Suppression of excitatory synaptic transmission in the centrolateral amygdala via presynaptic histamine H3 heteroreceptors.

The central histaminergic system has a pivotal role in emotional regulation and psychiatric disorders, including anxiety, depression and schizophrenia. However, the effect of histamine on neuronal activity of the centrolateral amygdala (CeL), an essential node for fear and anxiety processing, remains unknown. Here, using immunostaining and whole-cell patch clamp recording combined with optogenetic manipulation of histaminergic terminals in CeL slices prepared from histidine decarboxylase (HDC)-Cre rats, we show that histamine selectively suppresses excitatory synaptic transmissions, including glutamatergic transmission from the basolateral amygdala, on both PKC-δ- and SOM-positive CeL neurons. The histamine-induced effect is mediated by H3 receptors expressed on VGLUT1-/VGLUT2-positive presynaptic terminals in CeL. Furthermore, optoactivation of histaminergic afferent terminals from the hypothalamic tuberomammillary nucleus (TMN) also significantly suppresses glutamatergic transmissions in CeL via H3 receptors. Histamine neither modulates inhibitory synaptic transmission by presynaptic H3 receptors nor directly excites CeL neurons by postsynaptic H1, H2 or H4 receptors. These results suggest that histaminergic afferent inputs and presynaptic H3 heteroreceptors may hold a critical position in balancing excitatory and inhibitory synaptic transmissions in CeL by selective modulation of glutamatergic drive, which may not only account for the pathophysiology of psychiatric disorders but also provide potential psychotherapeutic targets. KEY POINTS: Histamine selectively suppresses the excitatory, rather than inhibitory, synaptic transmissions on both PKC-δ- and SOM-positive neurons in the centrolateral amygdala (CeL). H3 receptors expressed on VGLUT1- or VGLUT2-positive afferent terminals mediate the suppression of histamine on glutamatergic synaptic transmission in CeL. Optogenetic activation of hypothalamic tuberomammillary nucleus (TMN)-CeL histaminergic projections inhibits glutamatergic transmission in CeL via H3 receptors.

Study of Mechanical Properties of Sand Layer Grouting Reinforcement under Seawater Erosion.

Grouting serves as an effective method for mitigating geotechnical disasters in subsea tunnels. However, current theories and designs, primarily based on terrestrial tunnel contexts, seldom address the long-term effects of seawater ion erosion on reinforcement. An improved sand permeation grouting simulation test system was employed to examine the mechanical property evolution of sand layer grouting reinforcement under seawater erosion utilizing various grout types. The mechanical properties of grouting reinforcement, under varying curing conditions, were analyzed using a uniaxial compression test, permeability test, and scanning electron microscope (SEM) test. Test results indicate that seawater curing conditions initially enhance the strength and impermeability of grouting reinforcement; however, prolonged curing diminishes these mechanical benefits. The onset of this process occurs significantly sooner in cement-sodium silicate grout (28-56 days) compared to cement grout (56d to 90d). For the cement grouting reinforcement, the deformation modulus increases over time, albeit at a decreasing rate. The deformation modulus of cement-sodium silicate grouting reinforcement follows an increase-decrease-increase pattern, correlating with the volume ratio over time. The decline in mechanical properties of grouting reinforcement during the test's mid to late stages under seawater conditions results from the interplay between erosive ions, which inhibit mechanical growth and accelerate deterioration.

Radical Prostatectomy Without Prior Biopsy in Selected Patients Evaluated by 18F-Labeled Prostate-Specific Membrane Antigen-Ligand Positron Emission Tomography/Computed Tomography and Multiparameter Magnetic Resonance Imaging: A Single-Center, Prospective, Single-Arm Trial.

