Soil amendments reduce CH4 and CO2 but increase N2O and NH3 emissions in saline-alkali paddy fields.

Limited research has been conducted on ammonia (NH3) volatilization and greenhouse gases (GHGs) emissions in saline-alkali paddy fields, along with complex interaction involving various genes (16sRNA, amoA, narG, nirK, nosZ, and nifH). This study employed mesocosm-scale experiment to investigate NH3 volatilization and GHGs emissions, focusing on bacterial communities and genic abundance, in saline-alkali paddy fields with desulfurized gypsum (DG) and organic fertilizer (OF) amendments. Compared to the control (CK) treatment, DG and OF treatments reduced methane (CH4) and carbon dioxide (CO2) emissions by 78.05 % and 26.18 %, and 65.84 % and 11.62 %, respectively. However, these treatments increased NH3 volatilization by 26.26 % and 45.23 %, and nitrous oxide (N2O) emission by 41.00 % and 12.31 %. Notably, NH3 volatilization primarily stemmed from ammonia nitrogen (NH4+-N), rather than total nitrogen (TN) in soil and water. N2O was mainly produced from nitrate nitrogen (NO3--N) in soil and water, as well as NH4+-N in water. The increase in NH3 volatilization and N2O emission in DG and OF treatments, was attributed to the reduced competition among bacterial communities, rather than the increased bacterial activity and genic copies. These findings offer valuable insights for managing nutrient loss and gaseous emissions in saline-alkali paddy fields.

Neurological manifestations of SARS-CoV-2 infection in children in Taiwan: A cross-section, multicenter study.

BACKGROUND: The SARS-CoV-2 virus has been a global public health threat since December 2019. This study aims to investigate the neurological characteristics and risk factors of coronavirus disease 2019 (COVID-19) in Taiwanese children, using data from a collaborative registry. METHODS: A retrospective, cross-sectional, multi-center study was done using an online network of pediatric neurological COVID-19 cohort collaborative registry. RESULTS: A total of 11160 COVID-19-associated emergency department (ED) visits and 1079 hospitalizations were analyzed. Seizures were the most common specific neurological symptom, while encephalitis and acute disseminated encephalomyelitis (ADEM) was the most prevalent severe involvement. In ED patients with neurological manifestations, severe neurological diagnosis was associated with visual hallucination, seizure with/without fever, behavior change, decreased GCS, myoclonic jerk, decreased activity/fatigue, and lethargy. In hospitalized patients with neurological manifestations, severe neurological diagnosis was associated with behavior change, visual hallucination, decreased GCS, seizure with/without fever, myoclonic jerk, fatigue, and hypoglycemia at admission. Encephalitis/ADEM was the only risk factor for poor neurological outcomes at discharge in hospitalized patients. CONCLUSION: Neurological complications are common in pediatric COVID-19. Visual hallucination, seizure, behavior change, myoclonic jerk, decreased GCS, and hypoglycemia at admission are the most important warning signs of severe neurological involvement such as encephalitis/ADEM.

The core exosome proteome of Trichomonas vaginalis.

BACKGROUND: Trichomonas vaginalis is parasitic protozoan that causes human urogenital infections. Accumulated reports indicated that exosomes released by this parasite play a crucial role in transmitting information and substances between cells during host-parasite interactions. Current knowledge on the protein contents in T. vaginalis exosome is mainly generated from three previous studies that used different T. vaginalis isolates as an experimental model. Whether T. vaginalis exosomes comprise a common set of proteins (core exosome proteome) is still unclear. METHODS: To explore the core exosome proteome in T. vaginalis, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the contents of sucrose ultracentrifugation-enriched exosome and supernatant fractions isolated from six isolates. RESULTS: Transmission electron microscopy (TEM) confirmed the presence of exosomes in the enriched fraction. Proteomic analysis identified a total of 1870 proteins from exosomal extracts. There were 1207 exosomal-specific proteins after excluding 436 'non-core exosomal proteins'. Among these, 72 common exosomal-specific proteins were expressed in all six isolates. Compared with three published T. vaginalis exosome proteome datasets, we identified 16 core exosomal-specific proteins. These core exosomal-specific proteins included tetraspanin (TvTSP1), the classical exosome marker, and proteins mainly involved in catalytic activity and binding such as ribosomal proteins, ras-associated binding (Rab) proteins, and heterotrimeric G proteins. CONCLUSIONS: Our study highlighted the importance of using supernatant fraction from exosomal extract as a control to eliminate 'non-core exosomal proteins'. We compiled a reference core exosome proteome of T. vaginalis, which is essential for developing a fundamental understanding of exosome-mediated cell communication and host-parasite interaction.

