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Abstract Congenital heart disease (CHD) is the most common type of birth defect and the main noninfectious cause of death during the neonatal stage. The non-POU domain containing, octamer-binding gene, NONO, performs a variety of roles involved in DNA repair, RNA synthesis, transcriptional and post-transcriptional regulation. Currently, hemizygous loss-of-function mutation of NONO have been described as the genetic origin of CHD. However, essential effects of NONO during cardiac development have not been fully elucidated. In this study, we aim to understand role of Nono in cardiomyocytes during development by utilizing the CRISPR/Cas9 gene editing system to deplete Nono in the rat cardiomyocytes H9c2. Functional comparison of H9c2 control and knockout cells showed that Nono deficiency suppressed cell proliferation and adhesion. Furthermore, Nono depletion significantly affected the mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis, resulting in H9c2 overall metabolic deficits. Mechanistically we demonstrated that the Nono knockout impeded the cardiomyocyte function by attenuating phosphatidyl inositol 3 kinase-serine/threonine kinase (Pi3k/Akt) signaling via the assay for transposase-accessible chromatin using sequencing in combination with RNA sequencing. From these results we propose a novel molecular mechanism of Nono to influence cardiomyocytes differentiation and proliferation during the development of embryonic heart. We conclude that NONO may represent an emerging possible biomarkers and targets for the diagnosis and treatment of human cardiac development defects.

Abstract Excessive proliferation and migration of airway smooth muscle cell (ASMC) contribute to asthma pathogenesis. Long noncoding RNAs (lncRNAs) are reported to take part in asthma pathogenesis. This study is targeted at deciphering the role of the lncRNA antisense noncoding RNA in the INK4 locus (ANRIL) in ASMC proliferation, migration and extracellular matrix (ECM) deposition. qRT‐PCR was performed to determine ANRIL, miR‐98‐5p, and cyclin D1 (CCND1) mRNA expression levels in transforming growth factor‐β1 (TGF‐β1)‐treated ASMCs. CCK‐8 and Transwell assays were employed to examine ASMC proliferation and migration, respectively. Dual‐luciferase reporter gene assay and RNA immunoprecipitation assay were carried out for analyzing the targeted relationship of miR‐98‐5p with ANRIL or CCND1 mRNA 3′‐UTR. The levels of CCND1 and ECM proteins (such as fibronectin, COL3A1, and COL1A2) in ASMCs were detected through Western blot. In this work, we found that ANRIL and CCND1 were up‐regulated in TGF‐β1‐treated ASMCs, whereas miR‐98‐5p was down‐regulated. ANRIL overexpression facilitated the proliferation, ECM deposition and migration of TGF‐β1‐induced ASMCs, while knocking down ANRIL had the opposite effect. Furthermore, ANRIL targeted miR‐98‐5p directly, and CCND1 was miR‐98‐5p's downstream target. ANRIL indirectly increased CCND1 expression in ASMCs via competitively binding to miR‐98‐5p. MiR‐98‐5p inhibition or CCND1 overexpression counteracted the inhibiting effect that ANRIL knockdown had on TGF‐β1‐stimulated ASMC proliferation, migration and ECM deposition. In conclusion, ANRIL indirectly up‐regulates CCND1 expression by targeting miR‐98‐5p to promote ASMC proliferation, migration and ECM deposition, thus facilitating the pathogenesis of asthma.

Cryptococcus neoformans causes opportunistic infection and potentially lethal immunopathology but therapeutic options are limited. Here the authors implicate myeloid derived suppressor cells during C. neoformans infection and suggest targeting arginase-1 production as a potential therapeutic strategy.

ABSTRACT Chronic pulmonary aspergillosis (CPA) is a chronic and progressive fungal disease with high morbidity and mortality. Avoiding diagnostic delay and misdiagnosis are concerns for CPA patients. However, diagnostic practice is poorly evaluated, especially in resource-constrained areas where Aspergillus antibody testing tools are lacking. This study aimed to investigate the diagnostic laboratory findings in a retrospective CPA cohort and to evaluate the performance of a novel Aspergillus IgG lateral flow assay (LFA; Era Biology, Tianjin, China). During January 2016 and December 2021, suspected CPA patients were screened at the Center for Infectious Diseases at Huashan Hospital. A total of 126 CPA patients were enrolled. Aspergillus IgG was positive in 72.1% with chronic cavitary pulmonary aspergillosis, 75.0% with chronic necrotizing pulmonary aspergillosis, 41.7% with simple aspergilloma, and 30.3% with Aspergillus nodule(s). The cavitary CPA subtypes had significantly higher levels of Aspergillus IgG. Aspergillus IgG was negative in 52 patients, who were finally diagnosed by histopathology, respiratory culture, and metagenomic next-generation sequencing (mNGS). Sputum culture was positive in 39.3% (42/107) of patients and Aspergillus fumigatus was the most common species (69.0%, 29/42). For CPA cohort versus controls, the sensitivity and specificity of the LFA were 55.6% and 92.7%, respectively. In a subgroup analysis, the LFA was highly sensitive for A. fumigatus-associated chronic cavitary pulmonary aspergillosis (CCPA; 96.2%, 26/27). Given the complexity of the disease, a combination of serological and non-serological tests should be considered to avoid misdiagnosis of CPA. The novel LFA has a satisfactory performance and allows earlier screening and diagnosis of CPA patients. IMPORTANCE There are concerns on avoiding diagnostic delay and misdiagnosis for chronic pulmonary aspergillosis due to its high morbidity and mortality. A proportion of CPA patients test negative for Aspergillus IgG. An optimal diagnostic strategy for CPA requires in-depth investigation based on real-world diagnostic practice, which has been rarely discussed. We summarized the clinical and diagnostic laboratory findings of 126 CPA patients with various CPA subtypes. Aspergillus IgG was the most sensitive test for diagnosing CPA. However, it was negative in 52 patients, who were finally diagnosed by non-serological tests, including biopsy, respiratory culture, and metagenomic next-generation sequencing. We also evaluated a novel Aspergillus IgG lateral flow assay, which showed a satisfactory performance in cavitary CPA patients and was highly specific to Aspergillus fumigatus. This study gives a full picture of the diagnostic practice for CPA patients in Chinese context and calls for early diagnosis of CPA with combined approaches.