PURPOSE: This study aimed to verify the feasibility and short-term prognosis of prostatectomy without biopsy. MATERIALS AND METHODS: Patients with a rising PSA level ranging from 4 to 30 ng/mL were scheduled for multiparametric (mp) MRI and 18F-labeled prostate-specific membrane antigen (PSMA) positron emission tomography (PET). Forty-seven patients (cT2N0M0) with Prostate Imaging Reporting and Data System ≥ 4 and molecular imaging PSMA score ≥ 2 were enrolled. All candidates underwent robot-assisted laparoscopic radical prostatectomy without biopsy. Prostate cancer detection rate, index tumors localization correspondence rate, positive surgical margin, complications, postoperative hospital stay, and PSA level in a 6-week postoperative follow-up visit were collected. RESULTS: All the patients with positive mpMRI and PSMA PET were diagnosed with clinically significant prostate cancer. A total of 80 lesions were verified as cancer by pathology, of which 63 cancer lesions were clinically significant prostate cancer. Fifty-one lesions were simultaneously found by mpMRI and PSMA PET. A total of 23 lesions were invisible on either image, and all lesions were ≤ International Society of Urological Pathology 2 or ≤ 15 mm. Forty-five (95.7%) index tumors found by mpMRI combined with PSMA PET were consistent with pathology. Nine patients reported positive surgical margin. CONCLUSIONS: Biopsy-free prostatectomy is safe and feasible for patients with evaluation strictly by mpMRI combined with 18F-PSMA PET/CT.

Time trends in surgical provision and cancer-specific outcomes in patients with stage T2-3 kidney cancer: a SEER-based study.

Background and objective: Surgery is the primary therapy that crucially affects the survival of patients with kidney cancer (KC). However, pertinent surgical decision criteria for individuals with stage T2-3 KC are lacking. This study aimed to display the practical choices and evolving trends of surgical procedures and elucidate their implied value. Methods: Through the Surveillance, Epidemiology, and End Results (SEER) dataset, the levels and evolving trends of different surgical methods were examined to determine cancer-specific risk of death (CSRD). Additionally, stratification analysis and survival rate analysis were performed to explore the effectiveness of partial nephrectomy (PN). Results: In this study, 9.27% of patients opted for PN. Interestingly, an upward trend was observed in its decision, with an average annual percentage change (AAPC) of 7.0 (95% CI: 4.8-9.3, P < 0.05). Patients who underwent PN and were in a relatively less severe condition exhibited more favorable CSRD levels (0.17-0.36 vs. 0.50-0.67) and an improvement trend compared with those who underwent radical nephrectomy (RN) (AAPC: -1.9 vs. -0.8). Further analysis showed that the levels of CSRD and survival rates for patients opting for different surgical methods followed a similar pattern. Conclusions: This study showed that RN was still the most common surgical method. Patients with stage T2-3 KC had an increasing preference for PN and exhibited more favorable cancer-related survival outcomes, which underscores the need for further investigation and validation.

Greenhouse gas emissions and their driving factors among different flowering Chinese cabbage (Brassica campestris L.) varieties.

Crop cultivars have an influence on greenhouse gas (GHG) emissions, and there is variation between varieties. However, there are few reports available on the differences in GHG emissions and their driving factors among vegetable varieties. In this study, we conducted a field experiment to examine the variances in GHG emissions and their contributing factors among eight flowering Chinese cabbage varieties (considering growth period, leaf shape, and colour). The results showed significant differences in GHG emissions within varieties; early-maturing varieties exhibited GHG by 25.6% and 15.3%, respectively, when compared to mid- and late-maturing varieties. Among the different leaf types and color classifications, light-colored and sharp-leafed varieties had the lower global warming potential (GWP) overall. Cumulative CO2 emissions were influenced by leaf SPAD values and biomass, while cumulative N2O emissions were driven mainly by stem thickness, carbon accumulation, leaf SPAD values, and biomass. In summary, the selection of light-colored varieties with pointed leaves and shorter growth periods in actual production contributed positively to the reduction of carbon emissions from flowering Chinese cabbage production. Through efficient variety screening, this study provides a win-win strategy for achieving efficient vegetable production while also addressing the global climate challenge.

Suitability of the MP1000 Platform for Robot-assisted Prostatectomy: A Prospective Randomised Controlled Trial.