Microglia are not protective against cryptococcal meningitis.

Microglia provide protection against a range of brain infections including bacteria, viruses and parasites, but how these glial cells respond to fungal brain infections is poorly understood. We investigated the role of microglia in the context of cryptococcal meningitis, the most common cause of fungal meningitis in humans. Using a series of transgenic- and chemical-based microglia depletion methods we found that, contrary to their protective role during other infections, loss of microglia did not affect control of Cryptococcus neoformans brain infection which was replicated with several fungal strains. At early time points post-infection, we found that microglia depletion lowered fungal brain burdens, which was related to intracellular residence of C. neoformans within microglia. Further examination of extracellular and intracellular fungal populations revealed that C. neoformans residing in microglia were protected from copper starvation, whereas extracellular yeast upregulated copper transporter CTR4. However, the degree of copper starvation did not equate to fungal survival or abundance of metals within different intracellular niches. Taken together, these data show how tissue-resident myeloid cells may influence fungal phenotype in the brain but do not provide protection against this infection, and instead may act as an early infection reservoir.

Effect of Radiotherapy on Sino-nasal Function in Nasopharynx Cancer Patients.

Introduction: Radiotherapy causes significant nasal comorbidity in nasopharynx cancer (NPC) patients. However, the literature addressing the sino-nasal quality of life (QoL) of those patients, especially on structural and functional changes after radiotherapy, is limited. Method: It is a case-control study with 14 NPC groups and 14 healthy control group. The sino-nasal QoL, including the olfactory threshold using Butanol Threshold Test (BTT), the olfactory identification level using the University of Pennsylvania Smell Identification Test (UPSIT), nasal symptoms using the sino-nasal outcome test (SNOT-22) questionnaire, nasal cross-sectional area, nasal flow, and nasal resistance using the acoustic rhinometry and rhinomanometry, were measured and compared. Result: The mean BTT score of the control group was higher than that of the NPC group (5.17 vs 2.71). The UPSIT score of the control group was higher than that of the NPC group (31.93 vs 25.14). The mean SNOT-22 score of control group was lower than that of the NPC group of (16.71 vs 37.71). All 3 results are statistically significant (P < .05). However, there is no statistical difference in nasal cross-sectional area, nasal flow, and nasal resistance between these 2 groups. Conclusion: In this study, we concluded that NPC patients who received radiotherapy suffered a worsening of sino-nasal functional changes, including the olfaction threshold, olfaction identification, and nasal symptoms. However, the sino-nasal structural changes on nasal cross-sectional area, nasal flow, and nasal resistance after radiation remain questionable.

Highly efficient rare-earth free vanadate phosphors for WLEDs.

Yellow-green emitting phosphors of vanadate Ca5Mg4(VO4)6 (CMV) doped with different concentrations of Ta5+ ions were synthesized by a solid-state reaction method. The formation of single-phase compounds with a garnet structure was verified by X-ray diffraction (XRD), Rietveld refinement calculations and energy-dispersive X-ray spectroscopy. Different luminescence properties of CMV phosphors such as spectral shift, luminescence lifetime, quantum efficiency, color coordinates and Stokes shift were measured and have been discussed in detail. PLE and PL spectra showed that CMV : xTa5+ (0 ≤ x ≤ 5%) phosphors could match well to 365 nm LED chips, and showed bright yellow-green emission in the visible range of 400-750 nm, with a peak at 544 nm, which is attributed to the charge transfer (CT) of an electron from the 2p orbital of the oxygen atom to the vacant 3d orbital of V5+ ions in the tetrahedral [VO4]3- group. Compared with the CMV host, the integrated luminescence intensity of CMV : 0.5%Ta5+ increased by 26.31%, and the quantum efficiency increased by 15.98%. The phenomenon can be ascribed to the substitution of V5+ ions by the large Ta5+ ions, which resulted in the squeezed and distorted VO4 tetrahedron. Finally, the white light emitting diode (WLED) devices prepared with UV WLED chips and the CMV : 0.5%Ta5+ phosphor exhibited excellent color temperature (4083 K) and CIE coordinates (0.3677, 0.3409). The CMV : 0.5%Ta5+ phosphor can be considered as a potential yellow-green emitting phosphor in the solid-state lighting field.

