Dopamine Neuron-Specific Optogenetic Stimulation in Rhesus Macaques.

Optogenetic studies in mice have revealed new relationships between well-defined neurons and brain functions. However, there are currently no means to achieve the same cell-type specificity in monkeys, which possess an expanded behavioral repertoire and closer anatomical homology to humans. Here, we present a resource for cell-type-specific channelrhodopsin expression in Rhesus monkeys and apply this technique to modulate dopamine activity and monkey choice behavior. These data show that two viral vectors label dopamine neurons with greater than 95% specificity. Infected neurons were activated by light pulses, indicating functional expression. The addition of optical stimulation to reward outcomes promoted the learning of reward-predicting stimuli at the neuronal and behavioral level. Together, these results demonstrate the feasibility of effective and selective stimulation of dopamine neurons in non-human primates and a resource that could be applied to other cell types in the monkey brain.

RVFScan predicts virulence factor genes and hypervirulence of the clinical metagenome.

Bacterial infections often involve virulence factors that play a crucial role in the pathogenicity of bacteria. Accurate detection of virulence factor genes (VFGs) is essential for precise treatment and prognostic management of hypervirulent bacterial infections. However, there is a lack of rapid and accurate methods for VFG identification from the metagenomic data of clinical samples. Here, we developed a Reads-based Virulence Factors Scanner (RVFScan), an innovative user-friendly online tool that integrates a comprehensive VFG database with similarity matrix-based criteria for VFG prediction and annotation using metagenomic data without the need for assembly. RVFScan demonstrated superior performance compared to previous assembly-based and read-based VFG predictors, achieving a sensitivity of 97%, specificity of 98% and accuracy of 98%. We also conducted a large-scale analysis of 2425 clinical metagenomic datasets to investigate the utility of RVFScan, the species-specific VFG profiles and associations between VFGs and virulence phenotypes for 24 important pathogens were analyzed. By combining genomic comparisons and network analysis, we identified 53 VFGs with significantly higher abundances in hypervirulent Klebsiella pneumoniae (hvKp) than in classical K. pneumoniae. Furthermore, a cohort of 1256 samples suspected of K. pneumoniae infection demonstrated that RVFScan could identify hvKp with a sensitivity of 90%, specificity of 100% and accuracy of 98.73%, with 90% of hvKp samples consistent with clinical diagnosis (Cohen's kappa, 0.94). RVFScan has the potential to detect VFGs in low-biomass and high-complexity clinical samples using metagenomic reads without assembly. This capability facilitates the rapid identification and targeted treatment of hvKp infections and holds promise for application to other hypervirulent pathogens.

Oxytocin-dependent reopening of a social reward learning critical period with MDMA.

A critical period is a developmental epoch during which the nervous system is expressly sensitive to specific environmental stimuli that are required for proper circuit organization and learning. Mechanistic characterization of critical periods has revealed an important role for exuberant brain plasticity during early development, and for constraints that are imposed on these mechanisms as the brain matures1. In disease states, closure of critical periods limits the ability of the brain to adapt even when optimal conditions are restored. Thus, identification of manipulations that reopen critical periods has been a priority for translational neuroscience2. Here we provide evidence that developmental regulation of oxytocin-mediated synaptic plasticity (long-term depression) in the nucleus accumbens establishes a critical period for social reward learning. Furthermore, we show that a single dose of (+/-)-3,4-methylendioxymethamphetamine (MDMA) reopens the critical period for social reward learning and leads to a metaplastic upregulation of oxytocin-dependent long-term depression. MDMA-induced reopening of this critical period requires activation of oxytocin receptors in the nucleus accumbens, and is recapitulated by stimulation of oxytocin terminals in the nucleus accumbens. These findings have important implications for understanding the pathogenesis of neurodevelopmental diseases that are characterized by social impairments and of disorders that respond to social influence or are the result of social injury3.

AgSbF6-catalyzed C3 aza-Friedel-Crafts alkylation of N,O-acetals with indoles for the synthesis of N-α indole substituted pyrrolidine and piperidine derivatives.

