosensory and insular cortices.

The directional organization of multiple nociceptive regions, particularly within obscure operculoinsular areas, underlying multidimensional pain processing remains elusive. This study aims to establish the fundamental organization between somatosensory and insular cortices in routing nociceptive information. By employing an integrated multimodal approach of high-field fMRI, intracranial electrophysiology, and transsynaptic viral tracing in rats, we observed a hierarchically organized connection of S1/S2 → posterior insula → anterior insula in routing nociceptive information. The directional nociceptive pathway determined by early fMRI responses was consistent with that examined by early evoked LFP, intrinsic effective connectivity, and anatomical projection, suggesting fMRI could provide a valuable facility to discern directional neural circuits in animals and humans non-invasively. Moreover, our knowledge of the nociceptive hierarchical organization of somatosensory and insular cortices and the interface role of the posterior insula may have implications for the development of targeted pain therapies.

Pleiotropic immunoregulation by growth-blocking peptide in Ostrinia furnacalis.

Insects rely on their innate immune system to eliminate pathogenic microbes. As a system component, cytokines transmit intercellular signals to control immune responses. Growth-blocking peptide (GBP) is a member of the stress-responsive peptide family of cytokines found in several orders of insects, including Drosophila. However, the physiological role of GBP in defence against pathogens is not thoroughly understood. In this study, we explored the functions of GBP in a lepidopteran pest, Ostrinia furnacalis. Injection of recombinant O. furnacalis GBP (OfGBP) precursor (proGBP) and chemically synthesised GBP significantly induced the transcription of antimicrobial peptides (AMPs) and other immunity-related genes including immune deficiency (IMD) and Dorsal. The level of OfGBP mRNA was upregulated after bacterial infection. Knockdown of OfGBP expression led to a decrease in IMD, Relish, MyD88 and Dorsal mRNA levels. OfGBP induced phenoloxidase activity and affected hemocyte behaviours in O. furnacalis larvae. In summary, GBP is a potent cytokine, effectively regulating AMP synthesis, melanization response and cellular immunity to eliminate invading pathogens.

Identification and characteristic analysis of an extracellular signal-regulated kinase from Ostrinia furnacalis Guenée.

The extracellular signal-regulated kinase (ERK) pathway, a critical genetic determinant, controls diverse physiological functions, including innate immunity, development, and stress response. In the current study, a full-length cDNA (1592bp) encoding the ERK gene (OfERK) was cloned from Ostrinia furnacalis Guenée (GenBank accession number: MF797866). The open reading frame of the OfERK gene encoded 364 amino acids and shared 96.43%-98.08% amino acid identities with other insect mitogen-activated protein kinases. For spatiotemporal analysis of the expression pattern, OfERK exhibited a significant peak expression on the 3rd day of the pupa stage and showed the highest expression in hemocytes specifically. Indirect immunofluorescence assays and immuno-electron microscopy revealed a wide distribution of the OfERK protein in hemocytes and epidermis. Moreover, the results demonstrated that the Bt Cry1Ab-activated toxin significantly induces the expression of OfERK. Other genes related to immune response, development, and stress response exhibited dynamic changes in expression after Cry1Ab oral treatment. The expression of OfERK was downregulated through RNA interference, and the correlation of its expression with other related genes was verified using quantitative real-time polymerase chain reaction. Our study provides valuable insights into the regulatory mechanism of ERK in insects for future studies.

Cerebellar Purkinje cell firing promotes conscious recovery from anesthesia state through coordinating neuronal communications with motor cortex.