Update current understanding of neurometabolic disorders related to lysine metabolism.

Lysine, as an essential amino acid, predominantly undergoes metabolic processes through the saccharopine pathway, whereas a smaller fraction follows the pipecolic acid pathway. Although the liver is considered the primary organ for lysine metabolism, it is worth noting that lysine catabolism also takes place in other tissues and organs throughout the body, including the brain. Enzyme deficiency caused by pathogenic variants in its metabolic pathway may lead to a series of neurometabolic diseases, among which glutaric aciduria type 1 and pyridoxine-dependent epilepsy have the most significant clinical manifestations. At present, through research, we have a deeper understanding of the multiple pathophysiological mechanisms related to these diseases, including intracerebral accumulation of neurotoxic metabolites, imbalance between GABAergic and glutamatergic neurotransmission, energy deprivation due to metabolites, and the dysfunction of antiquitin. Because of the complexity of these diseases, their clinical manifestations are also diverse. The early implementation of lysine-restricted diets and supplementation with arginine and carnitine has reported positive impacts on the neurodevelopmental outcomes of patients. Presently, there is more robust evidence supporting the effectiveness of these treatments in glutaric aciduria type 1 compared with pyridoxine-dependent epilepsy.

Metabolomics analysis reveals changes related to pseudocyst formation induced by iron depletion in Trichomonas vaginalis.

BACKGROUND: Iron is an essential element for cellular functions, such as energy metabolism. Trichomonas vaginalis, a human urogenital tract pathogen, is capable of surviving in the environment without sufficient iron supplementation. Pseudocysts (cyst-like structures) are an environmentally tolerated stage of this parasite while encountering undesired conditions, including iron deficiency. We previously demonstrated that iron deficiency induces more active glycolysis but a drastic downregulation of hydrogenosomal energy metabolic enzymes. Therefore, the metabolic direction of the end product of glycolysis is still controversial. METHODS: In the present work, we conducted an LC‒MS-based metabolomics analysis to obtain accurate insights into the enzymatic events of T. vaginalis under iron-depleted (ID) conditions. RESULTS: First, we showed the possible digestion of glycogen, cellulose polymerization, and accumulation of raffinose family oligosaccharides (RFOs). Second, a medium-chain fatty acid (MCFA), capric acid, was elevated, whereas most detected C18 fatty acids were reduced significantly. Third, amino acids were mostly reduced, especially alanine, glutamate, and serine. Thirty-three dipeptides showed significant accumulation in ID cells, which was probably associated with the decrease in amino acids. Our results indicated that glycogen was metabolized as the carbon source, and the structural component cellulose was synthesized at same time. The decrease in C18 fatty acids implied possible incorporation in the membranous compartment for pseudocyst formation. The decrease in amino acids accompanied by an increase in dipeptides implied incomplete proteolysis. These enzymatic reactions (alanine dehydrogenase, glutamate dehydrogenase, and threonine dehydratase) were likely involved in ammonia release. CONCLUSION: These findings highlighted the possible glycogen utilization, cellulose biosynthesis, and fatty acid incorporation in pseudocyst formation as well as NO precursor ammonia production induced by iron-depleted stress.

Barley HOMOCYSTEINE METHYLTRANSFERASE 2 confers drought tolerance by improving polyamine metabolism.

