Oncolytic varicella-zoster virus engineered with ORF8 deletion and armed with drug-controllable interleukin-12.

BACKGROUND: The varicella-zoster virus (VZV), belonging to the group of human α-herpesviruses, has yet to be developed as a platform for oncolytic virotherapy, despite indications from clinical case reports suggesting a potential association between VZV infection and cancer remission. METHODS: Here, we constructed oncolytic VZV candidates based on the vaccine strain vOka and the laboratory strain Ellen. These newly engineered viruses were subsequently assessed for their oncolytic properties in the human MeWo melanoma xenograft model and the mouse B16-F10-nectin1 melanoma syngeneic model. RESULTS: In the MeWo xenograft model, both vOka and Ellen exhibited potent antitumor efficacy. However, it was observed that introducing a hyperfusogenic mutation into glycoprotein B led to a reduction in VZV's effectiveness. Notably, the deletion of ORF8 (encodes viral deoxyuridine triphosphatase) attenuated the replication of VZV both in vitro and in vivo, but it did not compromise VZV's oncolytic potency. We further armed the VZV Ellen-ΔORF8 vector with a tet-off controlled mouse single-chain IL12 (scIL12) gene cassette. This augmented virus was validated for its oncolytic activity and triggered systemic antitumor immune responses in the immunocompetent B16-F10-nectin1 model. CONCLUSIONS: These findings highlight the potential of using Ellen-ΔORF8-tet-off-scIL12 as a novel VZV-based oncolytic virotherapy.

Oncolytic α-herpesvirus and myeloid-tropic cytomegalovirus cooperatively enhance systemic antitumor responses.

Oncolytic virotherapy aims to activate host antitumor immunity. In responsive tumors, intratumorally injected herpes simplex viruses (HSVs) have been shown to lyse tumor cells, resulting in local inflammation, enhanced tumor antigen presentation, and boosting of antitumor cytotoxic lymphocytes. In contrast to HSV, cytomegalovirus (CMV) is nonlytic and reprograms infected myeloid cells, limiting their antigen-presenting functions and protecting them from recognition by natural killer (NK) cells. Here, we show that when co-injected into mouse tumors with an oncolytic HSV, mouse CMV (mCMV) preferentially targeted tumor-associated myeloid cells, promoted the local release of proinflammatory cytokines, and enhanced systemic antitumor immune responses, leading to superior control of both injected and distant contralateral tumors. Deletion of mCMV genes m06, which degrades major histocompatibility complex class I (MHC class I), or m144, a viral MHC class I homolog that inhibits NK activation, was shown to diminish the antitumor activity of the HSV/mCMV combination. However, an mCMV recombinant lacking the m04 gene, which escorts MHC class I to the cell surface, showed superior HSV adjuvanticity. CMV is a potentially promising agent with which to reshape and enhance antitumor immune responses following oncolytic HSV therapy.

NADH Photosynthesis System with Affordable Electron Supply and Inhibited NADH Oxidation.

Photosynthesis offers a green approach for the recycling of nicotinamide cofactors primarily NADH in bio-redox reactions. Herein, we report an NADH photosynthesis system where the oxidation of biomass derivatives is designed as an electron supply module (ESM) to afford electrons and superoxide dismutase/catalase (SOD/CAT) cascade catalysis is designed as a reactive oxygen species (ROS) elimination module (REM) to inhibit NADH degradation. Glucose as the electron donor guarantees the reaction sustainability accompanied with oxidative products of gluconic acid and formic acid. Meanwhile, enzyme cascades of SOD/CAT greatly eliminate ROS, leading to a ≈2.00-fold elevation of NADH yield (61.1 % vs. 30.7 %). The initial reaction rate and turnover frequency (TOF) increased by 2.50 times and 2.54 times, respectively, compared with those systems without REM. Our study establishes a novel and efficient platform for NADH photosynthesis coupled to biomass-to-chemical conversion.

Human cytomegalovirus infection perturbs neural progenitor cell fate via the expression of viral microRNAs.