Background: The neurobiological basis of gaining consciousness from unconscious state induced by anesthetics remains unknown. This study was designed to investigate the involvement of the cerebello-thalamus-motor cortical loop mediating consciousness transitions from the loss of consciousness (LOC) induced by an inhalational anesthetic sevoflurane in mice. Methods: The neural tracing and fMRI together with opto-chemogenetic manipulation were used to investigate the potential link among cerebello-thalamus-motor cortical brain regions. The fiber photometry of calcium and neurotransmitters, including glutamate (Glu), γ-aminobutyric acid (GABA) and norepinephrine (NE), were monitored from the motor cortex (M1) and the 5th lobule of the cerebellar vermis (5Cb) during unconsciousness induced by sevoflurane and gaining consciousness after sevoflurane exposure. Cerebellar Purkinje cells were optogenetically manipulated to investigate their influence on consciousness transitions during and after sevoflurane exposure. Results: Activation of 5Cb Purkinje cells increased the Ca2+ flux in the M1 CaMKIIα+ neurons, but this increment was significantly reduced by inactivation of posterior and parafascicular thalamic nucleus. The 5Cb and M1 exhibited concerted calcium flux, and glutamate and GABA release during transitions from wakefulness, loss of consciousness, burst suppression to conscious recovery. Ca2+ flux and Glu release in the M1, but not in the 5Cb, showed a strong synchronization with the EEG burst suppression, particularly, in the gamma-band range. In contrast, the Glu, GABA and NE release and Ca2+ oscillations were coherent with the EEG gamma band activity only in the 5Cb during the pre-recovery of consciousness period. The optogenetic activation of Purkinje cells during burst suppression significantly facilitated emergence from anesthesia while the optogenetic inhibition prolonged the time to gaining consciousness. Conclusions: Our data indicate that cerebellar neuronal communication integrated with motor cortex through thalamus promotes consciousness recovery from anesthesia which may likely serve as arousal regulation.

Natural bioactive lysosomes extracted from multiple cells for tumor therapy.

Nanoparticles of biological origin exhibit many unique properties in biological applications due to their exquisite structure, specific composition, and natural biological functionality. In this study, we obtained lysosomes from three distinct cell types (one normal cell and two activated immune cells) and demonstrated their potential as natural therapeutic nanoparticles for tumor therapy. In vitro experiments revealed that these lysosomes maintained their structural integrity, were well-distributed, and exhibited significant biological activity, which effectively induced cancer cell death by generating ROS and disrupting biological substrates. Additionally, in vivo investigations showed that these lysosomes could accumulate in tumor tissues after intravenous administration and exhibited exceptional therapeutic effects through the destruction of tumor blood vessels and the degradation of immunosuppressive proteins, with complete tumor disappearance in a single treatment. This research on the utilization of bioactive lysosomes for tumor treatment provides valuable insights into drug development and tumor treatment, particularly when conventional approaches have proven ineffective.

The analysis of HPV integration sites based on nanopore sequencing and the profiling changes along the course of photodynamic therapy.

OBJECTIVE: To detect the HPV genotype and integration sites in patients with high-risk HPV infection at different stages of photodynamic therapy using nanopore technology and to evaluate the treatment effect. METHODS: Four patients with HPV infection were selected and subjected to photodynamic therapy, and cervical exfoliated cell was sampled at before treatment, after three courses of treatment and six courses of treatment, their viral abundance and insertion sites were analyzed by nanopore technology, and pathological examinations were performed before and after treatment. In this study, we developed a novel assay that combined viral sequence enrichment and Nanopore sequencing for identification of HPV genotype and integration sites at once. The assay has obvious advantages over qPCR or NGS-based methods, as it has better sensitivity after viral sequences enrichment and can generate long-reads (kb to Mb) for better detection rate of structure variations, moreover, fast turn-around time for real-time viral sequencing and analysis. RESULTS: The pathological grade was reduced in all four patients after photodynamic therapy. Virus has been cleared in two cases after treatment, the virus amount reduced after treatment but not completely cleared in one case, and two type viruses were cleared and one type virus persisted after treatment in the last patient with multiple infection. Viral abundance and the number of integration sites were positively correlated. Gene enrichment analysis showed complete viral clearance in 1 patient and 3 patients required follow-up. CONCLUSION: Nanopore sequencing can effectively monitor the abundance of HPV viruses and integration sites to show the presence status of viruses, and combined with the results of gene enrichment analysis, the treatment effect can be dynamically assessed.

Developing a trans-multisynaptic tracer to map the neural circuit of recovered sciatic nerve after treatment with nerve growth factor.

