Deep learning model to discriminate diverse infection types based on pairwise analysis of host gene expression.

Accurate detection of pathogens, particularly distinguishing between Gram-positive and Gram-negative bacteria, could improve disease treatment. Host gene expression can capture the immune system's response to infections caused by various pathogens. Here, we present a deep neural network model, bvnGPS2, which incorporates the attention mechanism based on a large-scale integrated host transcriptome dataset to precisely identify Gram-positive and Gram-negative bacterial infections as well as viral infections. We performed analysis of 4,949 blood samples across 40 cohorts from 10 countries using our previously designed omics data integration method, iPAGE, to select discriminant gene pairs and train the bvnGPS2. The performance of the model was evaluated on six independent cohorts comprising 374 samples. Overall, our deep neural network model shows robust capability to accurately identify specific infections, paving the way for precise medicine strategies in infection treatment and potentially also for identifying subtypes of other diseases.

Multiplex nanozymatic biosensing of Salmonella on a finger-actuated microfluidic chip.

A colorimetric biosensor was elaboratively designed for fast, sensitive and multiplex bacterial detection on a single microfluidic chip using immune magnetic nanobeads for specific bacterial separation, immune gold@platinum palladium nanoparticles for specific bacterial labeling, a finger-actuated mixer for efficient immunoreaction and two coaxial rotatable magnetic fields for magnetic nanobead capture (outer one) and magnet-actuated valve control (inner one). First, preloaded bacteria, nanobeads and nanozymes were mixed through a finger actuator to form nanobead-bacteria-nanozyme conjugates, which were captured by the outer magnetic field. After the inner magnetic field was rotated to successively wash the conjugates and push the H2O2-TMB substrate for resuspending these conjugates, colorless TMB was catalyzed into blue TMBox products, followed by color analysis using ImageJ software for bacterial determination. This simple biosensor enabled multiplex Salmonella detection as low as 9 CFU per sample in 45 min.

Evaluation of deproteinised bovine bone matrix combined with absorbable biofilm for the preservation of extraction sites of mandibular impacted wisdom teeth.

BACKGROUND: Bone defects and deep periodontal pockets often exist distal to the second molar after mandibular third molar extraction, seriously threatening the periodontal health of the second molar. OBJECTIVE: To evaluate the effect of socket preservation with bone substitute materials on alveolar bone resorption and prevention of the distal periodontal defect of the adjacent tooth after mandibular impacted third molar extraction compared with natural healing. METHODS: Ninety-nine patients with mandibular impacted teeth, treated in our hospital from January 2018 to December 2020, were randomly divided into the control and experimental groups. The experimental group underwent minimally invasive tooth extraction and socket preservation using the deproteinised bovine bone mineral, Bio-Oss and the bioabsorbable collagen membrane, Bio-Gide. The control group healed naturally after minimally invasive tooth extraction. The alveolar ridge dimension of the extraction sites, the probing depth, tooth mobility and gingival index on the distal aspect of the mandibular second molars were examined and recorded before and six months after the operations. RESULTS: There was a significant difference between the experimental group and the control group in the alveolar bone width (P< 0.05) and height (P< 0.05) before and after surgery. The probing depth of the extraction sites in both groups was reduced. CONCLUSION: Using Bio-Oss and Bio-Gide to preserve extraction sites of impacted teeth can promote recovery more effectively than natural healing on the height of the distal alveolar bone and the width of the alveolar crest of the second molar and thus improve the periodontal status of the adjacent second molar.

Truncated Dyrk1A aggravates neuronal apoptosis by inhibiting ASF-mediated Bcl-x exon 2b inclusion.