Human cytomegalovirus (HCMV) preferentially targets neural progenitor cells (NPCs) in congenitally infected fetal brains, inducing neurodevelopmental disorders. While HCMV expresses several microRNAs (miRNAs) during infection, their roles in NPC infection are unclear. Here, we characterized expression of cellular and viral miRNAs in HCMV-infected NPCs during early infection by microarray and identified seven differentially expressed cellular miRNAs and six significantly upregulated HCMV miRNAs. Deep learning approaches were used to identify potential targets of significantly upregulated HCMV miRNAs against differentially expressed cellular messenger RNA (mRNAs), and the associations with miRNA-mRNA expression changes were observed. Gene ontology enrichment analysis indicated cellular gene targets were significantly enriched in pathways involved in neurodevelopment and cell-cycle processes. Viral modulation of selected miRNAs and cellular gene targets involved in neurodevelopmental processes were further validated by real-time quantitative reverse transcription polymerase chain reaction. Finally, a predicted 3' untranslated region target site of hcmv-miR-US25-1 in Jag1, a factor important for neurogenesis, was confirmed by mutagenesis. Reduction of Jag1 RNA and protein levels in NPCs was observed in response to transient expression of hcmv-miR-US25-1. A hcmv-miR-US25-1 mutant virus (ΔmiR-US25) displayed limited ability to downregulate Jag1 mRNA levels and protein levels during the early infection stage compared with the wild type virus. Our collective experimental and computational investigation of miRNAs and cellular mRNAs expression in HCMV-infected NPCs yields new insights into the roles of viral miRNAs in regulating NPC fate and their contributions to HCMV neuropathogenesis.

Single Solution-Phase Synthesis of Charged Covalent Organic Framework Nanosheets with High Volume Yield.

Covalent organic framework nanosheets (COF-NSs) are emerging building blocks for functional materials, and their scalable fabrication is highly desirable. Current synthetic methods suffer from low volume yields resulting from confined on-surface/at-interface growth space and complex multiple-phase synthesis systems. Herein, we report the synthesis of charged COF-NSs in open space using a single-phase organic solution system, achieving magnitudes higher volume yields of up to 18.7 mg mL-1 . Charge-induced electrostatic repulsion forces enable in-plane anisotropic secondary growth from initial discrete and disordered polymers into large and crystalline COF-NSs. The charged COF-NS colloidal suspensions are cast into thin and compact proton exchange membranes (PEMs) with lamellar morphology and oriented crystallinity, displaying outstanding proton conductivity, negligible dimensional swelling, and good H2 /O2 fuel cell performance.

Covalent organic framework membranes for efficient separation of monovalent cations.

Covalent organic frameworks (COF), with rigid, highly ordered and tunable structures, can actively manipulate the synergy of entropic selectivity and enthalpic selectivity, holding great potential as next-generation membrane materials for ion separations. Here, we demonstrated the efficient separation of monovalent cations by COF membrane. The channels of COF membrane are decorated with three different kinds of acid groups. A concept of confined cascade separation was proposed to elucidate the separation process. The channels of COF membrane comprised two kinds of domains, acid-domains and acid-free-domains. The acid-domains serve as confined stages, rendering high selectivity, while the acid-free-domains preserve the pristine channel size, rendering high permeation flux. A set of descriptors of stage properties were designed to elucidate their effect on selective ion transport behavior. The resulting COF membrane acquired high ion separation performances, with an actual selectivity of 4.2-4.7 for K+/Li+ binary mixtures and an ideal selectivity of ~13.7 for K+/Li+.

Coordination-driven structure reconstruction in polymer of intrinsic microporosity membranes for efficient propylene/propane separation.

Polymers of intrinsic microporosity (PIMs), integrating unique microporous structure and solution-processability, are one class of the most promising membrane materials for energy-efficient gas separations. However, the micropores generated from inefficient chain packing often exhibit wide pore size distribution, making it very challenging to achieve efficient olefin/paraffin separations. Here, we propose a coordination-driven reconstruction (CDR) strategy, where metal ions are incorporated into amidoxime-functionalized PIM-1 (AO-PIM) to in situ generate coordination crosslinking networks. By varying the type and content of metal ions, the resulting crosslinking structures can be optimized, and the molecular sieving capability of PIM membranes can be dramatically enhanced. Particularly, the introduction of alkali or alkaline earth metals renders more precise micropores contributing to superior C3H6/C3H8 separation performance. K+ incorporated AO-PIM membranes exhibit a high ideal C3H6/C3H8 selectivity of 50, surpassing almost all the reported polymer membranes. Moreover, the coordination crosslinking structure significantly improves the membrane stability under higher pressure as well as the plasticization resistant performance. We envision that this straightforward and generic CDR strategy could potentially unlock the potentials of PIMs for olefin/paraffin separations and many other challenging gas separations.