Drought stress poses a serious threat to crop production worldwide. Genes encoding homocysteine methyltransferase (HMT) have been identified in some plant species in response to abiotic stress, but its molecular mechanism in plant drought tolerance remains unclear. Here, transcriptional profiling, evolutionary bioinformatics, and population genetics were conducted to obtain insight into the involvement of HvHMT2 from Tibetan wild barley (Hordeum vulgare ssp. agriocrithon) in drought tolerance. We then performed genetic transformation coupled with physio-biochemical dissection and comparative multiomics approaches to determine the function of this protein and the underlying mechanism of HvHMT2-mediated drought tolerance. HvHMT2 expression was strongly induced by drought stress in tolerant genotypes in a natural Tibetan wild barley population and contributed to drought tolerance through S-adenosylmethionine (SAM) metabolism. Overexpression of HvHMT2 promoted HMT synthesis and efficiency of the SAM cycle, leading to enhanced drought tolerance in barley through increased endogenous spermine and less oxidative damage and growth inhibition, thus improving water status and final yield. Disruption of HvHMT2 expression led to hypersensitivity under drought treatment. Application of exogenous spermine reduced accumulation of reactive oxygen species (ROS), which was increased by exogenous mitoguazone (inhibitor of spermine biosynthesis), consistent with the association of HvHMT2-mediated spermine metabolism and ROS scavenging in drought adaptation. Our findings reveal the positive role and key molecular mechanism of HvHMT2 in drought tolerance in plants, providing a valuable gene not only for breeding drought-tolerant barley cultivars but also for facilitating breeding schemes in other crops in a changing global climate.

Adoptive Transfer of Cryptococcus neoformans-Specific CD4 T-Cells to Study Anti-fungal Lymphocyte Responses In Vivo.

CD4 T-cells are important for long-term control and clearance of several fungal infections in humans, particularly those caused by Cryptococcus species. Understanding the mechanisms underlying protective T-cell immunity against fungal infection is critical for developing mechanistic insights into the pathogenesis of the disease. Here, we describe a protocol that enables analysis of fungal-specific CD4 T-cell responses in vivo, using adoptive transfer of fungal-specific T-cell receptor (TCR) transgenic CD4 T-cells. While the protocol here uses a TCR transgenic model reactive to peptide deriving from Cryptococcus neoformans, this method could be adapted to other fungal infection experimental settings.

Identification of key genes, biological functions, and pathways of empagliflozin by network pharmacology and its significance in the treatment of type 2 diabetes mellitus.

Background: To explore the key genes, biological functions, and pathways of empagliflozin in the treatment of type 2 diabetes mellitus (T2DM) through network pharmacology. Methods: The TCMSP (a traditional Chinese medicine system pharmacology database and analysis platform) was used to screen empagliflozin's active components and targets. The target genes of T2DM were screened according to the GeneCards and OMIM databases, and a Venn diagram was constructed to obtain the target for T2DM treatment. Cytoscape 3.7.2 software was adopted to construct the drug-component-target-disease network. Functional annotation of Gene Ontology (GO) and enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed using R software. Results: Target genes with a probability >0 were selected, among which Compound 012, Compound 060, Compound 093, Compound 111, and Compound 119 Swiss Target Prediction suggested that no similar active substances or predictable target genes were found. A "compound-target gene-disease" network was constructed, in which SLC5A2, SLC5A1, SLC5A4, SLC5A11, ADK, and ADORA2A were the core genes of T2DM. The key factors of the GO summary map included chemical reaction, membrane organelle, protein binding, and so on. The KEGG pathway summary map included the AMPK pathway, insulin resistance, the MAPK pathway, longevity-related pathway regulation, and so on. The top 10 pathways were endocrine resistance, the NF-κB signaling pathway, the HIF-1 signaling pathway, apoptosis, cell senescence, the Ras signaling pathway, the MAPK signaling pathway, the FoxO signaling pathway, the P13K-Akt signaling pathway, and the p53 signaling pathway. The binding of active compounds to key proteins was verified based on the Swiss Dock database, and the molecular docking of 193 bioactive compounds was finally verified. Among them, SLC5A2, SLC5A1, LDHA, KLK1, KLF5, and GSTP1 had better binding to the protein molecules. Conclusions: Empagliflozin may regulate the targets of SLC5A2, SLC5A1, LDHA, KLK1, KLF5, and GSTP1. There are numerous ways of treating T2DM with empagliflozin, including by regulating apoptosis, cell aging, as well as the NF-κB, HIF-1HIF-1, Ras, MAPK, FoxO, P13K-Akt, and p53 pathways.