AIM: Aggravated neuronal loss, caused mainly by neuronal apoptosis, is observed in the brain of patients with Alzheimer's disease (AD) and animal models of AD. A truncated form of Dual-specific and tyrosine phosphorylation-regulated protein kinase 1A (Dyrk1A) plays a vital role in AD pathogenesis. Downregulation of anti-apoptotic Bcl-xL is tightly correlated with neuronal loss in AD. However, the molecular regulation of neuronal apoptosis and Bcl-x expression by Dyrk1A in AD remains largely elusive. Here, we aimed to explore the role and molecular mechanism of Dyrk1A in apoptosis. METHODS: Cell Counting Kit-8 (CCK8), flow cytometry, and TdT-mediated dUTP Nick-End Labeling (TUNEL) were used to check apoptosis. The cells, transfected with Dyrk1A or/and ASF with Bcl-x minigene, were used to assay Bcl-x expression by RT-PCR and Western blots. Co-immunoprecipitation, autoradiography, and immunofluorescence were conducted to check the interaction of ASF and Dyrk1A. Gene set enrichment analysis (GSEA) of apoptosis-related genes was performed in mice overexpressing Dyrk1A (TgDyrk1A) and AD model 5xFAD mice. RESULTS: Dyrk1A promoted Bcl-xS expression and apoptosis. Splicing factor ASF promoted Bcl-x exon 2b inclusion, leading to increased Bcl-xL expression. Dyrk1A suppressed ASF-mediated Bcl-x exon 2b inclusion via phosphorylation. The C-terminus deletion of Dyrk1A facilitated its binding and kinase activity to ASF. Moreover, Dyrk1a1-483 further suppressed the ASF-mediated Bcl-x exon 2b inclusion and aggravated apoptosis. The truncated Dyrk1A, increased Bcl-xS, and enrichment of apoptosis-related genes was observed in the brain of 5xFAD mice. CONCLUSIONS: We speculate that increased Dyrk1A and truncated Dyrk1A may aggravate neuronal apoptosis by decreasing the ratio of Bcl-xL/Bcl-xS via phosphorylating ASF in AD.

Magnetic nanobead chain-assisted real-time impedance monitoring using PCB interdigitated electrode for Salmonella detection.

Pathogen testing is effective to prevent food poisoning. Here, an electrochemical biosensor was explored for Salmonella detection by combining magnetic grid based bacterial separation with enzymatic catalysis based signal amplification on a PCB interdigitated electrode in a microfluidic chip. First, immune magnetic nanobeads, target bacteria, and immune polystyrene microspheres decorated with glucose oxidase were sufficiently mixed to form nanobead-bacteria-microsphere sandwich conjugates. Then, these conjugates were injected into the chip to form conjugate chains right over the electrode under an iron grid enhanced magnetic field. After non-conductive glucose was injected and catalyzed by glucose oxidase on the conjugate chains, conductive glucose acid and non-conductive hydrogen peroxide were continuously produced and rapidly diffused from the conjugate chains to the electrode. Finally, the impedance change was real-timely monitored and used to determine the bacterial amount. This sensor enabled detection of 50 CFU/mL Salmonella typhimurium in 1 h.

Exploring the Role of Circulating Cell-Free RNA in the Development of Colorectal Cancer.

Circulating tumor RNA (ctRNA) has recently emerged as a novel and attractive liquid biomarker. CtRNA is capable of providing important information about the expression of a variety of target genes noninvasively, without the need for biopsies, through the use of circulating RNA sequencing. The overexpression of cancer-specific transcripts increases the tumor-derived RNA signal, which overcomes limitations due to low quantities of circulating tumor DNA (ctDNA). The purpose of this work is to present an up-to-date review of current knowledge regarding ctRNAs and their status as biomarkers to address the diagnosis, prognosis, prediction, and drug resistance of colorectal cancer. The final section of the article discusses the practical aspects involved in analyzing plasma ctRNA, including storage and isolation, detection technologies, and their limitations in clinical applications.

Cell-free circulating tumor RNAs in plasma as the potential prognostic biomarkers in colorectal cancer.