Neuropsychiatric symptoms and altered sleep quality in cerebral small vessel disease.

Background: Sleep disturbance and neuropsychiatric symptoms are common clinical symptoms of cerebral small vessel disease (CSVD), but the underlying mechanism is unclear. Here, we investigated the relationship between sleep quality and neuropsychiatric performance in patients with CSVD. Methods: A total of 30 patients with CSVD and 35 healthy controls (HCs) were recruited. The 13-item Beck Depression Inventory (BDI-13), Beck Anxiety Inventory (BAI), and Symptom Check List 90 (SCL90) were used to assess depression, anxiety, and other psychological symptoms, respectively. Sleep quality was assessed using Pittsburgh Sleep Quality Index (PSQI), and cognitive function was tested using Montreal Cognitive Assessment (MoCA). Results: When compared to the HC group, the patients with CSVD showed increased anxiety and neuropsychiatric symptoms, worse sleep quality, and impaired cognition (p < 0.05). The prevalence of comorbid poor sleep quality in the patients with CSVD was approximately 46%. The patients with CSVD with poor sleep quality also had more severe neuropsychiatric symptoms. After controlling for demographic variables, sex and anxiety significantly predicted sleep quality. Conclusion: This study suggests that the prevalence of CSVD with poor sleep quality is high, and that sex and anxiety are independent risk factors for CSVD comorbid sleep quality.

A semantic information-driven stepwise landslide displacement prediction model.

Landslide prediction is critical for the early warning of a landslide occurrence. Existing stepwise landslide displacement prediction methods are mostly data-driven approaches. However, these models are vulnerable to overfitting, and the low-dimensional numerical features with high numerical volatility prevent them from precisely quantifying the rapid increase in daily displacement in the acceleration phase. Therefore, we propose a semantic information-driven stepwise landslide displacement prediction model comprising an identifier in the displacement phase and a predictor in the acceleration phase. First, the raw landslide monitoring data are converted into text-based semantic information and the semantic features are fused. Subsequently, based on the daily displacement and velocity, we propose a sliding window phase division algorithm to divide the stepwise landslide phase into stationary and acceleration phases. Finally, the landslide displacement phase is identified, and the displacement during the acceleration phase is predicted. The experimental results of the model on the Xinpu and Qingshi landslides in Chongqing, China, show that the proposed model exploits the derived semantic information to identify the landslide acceleration phase qualitatively, and predict the daily displacement of the acceleration phase quantitatively. The proposed model provides a valuable reference for the early warning of stepwise landslides.

Missing-linker Defects in Covalent Organic Framework Membranes for Efficient CO2 Separation.

Covalent organic framework (COF) membranes with tunable ordered channels and free organic groups hold great promise in molecular separations owing to the synergy of physical and chemical microenvironments. Herein, we develop a defect engineering strategy to fabricate COF membranes for efficient CO2 separation. Abundant amino groups are in situ generated on the COF nanosheets arising from the missing-linker defects during the reactive assembly of amine monomer and mixed aldehyde monomers. The COF nanosheets are assembled to fabricate COF membranes. Amino groups, as the CO2 facilitated transport carriers, along with ordered channels endow COF membrane with high CO2 permeances exceeding 300 GPU and excellent separation selectivity of 80 for CO2 /N2 , and 54 for CO2 /CH4 mixed gas under humidified state. Our defect engineering strategy offers a facile approach to generating free organic functional groups in COF membranes and other organic framework membranes for diverse chemical separations.

MOF-COF "Alloy" Membranes for Efficient Propylene/Propane Separation.

Molecular-sieving membranes from metal-organic frameworks (MOFs) are promising candidates for separating olefin/paraffin mixtures, a critical demand in sustainable chemical processes and a grand challenge in molecular separation. Currently, the inherent lattice flexibility of MOFs severely compromises their precise sieving ability. Here, a proof-of-concept of "alloy" membranes (AMs), which are fabricated by incorporating quaternary ammonium (QA)-functionalized covalent organic frameworks (COFs) into a zeolitic imidazolate framework-8 (ZIF-8) matrix is demonstrated. The Coulomb force between the COFs and the ZIF-8 restricts the linker rotation of the ZIF-8, generating a distinct alloying effect, by which the lattice rigidity of ZIF-8 can be conveniently tuned through varying the content of the COFs, similar to the flexible-to-rigid transition in aluminum alloy manufacturing. Such an alloying effect confers the AM's superior propylene/propane separation performance, with a propylene/propane separation factor surpassing 200 and a propylene permeance of 168 GPU. Hopefully, the AMs concept and the concomitant alloying effect can update the connotation of mixed matrix membranes and stimulate the re-envisioning about the design paradigm and development of advanced membranes for energy-efficient separations.