The cerebrospinal fluid (CSF) immune responses in HIV-uninfected cryptococcal meningitis (CM) have not been well studied. In this study, we aimed to explore the phenotype of CSF immune response during the course of disease and to examine relationships between phenotypes and disease severity. We profiled the CSF immune response in 128 HIV-uninfected CM and 30 pulmonary cryptococcosis patients using a 27-plex Luminex cytokine kit. Principal component analyses (PCA) and logistic regression model were performed. Concentrations of 23 out of 27 cytokines and chemokines in baseline CSF were significantly elevated in CM patients compared with pulmonary cryptococcosis cases. In CM patients with Cryptococcus neoformans infection, IL-1ra, IL-9, and VEGF were significantly elevated in immunocompetent cases. Cytokine levels usually reached peaks within the first 2 weeks of antifungal treatment and gradually decreased over time. PCA demonstrated a co-correlated CSF cytokine and chemokine response consisting of Th1, Th2, and Th17 type cytokines. Prognostic analysis showed that higher scores for the PCs loading pro-inflammatory cytokines, IFN-γ, TNF-α, and IL-12; and anti-inflammatory cytokine, IL-4; and chemokines, Eotaxin, FGF-basis, and PDGF-bb; as well as lower scores for the PCs loading RANTES were associated with disease severity, as defined by a Glasgow Coma Scale of

As a new and safe food resource, Cyclocarya paliurus (CP), rich in flavonoids, exhibits hypouricemic, anti-inflammatory and antioxidative properties. However, whether CP aqueous extract (CPA) still possesses the ameliorative effects on hyperuricemia (HUA) and urate-induced renal inflammation remains vacant. In HUA mice, CPA could significantly reduce serum uric acid (UA), alleviate kidney damage and liver steatosis, inhibit xanthine oxidase (XOD) activity, ameliorate renal urate transporters and renal inflammation by inhibiting NLRP3 inflammasome and interleukin-1β (IL-1β). Moreover, the flavonoids quercetin-3-O-β-D-galactopyranoside, quercetin-3-O-β-D-glucopyranoside, and quercetin-3-O-α-L-rhamnopyranoside from CPA could significantly reduce IL-1β and interleukin-6 (IL-6) in MSU-challenged THP-1 cells. Noticeably, quercetin-3-O-β-D-galactopyranoside could inhibit the intracellular XOD activity, reduce intracellular ROS and the expression of pro-IL-1β, IL-1β, and NLRP3 inflammasome. Collectively, CPA shows appreciable therapeutic effects on HUA and evoked renal inflammation. Quercetin-3-O-β-D-galactopyranoside, quercetin-3-O-β-D-glucopyranoside, and quercetin-3-O-α-L-rhamnopyranoside might be the major active compounds of CPA, which obtain the favorable effects through blocking the activation of ROS/NLRP3/IL-1β pathway.

Summary Diallyl sulfide (DAS) and diallyl disulfide (DADS), two constituents of garlic, can inhibit quorum sensing (QS) systems of Pseudomonas aeruginosa. However, the differences in the mechanism of QS inhibition between DAS and DADS, and the functional chemical groups of these sulfides that contribute in QS inhibition have not been elucidated yet. We assumed that the sulfide group might play a key role in QS inhibition. To prove this hypothesis and to clarify these unsolved problems, in this study, we synthesized diallyl ether (DAE), and compared and investigated the effects of DAS and DAE on the growth and production of virulence factors, including Pseudomonas quinolone signal (PQS), elastase and pyocyanin, of P. aeruginosa PAO1. Transcriptome analysis and qRT‐PCR were used to compare and analyse the differentially expressed genes between the different treatment groups (DAS, DAE and control). The results indicated that DAS did not affect the growth dynamics of P. aeruginosa PAO1; however, DAS inhibited transcription of most of the QS system genes, including lasR, rhlI/rhlR and pqsABCDE/pqsR; thus, biosynthesis of the signal molecules C4‐HSL (encoded by rhlI) and PQS (encoded by pqsABCDE) was inhibited. Furthermore, DAS inhibited the transcription of virulence genes regulated by the QS systems, including rhlABC, lasA, lasB, lecA and phzAB, phzDEFG, phzM and phzS that encode for rhamnolipid, exoprotease, elastase, lectin and pyocyanin biosynthesis respectively. DAS also enhanced the expression of the key genes involved in the biosynthesis of three B vitamins: folate, thiamine and riboflavin. In conclusion, DAS suppressed the production of some virulence factors toxic to the host and enhanced the production of some nutrition factors beneficial to the host. These actions of DAS may be due to its thioether group. These findings would be significant for development of an effective drug to control the virulence and pathogenesis of the opportunistic pathogen P. aeruginosa.