Background: Cell free RNA (cfRNA) contains transcript fragments from multiple cell types, making it useful for cancer detection in clinical settings. However, the pathophysiological origins of cfRNAs in plasma from colorectal cancer (CRC) patients remain unclear. Methods: To identify the tissue-specific contributions of cfRNAs transcriptomic profile, we used a published single-cell transcriptomics profile to deconvolute cell type abundance among paired plasma samples from CRC patients who underwent tumor-ablative surgery. We further validated the differentially expressed cfRNAs in 5 pairs of CRC tumor samples and adjacent tissue samples as well as 3 additional CRC tumor samples using RNA-sequencing. Results: The transcriptomic component from intestinal secretory cells was significantly decreased in the in-house post-surgical cfRNA. The HPGD, PACS1, and TDP2 expression was consistent across cfRNA and tissue samples. Using the Cancer Genome Atlas (TCGA) CRC datasets, we were able to classify the patients into two groups with significantly different survival outcomes. Conclusions: The three-gene signature holds promise in applying minimal residual disease (MRD) testing, which involves profiling remnants of cancer cells after or during treatment. Biomarkers identified in the present study need to be validated in a larger cohort of samples in order to ascertain their possible use in early diagnosis of CRC.

Two simple assays for assessing the seeding activity of proteopathic tau.

The regional distribution of neurofibrillary tangles of hyperphosphorylated tau aggregates is associated with the progression of Alzheimer's disease (AD). Misfolded proteopathic tau recruits naïve tau and templates its misfolding and aggregation in a prion-like fashion, which is believed to be the molecular basis of propagation of tau pathology. A practical way to assess tau seeding activity is to measure its ability to recruit/bind other tau molecules and to induce tau aggregation. Based on the properties of proteopathic tau, here we report the development of two simple assays to assess tau seeding activity ----- capture assay in vitro and seeded-tau aggregation assay in cultured cells. In the capture assay, proteopathic tau was applied onto a nitrocellulose membrane and the membrane was incubated with cell lysate containing HA-tagged tau151-391 (HA-tau151-391). The captured tau on the membrane was determined by immuno-blots developed with anti-HA. For the seeded-tau aggregation assay, HEK-293FT cells transiently expressing HA-tau151-391 were treated with proteopathic tau in the presence of Lipofectamine 2000 and then lysed with RIPA buffer. RIPA-insoluble fraction containing aggregated tau was obtained by ultracentrifugation and analyzed by immuno-blot developed with anti-HA. To validate these two assays, we assessed the seeding activity of tau in the middle frontal gyrus, middle temporal gyrus and basal forebrain of AD and control brains and found that AD, but not control, brain extracts effectively captured and seeded tau151-391 aggregation. Basal forebrain contained less phospho-tau and tau seeding activity. The levels of captured tau or seeded-tau aggregates were positively correlated to the levels of phospho-tau, Braak stages and tangle sores. These two assays are specific and sensitive and can be carried out in a regular biomedical laboratory setting by using routine biochemical techniques.

Semi-circle magnetophoretic separation under rotated magnetic field for colorimetric biosensing of Salmonella.

Magnetic separation was often applied to isolate and concentrate foodborne bacteria using immunomagnetic nanobeads before downstream bacterial detection. However, nanobead-bacteria conjugates (magnetic bacteria) were coexisting with excessive unbound nanobeads, limiting these nanobeads on magnetic bacteria to further act as signal probes for bacterial detection. Here, a new microfluidic magnetophoretic biosensor was elaboratively developed using a rotated high gradient magnetic field and platinum modified immunomagnetic nanobeads for continuous-flow isolation of magnetic bacteria from free nanobeads, and combined with nanozyme signal amplification for colorimetric biosensing of Salmonella. First, the platinum modified immunomagnetic nanobeads were mixed with the bacterial sample to form the magnetic bacteria, and magnetically separated to eliminate non-magnetic background. Then, the mixture of free immunomagnetic nanobeads and magnetic bacteria was injected with sheath flow (PBS) at higher flowrate into the semi-circle magnetophoretic separation channel under rotated magnetic field, which was generated by two repulsive cylindric magnets and their in-between ring iron gear, leading to continuous-flow isolation of magnetic bacteria from free immunomagnetic nanobeads because they suffered from different magnetic forces and thus had different deviating positions at the outlet. Finally, the separated magnetic bacteria and unbound magnetic nanobeads were respectively collected and used to catalyze coreless substrate into blue product, which was further analyzed using the microplate reader to obtain bacterial amount. This biosensor could determinate Salmonella as low as 41 CFU/mL in 40 min.