Oil-Water-Oil Triphase Synthesis of Ionic Covalent Organic Framework Nanosheets.

Ionic covalent organic framework nanosheets (iCOFNs) with long-range ordered and mono-dispersed ionic groups hold great potential in many advanced applications. Considering the inherent drawbacks of oil-water biphase method, herein, we explore an oil-water-oil triphase method based on phase engineering strategy for the bottom-up synthesis of iCOFNs. The middle water phase serves as a confined reaction region, and the two oil phases are reservoirs for storing and supplying monomers to the water phase. A large aqueous space and low monomer concentration lead to the anisotropic gradual growth of iCOFNs into few-layer thickness, large lateral size, and high crystallinity. Notably, the resulting three cationic and anionic iCOFNs exhibit ultra-high aspect ratios of up to 20,000. We further demonstrate their application potential by processing into ultrathin defect-free COF membranes for efficient biogas separation. Our triphase method may offer an alternative platform technology for the synthesis and innovative applications of iCOFNs.

Susceptibility-diffusion mismatch correlated with leptomeningeal collateralization in large vessel occlusion stroke.

OBJECTIVE: To investigate the relationship between asymmetric prominent hypointense vessels (prominent vessel sign, PVS) on susceptibility-weighted imaging (SWI) and leptomeningeal collateralization in patients with acute ischemic stroke due to large vessel occlusion. METHODS: We retrospectively enrolled patients with M1 segment occlusion of the middle cerebral artery who underwent emergency magnetic resonance imaging and digital subtraction angiography within 24 hours from stroke onset. The extent of PVS on SWI was assessed using the Alberta Stroke Program Early CT Score (ASPECTS). Leptomeningeal collateralization on digital subtraction angiography images was assessed using the American Society of Interventional and Therapeutic Neuroradiology/Society of Interventional Radiology (ASITN/SIR) scale. Spearman's rank correlation test was performed to explore the correlation of ASITN/SIR scores with SWI-ASPECTS and SWI-diffusion-weighted imaging (DWI) mismatch scores. RESULTS: Thirty-five patients were enrolled. There was no significant correlation between SWI-ASPECTS and ASITN/SIR scores. However, SWI-DWI mismatch scores were positively correlated with ASITN/SIR scores. CONCLUSION: The range of PVS on SWI did not closely reflect the collateral status, while the range of SWI-DWI mismatch was significantly correlated with the leptomeningeal collateralization. In patients with acute anterior circulation stroke due to large vessel occlusion, larger SWI-DWI mismatch was associated with better leptomeningeal collaterals.

Factors associated with prominent vessel sign on susceptibility-weighted imaging in acute ischemic stroke.

The prominent vessel sign (PVS) on susceptibility-weighted imaging (SWI) is not displayed in all cases of acute ischemia. We aimed to investigate the factors associated with the presence of PVS in stroke patients. Consecutive ischemic stroke patients admitted within 24 h from symptom onset underwent emergency multimodal MRI at admission. Associated factors for the presence of PVS were analyzed using univariate analyses and multivariable logistic regression analyses. A total of 218 patients were enrolled. The occurrence rate of PVS was 55.5%. Univariate analyses showed significant differences between PVS-positive group and PVS-negative group in age, history of coronary heart disease, baseline NIHSS scores, total cholesterol, hemoglobin, anterior circulation infarct, large vessel occlusion, and cardioembolism. Multivariable logistic regression analyses revealed that the independent factors associated with PVS were anterior circulation infarct (odds ratio [OR] 13.7; 95% confidence interval [CI] 3.5-53.3), large vessel occlusion (OR 123.3; 95% CI 33.7-451.5), and cardioembolism (OR 5.6; 95% CI 2.1-15.3). Anterior circulation infarct, large vessel occlusion, and cardioembolism are independently associated with the presence of PVS on SWI.