We conducted a systematic literature review to obtain risk population–based fungal disease incidence or prevalence data from China. Data were categorized by risk factors and extrapolated by using most recent demographic figures. A total of 71,316,101 cases (5.0% of the population) were attributed to 12 risk factors and 17 fungal diseases. Excluding recurrent Candida vaginitis (4,057/100,000 women) and onychomycosis (2,600/100,000 persons), aspergillosis (317/100,000 persons) was the most common problem; prevalence exceeded that in most other countries. Cryptococcal meningitis, an opportunistic infection, occurs in immunocompetent persons almost twice as often as AIDS. The pattern of fungal infections also varies geographically; Talaromyces marneffei is distributed mainly in the Pearl River Basin, and the Yangtze River bears the greatest histoplasmosis burden. New host populations, new endemic patterns, and high fungal burdens in China, which caused a huge impact on public health, underscore the urgent need for building diagnostic and therapeutic capacity.

Objective To summarize and analyze the etiology and clinical features of the third nervepalsy(TNP). Methods and Results The clinical data of patients with TNP diagnosed from January to October 2019 were retrospectively analyzed. A total of 32 patients with TNP were included. There were 17 cases (53.12%) of acute onset and 15 cases (46.88%) of subacute onset. Monocular involvement was found in 30 cases (93.75%) and binocular involvement in 2 cases (6.25%). There were 10 cases (31.25%) with headache, and 8 cases(25%)with pupil involvement. Theetiology wasdiagnosed as microcirculation disorder (16cases,50%), non⁃specific inflammation (11cases,34.38%), posterior communication aneurysm (2 cases, 6.25%), midbrain infarction (1 case, 3.13%), brain stem demystification (1 case, 3.13%) and eosinophilia (1 case, 3.13% ). Improvement of circulation (microcirculation disorder and midbrain infarction), anti⁃inflammatory and suppressive inflammatory response (non⁃specific inflammation, brainstem demyelination and eosinophilia) or interventional therapy (posterior communication aneurysm) were performed. After treatment, 31 cases (96.88%) showed remission of symptoms and 1 case (3.13%) had poor prognosis. Conclusions Microcirculation disorder is the main cause of TNP, and intracranial aneurysm is the most dangerous cause. Eye pain and pupil involvement are not the specific symptoms of intracranial aneurysms. Relevant examinations can actively improve the determination of the cause and appropriate treatment. Most patients have a good prognosis. DOI：10.3969/j.issn.1672⁃6731.2020.08.016

Abstract Cryptococcosis is one of the most common opportunistic infections in both immunocompetent and immunocompromised hosts. Although the cryptococcal antigen (CrAg) lateral flow assay (LFA) has been widely used in clinical settings due to its high sensitivity and specificity, the diagnostic value of a low CrAg LFA titers remains unclear. In this study, we performed a retrospective analysis of 149 HIV-negative patients with low CrAg LFA titers (≤1:10) in a Chinese tertiary hospital from January 2013 to December 2017, to evaluate the diagnostic value of low CrAg LFA titers in serum and cerebrospinal fluid (CSF) at different thresholds. Sensitivity and specificity of low CrAg LFA titers in patients with definitive diagnoses of cryptococcosis were 39.6% (95% CI, 29.7–50.1%) and 100% (95% CI, 69.2–100%), respectively, at a threshold of 1:10 in serum. A sensitivity of 72.9% (95% CI, 62.9–81.5%) and a decreased specificity of 70.0% (95% CI, 34.8–93.3%) were observed at a threshold of 1:5 in serum. No false-positive cases were identified in patients with low CrAg titers in CSF and all positive predictive values (PPVs) were 100%. Among the cases with low serum CrAg titers, lumbar puncture was performed in 97 patients and positive CSF CrAg titers were reported in 6 patients. In conclusion, the results of this study imply that low CrAg LFA titer, either in serum or CSF, is crucial for early diagnosis of cryptococcosis in HIV-negative patients, and lumbar puncture is recommended to be performed routinely for CSF testing when a positive low serum titer is reported. Cryptococcal meningitis should be considered seriously when the CSF CrAg titer is positive.

Arjunolic acid (AA), as the most abundant triterpenoid component in Cyclocarya paliurus (Batal) Iljinskaja, previously displayed delipidating effects in free fatty acid (FFA)-induced HepG2 steatosis cells. However, whether AA still possesses the ameliorative effects on nonalcoholic fatty liver disease (NAFLD) in normal hepatocytes and in vivo remains vacant and the detailed mechanisms are not defined. In vitro, AA dose-dependently alleviated lipid accumulation in FFA-challenged primary hepatocytes without cytotoxicity. In vivo, AA showed pleiotropic benefits, including attenuating adiposity, mitigating hepatic steatosis and inflammation, improving lipid disorders and insulin resistance, and restoring the impaired gut barrier. Mechanically, we found that AA indirectly activated Sirt1/AMPK-modulated lipid metabolism through elevating NAMPT-mediated NAD+ level, and triggering autophagy, together mediating the lipid-lowering effects. Collectively, our results convey that AA can substantially mitigate NAFLD via indirectly activating Sirt1/AMPK signaling, inducing autophagy and restoring gut barrier, and will be considered as a promising candidate for NAFLD therapy.