The clinical characteristics of pediatric patients infected by SARS-CoV-2 Omicron variant and whole viral genome sequencing analysis.

Pediatric population was generally less affected clinically by SARS-CoV-2 infection. Few pediatric cases of COVID-19 have been reported compared to those reported in infected adults. However, a rapid increase in the hospitalization rate of SARS-CoV-2 infected pediatric patients was observed during Omicron variant dominated COVID-19 outbreak. In this study, we analyzed the B.1.1.529 (Omicron) genome sequences collected from pediatric patients by whole viral genome amplicon sequencing using Illumina next generation sequencing platform, followed by phylogenetic analysis. The demographic, epidemiologic and clinical data of these pediatric patients are also reported in this study. Fever, cough, running nose, sore throat and vomiting were the more commonly reported symptoms in children infected by Omicron variant. A novel frameshift mutation was found in the ORF1b region (NSP12) of the genome of Omicron variant. Seven mutations were identified in the target regions of the WHO listed SARS-CoV-2 primers and probes. On protein level, eighty-three amino acid substitutions and fifteen amino acid deletions were identified. Our results indicate that asymptomatic infection and transmission among children infected by Omicron subvariants BA.2.2 and BA.2.10.1 are not common. Omicron may have different pathogenesis in pediatric population.

Waldenstrom Macroglobulinemia Arising During Maintenance Therapy of Plasma Cell Myeloma.

BACKGROUND: Both plasma cell myeloma (PCM) and Waldenstrom's macroglobulinemia (WM) are mature B-cell neoplasms commonly involving bone marrow and usually related to paraproteinemia. METHODS: Secondary WM in a patient with PCM during maintenance therapy has not been previously reported. We herein report the first case of WM arising during maintenance therapy of PCM. RESULTS: The diagnosis of secondary WM during maintenance therapy of PCM was based on combination of medical history, morphology, flow cytometry, immunofixation electrophoresis, and molecular genetics. CONCLUSIONS: This case highlights the importance of an integrated diagnostic work-up, with an interesting role for morphology and flow immunotyping.

Casein Kinase 1δ Phosphorylates TDP-43 and Suppresses Its Function in Tau mRNA Processing.

BACKGROUND: Neurofibrillary tangle aggregated from anomalous hyperphosphorylated tau is a hallmark of Alzheimer's disease (AD). Trans-active response DNA-binding protein of 43 kDa (TDP-43) enhances the instability and exon (E) 10 inclusion of tau mRNA. Cytoplasmic inclusion of hyperphosphorylated TDP-43 in the neurons constitutes the third most prevalent proteinopathy of AD. Casein kinase 1δ (CK1δ) is elevated in AD brain and phosphorylates TDP-43 in vitro. OBJECTIVE: To determine the roles of CK1δ in phosphorylation, aggregation, and function of TDP-43 in the processing of tau mRNA. METHODS: The interaction and colocalization of TDP-43 and CK1δ were analyzed by co-immunoprecipitation and immunofluorescence staining. TDP-43 phosphorylation by CK1δ was determined in vitro and in cultured cells. RIPA-insoluble TDP-43 aggregates obtained by ultracentrifugation were analyzed by immunoblots. The instability and E10 splicing of tau mRNA were studied by using a reporter of green fluorescence protein tailed with 3'-untranslational region of tau mRNA and a mini-tau gene and analyzed by real-time quantitative PCR and reverse transcriptional PCR. RESULTS: We found that CK1δ interacted and co-localized with TDP-43. TDP-43 was phosphorylated by CK1δ at Ser379, Ser403/404, and Ser409/410 in vitro and in cultured cells, which was mutually enhanced. CK1δ overexpression promoted the aggregation of TDP-43 and suppressed its activity in enhancing the instability and E10 inclusion of tau mRNA. CONCLUSION: CK1δ phosphorylates TDP-43, promotes its aggregation, and inhibits its activity in promoting the instability of tau mRNA and inclusion of tau E10. Elevated CK1δ in AD brain may contribute to TDP-43 and tau pathologies directly or indirectly.