An efficient approach to access chiral N-α indole substituted pyrrolidine and piperidine skeletons has been developed through a AgSbF6-catalyzed N-α aza-Friedel-Crafts alkylation of N,O-acetals 6a, 6b, 9, and 11a-11d with indoles. As a result, a series of 2,3-trans N-α indole substituted pyrrolidines 8a-8x and piperidines 10a-10j were prepared in moderate to excellent yields and with excellent diastereoselectivities (dr up to 99 : 1). Moreover, several 2,5-cis-N-α indole substituted pyrrolidine derivatives 12a-12k were synthesized according to this strategy with moderate to good yields and diastereoselectivities (dr up to 99 : 1).

How Public Health Agencies Break through COVID-19 Conversations: A Strategic Network Approach to Public Engagement.

In times of public health emergencies, health agencies need to engage and communicate with the public in real-time to share updates and accurate information. This is especially the case for the COVID-19 pandemic where public engagement can potentially save lives and flatten the curve. This paper considers how the use of interactive features and strategic network positions of health agencies on social media influenced their public engagement outcomes. Specifically, we analyzed 203 U.S. public health agencies' Twitter activity and the public engagement they received by extracting data from a large-scale Twitter dataset collected from January 21st to May 31st, 2020. Results show that health agencies' network position in addition to their two-way communication strategy greatly influenced the level of public engagement with their COVID-19 related content on Twitter. Findings highlight the benefits of strategic social media communication of public health agencies resides not only in how agencies use social media but also in their formation of network position to amplify their visibility. As official sources of health and risk information, public health agencies should coordinate their social media communication efforts to strategically position themselves in advantageous network positions to augment public engagement outcomes.

Tabersonine Inhibits the Lipopolysaccharide-Induced Neuroinflammatory Response in BV2 Microglia Cells via the NF-κB Signaling Pathway.

The occurrence and development of neurodegenerative diseases is related to a variety of physiological and pathological changes. Neuroinflammation is one of the major factors that induces and aggravates neurodegenerative diseases. The most important manifestation of neuroinflammation is the activation of microglia. Therefore, inhibiting the abnormal activation of microglia is an important way to alleviate the occurrence of neuroinflammatory diseases. In this research, the inhibitory effect of tabersonine (Tab) on neuroinflammation was evaluated by establishing the BV2 neuroinflammation model induced by lipopolysaccharide (LPS). It was found that Tab significantly inhibited the production and expression of nitric oxide (NO), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and reactive oxygen species (ROS) in BV-2 cells stimulated by LPS. In addition, Tab can also inhibit the activation of nuclear factor-κB (NF-κB) induced by LPS, thus regulating inflammatory mediators such as inducible nitric oxide synthase (iNOS). These results indicated that Tab regulated the release of inflammatory mediators such as NO, IL-1ß, TNF-α, and IL-6 by inhibiting NF-κB signaling pathway, and exerting its anti-neuroinflammatory effect. This is the first report regarding the inhibition on LPS-induced neuroinflammation in BV2 microglia cells of Tab, which indicated the drug development potential of Tab for the treatment of neurodegenerative diseases.

U.S. Fortune 500's stakeholders engagement during the COVID-19 pandemic: Evidence for proactive approaches.

In times of a national crisis such as COVID-19, it is important for organizations to show that they are good corporate citizens. At the same time, organizations should carefully select the type of messages that resonate with stakeholders so as to reduce stakeholder skepticism. This study examines how U.S. Fortune 500 companies discussed their COVID-19 pandemic CSR actions on Facebook over 15 months and how the public responded to such messages. We identified three CSR themes: internal stakeholder proactive CSR, external stakeholder proactive CSR, and external stakeholder accommodative CSR. When publics engaged, external stakeholder proactive CSR was significantly associated with better behavioral engagement outcomes, more positive emotional engagement outcomes, and less negative emotions. However, such effects are moderated by industry type. Our findings inform public relations theory and practice and suggest that in times of major crises, organizations should prioritize proactive approaches to engage external stakeholders while being mindful of specific institutional contexts.

Zn(OTf)2-catalyzed hydroamination of ynamides with aromatic amines.

The Zn(OTf)2-catalyzed hydroamination of ynamides 2a-2l with aromatic amines 1a-1r was developed. This protocol features broad substrate scope of aromatic amines, good functional group tolerance for ynamides, and excellent regioselectivities. As a result, a variety of substituted amidine compounds 3aa-3oa, 3ab-3al and 3pa-3rk were prepared in moderate to excellent yields and with high regioselectivities.