Background and objective: Robot-assisted radical prostatectomy (RARP) is widely used because of the many advantages of a robotic approach. The da Vinci Si robot is one of the most commonly used surgical robot systems, but it may be associated with higher costs owing to the use of consumable surgical supplies. Our aim was to conduct a preliminary investigation of the capability of the MP1000 system for RARP. Methods: In this prospective, multicentre, single-blinded study, we randomly assigned 42 patients scheduled to undergo RARP between April and September 2021 to a da Vinci Si group (control) or an MP1000 group (intervention). Patients underwent RARP performed using the assigned robotic system and were followed up at 3-mo intervals. The primary outcome was the rate of conversion to open/laparoscopic surgery. Secondary outcomes were installation and operation times, intraoperative blood loss, postoperative surgical margin status, hospital stay, incontinence, complications, safety indicators, and surgeon ergonomics. Key findings and limitations: All procedures were successfully completed without conversion to open/laparascopic surgery or major complications. Secondary outcomes, including oncological and ergonomic indicators, did not differ significantly between the groups over the study period. One patient in the control group experienced dysuria (Clavien-Dindo grade 3). No patients had incontinence at 3 mo. A limitation of the study is the small sample size. Conclusions and clinical implications: RARP with the MP1000 system is feasible, safe, and effective in the management of localised prostate cancer. Patient summary: We assessed the effectiveness and safety of the new MP1000 robot system for robot-assisted removal of the prostate in comparison to the da Vinci Si robot. We found no difference in effectiveness or safety among 42 patients with prostate cancer who were assigned randomly to one of the two systems. We conclude that the MP1000 is a suitable robot for this surgery.

Targeting STING elicits GSDMD-dependent pyroptosis and boosts anti-tumor immunity in renal cell carcinoma.

While Stimulator-of-interferon genes (STING) is an innate immune adapter cruicial for sensing cytosolic DNA and modulating immune microenvironment, its tumor-promoting role in tumor survival and immune evasion remains largely unknown. Here we reported that renal cancer cells are exceptionally dependent on STING for survival and evading immunosurveillance via suppressing ER stress-mediated pyroptosis. We found that STING is significantly amplified and upregulated in clear cell renal cell carcinoma (ccRCC), and its elevated expression is associated with worse clinical outcomes. Mechanically, STING depletion in RCC cells specifically triggers activation of the PERK/eIF2α/ATF4/CHOP pathway and activates cleavage of Caspase-8, thereby inducing GSDMD-mediated pyroptosis, which is independent of the innate immune pathway of STING. Moreover, animal study revealed that STING depletion promoted infiltration of CD4+ and CD8+ T cells, consequently boosting robust antitumor immunity via pyroptosis-induced inflammation. From the perspective of targeted therapy, we found that Compound SP23, a PROTAC STING degrader, demonstrated comparable efficacy to STING depletion both in vitro and in vivo for treatment of ccRCC. These findings collectively unveiled an unforeseen function of STING in regulating GSDMD-dependent pyroptosis, thus regulating immune response in RCC. Consequently, pharmacological degradation of STING by SP23 may become an attractive strategy for treatment of advanced RCC.

Suitability of the MP1000 system for robot-assisted partial nephrectomy: a multicenter randomized controlled noninferiority trial.

PURPOSE: This study aimed to compare the safety and effectiveness of the MP1000 surgical system with the da Vinci Si robot system in robot-assisted partial nephrectomy (RAPN) through a prospective, single-blinded, randomized controlled trial. MATERIALS AND METHODS: A total of 62 patients who were scheduled to undergo RAPN were randomly assigned to either the da Vinci Si robot or MP1000 group. A noninferiority test was conducted with a noninferior intermediate value of 10%. The study compared installation and operation times, estimated blood loss, warm ischemia time, postoperative surgical margin, rate of conversion to open surgery, eGFR level, complications, and other safety indicators between the two groups. RESULTS: All procedures were successfully completed without the need for conversion to open or laparoscopic surgery, and no major complications were observed during the process. The test of noninferiority was achieved. There were no significant differences in median installation time, operation time, complication rate at 3 months, rate of positive surgical margin, and eGFR level at 3 months between the groups. Additionally, no evidence of recurrence was found on imaging in both groups. No difference in National Aeronautics and Space Administration task load index results for ergonomic considerations. A limitation of this study was its small sample size. CONCLUSIONS: The MP1000 system is a suitable platform for RAPN with safety and effectiveness compared with da Vinci Si system.