Similar evolutionary trajectories in an environmental Cryptococcus neoformans isolate after human and murine infection.

A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from an immunocompromised patient with cryptococcosis based on molecular analyses available in 2000. Here, we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MATα strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including eight nonsynonymous changes involving seven genes. To ascertain whether changes in these genes are selected for during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frameshift mutation in one of the seven genes altered in the human sample (LQVO5_000317), a gene predicted to encode an SWI-SNF chromatin-remodeling complex protein. In addition, both cockatoo and patient strains as well as mouse-passaged isolates obtained from brain tissue had a premature stop codon in a homologue of ZFC3 (LQVO5_004463), a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and reverted to a full-length protein in the mouse-passaged isolates obtained from lung tissue. The patient strain and mouse-passaged isolates show variability in virulence factors, with differences in capsule size, melanization, rates of nonlytic expulsion from macrophages, and amoeba predation resistance. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.

Identification of Endosymbiotic Virus in Small Extracellular Vesicles Derived from Trichomonas vaginalis.

Accumulated evidence suggests that the endosymbiotic Trichomonasvirus (TVV) may play a role in the pathogenesis and drug susceptibility of Trichomonas vaginalis. Several reports have shown that extracellular vesicles (EVs) released from TVV-positive (TVV+) trichomonads can modulate the immune response in human vaginal epithelial cells and animal models. These results prompted us to examine whether EVs released from TVV+ isolates contained TVV. We isolated small extracellular vesicles (sEVs) from six T. vaginalis isolates that were either TVV free (ATCC 50143), harbored a single (ATCC 30236, ATCC 30238, T1), two (ATCC PRA-98), or three TVV subspecies (ATCC 50148). The presence of TVV subspecies in the six isolates was observed using reverse transcription-polymerase chain reaction (RT-PCR). Transmission electron microscopy (TEM) confirmed the presence of cup-shaped sEVs with a size range from 30-150 nm. Trichomonas vaginalis tetraspanin (TvTSP1; TVAG_019180), the classical exosome marker, was identified in all the sEV preparations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that all the sEVs isolated from TVV+ isolates contain viral capsid proteins derived from the same TVV subspecies in that isolate as demonstrated by RT-PCR. To provide more comprehensive information on the TVV subspecies population in other T. vaginalis isolates, we investigated the distribution of TVV subspecies in twenty-four isolates by mining the New-Generation Sequencing (NGS) RNAseq datasets. Our results should be beneficial for future studies investigating the role of TVV on the pathogenicity of T. vaginalis and the possible transmission of virus subspecies among different isolates via sEVs.

Optimizing safety-measure combinations to address construction risks.

Objectives. Most methods used to develop construction risk responses address the risk-mitigation optimization problem by solving the objective functions. They are passively achieved by satisfying constraint conditions, which are not adequate for efficient construction management. This study aims to provide an active optimization strategy for selecting risk responses. Methods. We combined set pair analysis (SPA) with the technique for order preference by similarity to an ideal solution (TOPSIS) to control the construction risks to an acceptable level instead of excessively to the minimum level. SPA is employed to assess the pre-mitigation and post-mitigation risk levels based on the uncertainty theory, and the TOPSIS is used to rank safety measures based on their risk-mitigation effects. A case study of concrete pumping for a super high-rise building was used to exemplify how the proposed optimization model assists risk control and to validate its reasonability. Conclusion. The developed TOPSIS-SPA-based method figures out the optimal safety-measure combination reducing construction risks economically to an acceptable level with the fewest number of measures. The findings can assist decision-makers in formulating cost-effective risk-control schemes.