Abstract Background The 2010 Infectious Diseases Society of America (IDSA) guidelines for management of cryptococcal diseases recommend high dose fluconazole (≥ 800 mg/day), either alone or with other antifungal drugs, as alternative anticryptococcal choices. But evidence for its use in the treatment of HIV-uninfected cryptococcal meningitis (CM) remains sparse. Methods A retrospective analysis of HIV-uninfected CM patients who received fluconazole 800 mg/day for salvage therapy from January 2011 to December 2016 at Huashan Hospital, Shanghai, China was performed. Efficacy and safety were assessed, and mortality and prognostic factors evaluated. Results A total of 44 patients were studied including 19 refractory to amphotericin B induction therapy, 8 refractory to fluconazole consolidation therapy (400 mg/d), and 17 intolerant of antifungal drugs. For salvage, 11 patients received triple therapy of high dose fluconazole, amphotericin B and flucytosine, 20 received dual therapy of high dose fluconazole and flucytosine, 13 received monotherapy of high dose fluconazole. Median duration of high dose fluconazole in salvage regimens was 136.5 days (range, 1–667 days). Clinical response rates were 72.1% (31/43) and 83.7% (36/43) when assessed at 2 weeks and the end of salvage therapy, respectively. Adverse events possibly related to high dose fluconazole occurred in 54.5% (24/44) of the patients, and all were mild or moderate. From the initiation of salvage therapy, 1-year all-cause mortality was 13.6% (6 of 44 patients) among the study population with no significant difference in refractory or intolerant patients. Conclusions Adherence to guideline recommendations of high dose fluconazole, alone or in combination with other antifungals, was safe and often effective for salvage therapy of HIV-uninfected CM patients.

Abstract Background Thus far, the incidence, mortality, and temporal trend data of oropharyngeal cancers (OPC) in China were few. We estimated the incidence, mortality, and temporal patterns of OPC in China during 2008–2012 according to the data from 135 population-based cancer registries to better understand the epidemiological pattern of OPC and to provide more precise information for OPC control in China. Methods According to the data of diagnosed OPC reported to 135 cancer registries during 2008–2012, we calculated age-standardized rate of incidence and mortality by 2000 Chinese standard population (ASRIC and ASRMC) and by 1985 Segi’s world standard population (ASRIW and ASRMW) by age, sex, and geographic regions; annual percentage changes of OPC incidence and mortality were calculated using Joinpoint trend analysis. Results ASRIW and ASRMW were 2.22/100,000 person-years and 0.94/100,000 person-years, respectively. The incidence and mortality in urban areas were higher than those in rural areas. ASRIC and ASRIW of males were higher than those of females. The overall ASRIC of OPC was significantly increased by 6.2% annually between 2003 and 2006 (P = 0.038), but remained stable between 2007 and 2012 (P = 0.392). ASRIC and ASRMC of males and in rural areas were significantly increased in the last decade (P 0.05). Conclusions Across multiple cancer registries in China, there was an obvious increase in OPC in the recent decade, especially for incidence and mortality of males and in rural areas, whereas the rates of females remained stable. A healthy lifestyle should be advocated and early diagnosis and early treatment of OPC should be enhanced.

Abstract Background Butadiene is a platform chemical used as an industrial feedstock for the manufacture of automobile tires, synthetic resins, latex and engineering plastics. Currently, butadiene is predominantly synthesized as a byproduct of ethylene production from non-renewable petroleum resources. Although the idea of biological synthesis of butadiene from sugars has been discussed in the literature, success for that goal has so far not been reported. As a model system for methanol assimilation, Methylobacterium extorquens AM1 can produce several unique metabolic intermediates for the production of value-added chemicals, including crotonyl-CoA as a potential precursor for butadiene synthesis. Results In this work, we focused on constructing a metabolic pathway to convert crotonyl-CoA into crotyl diphosphate, a direct precursor of butadiene. The engineered pathway consists of three identified enzymes, a hydroxyethylthiazole kinase (THK) from Escherichia coli, an isopentenyl phosphate kinase (IPK) from Methanothermobacter thermautotrophicus and an aldehyde/alcohol dehydrogenase (ADHE2) from Clostridium acetobutylicum. The K m and k cat of THK, IPK and ADHE2 were determined as 8.35 mM and 1.24 s−1, 1.28 mM and 153.14 s−1, and 2.34 mM and 1.15 s−1 towards crotonol, crotyl monophosphate and crotonyl-CoA, respectively. Then, the activity of one of rate-limiting enzymes, THK, was optimized by random mutagenesis coupled with a developed high-throughput screening colorimetric assay. The resulting variant (THKM82V) isolated from over 3000 colonies showed 8.6-fold higher activity than wild-type, which helped increase the titer of crotyl diphosphate to 0.76 mM, corresponding to a 7.6% conversion from crotonol in the one-pot in vitro reaction. Overexpression of native ADHE2, IPK with THKM82V under a strong promoter mxaF in M. extorquens AM1 did not produce crotyl diphosphate from crotonyl-CoA, but the engineered strain did generate 0.60 μg/mL of intracellular crotyl diphosphate from exogenously supplied crotonol at mid-exponential phase. Conclusions These results represent the first step in producing a butadiene precursor in recombinant M. extorquens AM1. It not only demonstrates the feasibility of converting crotonol to key intermediates for butadiene biosynthesis, it also suggests future directions for improving catalytic efficiency of aldehyde/alcohol dehydrogenase to produce butadiene precursor from methanol.