Multidimensional screening yields channelrhodopsin variants having improved photocurrent and order-of-magnitude reductions in calcium and proton currents.

Channelrhodopsins (ChRs) are light-gated ion channels in widespread use in neuroscience for mediating the genetically targetable optical control of neurons (optogenetics). ChRs pass multiple kinds of ions, and although nonspecific ChR-mediated conductance is not an issue in many neuroscience studies, conductance of calcium and protons, which can mediate diverse cellular signals, may be undesirable in some instances. Here, we turned our attention to the creation of ChRs that have high cation photocurrent but pass fewer calcium ions and protons. We developed an automated, time-resolved screening method capable of rapidly phenotyping channelrhodopsin-2 (ChR2) variants. We found substitution mutations throughout ChR2 that could boost current while altering ion selectivity and observed that the mutations that reduced calcium or proton conductance have additive effects. By combining four mutations, we obtained a ChR, ChromeQ, with improved photocurrent that possesses order-of-magnitude reductions in calcium and proton conductance and high fidelity in driving repetitive action potentials in neurons. The approach presented here offers a viable pathway toward customization of complex physiological properties of optogenetic tools. We propose that our screening method not only enables elucidation of new ChR variants that affect microbial opsin performance but may also reveal new principles of optogenetic protein engineering.

Melanopsin for precise optogenetic activation of astrocyte-neuron networks.

Optogenetics has been widely expanded to enhance or suppress neuronal activity and it has been recently applied to glial cells. Here, we have used a new approach based on selective expression of melanopsin, a G-protein-coupled photopigment, in astrocytes to trigger Ca2+ signaling. Using the genetically encoded Ca2+ indicator GCaMP6f and two-photon imaging, we show that melanopsin is both competent to stimulate robust IP3-dependent Ca2+ signals in astrocyte fine processes, and to evoke an ATP/Adenosine-dependent transient boost of hippocampal excitatory synaptic transmission. Additionally, under low-frequency light stimulation conditions, melanopsin-transfected astrocytes can trigger long-term synaptic changes. In vivo, melanopsin-astrocyte activation enhances episodic-like memory, suggesting melanopsin as an optical tool that could recapitulate the wide range of regulatory actions of astrocytes on neuronal networks in behaving animals. These results describe a novel approach using melanopsin as a precise trigger for astrocytes that mimics their endogenous G-protein signaling pathways, and present melanopsin as a valuable optical tool for neuron-glia studies.

Enhancement of Ferroelectricity for Orthorhombic (Tb0.861Mn0.121)MnO3-δ by Copper Doping.

Copper-doped (Tb0.861Mn0.121)MnO3-δ has been synthesized by the conventional solid state reaction method. X-ray, neutron, and electron diffraction data indicate that they crystallize in Pnma space group at room temperature. Two magnetic orderings are found for this series by neutron diffraction. One is the ICAM (incommensurate canted antiferromagnetic) ordering of Mn with a wave vector qMn = (∼0.283, 0, 0) with a ≈ 5.73 Å, b ≈ 5.31 Å, and c ≈ 7.41 Å, and the other is the CAM (canted antiferromagnetic) ordering of both Tb and Mn in the magnetic space group Pn'a21' with a ≈ 5.73 Å, b ≈ 5.31 Å, and c ≈ 7.41 Å. A dielectric peak around 40 K is found for the samples doped with Cu, which is higher than that for orthorhombic TbMnO3.

The Immunotherapeutic Role of Bacterial Lysates in a Mouse Model of Asthma.

INTRODUCTION: Asthma is the most common chronic lower respiratory disease in childhood throughout the world. Recurrent respiratory tract infections in young children, especially viral infections, are the major cause of acute asthmatic exacerbations and contribute to development of asthma. Bacterial extracts have been used to improve the immune defenses of the respiratory tract. However, seldom studies have examined the effect of bacterial lysates on childhood asthma. In this study, we examined whether bacterial lysates (OM-85) will improve symptoms of asthmatic mice via modulation of the immune response. METHODS: Asthmatic mice models were established with OVA challenge and treated with oral administration of Broncho-Vaxom (OM-85). Next, infiltrations of inflammatory cells including eosinophil and neutrophils were examined. Pulmonary tissues in asthmatic mice models were analyzed by hematoxylin and eosin (HE) staining. The levels of Th1/Th2-typed cytokines in bronchoalveolar lavage fluid (BALF) of asthmatic mice models were examined by enzyme-linked immunosorbent assay. RESULTS: Compared to control group, we found significant reduction of airway wall thickness, luminal stenosis, and mucus plug formation in asthmatic mice models after oral administration of OM-85. The infiltrations of eosinophil were also significantly decreased in BALF in asthmatic mice models. Oral administration of OM-85 was shown to suppress Th2-type cytokine levels. CONCLUSION: Our findings provide evidence that oral administration of OM-85 is capable of attenuating airway inflammation in asthmatic mice models. Oral administration of OM-85 may have a positive impact in terms of asthma severity.