HSV-1 H129-Derived Anterograde Neural Circuit Tracers: Improvements, Production, and Applications.

Anterograde viral tracers are powerful and essential tools for dissecting the output targets of a brain region of interest. They have been developed from herpes simplex virus 1 (HSV-1) strain H129 (H129), and have been successfully applied to map diverse neural circuits. Initially, the anterograde polysynaptic tracer H129-G4 was used by many groups. We then developed the first monosynaptic tracer, H129-dTK-tdT, which was highly successful, yet improvements are needed. Now, by inserting another tdTomato expression cassette into the H129-dTK-tdT genome, we have created H129-dTK-T2, an updated version of H129-dTK-tdT that has improved labeling intensity. To help scientists produce and apply our H129-derived viral tracers, here we provide the protocol describing our detailed and standardized procedures. Commonly-encountered technical problems and their solutions are also discussed in detail. Broadly, the dissemination of this protocol will greatly support scientists to apply these viral tracers on a large scale.

Interactive effect of susceptibility-diffusion mismatch and recanalization status on clinical outcome in large vessel occlusion stroke.

OBJECTIVE: To investigate the interactive effect of susceptibility-diffusion mismatch and recanalization status on clinical outcome in patients with acute ischemic stroke due to large vessel occlusion. METHODS: In this prospective study, consecutive ischemic stroke patients admitted within 24 h from symptom onset underwent emergency multimodal MRI at admission, including diffusion-weighted imaging (DWI), susceptibility-weighted imaging (SWI), and time-of-flight magnetic resonance angiography (TOF-MRA). Patients with large vessel occlusion within the anterior circulation were recruited. Follow-up MRI was performed within 24 h after recanalization therapy (intravenous thrombolysis, endovascular therapy, or both). Multivariable logistic regression analysis was performed to estimate the interaction between SWI-DWI mismatch score and recanalization status on clinical outcome. RESULTS: A total of 90 patients were enrolled. A multiplicative interaction between SWI-DWI mismatch score and recanalization status on clinical outcome was observed (P=0.037). The interaction term "SWI-DWI mismatch score × successful recanalization" was significantly associated with favorable outcome (modified Rankin Scale score of 0-2 at 90 days; adjusted odds ratio [aOR], 2.162; 95% confidence interval [CI], 1.046-4.468). Stratified analysis showed that the likelihood of favorable outcome increased with the increase of SWI-DWI mismatch score in the successful recanalization group (OR, 2.140; 95% CI, 1.376-3.326), while there was no significant relationship between SWI-DWI mismatch score and clinical outcome in the unsuccessful recanalization group (OR, 1.212; 95% CI, 0.933-1.574). CONCLUSIONS: The effects of SWI-DWI mismatch and recanalization status on clinical outcome were realized through their interaction. In anterior circulation stroke due to large vessel occlusion, patients with both high SWI-DWI mismatch scores and successful recanalization were more likely to achieve a favorable outcome, while patients with unsuccessful recanalization, or with successful recanalization but low SWI-DWI mismatch scores, were less likely to have a good prognosis.

The emerging role of fibroblast-like synoviocytes-mediated synovitis in osteoarthritis: An update.

Osteoarthritis (OA), the most ubiquitous degenerative disease affecting the entire joint, is characterized by cartilage degradation and synovial inflammation. Although the pathogenesis of OA remains poorly understood, synovial inflammation is known to play an important role in OA development. However, studies on OA pathophysiology have focused more on cartilage degeneration and osteophytes, rather than on the inflamed and thickened synovium. Fibroblast-like synoviocytes (FLS) produce a series of pro-inflammatory regulators, such as inflammatory cytokines, nitric oxide (NO) and prostaglandin E2 (PGE2 ). These regulators are positively associated with the clinical symptoms of OA, such as inflammatory pain, joint swelling and disease development. A better understanding of the inflammatory immune response in OA-FLS could provide a novel approach to comprehensive treatment strategies for OA. Here, we have summarized recently published literatures referring to epigenetic modifications, activated signalling pathways and inflammation-associated factors that are involved in OA-FLS-mediated inflammation. In addition, the current related clinical trials and future perspectives were also summarized.

An Excitatory Neural Assembly Encodes Short-Term Memory in the Prefrontal Cortex.