Background: Cryptococcal meningitis (CM) is a significant source of mortality, the pathogenesis of which has not been fully understood, especially in non-HIV infected populations. We aimed to explore the potential genetic influence of Toll-like receptor (TLR) on non-HIV CM. Methods: This observational cohort study was done in two stages: a discovery stage and a validation stage. A case-control genetic association study was conducted between 159 non-HIV CM patients and 468 healthy controls. TLR SNPs significantly related to susceptibility went further validation in a second cohort of 583 subjects from a certain district. Associations among TLR SNPs, cerebrospinal fluid (CSF) cytokine concentrations, and clinical severity were explored in a third cohort of 99 previously untreated non-HIV CM patients. Logistic regression model was used to determine the independent predictors for disease severity. Findings: In the discovery stage, eight TLR SNPs exhibited significant genetic susceptibility to non-HIV CM, one of which was validated in a population validation of HIV-infected cases while none survived in non-HIV cases. CSF cytokine detections showed that 18 cytokines were significantly over-expressed in severely ill patients. Two of the 8 SNPs (rs5743604 and rs3804099) were also significantly associated with disease severity. Specifically, the rs3804099 C/T genotype was further found to be correlated to 12 of the 18 up-regulated cytokines in severe patients. In addition, high levels of interleukin (IL)-10 in CSF (OR 2·97, 95% CI 1·49–5·90; p = 0·002) was suggested as an independent predictor for severity after adjusted for possible confounders. Interpretation: TLR participates in both the occurrence and the pathogenesis of non-HIV CM. The in situ immune responses of CM were under genetic influence of TLR and contributed to disease severity. Fund: National Natural Science Foundation of China and National Key Basic Research Program of China (973 Program). Keywords: Toll-like receptor, Non-HIV cryptococcal meningitis, Genetic susceptibility, Disease severity, CSF cytokine

Abstract Background 3-Hydroxypropionic acid (3-HP) is an important platform chemical, serving as a precursor for a wide range of industrial applications such as the production of acrylic acid and 1,3-propanediol. Although Escherichia coli or Saccharomyces cerevisiae are the primary industrial microbes for the production of 3-HP, alternative engineered hosts have the potential to generate 3-HP from other carbon feedstocks. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating 3-HP from one-carbon feedstock methanol. Results Here we constructed a malonyl-CoA pathway by heterologously overexpressing the mcr gene to convert methanol into 3-HP in M. extorquens AM1. The engineered strains demonstrated 3-HP production with initial titer of 6.8 mg/l in shake flask cultivation, which was further improved to 69.8 mg/l by increasing the strength of promoter and mcr gene copy number. In vivo metabolic analysis showed a significant decrease of the acetyl-CoA pool size in the strain with the highest 3-HP titer, suggesting the supply of acetyl-CoA is a potential bottleneck for further improvement. Notably, 3-HP was rapidly degraded after the transition from exponential phase to stationary phase. Metabolomics analysis showed the accumulation of intracellular 3-hydroxypropionyl-CoA at stationary phase with the addition of 3-HP into the cultured medium, indicating 3-HP was first converted to its CoA derivatives. In vitro enzymatic assay and β-alanine pathway dependent 13C-labeling further demonstrated that a reductive route sequentially converted 3-HP-CoA to acrylyl-CoA and propionyl-CoA, with the latter being reassimilated into the ethylmalonyl-CoA pathway. The deletion of the gene META1_4251 encoding a putative acrylyl-CoA reductase led to reduced degradation rate of 3-HP in late stationary phase. Conclusions We demonstrated the feasibility of constructing the malonyl-CoA pathway in M. extorquens AM1 to generate 3-HP. Furthermore, we showed that a reductive route coupled with the ethylmalonyl-CoA pathway was the major channel responsible for degradation of the 3-HP during the growth transition. Engineered M. extorquens AM1 represents a good platform for 3-HP production from methanol.

Abstract Background Cryptococcal infection has become a public health challenge globally. However, information about cryptococcal infection in patients with hematological diseases remains relatively rare. Methods HIV-uninfected cryptococcosis cases with hematological diseases admitted to Huashan Hospital from January 2001 to December 2014 were reviewed. Results In total, 33 cryptococcosis patients were enrolled, including 12 malignant and 21 non-malignant hematological cases. Twenty-six patients had central nervous system (CNS) involvement, which was observed more often in patients with non-malignancies than with malignancies (20/21 vs. 6/12, P = 0.001) Most patients (25/26) with CNS infection were confirmed by cerebrospinal fluid (CSF) culture or smear, and 100% (20/20) of them tested positive for the CSF cryptococcal antigen test. Eighteen out of 26 cryptococcal meningitis patients were treated with amphotericin B (AmB)-based therapy, 16 of them with AmB deoxycholate (d-AmB) and 2 patients with liposomal AmB. The clinical success rate was 55.6%. D-AmB was well-tolerated at 0.35–0.59 mg/kg/d (median 0.43 mg/kg/d) and only 12 patients had mild adverse events. Conclusions CNS cryptococcal infection was more frequent in patients with hematological non-malignancies, and cryptococcal antigen test as well as the CSF fungal culture or smear are suggested for early diagnosis. D-AmB could be used as an alternative therapy for CNS-infected patients with hematological diseases.