Another Look at Pyrroloiminoquinone Alkaloids-Perspectives on Their Therapeutic Potential from Known Structures and Semisynthetic Analogues.

This study began with the goal of identifying constituents from Zyzzya fuliginosa extracts that showed selectivity in our primary cytotoxicity screen against the PANC-1 tumor cell line. During the course of this project, which focused on six Z. fuliginosa samples collected from various regions of the Indo-Pacific, known compounds were obtained consisting of nine makaluvamine and three damirone analogues. Four new acetylated derivatives were also prepared. High-accuracy electrospray ionization mass spectrometry (HAESI-MS) m/z ions produced through MS² runs were obtained and interpreted to provide a rapid way for dereplicating isomers containing a pyrrolo[4,3,2-de]quinoline core. In vitro human pancreas/duct epithelioid carcinoma (PANC-1) cell line IC50 data was obtained for 16 compounds and two therapeutic standards. These results along with data gleaned from the literature provided useful structure activity relationship conclusions. Three structural motifs proved to be important in maximizing potency against PANC-1: (i) conjugation within the core of the ABC-ring; (ii) the presence of a positive charge in the C-ring; and (iii) inclusion of a 4-ethyl phenol or 4-ethyl phenol acetate substituent off the B-ring. Two compounds, makaluvamine J (9) and 15-O-acetyl makaluvamine J (15), contained all three of these frameworks and exhibited the best potency with IC50 values of 54 nM and 81 nM, respectively. These two most potent analogs were then tested against the OVCAR-5 cell line and the presence of the acetyl group increased the potency 14-fold from that of 9 whose IC50 = 120 nM vs. that of 15 having IC50 = 8.6 nM.

[ORMDL3 polymorphisms and their relationship with OPN and TGF-ß1 levels in children with asthma in Hunan, China: an analysis of 98 cases].

OBJECTIVE: To investigate ORMDL3 polymorphisms in children with asthma in Hunan, China, and to determine the relationship between ORMDL3 polymorphisms and serum osteopontin (OPN) and transforming growth factor-ß1 (TGF-ß1) levels. METHODS: Peripheral blood samples were collected in children with asthma (n=98; astma group) or without asthma (n=30; control group) from Hunan, China. The asthma group was subdivided into atopic (n=62) and non-atopic (n=36) subgroups. Single nucleotide polymorphism (SNP) analysis was performed, and serum OPN and TGF-ß1 levels were measured. RESULTS: There were no significant differences in genotype and allele frequencies of rs7216389 of the ORMDL3 gene between the asthma and control groups. The serum level of OPN in the asthma group was significantly higher than in the control group (P<0.05). Both the atopic and non-atopic subgroups showed increased serum levels of OPN compared with the control group (P<0.05). The serum level of TGF-ß1 in the atopic subgroup was significantly higher than in the control group (P<0.05). The serum levels of OPN and TGF-ß1 showed no significant differences in asthmatic children with different genotypes. The serum levels of OPN and TGF-ß1 were in a positive linear correlation in the asthma group (r=0.620; P<0.01) and its two subgroups (r=0.734, 0.649 respectively; P<0.01). CONCLUSIONS: In children from Hunan, China, the SNP (rs7216389) of ORMDL3 is not related to asthma susceptibility. OPN and TGF-ß1 may be involved in the development of asthma, and they are in a positive linear correlation. The SNP (rs7216389) of ORMDL3 does not influence the expression of OPN and TGF-ß1, suggesting that it may not be associated with airway remodeling.

Microchip amplifier for in vitro, in vivo, and automated whole cell patch-clamp recording.