A role for the cerebellum in motor-triggered alleviation of anxiety.

Physical exercise is known to reduce anxiety, but the underlying brain mechanisms remain unclear. Here, we explore a hypothalamo-cerebello-amygdalar circuit that may mediate motor-dependent alleviation of anxiety. This three-neuron loop, in which the cerebellar dentate nucleus takes center stage, bridges the motor system with the emotional system. Subjecting animals to a constant rotarod engages glutamatergic cerebellar dentate neurons that drive PKCδ+ amygdalar neurons to elicit an anxiolytic effect. Moreover, challenging animals on an accelerated rather than a constant rotarod engages hypothalamic neurons that provide a superimposed anxiolytic effect via an orexinergic projection to the dentate neurons that activate the amygdala. Our findings reveal a cerebello-limbic pathway that may contribute to motor-triggered alleviation of anxiety and that may be optimally exploited during challenging physical exercise.

Distinguishing host responses, extensive viral dissemination and long-term viral RNA persistence in domestic sheep experimentally infected with Crimean-Congo haemorrhagic fever virus Kosovo Hoti.

Crimean-Congo haemorrhagic fever orthonairovirus (CCHFV) is a tick-borne, risk group 4 pathogen that often causes a severe haemorrhagic disease in humans (CCHF) with high case fatality rates. The virus is believed to be maintained in a tick-vertebrate-tick ecological cycle involving numerous wild and domestic animal species; however the biology of CCHFV infection in these animals remains poorly understood. Here, we experimentally infect domestic sheep with CCHFV Kosovo Hoti, a clinical isolate representing high pathogenicity to humans and increasingly utilized in current research. In the absence of prominent clinical signs, the infection leads to an acute viremia and coinciding viral shedding, fever and markers for potential impairment in liver and kidney functions. A number of host responses distinguish the subclinical infection in sheep versus fatal infection in humans. These include an early reduction of neutrophil recruitment and its chemoattractant, IL-8, in the blood stream of infected sheep, whereas neutrophil infiltration and elevated IL-8 are features of fatal CCHFV infections reported in immunodeficient mice and humans. Several inflammatory cytokines that correlate with poor disease outcomes in humans and have potential to cause vascular dysfunction, a primary hallmark of severe CCHF, are down-regulated or restricted from increasing in sheep. Of particular interest, the detection of CCHFV RNA (including full-length genome) in a variety of sheep tissues long after the acute phase of infection indicates a widespread viral dissemination in the host and suggests a potentially long-term persisting impact of CCHFV infection. These findings reveal previously unrecognized aspects of CCHFV biology in animals.

Biochar's dual role in greenhouse gas emissions: Nitrogen fertilization dependency and mitigation potential.

Biochar was popularly used for reducing greenhouse gas (GHG) emissions in vegetable production, but using biochar does not necessarily guarantee a reduction in GHG emissions. Herein, it's meaningful to elucidate the intricate interplay among biochar properties, soil characteristics, and GHG emissions in vegetable production to provide valuable insights for informed and effective mitigation strategies. Therefore, in current research, a meta-analysis of 43 publications was employed to address these issues. The boost-regression analysis results indicated that the performance of biochar in inhibiting N2O emissions was most affected by the N application rate both in high and low N application conditions. Besides, biochar had dual roles and showed well performance in reducing GHG emissions under low N input (≤300 kg N ha-1), while having the opposite effect during high N input (>300 kg N ha-1). Specifically, applying biochar under low N fertilization input could obviously reduce soil N2O emissions, CO2 emissions, and CH4 emissions by 18.7 %, 17.9 %, and 16.9 %, respectively. However, the biochar application under high N fertilization input significantly (P < 0.05) increased soil N2O emissions, CO2 emissions, and CH4 emissions by 39.7 %, 43.0 %, and 27.7 %, respectively. Except for the N application rate, the soil pH, SOC, biochar C/N ratio, biochar pH, and biochar pyrolysis temperature are also the key factors affecting the control of GHG emissions in biochar-amended soils. The findings of this study will contribute to deeper insights into the potential application of biochar in regulating GHG under consideration of N input, offering scientific evidence and guidance for sustainable agriculture management.