A microfluidic biosensor based on finger-driven mixing and nuclear track membrane filtration for fast and sensitive detection of Salmonella.

A novel colorimetric biosensor was explored for fast, sensitive, and on-site detection of Salmonella on a microfluidic chip employing immune gold@platinum nanoparticles (Au@Pt NPs) for specific bacterial labeling, a finger-driven mixer with two serial air chambers for efficient immunoreaction and a nuclear track membrane as specific-size microfilter for effective bacterial isolation from excessive immune Au@Pt NPs. First, the bacterial sample and immune Au@Pt NPs were pipetted into the mixing chamber and mixed sufficiently through repeated press-release actions on the small air chamber which could precisely control the air volume, leading to the formation of bacteria-immune Au@Pt NP conjugates. Then, the small and large air chambers were pressed simultaneously to push all the solution in the mixing chamber to flow through the microfilter for trap of the formed larger-size conjugates on the membrane and removal of the unbound smaller-size immune Au@Pt NPs. After the H2O2-TMB substrate was pipetted into the microfilter and catalyzed by the conjugates, ImageJ was used to analyze the color change for bacterial determination. This simple biosensor enabled Salmonella detection as low as 168 CFU/mL in 25 min.

MrGPS: an m6A-related gene pair signature to predict the prognosis and immunological impact of glioma patients.

N6-methyladenosine (m6A) RNA methylation is the predominant epigenetic modification for mRNAs that regulates various cancer-related pathways. However, the prognostic significance of m6A modification regulators remains unclear in glioma. By integrating the TCGA lower-grade glioma (LGG) and glioblastoma multiforme (GBM) gene expression data, we demonstrated that both the m6A regulators and m6A-target genes were associated with glioma prognosis and activated various cancer-related pathways. Then, we paired m6A regulators and their target genes as m6A-related gene pairs (MGPs) using the iPAGE algorithm, among which 122 MGPs were significantly reversed in expression between LGG and GBM. Subsequently, we employed LASSO Cox regression analysis to construct an MGP signature (MrGPS) to evaluate glioma prognosis. MrGPS was independently validated in CGGA and GEO glioma cohorts with high accuracy in predicting overall survival. The average area under the receiver operating characteristic curve (AUC) at 1-, 3- and 5-year intervals were 0.752, 0.853 and 0.831, respectively. Combining clinical factors of age and radiotherapy, the AUC of MrGPS was much improved to around 0.90. Furthermore, CIBERSORT and TIDE algorithms revealed that MrGPS is indicative for the immune infiltration level and the response to immune checkpoint inhibitor therapy in glioma patients. In conclusion, our study demonstrated that m6A methylation is a prognostic factor for glioma and the developed prognostic model MrGPS holds potential as a valuable tool for enhancing patient management and facilitating accurate prognosis assessment in cases of glioma.

Tau antibody 77G7 targeting microtubule binding domain suppresses proteopathic tau to seed tau aggregation.