Analysis of Zuojinwan Based on Ancient Literature / 中国实验方剂学杂志

Zuojinwan originated from Danxi′s Experiential Therapy （《丹溪心法》） in the Yuan dynasty. It is a representative prescription for the treatment of liver fire invading stomach syndrome， and is also one of the typical prescriptions of the anti-adjuvant method of traditional Chinese medicine （TCM）. In this paper， the method of bibliometrics was used to systematically sort out the ancient books of Zuojinwan， and 729 relevant literature data were obtained. After certain retrieval and screening， 57 ancient books of TCM were finally obtained. The statistics and analysis were carried out from the aspects of prescription source， historical evolution， composition， functions， evolution of prescription meaning， prescription dose， and preparation and usage of Zuojinwan. It was found that Zuojinwan was composed of Coptis chinensis rhizoma and Euodia rutaecarpa fructus in a ratio of 6∶1. It was mainly used for the treatment of liver fire invading stomach syndrome. The symptoms included pain in chest and hypochondrium， vomiting and bitter mouth， noisy acid-swallowing， red tongue coating yellow， and pulse string number. Later medical records recorded that Zuojinwan was mostly consistent with the original prescription. It mainly treated various diseases caused by liver fire， including left by liver fire， including left hypochondriac pain， swallowing acid and vomiting acid， tendon hernia and lump， epigastric pain， bitter mouth pulse string， head pain， diarrhea， gonorrhea， cold and hot， abdominal pain， alcohol wet yellowing， silence of oral dysentery and so on. There was little controversy in the analysis of relevant prescriptions. In the past dynasties， pills was mainly used， which was consistent with the original prescription. In modern times， it is mainly water flooding for pills or steamed cakes for pills， warm boiling water to serve 6 g， taking 2-3 g per time， the history is basically the same. In this paper， through the excavation， collation and systematic analysis of the ancient literature of TCM that recorded Zuojinwan， we hope to provide the literature basis for the development， inheritance and utilization of this famous classical formulas.

Sporadic Dissemination of tet(X3) and tet(X6) Mediated by Highly Diverse Plasmidomes among Livestock-Associated Acinetobacter.

The emergence of high-level tigecycline resistance mediated by plasmid-borne tet(X) genes greatly threatens the clinical effectiveness of tigecycline. However, the dissemination pattern of plasmid-borne tet(X) genes remains unclear. We here recovered tet(X)-positive Acinetobacter isolates from 684 fecal and environmental samples collected at six livestock farms. Fifteen tet(X)-positive Acinetobacter isolates were identified, mainly including 9 tet(X3)- and 5 tet(X6)-positive Acinetobacter towneri isolates. A clonal dissemination of tet(X3)-positive A. towneri was detected in a swine farm, while the tet(X6)-positive A. towneri isolates mainly disseminated sporadically in the same farm. A tet(X3)-carrying plasmid (pAT181) was self-transmissible from a tigecycline-susceptible A. towneri strain to Acinetobacter baumannii strain ATCC 17978, causing 64- to 512-fold increases in the MIC values of tetracyclines (including tigecycline). Worrisomely, pAT181 was stably maintained and increased the growth rate of strain ATCC 17978. Further identification of tet(X) genes in 10,680 Acinetobacter genomes retrieved from GenBank revealed that tet(X3) (n = 249), tet(X5)-like (n = 61), and tet(X6) (n = 53) were the prevalent alleles mainly carried by four species, and most of them were livestock associated. Phylogenetic analysis showed that most of the tet(X3)- and tet(X6)-positive isolates disseminated sporadically. The structures of the tet(X3), and tet(X6) plasmidomes were highly diverse, and no epidemic plasmids were detected. However, cross-species and cross-region transmissions of tet(X3) might have been mediated by several plasmids in a small proportion of strains. Our study implies that horizontal plasmid transfer may be insignificant for the current dissemination of tet(X3) and tet(X6) in Acinetobacter strains. Continuous surveillance for tet(X) genes in the context of One Health is necessary to prevent them from transmitting to humans. IMPORTANCE Recently identified plasmid-borne tet(X) genes have greatly challenged the efficiency of tigecycline, a last-resort antibiotic for severe infection, while the dissemination pattern of the plasmid-borne tet(X) genes remains unclear. In this study, we identified a clonal dissemination of tet(X3)-positive A. towneri isolates on a swine farm, while the tet(X6)-positive A. towneri strains mainly disseminated sporadically on the same farm. Of more concern, a tet(X3)-carrying plasmid was found to be self-transmissible, resulting in enhanced tigecycline resistance and growth rate of the recipient. Further exploration of a global data set of tet(X)-positive Acinetobacter genomes retrieved from GenBank revealed that most of the tet(X3)- and tet(X6)-positive isolates shared a highly distant relationship, and the structures of tet(X3) and tet(X6) plasmidomes exhibited high mosaicism. Notably, some of the isolates belong to Acinetobacter species that are opportunistic pathogens and have been identified as sources of nosocomial infections, raising concerns about transmission to humans in the future. Our study evidenced the sporadic dissemination of tet(X3) and tet(X6) in Acinetobacter strains and the necessity of continuous surveillance for tet(X) genes in the context of One Health.