Fast and sensitive differential diagnosis of pseudorabies virus-infected versus pseudorabies virus-vaccinated swine using CRISPR-Cas12a.

IMPORTANCE: Pseudorabies virus (PRV) causes high mortality and miscarriage rates in the infected swine, and the eradication policy coupled with large-scale vaccination of live attenuated vaccines has been adopted globally against PRV. Differential diagnosis of the vaccinated and infected swine is highly demanded. Our multienzyme isothermal rapid amplification (MIRA)-Cas12a detection method described in this study can diagnose PRV with a superior sensitivity comparable to the quantitative PCR (qPCR) and a competitive detection speed (only half the time as qPCR needs). The portable feature and the simple procedure of MIRA-Cas12a make it easier to deploy for clinical diagnosis, even in resource-limited settings. The MIRA-Cas12a method would provide immediate and accurate diagnostic information for policymakers to respond promptly.

Advances in HER2-Targeted Treatment for Advanced/Metastatic Urothelial Carcinoma.

Urothelial carcinoma (UC) represents a common malignancy of the urinary system that can involve the kidneys, ureter, bladder, and urethra. Advanced/metastatic UC (mUC) tends to have a poor prognosis. UC ranks third in terms of human epidermal growth factor receptor 2 (HER2) overexpression among all tumors. However, multiple studies found that, unlike breast cancer, variable degrees of HER2 positivity and poor consistency between HER2 protein overexpression and gene amplification have been found. Trials involving trastuzumab, pertuzumab, lapatinib, afatinib, and neratinib have failed to prove their beneficial effect in patients with HER2-positive mUC, and a clinical trial on T-DM1 (trastuzumab emtansine) was terminated prematurely because of the adverse reactions. However, a phase II trial showed that RC48-ADC was effective. In this review, we provided an in-depth overview of the advances in the research regarding HER2-targeted therapy and the role of HER2 in mUC. Furthermore, we also discussed the prospects of potential strategies aimed at overcoming anti-HER2 resistance, and summarize the novel anti-HER2 approaches for the management of mUC used in recent clinical trials.

Henipavirus zoonosis: outbreaks, animal hosts and potential new emergence.

Hendra virus (HeV) and Nipah virus (NiV) are biosafety level 4 zoonotic pathogens causing severe and often fatal neurological and respiratory disease. These agents have been recognized by the World Health Organization as top priority pathogens expected to result in severe future outbreaks. HeV has caused sporadic infections in horses and a small number of human cases in Australia since 1994. The NiV Malaysia genotype (NiV-M) was responsible for the 1998-1999 epizootic outbreak in pigs with spillover to humans in Malaysia and Singapore. Since 2001, the NiV Bangladesh genotype (NiV-B) has been the predominant strain leading to outbreaks almost every year in Bangladesh and India, with hundreds of infections in humans. The natural reservoir hosts of HeV and NiV are fruit bats, which carry the viruses without clinical manifestation. The transmission pathways of henipaviruses from bats to humans remain poorly understood. Transmissions are often bridged by an intermediate animal host, which amplifies and spreads the viruses to humans. Horses and pigs are known intermediate hosts for the HeV outbreaks in Australia and NiV-M epidemic in Malaysia and Singapore, respectively. During the NiV-B outbreaks in Bangladesh, following initial spillover thought to be through the consumption of date palm sap, the spread of infection was largely human-to-human transmission. Spillover of NiV-B in recent outbreaks in India is less understood, with the primary route of transmission from bat reservoir to the initial human infection case(s) unknown and no intermediate host established. This review aims to provide a concise update on the epidemiology of henipaviruses covering their previous and current outbreaks with emphasis on the known and potential role of livestock as intermediate hosts in disease transmission. Also included is an up-to-date summary of newly emerging henipa-like viruses and animal hosts. In these contexts we discuss knowledge gaps and new challenges in the field and propose potential future directions.