Patch clamping is a gold-standard electrophysiology technique that has the temporal resolution and signal-to-noise ratio capable of reporting single ion channel currents, as well as electrical activity of excitable single cells. Despite its usefulness and decades of development, the amplifiers required for patch clamping are expensive and bulky. This has limited the scalability and throughput of patch clamping for single-ion channel and single-cell analyses. In this work, we have developed a custom patch-clamp amplifier microchip that can be fabricated using standard commercial silicon processes capable of performing both voltage- and current-clamp measurements. A key innovation is the use of nonlinear feedback elements in the voltage-clamp amplifier circuit to convert measured currents into logarithmically encoded voltages, thereby eliminating the need for large high-valued resistors, a factor that has limited previous attempts at integration. Benchtop characterization of the chip shows low levels of current noise [1.1 pA root mean square (rms) over 5 kHz] during voltage-clamp measurements and low levels of voltage noise (8.2 µV rms over 10 kHz) during current-clamp measurements. We demonstrate the ability of the chip to perform both current- and voltage-clamp measurement in vitro in HEK293FT cells and cultured neurons. We also demonstrate its ability to perform in vivo recordings as part of a robotic patch-clamping system. The performance of the patch-clamp amplifier microchip compares favorably with much larger commercial instrumentation, enabling benchtop commoditization, miniaturization, and scalable patch-clamp instrumentation.

[Effect of budesonide aerosol treatment on expression of glucocorticoid receptor and nuclear factor-κB in asthmatic mice].

OBJECTIVE: To study the effect of budesonide aerosol inhalation on the expression of glucocorticoid receptor (GR) and nuclear factor (NF)-κB in asthmatic mice. METHODS: Twenty-four healthy male BALB/c mice aged 6 to 8 weeks were randomly divided into three groups (n=8 each): normal saline (control group), asthma model (asthma group) and budesonide-treated asthma (BUD group). Asthma was induced by intraperitoneal injection of ovalbumin (OVA) and aluminium hydroxide suspension and aerosol inhalation of OVA solution. Mice were sacrificed 24 hours after the last challenge. Eosinophil count in the bronchoalveolar lavage fluid (BALF) was determined. Pathological examination of the lung tissues was performed and the expression levels of GR and NF-κB were measured by immunohistochemical analysis. RESULTS: Eosinophil count in the BALF was significantly higher in the asthma and BUD groups than in the control group (P<0.05). BUD treatment decreased eosinophil count in the BALF compared with the asthma group (P<0.05). The lung tissues in the BUD group showed a less severe infiltration of eosinophils and lymphocytes compared with the asthma group. The percentage of GR-positive cells in the asthma group decreased significantly compared with the control group (P<0.05), and the percentage of GR-positive cells in the BUD group increased significantly compared with the asthma group (P<0.05). Compared with the control group, the percentage of NF-κB-positive cells increased significantly in the asthma group (P<0.05), and the percentage of NF-κB positive cells in the BUD group was significantly reduced compared with the asthma group (P<0.05). CONCLUSIONS: The action mechanism of budesonide in treating asthmatic mice may be related to the upregulation of GR expression and the inhibition of NF-κB activity.

Chemical constituents from Notopterygium incisum and their anti-neuroinflammatory activity.