Objectives: The increase in the prevalence of obesity presents a significant health and economic problem. Obesity has been reported to be a major contributor to variety of chronic diseases. Childhood obesity has been rising over the past decades leading to various complications in health. Millions of infants and children undergo surgery every year on various health grounds. The present investigation was undertaken to evaluate the effect of spinal anesthesia of equipotent doses of ropivacaine and bupivacaine on over-weight neonatal rats. Methods: The Sprague-Dawley rat pups were overfed on high fat diet to induce obesity. Behavioral assessments for sensory and motor blockade was made by evaluating thermal and mechanical withdrawal latencies at various time intervals following intrathecal injections of bupivacaine (5.0 mg·kg−1) and ropivacaine (7.5 mg·kg−1) in P14 rats. Spinal tissue was analyzed for apoptosis by determination of activated caspase-3 using monoclonal anti-activated caspase-3 and Fluoro-Jade C staining. Long-term spinal function in P30 rat pups was evaluated. Results: Exposure to intrathecal anesthesia in P14 increased thermal and mechanical latencies and was observed to increase apoptosis as presented by increase in activated caspase-3 and Fluro-Jade C positive cells. Significant alterations in spinal function were observed in high fat diet-fed pups as against non-obese control pups that were on standard diet. Bupivacaine produced more pronounced apoptotic effects on P14 pups; ropivacaine however produced long lasting effects as evidenced in motor function tests at P30. Conclusion: Ropivacaine and bupivacaine induced spinal toxicity that was more pronounced in over-fed rat pups as against normal controls. Resumo: Objetivos: O aumento da prevalência da obesidade é um problema sério de saúde e econômico. A obesidade tem sido relatada como um dos principais contribuintes para uma variedade de doenças crônicas. A obesidade infantil tem aumentado nas últimas décadas, levando a várias complicações de saúde. Milhões de bebês e crianças são submetidos à cirurgia todos os anos por diversos motivos de saúde. O presente estudo foi realizado para avaliar o efeito da raquianestesia com doses equipotentes de ropivacaína e bupivacaína em ratos recém-nascidos com sobrepeso. Métodos: As crias de ratos Sprague-Dawley foram alimentadas em excesso com dieta rica em gordura para induzir obesidade. Avaliações comportamentais para bloqueio sensorial e motor foram feitas através da avaliação das latências de retirada térmicas e mecânicas em vários intervalos de tempo após injeções por via intratecal de bupivacaína (5,0 mg·kg−1) e ropivacaína (7,5 mg·kg−1) em ratos P14. Tecido medular foi analisado para apoptose por determinação da caspase-3 ativada, usando anticorpo monoclonal anti-caspase 3 ativada e ecoloração com Fluoro-Jade C. A função da coluna vertebral a longo prazo em filhotes de ratos P30 foi avaliada. Resultados: A exposição à anestesia intratecal em P14 aumentou as latências térmicas e mecânicas e observamos aumento da apoptose, como apresentado pelo aumento da caspase-3 ativada e células positivas para Fluro-Jade C. Alterações significativas da função da coluna vertebral foram observadas em filhotes alimentados com dieta rica em gordura versus filhotes controles não-obesos em dieta padrão. Bupivacaína produziu efeitos apoptóticos mais pronunciados sobre os filhotes P14; ropivacaína, entretanto, produziu efeitos duradouros como evidenciado nos testes de função motora em P30. Conclusão: Ropivacaína e bupivacaína induziram toxicidade medular mais pronunciada nos filhotes de ratos sobrealimentados que nos controles normais. Keywords: Bupivacaine, Ropivacaine, Intrathecal anesthesia, Spinal toxicity, Obesity, Palavras-chave: Bupivacaína, Ropivacaína, Anestesia intratecal, Toxicidade medular, Obesidade

Probing the decomposition products of Sulfur hexafluoride (SF6) under partial discharge provides important information for fault diagnosis of Gas Insulated Switchgear (GIS). Here, the effects of discharging time, gas pressure, and discharging voltage on SF6 decomposition products have been investigated by Fourier transform infrared (FTIR) spectroscopy. The infrared spectra of decomposition products such as CF4, SOF2, and SO2F2 have been obtained. It can be found that the CF4, SOF2, and SO2F2 concentrations increase over increased discharging time up to 96 hours. The SO2F2 concentration increases while the discharging voltage is raised from 20 kV to 46 kV, due to the rapid deceleration to the lower energy of electron capture and dissociative attachment. The SO2F2 concentration is reduced while the gas pressure is increased from 0.3 MPa to 0.4 MPa. The present results about the SF6 decomposition can be useful for electrical fault diagnosis.

As a widely used gas insulator, sulfur hexafluoride (SF6) has a large cross section for electron absorption, which may make the molecule ionized to the -1 charge state in the high-voltage environment. Using ab initio calculations, we show that the absorbed electron is located averagely on the six F atoms, occupying the antibonding level of the s-p σ bonds and increasing the S-F bond length. The ionized SF6- molecule decreases its decomposition energy to only 1.5 eV, much lower than that of the neutral molecule (4.8 eV), which can be understood according to the occupying of the antibonding orbital and thus weakening of the s-p σ bonds. The weakening of the bonds results in an obvious red shift in the vibrational modes of the ionized SF6- molecule by 120-270 cm-1, compared to those of the neutral molecule. The detailed origin of these vibrational modes is analyzed. Since the appearance of the ionized SF6- molecules is before the decomposition reaction of the SF6- molecule into low-fluoride sulfides, this method may improve the sensitivity of the defection of the partial discharge and save more time for the prevention of the insulation failure in advance.

The co-culture of Trametes versicolor and Ganoderma applanatum is a model of intense basidiomycete interaction, which induces many newly synthesized or highly produced features. Currently, one of the major challenges is an identification of the origin of induced features during the co-culture. Herein, we report a 13C-dynamic labeling analysis used to determine an association of induced features and corresponding fungus even if the identities of metabolites were not available or almost nothing was known of biochemical aspects. After the co-culture of T. versicolor and G. applanatum for 10 days, the mycelium pellets of T. versicolor and G. applanatum were sterilely harvested and then mono-cultured in the liquid medium containing half fresh medium with 13C-labeled glucose as carbon source and half co-cultured supernatants collected on day 10. 13C-labeled metabolome analyzed by LC-MS revealed that 31 induced features including 3-phenyllactic acid and orsellinic acid were isotopically labeled in the mono-culture after the co-culture stimulation. Twenty features were derived from T. versicolor, 6 from G. applanatum, and 5 features were synthesized by both T. versicolor and G. applanatum. 13C-labeling further suggested that 12 features such as previously identified novel xyloside [N-(4-methoxyphenyl)formamide 2-O-beta-D-xyloside] were likely induced through the direct physical interaction of mycelia. Use of molecular network analysis combined with 13C-labeling provided an insight into the link between the generation of structural analogs and producing fungus. Compound 1 with m/z 309.0757, increased 15.4-fold in the co-culture and observed 13C incorporation in the mono-culture of both T. versicolor and G. applanatum, was purified and identified as a phenyl polyketide, 2,5,6-trihydroxy-4, 6-diphenylcyclohex-4-ene-1,3-dione. The biological activity study indicated that this compound has a potential to inhibit cell viability of leukemic cell line U937. The current work sets an important basis for further investigations including novel metabolites discovery and biosynthetic capacity improvement.