The Cyclin Cln1 Controls Polyploid Titan Cell Formation following a Stress-Induced G2 Arrest in Cryptococcus.

The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the in vivo and in vitro cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells. We identified unbudded 2C cells, referred to as a G2 arrest, produced both in vivo and in vitro in response to various stresses. Deletion of the nonessential cyclin Cln1 resulted in overproduction of titan cells in vivo and transient morphology defects upon release from stationary phase in vitro. Using a copper-repressible promoter PCTR4-CLN1 strain and a two-step in vitro titan cell formation assay, our in vitro studies revealed Cln1 functions after the G2 arrest. These studies highlight unique cell cycle alterations in C. neoformans that ultimately promote genomic diversity and virulence in this important fungal pathogen. IMPORTANCE Dysregulation of the cell cycle underlies many human genetic diseases and cancers, yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen Cryptococcus neoformans generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease. We analyzed the C. neoformans in vivo and in vitro cell cycles to identify changes required to generate the polyploid titan cells. C. neoformans paused cell cycle progression in response to various environmental stresses after DNA replication and before morphological changes associated with cell division, referred to as a G2 arrest. Release from this G2 arrest was coordinated by the cyclin Cln1. Reduced CLN1 expression after the G2 arrest was associated with polyploid titan cell production. These results demonstrate a mechanism to generate genomic diversity in eukaryotic cells through manipulation of the cell cycle that has broad disease implications.

Amoeba Predation of Cryptococcus neoformans Results in Pleiotropic Changes to Traits Associated with Virulence.

Amoeboid predators, such as amoebae, are proposed to select for survival traits in soil microbes such as Cryptococcus neoformans; these traits can also function in animal virulence by defeating phagocytic immune cells, such as macrophages. Consistent with this notion, incubation of various fungal species with amoebae enhanced their virulence, but the mechanisms involved are unknown. In this study, we exposed three strains of C. neoformans (1 clinical and 2 environmental) to predation by Acanthamoeba castellanii for prolonged times and then analyzed surviving colonies phenotypically and genetically. Surviving colonies comprised cells that expressed either pseudohyphal or yeast phenotypes, which demonstrated variable expression of traits associated with virulence, such as capsule size, urease production, and melanization. Phenotypic changes were associated with aneuploidy and DNA sequence mutations in some amoeba-passaged isolates, but not in others. Mutations in the gene encoding the oligopeptide transporter (CNAG_03013; OPT1) were observed among amoeba-passaged isolates from each of the three strains. Isolates derived from environmental strains gained the capacity for enhanced macrophage toxicity after amoeba selection and carried mutations on the CNAG_00570 gene encoding Pkr1 (AMP-dependent protein kinase regulator) but manifested reduced virulence in mice because they elicited more effective fungal-clearing immune responses. Our results indicate that C. neoformans survival under constant amoeba predation involves the generation of strains expressing pleiotropic phenotypic and genetic changes. Given the myriad potential predators in soils, the diversity observed among amoeba-selected strains suggests a bet-hedging strategy whereby variant diversity increases the likelihood that some will survive predation.IMPORTANCECryptococcus neoformans is a ubiquitous environmental fungus that is also a leading cause of fatal fungal infection in humans, especially among immunocompromised patients. A major question in the field is how an environmental yeast such as C. neoformans becomes a human pathogen when it has no need for an animal host in its life cycle. Previous studies showed that C. neoformans increases its pathogenicity after interacting with its environmental predator amoebae. Amoebae, like macrophages, are phagocytic cells that are considered an environmental training ground for pathogens to resist macrophages, but the mechanism by which C. neoformans changes its virulence through interactions with protozoa is unknown. Our study indicates that fungal survival in the face of amoeba predation is associated with the emergence of pleiotropic phenotypic and genomic changes that increase the chance of fungal survival, with this diversity suggesting a bet-hedging strategy to ensure that some forms survive.