INTRODUCTION: Neurofibrillary tangle (NFT) of hyperphosphorylated tau is a hallmark of Alzheimer's disease (AD) and related tauopathies. Tau lesion starts in the trans-entorhinal cortex, from where it spreads to limbic regions, followed by neocortical areas. The regional distribution of NFTs associates with the progression of AD. Accumulating evidence suggests that proteopathic tau can seed tau aggregation in a prion-like fashion in vitro and in vivo. Inhibition of tau seeding activity could provide a potential therapeutic opportunity to block the propagation of tau pathology in AD and related tauopathies. AIMS: In the present study, we investigated the role of 77G7, a monoclonal tau antibody to the microtubule-binding repeats, in repressing the seeding activity of proteopathic tau. RESULTS: We found that 77G7 had a higher affinity toward aggregated pathological tau fractions than un-aggregated tau derived from AD brain. 77G7 inhibited the internalization of tau aggregates by cells, blocked AD O-tau to capture normal tau, and to seed tau aggregation in vitro and in cultured cells. Tau pathology induced by hippocampal injection of AD O-tau in 3xTg-AD mice was suppressed by mixing 77G7 with AD O-tau. Intravenous administration of 77G7 ameliorated site-specific hyperphosphorylation of tau induced by AD O-tau in the hippocampi of Tg/hTau mice. CONCLUSION: These findings indicate that 77G7 can effectively suppress the seeding activity of AD O-tau and thus could be developed as a potential immunotherapeutic drug to inhibit the propagation of tau pathology in AD and related tauopathies.

Tau seeding activity in various regions of down syndrome brain assessed by two novel assays.

Propagation of tau pathology via the seeding of naive tau aggregation underlies the progression of Alzheimer's disease (AD) and related tauopathies. Individuals with Down syndrome (DS) develop tau pathology at the fourth decade of life, but tau seeding activity in DS brain has not yet been determined. To measure tau seeding activity, we developed capture assay and seeded-tau aggregation assay with truncated tau151-391. By using brain extracts from AD and related tauopathies, we validated these two methods and found that the brain extracts from AD and related tauopathies, but not from controls and the diseases in which tau was not hyperphosphorylated, captured in vitro and seeded 3R-tau151-391 and 4R-tau151-391 to aggregate in cultured cells similarly. Captured tau151-391 levels were strongly correlated with the seeded-tau151-391 aggregation. Employing these two newly developed assays, we analyzed tau seeding activity in the temporal (TC), frontal (FC), and occipital cortex (OC); corpus callosum (CC); and cerebellar cortex (CBC) of DS and control brains. We found that the extracts of TC, FC, or OC, but not the CC or CBC of DS or the corresponding brain regions of control cases, captured tau151-391. Levels of the captured tau151-391 by brain extracts were positively correlated with their levels of phosphorylated tau. Extracts of cerebral cortex and CC, but not CBC of DS with a similar tau level, induced more tau151-391 aggregation than did the corresponding samples from the control cases. Thus, higher tau seeding activity associated with tau hyperphosphorylation was found in the TC, FC, and OC of DS compared with the corresponding control regions as well as with the CBC and CC of DS. Of note, these two assays are sensitive, specific, and repeatable at a low cost and provide a platform for measuring tau seeding activity and for drug screening that targets tau propagation.

Power-free microfluidic biosensing of Salmonella with slide multivalve and disposable syringe.

In this study, a power-free biosensor was presented to detect Salmonella typhimurium on a microfluidic chip using a slide multivalve for channel selection and a disposable syringe for fluidic transfer. First, bacterial sample with immunomagnetic nanoparticles (IMNPs) and glucose oxidase (GOx) modified immune polystyrene nanoparticles (IPNPs), washing buffer, glucose, and peroxide test strip (PTS) were preloaded in their respective chambers at the periphery of chip. After the slide multivalve was selected to connect sample chamber with common separation chamber, which was connected with a syringe, the mixture of Salmonella, IMNPs and IPNPs was back and forth moved through 3D Tesla-structure micromixer using the syringe, resulting in the formation of IMNP-Salmonella-IPNP complexes, which were captured in the separation chamber using a magnet. Then, two washing chambers were selectively connected respectively to remove sample background and excessive IPNPs, and glucose chamber was connected, allowing the GOx to catalyze glucose to produce hydrogen peroxide in the separation chamber. Finally, PTS chamber was connected and the catalysate was transferred from the separation chamber to the PTS chamber, leading to the color change of PTS, followed by using smartphone App to collect and analyze the image of PTS for bacterial determination. The simple biosensor enabled simple detection of Salmonella as few as 130 CFU/mL within 60 min and is promising for practical applications in the resource-limited regions due to its low cost, simple operation, and small size.