Nerve growth factor (NGF) has been shown to support the survival and differentiation of neurons. In this study, we first developed a retrograde trans-multisynaptic tracer PRV580 expressing the mCherry fluorescent protein based on pseudorabies virus Bartha strain to map the neural circuit of sciatic nerve. Secondly, the newly developed PRV580 was used to map the neural circuit of the recovering sciatic nerve upon treatment with NGF. Our results showed that red signals from PRV580 were observed in various brain regions. Among these regions, many areas of the pyramidal system and the extra-pyramidal system had been mapped, accounting for as much as 56.8 % of the total inputs. Furthermore, we found that NGF could significantly increase the ratio of total input (29.05 %) compared to PBS (3.65 %), indicating that NGF indeed can aid in the repair of injured sciatic nerve. These findings indicated that NGF has therapeutic ability for the treatment of peripheral nerve injuries and virus-based tracers can be used to monitor the recovery.

Oncolytic Viral Therapy for Glioma by Recombinant Sindbis Virus.

BACKGROUND: The characteristics of glioblastoma, such as drug resistance during treatment, short patient survival, and high recurrence rates, have made patients with glioblastoma more likely to benefit from oncolytic therapy. METHODS: In this study, we investigated the safety of the sindbis virus by injecting virus intravenously and intracranially in mice and evaluated the therapeutic effect of the virus carrying different combinations of IL-12, IL-7, and GM-CSF on glioma in a glioma-bearing mouse model. RESULTS: SINV was autologously eliminated from the serum and organs as well as from neural networks after entering mice. Furthermore, SINV was restricted to the injection site in the tree shrew brain and did not spread throughout the whole brain. In addition, we found that SINV-induced apoptosis in conjunction with the stimulation of the immune system by tumor-killing cytokines substantially suppressed tumor development. It is worth mentioning that SINV carrying IL-7 and IL-12 had the most notable glioma-killing effect. Furthermore, in an intracranial glioma model, SINV containing IL-7 and IL-12 effectively prolonged the survival time of mice and inhibited glioma progression. CONCLUSIONS: These results suggest that SINV has a significant safety profile as an oncolytic virus and that combining SINV with cytokines is an efficient treatment option for malignant gliomas.

Paraventricular Hypothalamic Nucleus Upregulates Intraocular Pressure Via Glutamatergic Neurons.

Purpose: The neuroregulatory center of intraocular pressure (IOP) is located in the hypothalamus. An efferent neural pathway exists between the hypothalamic nuclei and the autonomic nerve endings in the anterior chamber of the eye. This study was designed to investigate whether the paraventricular hypothalamic nucleus (PVH) regulates IOP as the other nuclei do. Methods: Optogenetic manipulation of PVH neurons was used in this study. Light stimulation was applied via an optical fiber embedded over the PVH to activate projection neurons after AAV2/9-CaMKIIα-hChR2-mCherry was injected into the right PVH of C57BL/6J mice. The same methods were used to inhibit projection neurons after AAV2/9-CaMKIIα-eNpHR3.0-mCherry was injected into the bilateral PVH of C57BL/6J mice. AAV2/9-EF1α-DIO-hChR2-mCherry was injected into the right PVH of Vglut2-Cre mice to elucidate the effect of glutamatergic neuron-specific activation. IOP was measured before and after light manipulation. Associated nuclei activation was clarified by c-Fos immunohistochemical staining. Only mice with accurate viral expression and fiber embedding were included in the statistical analysis. Results: Activation of projection neurons in the right PVH induced significant bilateral IOP elevation (n = 11, P < 0.001); the ipsilateral IOP increased more noticeably (n = 11, P < 0.05); Bilateral inhibition of PVH projection neurons did not significantly influence IOP (n = 5, P > 0.05). Specific activation of glutamatergic neurons among PVH projection neurons also induced IOP elevation in both eyes (n = 5, P < 0.001). The dorsomedial hypothalamic nucleus, ventromedial hypothalamic nucleus, locus coeruleus and basolateral amygdaloid nucleus responded to light stimulation of PVH in AAV-ChR2 mice. Conclusions: The PVH may play a role in IOP upregulation via glutamatergic neurons.