Cryptococcus neoformans-Infected Macrophages Release Proinflammatory Extracellular Vesicles: Insight into Their Components by Multi-omics.

Cryptococcus neoformans causes deadly mycosis in immunocompromised individuals. Macrophages are key cells fighting against microbes. Extracellular vesicles (EVs) are cell-to-cell communication mediators. The roles of EVs from infected host cells in the interaction with Cryptococcus remain uninvestigated. Here, EVs from viable C. neoformans-infected macrophages reduced fungal burdens but led to shorter survival of infected mice. In vitro, EVs induced naive macrophages to an inflammatory phenotype. Transcriptome analysis showed that EVs from viable C. neoformans-infected macrophages activated immune-related pathways, including p53 in naive human and murine macrophages. Conserved analysis demonstrated that basic cell biological processes, including cell cycle and division, were activated by infection-derived EVs from both murine and human infected macrophages. Combined proteomics, lipidomics, and metabolomics of EVs from infected macrophages showed regulation of pathways such as extracellular matrix (ECM) receptors and phosphatidylcholine. This form of intermacrophage communication could serve to prepare cells at more distant sites of infection to resist C. neoformans infection.IMPORTANCECryptococcus neoformans causes cryptococcal meningitis, which is frequent in patients with HIV/AIDS, especially in less-developed countries. The incidence of cryptococcal meningitis is close to 1 million each year globally. Macrophages are key cells that protect the body against microbes, including C. neoformans Extracellular vesicles are a group of membrane structures that are released from cells such as macrophages that modulate cell activities via the transfer of materials such as proteins, lipids, and RNAs. In this study, we found that Cryptococcus neoformans-infected macrophages produce extracellular vesicles that enhance the inflammatory response in Cryptococcus-infected mice. These Cryptococcus neoformans-infected macrophage vesicles also showed higher fungicidal biological effects on inactivated macrophages. Using omics technology, unique protein and lipid signatures were identified in these extracellular vesicles. Transcriptome analysis showed that these vesicles activated immune-related pathways like p53 in naive macrophages. The understanding of this intermacrophage communication could provide potential targets for the design of therapeutic agents to fight this deadly mycosis.

CMAP changes upon symptom onset and during treatment in spinal muscular atrophy patients: lessons learned from newborn screening.

PURPOSE: Early identification and treatment of spinal muscular atrophy (SMA) are crucial but difficult. In this study, we aimed to assess the significance of compound motor action potential (CMAP) amplitude in patients identified through a newborn screening program. METHODS: We initiated a large-scale population newborn screening program for SMA in Taiwan in 2014. Patients had access to treatment through clinical trials or expanded use programs. Symptomatic patients were evaluated regularly, including CMAP exams. RESULTS: Among 364,000 screened newborns, 21 were diagnosed with SMA. The incidence of SMA was around 1 in 17,000 live births, and 70% developed SMA type 1. All infants with two SMN2 copies became symptomatic before the age of 1 month. CMAP amplitudes of 12 newborns were available, including 6 who were subsequently treated with nusinersen. We found that a rapid decrease of CMAP amplitude was an early predictor of symptom onset. Pretreatment CMAP and rapid increment of post-treatment CMAP could predict better treatment outcomes. CONCLUSION: This study prospectively demonstrated the incidence of SMA and its types. Our results imply the importance of pretreatment CMAP amplitude and rapid reversal of post-treatment CMAP amplitude with regard to disease presentation and also treatment outcomes.