Magnetic/electric field intervention on oil-rich filamentous algae production in the application of acrylonitrile butadiene styrene based wastewater treatment.

Globally, the release of acrylonitrile-butadienestyrene (ABS) wastewater from numerous industries is a serious concern. Recently, oil-rich filamentous algae Tribonema sp has been grown utilizing toxic but nutrient-rich ABS effluent. Here, Tribonema sp. was cultivated under intervention of different magneto-electric combinatory fields (MCFs) (control, 0.6 V/cm, 1 h/d-1.2 V/cm, 1 h/d-0.6 V/cm, and 1 h/d-1.2 V/cm). Results showed MCF (1 h/d-0.6 V/cm) intervention increased the biomass by 9.7% (2.4 g/L) combined with high removal efficiencies (95% and 99%) of ammonium nitrogen and total phosphorus. The chemical oxygen demand (COD) removal rate increased to 82%, 6% higher than the control. Moreover, MCF of 1 h/d-0.6 V/cm significantly increased lipid and carbohydrate by 7.71% and 4.73% respectively. MCF increased premium fatty acid content such as palmitic acid (C16:0), myristic acid (C14: 0), and hexadecenoic acid (C16:1). MCF intervention also supported a diverse microbial flora, offering a favorable solution for ABS wastewater treatment.

The clinical application of metagenomic next-generation sequencing for detecting pathogens in bronchoalveolar lavage fluid: case reports and literature review.

INTRODUCTION: Clinical metagenomic next-generation sequencing (mNGS) allows a comprehensive genetic analysis of microbial materials. Different from other traditional target-driven molecular diagnostic tests, such as PCR, mNGS is a hypothesis-free diagnostic approach that allows a comprehensive genetic analysis of the clinical specimens that cover nearly any common, rare, and new pathogens ranging broadly from viruses, bacteria, fungi to parasites. AREAS COVERED: In this article, we discussed the clinical application of the mNGS using two clinical cases as examples and described the use of mNGS to assist the diagnosis of parasitic pulmonary infection. The advantages and challenges in implementing mNGS in clinical microbiology are also discussed. EXPERT OPINION: mNGS is a promising technology that allows quick diagnosis of infectious diseases. Currently, a plethora of sequencing and analysis methods exists for mNGS, each with individual merits and pitfalls. While standards and best practices were proposed by various metagenomics working groups, they are yet to be widely adopted in the community. The development of a consensus set of guidelines is necessary to guide the usage of this new technology and the interpretation of NGS results before clinical adoption of mNGS testing.

Excess folic acid supplementation before and during pregnancy and lactation activates ß-catenin in the brain of male mouse offspring.

Folic acid (FA) supplementation in early pregnancy is recommended to protect against birth defects. But excess FA has exhibited neurodevelopmental toxicity. We previously reported that the mice treated with 2.5-fold the dietary requirement of FA one week before mating and throughout pregnancy and lactation displayed abnormal behaviors in the offspring. Here we found the levels of non-phosphorylated ß-catenin (active) were increased in the brains of weaning and adult FA-exposed offspring. Meanwhile, demethylation of protein phosphatase 2 A catalytic subunit (PP2Ac), which suppresses its enzyme activity in regulatory subunit dependent manner, was significantly inhibited. Among the upstream regulators of ß-catenin, PI3K/Akt/GSK-3ß but not Wnt signaling was stimulated in FA-exposed brains only at weaning. In mouse neuroblastoma N2a cells, knockdown of PP2Ac or leucine carboxyl methyltransferase-1 (LCMT-1), or overexpression of PP2Ac methylation-deficient mutant decreased ß-catenin dephosphorylation. These results suggest that excess FA may activate ß-catenin via suppressing PP2Ac demethylation, providing a novel mechanism for the influence of FA on neurodevelopment.