In the original publication of this article [1], the number of death cases in ‘Materials and methods’ section is incorrect.

BACKGROUND: Multilocus PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is a new strategy for pathogen identification, but information about its application in fungal identification remains sparse. METHODS: One-hundred and twelve strains and isolates of clinically important fungi and Prototheca species were subjected to both rRNA gene sequencing and PCR/ESI-MS. Three regions of the rRNA gene were used as targets for sequencing: the 5' end of the large subunit rRNA gene (D1/D2 region), and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). Microbial identification (Micro ID), acquired by combining results of phenotypic methods and rRNA gene sequencing, was used to evaluate the results of PCR/ESI-MS. RESULTS: For identification of yeasts and filamentous fungi, combined sequencing of the three regions had the best performance (species-level identification rate of 93.8% and 81.8% respectively). The highest species-level identification rate was achieved by sequencing of D1/D2 for yeasts (92.2%) and ITS2 for filamentous fungi (75.8%). The two Prototheca species could be identified to species level by D1/D2 sequencing but not by ITS1 or ITS2. For the 102 strains and isolates within the coverage of PCR/ESI-MS identification, 87.3% (89/102) achieved species-level identification, 100% (89/89) of which were concordant to Micro ID on species/complex level. The species-level identification rates for yeasts and filamentous fungi were 93.9% (62/66) and 75% (27/36) respectively. CONCLUSIONS: rRNA gene sequencing provides accurate identification information, with the best results obtained by a combination of ITS1, ITS2 and D1/D2 sequencing. Our preliminary data indicated that PCR/ESI-MS method also provides a rapid and accurate identification for many clinical relevant fungi.

OBJECTIVES: To quantitatively analyze placental perfusion in a rat model at different gestation time and different portions of placenta by real-time contrast-enhanced ultrasound (CEUS) and parametric imaging analysis. MATERIALS AND METHODS: Sixty pregnant rats at different gestation time (15 dys,17 days and 20 days) were injected intravenously with microbubbles (5×10(5) microbubbles /ml, 1.0 ml/kg), and cadence contrast pulse sequencing (transmission frequency of 7 MHz, mechanical index 0.18) was performed. Dynamic enhancement changes in placenta at different gestation time and different portions of placenta were measured and enhancement parameters analyzed with software. Correlation between enhancement parameters and average area densities of placenta vascular compartment was compared. RESULTS: The pattern and real-time sequence of enhancement in uterus and placenta were clearly depicted by CEUS. The time-to-peak enhancement was earlier in central portion than that in peripheral portion (12.30±6.33s vs 36.26±10.65 s, p = 0.005), and peak intensity of enhancement is much higher in central portion than that in peripheral portion (30.20±2.85 dB vs 20.95±6.25 dB, p = 0.000). The peak intensity of enhancement at day 15 (27.70±4.47 dB) was lower than that at day 17 (30.20±2.85 dB, p = 0.042) and at day 20 (31.85±4.41 dB, p = 0.015) of gestation. Significant correlation between average area densities of vascular compartment and the peak intensity of enhancement was identified in placenta at different gestation time (p

BACKGROUND: As important regulators of the immune system, the human Fcγ receptors (FcγRs) have been demonstrated to play important roles in the pathogenesis of various infectious diseases. The aim of the present study was to identify the association between FCGR polymorphisms and cryptococcal meningitis. METHODOLOGY/PRINCIPAL FINDINGS: In this case control genetic association study, we genotyped four functional polymorphisms in low-affinity FcγRs, including FCGR2A 131H/R, FCGR3A 158F/V, FCGR3B NA1/NA2, and FCGR2B 232I/T, in 117 patients with cryptococcal meningitis and 190 healthy controls by multiplex SNaPshot technology. Among the 117 patients with cryptococcal meningitis, 59 had predisposing factors. In patients with cryptococcal meningitis, the FCGR2B 232I/I genotype was over-presented (OR = 1.652, 95% CI [1.02-2.67]; P = 0.039) and the FCGR2B 232I/T genotype was under-presented (OR = 0.542, 95% CI [0.33-0.90]; P = 0.016) in comparison with control group. In cryptococcal meningitis patients without predisposing factors, FCGR2B 232I/I genotype was also more frequently detected (OR = 1.958, 95% CI [1.05-3.66]; P = 0.033), and the FCGR2B 232I/T genotype was also less frequently detected (OR = 0.467, 95% CI [0.24-0.91]; P = 0.023) than in controls. No significant difference was found among FCGR2A 131H/R, FCGR3A 158F/V, and FCGR3B NA1/NA2 genotype frequencies between patients and controls. CONCLUSION/SIGNIFICANCE: We found for the first time associations between cryptococcal meningitis and FCGR2B 232I/T genotypes, which suggested that FcγRIIB might play an important role in the central nervous system infection by Cryptococcus in HIV-uninfected individuals.