Venous tumor thrombus consistency: is it a prognostic factor of survival for patients with renal cell carcinoma?

OBJECTIVES: To evaluate whether venous tumor thrombus (VTT) consistency is a risk factor for the patient's prognosis with renal cell carcinoma (RCC). MATERIALS AND METHODS: A total of 190 RCC patients with VTT, who were treated at Department of Urology, Chinese PLA General Hospital, were retrospectively analyzed in this study. The baseline clinical characteristics, postoperative outcomes, and pathological findings were analyzed. Tumor thrombus was classified as solid and friable based on their respective characteristics. Survival curves were estimated using the Kaplan-Meier survival curve analysis, and univariable and multivariable cox proportional hazard regression models were used. RESULTS: Among the total 190 patients included in this study, 145 (76.3%) patients had solid VTT, and 45 (23.7%) patients had friable VTT in their renal veins and inferior vena cava (IVC). There were no significant differences in the age, gender, BMI, symptoms, complex diseases, tumor side, tumor size, TNM stage, Mayo stage, tumor grade, sarcomatous differentiation, pelvic invasion, and sinus fat invasion of patients. Solid VTT consistency was more likely to have a capsule as compared to those with friable VTT (P = 0.007). Kaplan-Meier survival curve analysis demonstrated no statistically significant differences in the overall survival (OS) (P = 0.973) and progression-free survival (PFS) (P = 0.667) of patients. Moreover, VTT consistency was not associated with OS (P = 0.706) of PFS (P = 0.504) in multivariate cox regression analysis. CONCLUSIONS: RCC VTT consistency was not a prognostic risk factor for predicting the OS and PFS of patients.

Safe Production Strategies for Soil-Covered Cultivation of Morel in Heavy Metal-Contaminated Soils.

Morel is a popular edible mushroom with considerable medicinal and economic value which has garnered global popularity. However, the increasing heavy metal (HM) pollution in the soil presents a significant challenge to morels cultivation. Given the susceptibility of morels to HM accumulation, the quality and output of morels are at risk, posing a serious food safety concern that hinders the development of the morel industry. Nonetheless, research on the mechanism of HM enrichment and mitigation strategies in morel remains scarce. The morel, being cultivated in soil, shows a positive correlation between HM content in its fruiting body and the HM content in the soil. Therefore, soil remediation emerges as the most practical and effective approach to tackle HM pollution. Compared to physical and chemical remediation, bioremediation is a low-cost and eco-friendly approach that poses minimal threats to soil composition and structure. HMs easily enriched during morels cultivation were examined, including Cd, Cu, Hg, and Pb, and we assessed soil passivation technology, microbial remediation, strain screening and cultivation, and agronomic measures as potential approaches for HM pollution prevention. The current review underscores the importance of establishing a comprehensive system for preventing HM pollution in morels.

Strategies to Improve Multi-enzyme Compatibility and Coordination in One-Pot SHERLOCK.

While molecular diagnostics generally require heating elements that supply high temperatures such as 95 °C in polymerase chain reaction and 60-69 °C in loop-mediated isothermal amplification, the recently developed CRISPR-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can operate at 37 °C or a similar ambient temperature. This unique advantage may be translated into highly energy-efficient or equipment-free molecular diagnostic systems with unrestricted deployability. SHERLOCK is characterized by ultra-high sensitivity when performed in a traditional two-step format. For RNA sensing, the first step combines reverse transcription with recombinase polymerase amplification, while the second step consists of T7 transcription and CRISPR-Cas13a detection. The sensitivity drops dramatically, however, when all these components are combined into a single reaction mixture, and it largely remains an unmet need in the field to establish a high-performance one-pot SHERLOCK assay. An underlying challenge, conceivably, is the extremely complex nature of a one-pot formulation, crowding a large number of reaction types using at least eight enzymes/proteins. Although previous work has made substantial improvements by serving individual enzymes/reactions with accommodating conditions, we reason that the interactions among different enzymatic reactions could be another layer of complicating factors. In this study, we seek optimization strategies by which inter-enzymatic interference may be eliminated or reduced and cooperation created or enhanced. Several such strategies are identified for SARS-CoV-2 detection, each leading to a significantly improved reaction profile with faster and stronger signal amplification. Designed based on common molecular biology principles, these strategies are expected to be customizable and generalizable with various buffer conditions or pathogen types, thus holding broad applicability for integration into future development of one-pot diagnostics in the form of a highly coordinated multi-enzyme reaction system.