Short-term memory (STM) is crucial for animals to hold information for a small period of time. Persistent or recurrent neural activity, together with neural oscillations, is known to encode the STM at the cellular level. However, the coding mechanisms at the microcircuitry level remain a mystery. Here, we performed two-photon imaging on behaving mice to monitor the activity of neuronal microcircuitry. We discovered a neuronal subpopulation in the medial prefrontal cortex (mPFC) that exhibited emergent properties in a context-dependent manner underlying a STM-like behavior paradigm. These neuronal subpopulations exclusively comprise excitatory neurons and mainly represent a group of neurons with stronger functional connections. Microcircuitry plasticity was maintained for minutes and was absent in an animal model of Alzheimer's disease (AD). Thus, these results point to a functional coding mechanism that relies on the emergent behavior of a functionally defined neuronal assembly to encode STM.

Impaired glutamatergic projection from the motor cortex to the subthalamic nucleus in 6-hydroxydopamine-lesioned hemi-parkinsonian rats.

The glutamatergic projection from the motor cortex to the subthalamic nucleus (STN) constitutes the cortico-basal ganglia circuit and plays a critical role in the control of movement. Emerging evidence shows that the cortico-STN pathway is susceptible to dopamine depletion. Specifically in Parkinson's disease (PD), abnormal electrophysiological activities were observed in the motor cortex and STN, while the STN serves as a key target of deep brain stimulation for PD therapy. However, direct morphological changes in the cortico-STN connectivity in response to PD progress are poorly understood at present. In the present study, we used a trans-synaptic anterograde tracing method with herpes simplex virus-green fluorescent protein (HSV-GFP) to monitor the cortico-STN connectivity in a rat model of PD. We found that the connectivity from the primary motor cortex (M1) to the STN was impaired in parkinsonian rats as manifested by a marked decrease in trans-synaptic infection of HSV-GFP from M1 neurons to STN neurons in unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats. Ultrastructural analysis with electron microscopy revealed that excitatory synapses in the STN were also impaired in parkinsonian rats. Glutamatergic terminals identified by a specific marker (vesicular glutamate transporter 1) were reduced in the STN, while glutamatergic neurons showed an insignificant change in their total number in both the M1 and STN regions. These results indicate that the M1-STN glutamatergic connectivity is downregulated in parkinsonian rats. This downregulation is mediated probably via a mechanism involving the impairments of excitatory terminals and synapses in the STN.

Anterograde monosynaptic transneuronal tracers derived from herpes simplex virus 1 strain H129.

BACKGROUND: Herpes simplex virus type 1 strain 129 (H129) has represented a promising anterograde neuronal circuit tracing tool, which complements the existing retrograde tracers. However, the current H129 derived tracers are multisynaptic, neither bright enough to label the details of neurons nor capable of determining direct projection targets as monosynaptic tracer. METHODS: Based on the bacterial artificial chromosome of H129, we have generated a serial of recombinant viruses for neuronal circuit tracing. Among them, H129-G4 was obtained by inserting binary tandemly connected GFP cassettes into the H129 genome, and H129-ΔTK-tdT was obtained by deleting the thymidine kinase (TK) gene and adding tdTomato coding gene to the H129 genome. Then the obtained viral tracers were tested in vitro and in vivo for the tracing capacity. RESULTS: H129-G4 is capable of transmitting through multiple synapses, labeling the neurons by green florescent protein, and visualizing the morphological details of the labeled neurons. H129-ΔTK-tdT neither replicates nor spreads in neurons alone, but transmits to and labels the postsynaptic neurons with tdTomato in the presence of complementary expressed TK from a helper virus. H129-ΔTK-tdT is also capable to map the direct projectome of the specific neuron type in the given brain regions in Cre transgenic mice. In the tested brain regions where circuits are well known, the H129-ΔTK-tdT tracing patterns are consistent with the previous results. CONCLUSIONS: With the assistance of the helper virus complimentarily expressing TK, H129-ΔTK-tdT replicates in the initially infected neuron, transmits anterogradely through one synapse, and labeled the postsynaptic neurons with tdTomato. The H129-ΔTK-tdT anterograde monosynaptic tracing system offers a useful tool for mapping the direct output in neuronal circuitry. H129-G4 is an anterograde multisynaptic tracer with a labeling signal strong enough to display the details of neuron morphology.