AIM: To investigate the related factors and treatment of choroidal detachment after glaucoma filtering surgery.METHODS: The clinical data of 42 eyes with choroidal detachment of 457 eyes undergoing glaucoma filtering were retrospectively analyzed. RESULTS: Choroidal detachment occurred at 3-7 days after glaucoma filtering surgery, with mean 4.51±1.14 days. The patient's average age was 65.30±3.50 years. Chroidal detachment was reformed by corticosteroids medicine mydriatic drugs hypertonic agent and all recovered, within 16.50±2.75 days.CONCLUSION: Chroidal detachment is a common complication of glaucoma filtering surgery. The related factors include high intraocular pressure and eyeball congestion before surgery, postoperative low intraocular pressure, diabetes, hypertension and the aged.

A heuristic optimal discrete bit allocation algorithm is proposed for solving the margin maximization problem in discrete multitone (DMT) systems. Starting from an initial equal power assignment bit distribution, the proposed algorithm employs a multistaged bit rate allocation scheme to meet the target rate. If the total bit rate is far from the target rate, a multiple-bits loading procedure is used to obtain a bit allocation close to the target rate. When close to the target rate, a parallel bit-loading procedure is used to achieve the target rate and this is computationally more efficient than conventional greedy bit-loading algorithm. Finally, the target bit rate distribution is checked, if it is efficient, then it is also the optimal solution; else, optimal bit distribution can be obtained only by few bit swaps. Simulation results using the standard asymmetric digital subscriber line (ADSL) test loops show that the proposed algorithm is efficient for practical DMT transmissions.

Abstract Objectives: The increase in the prevalence of obesity presents a significant health and economic problem. Obesity has been reported to be a major contributor to variety of chronic diseases. Childhood obesity has been rising over the past decades leading to various complications in health. Millions of infants and children undergo surgery every year on various health grounds. The present investigation was undertaken to evaluate the effect of spinal anesthesia of equipotent doses of ropivacaine and bupivacaine on over-weight neonatal rats. Methods: The Sprague-Dawley rat pups were overfed on high fat diet to induce obesity. Behavioral assessments for sensory and motor blockade was made by evaluating thermal and mechanical withdrawal latencies at various time intervals following intrathecal injections of bupivacaine (5.0 mg·kg-1) and ropivacaine (7.5 mg·kg-1) in P14 rats. Spinal tissue was analyzed for apoptosis by determination of activated caspase-3 using monoclonal anti-activated caspase-3 and Fluoro-Jade C staining. Long-term spinal function in P30 rat pups was evaluated. Results: Exposure to intrathecal anesthesia in P14 increased thermal and mechanical latencies and was observed to increase apoptosis as presented by increase in activated caspase-3 and Fluro-Jade C positive cells. Significant alterations in spinal function were observed in high fat diet-fed pups as against non-obese control pups that were on standard diet. Bupivacaine produced more pronounced apoptotic effects on P14 pups; ropivacaine however produced long lasting effects as evidenced in motor function tests at P30. Conclusion: Ropivacaine and bupivacaine induced spinal toxicity that was more pronounced in over-fed rat pups as against normal controls.

Purpose • To study the risk factors affecting amputation and survival in patients with diabetic foot (DF) and to construct a predictive model using the machine learning technique for DF foot amputation and survival and evaluate its effectiveness. Materials and Methods • A total of 200 patients with DF hospitalized in the First Affiliated Hospital of Shantou University Medical College in China were selected via cluster analysis screening, Kaplan-Meier survival calculation, amputation rate and Cox proportional hazards model investigation of risk factors associated with amputation and death. In addition, we constructed various models, including Cox proportional hazards regression analysis, the deep learning method convolution neural network (CNN) model, backpropagation (BP) neural network model, and backpropagation neural network prediction model after optimizing the genetic algorithm. The accuracy of the 4 prediction models for survival and amputation was assessed, and we evaluated the reliability of these computational models based on the size of the area under the ROC curve (AUC), sensitivity and specificity. Results• We found that the 1-year survival rate in patients with DF was 88.5%, and the 1-year amputation rate was 12.5%. Wagner’s Classification of Diabetic Foot Ulcers grade, ankle-brachial index (ABI), low-density lipoprotein (LDL), and percutaneous oxygen partial pressure (TcPO2 ) were independent risk factors for amputation in patients with DF, while cerebrovascular disease, Sudoscan sweat gland function score, glycated hemoglobin (HbA1c) and peripheral artery disease (PAD) were independent risk factors for death in patients with DF. In addition, our results showed that in the case of amputation, the COX regression predictive model revealed an AUC of 0.788, sensitivity of 74.1% and specificity of 83.6%. The BP neural network predictive model identified an AUC of 0.874, sensitivity of 87.0% and specificity of 87.7%. An AUC of 0.909, sensitivity of 90.7% and specificity of 91.1% were found after optimizing the BP neural network prediction model via genetic algorithm. In the deep learning CNN model, the AUC, sensitivity and specificity were 0.939, 92.6%, and 95.2%, respectively. In the analysis of risk factors for death, the COX regression predictive model identified the AUC, sensitivity and specificity as 0.800, 74.1% and 85.9%, respectively. The BP neural network predictive model revealed an AUC, sensitivity and specificity of 0.937, 93.1% and 94.4%, respectively. Genetic algorithm-based optimization of the BP neural network predictive model identified an AUC, sensitivity and specificity of 0.932, 91.4% and 95.1%, respectively. The deep learning CNN model found the AUC, sensitivity and specificity to be 0.861, 82.8% and 89.4%, respectively. Conclusion • To identify risk factors for death, the BP neural network predictive model and genetic algorithmbased optimizing BP neural network predictive model have higher sensitivity and specificity than the deep learning method CNN predictive model and COX regression analysis. [ABSTRACT FROM AUTHOR]