Comparative characterization of Crimean-Congo hemorrhagic fever virus cell culture systems with application to propagation and titration methods.

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is a biosafety level 4 and World Health Organization top priority pathogen. Infection leads to an often fatal hemorrhagic fever disease in humans. The tick-borne virus is endemic in countries across Asia, Europe and Africa, with signs of spreading into new regions. Despite the severity of disease and the potential of CCHFV geographic expansion to cause widespread outbreaks, no approved vaccine or treatment is currently available. Critical for basic research and the development of diagnostics or medical countermeasures, CCHFV viral stocks are commonly produced in Vero E6 and SW-13 cell lines. While a variety of in-house methods are being used across different laboratories, there has been no clear, specific consensus on a standard, optimal system for CCHFV growth and titration. In this study, we perform a systematic, side-by-side characterization of Vero E6 and SW-13 cell lines concerning the replication kinetics of CCHFV under different culture conditions. SW-13 cells are typically cultured in a CO2-free condition (SW-13 CO2-) according to the American Type Culture Collection. However, we identify a CO2-compatible culture condition (SW-13 CO2+) that demonstrates the highest viral load (RNA concentration) and titer (infectious virus concentration) in the culture supernatants, in comparison to SW-13 CO2- and Vero E6 cultures. This optimal viral propagation system also leads to the development of two titration methods: an immunostaining-based plaque assay using a commercial CCHFV antibody and a colorimetric readout, and an antibody staining-free, cytopathic effect-based median tissue culture infectious dose assay using a simple excel calculator. These are anticipated to serve as a basis for a reproducible, standardized and user-friendly platform for CCHFV propagation and titration.

Proinflammatory activation of microglia in the cerebellum hyperexcites Purkinje cells to trigger ataxia.

Specific medications to combat cerebellar ataxias, a group of debilitating movement disorders characterized by difficulty with walking, balance and coordination, are still lacking. Notably, cerebellar microglial activation appears to be a common feature in different types of ataxic patients and rodent models. However, direct evidence that cerebellar microglial activation in vivo is sufficient to induce ataxia is still lacking. Here, by employing chemogenetic approaches to manipulate cerebellar microglia selectively and directly, we found that specific chemogenetic activation of microglia in the cerebellar vermis directly leads to ataxia symptoms in wild-type mice and aggravated ataxic motor deficits in 3-acetylpyridine (3-AP) mice, a classic mouse model of cerebellar ataxia. Mechanistically, cerebellar microglial proinflammatory activation induced by either chemogenetic M3D(Gq) stimulation or 3-AP modeling hyperexcites Purkinje cells (PCs), which consequently triggers ataxia. Blockade of microglia-derived TNF-α, one of the most important proinflammatory cytokines, attenuates the hyperactivity of PCs driven by microglia. Moreover, chemogenetic inhibition of cerebellar microglial activation or suppression of cerebellar microglial activation by PLX3397 and minocycline reduces the production of proinflammatory cytokines, including TNF-α, to effectively restore the overactivation of PCs and alleviate motor deficits in 3-AP mice. These results suggest that cerebellar microglial activation may aggravate the neuroinflammatory response and subsequently induce dysfunction of PCs, which in turn triggers ataxic motor deficits. Our findings thus reveal a causal relationship between proinflammatory activation of cerebellar microglia and ataxic motor symptoms, which may offer novel evidence for therapeutic intervention for cerebellar ataxias by targeting microglia and microglia-derived inflammatory mediators.