Phytochemical research on an extract of Notopterygium incisum yielded fifteen compounds (1-15), including four previously undescribed compounds (10-13). The structures of the unreported compounds were elucidated by spectroscopic and spectrometric data analysis such as 1D and 2D NMR, IR and HR-ESI-MS. Compounds 1-5 and 10-14 were isolated from N. incisum for the first time. 7S⁎,8R⁎-Phenethyl-(7-methoxy-8-isoeugenol)-ferulate (10), 7S⁎,8R⁎-p-hydroxyphenethyl-(7-methoxy-8-isoeugenol)-ferulate (11), 7S⁎,8R⁎-benzyl-(7-methoxy-8-isoeugenol)-ferulate (12) and p-hydroxyphenethyl-(4-benzoy-3-methoxy)-cinnamate (13) are the undescribed ferulic acid derivatives. Additionly, the anti-neuroinflammatory effects of compounds were evaluated in lipopolysaccharide (LPS)-induced BV2 cells. The pharmacological results showed that 6ß,10ß-epoxy-4α-hydroxy-guaiane (6), teuclatriol (7) and 7S⁎,8R⁎-p-hydroxyphenethyl-(7-methoxy-8-isoeugenol)-ferulate (11) inhibited the production and expression of nitric oxide (NO) in the LPS-induced BV2 cells in a concentration-dependent manner. Acorusnol (4), teucladiol (9), 7S⁎,8R⁎-benzyl-(7-methoxy-8-isoeugenol)-ferulate (12) and p-hydroxyphenethyl-(4-benzoy-3-methoxy)-cinnamate (13) only inhibited the release of NO at concentration of 20 µM. Moreover, 7S⁎,8R⁎-p-hydroxyphenethyl-(7-methoxy-8-isoeugenol)-ferulate (11) reduced the level of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in LPS-stimulated BV2 cells. The results demonstrated 7S⁎,8R⁎-p-hydroxyphenethyl-(7-methoxy-8-isoeugenol)-ferulate (11) could be a potential anti-neuroinflammatory agent and is worthy of further study.

[Extraction and purification of isocorydine from Dicranostigma leptopodum].

OBJECTIVE: To optimize the conditions of extraction and purification of isocorydine from Dicranostigma leptopodum. METHODS: Extraction conditions of isocorydine were selected on the basis of orthogonal experimental design, the static adsorption/desorption experiments were used to evaluate the optimum resin. RESULTS: The optimum extraction parameters were as follows: the ratio of raw materials to solvent 1:15 (g/mL), extraction solvent 1% vitriol, the extraction three times and 1 h each time. LX28 resin exhibited higher adsorption efficiency. CONCLUSION: Under the above optimum conditions, the extraction yield of isocorydine is 0.88%. The purity of isocorydine can reach 85.34% with a yield rate of 68.64%.

[Mycoplasma pneumoniae infection and drug resistance in children: an analysis of 1026 cases].

OBJECTIVE: To investigate the Mycoplasma pneumoniae (MP) infection and drug resistance in children with respiratory tract infection and to provide a rational basis for the clinical diagnosis and treatment of MP infection. METHODS: Throat swabs were collected from 3529 children with respiratory tract infection, who visited the pediatric outpatient department or received treatment in the pediatric ward of our hospital from September 2010 to September 2011. The swabs were cultured to detect MP. The drug sensitivity of MP to azithromycin, roxithromycin, erythromycin, acetylspiramycin and clarithromycin was evaluated. RESULTS: Of the 3529 children with respiratory tract infection, 1026 (29.07%) were MP-positive. There were cases of MP infection in all four seasons of the year but infection rates in summer and autumn were significantly higher than in spring and winter (P < 0.05). The infection rate in females was higher than in males (30.43% vs 28.32%; P > 0.05). The infection rate was negatively correlated with age in these children, and there were significant differences in the infection rate among all age groups (P < 0.05). For macrolide antibiotics suitable for children, the cultured MP developed the highest resistance to roxithromycin, followed by erythromycin, acetylspiramycin, clarithromycin, and azithromycin, with significant differences among them (P < 0.01). CONCLUSIONS: MP infection rate is very high among children with respiratory tract infection. The incidence of MP infection is relatively low among school-age children and children are more susceptible to MP infection in summer and autumn than in spring and winter. Throat swabs should be cultured and drug sensitivity tests should be performed as early as possible in children with respiratory tract infection, so that proper intervention can be undertaken in time to reduce drug-resistant strains of MP.

Diagnosis by metagenomic next-generation sequencing of invasive pulmonary aspergillosis in an infant with chronic granulomatous disease.

Invasive pulmonary aspergillosis (IPA) is a serious fungal infection, with a high degree of mortality in immunocompromised individuals. Diagnosis of IPA is challenging in that clinical manifestations are not specific, with sensitivity of traditional detection procedures low. We report a case of IPA in a chronic granulomatous disease (CGD) infant who was initially suspected to have a lung tumor. Aspergillus fumigatus was identified as the pathogen in bronchoalveolar lavage fluid (BALF) by next-generation sequencing (mNGS). The patient recovered rapidly following a change of appropriate antifungal treatment and was discharged. This case highlights the additional value of BALF-mNGS for the diagnosis of pediatric invasive pulmonary fungal infection in immune-deficient children.