The convoluted process of diagnosing pulmonary mycosis caused by Exophiala dermatitidis: a case report.

BACKGROUND: Etiological diagnosis is a key step in the treatment of patients with rare pulmonary mycosis, and the lack of understanding of this disease and lack of specific markers for the detection of rare species, such as Exophiala dermatitidis, add to the difficulty in diagnosing the condition. Therefore, improving the diagnostic strategies for this disease is very important. CASE PRESENTATION: A 52-year-old man presented with cough, sputum production and hemoptysis; chest computed tomography (CT) revealed multiple bilateral lesions. The pathogen was unable to be identified after three biopsies. Subsequently, we performed combined tissue metagenomic next-generation sequencing (mNGS). The results of mNGS and a good therapeutic response helped to identify the causative pathogen as Exophiala dermatitidis. Finally, the patient was diagnosed with Exophiala dermatitidis pneumonia. CONCLUSIONS: Combining molecular techniques, such as mNGS, with clinical microbiological tests will improve the rate of positivity in the diagnosis of rare fungal infections, and the importance of follow-up should be emphasized.

Palladium-catalyzed highly regioselective hydroaminocarbonylation of aromatic alkenes to branched amides.

Pd(t-Bu3P)2 has been successfully identified as an efficient catalyst for the hydroaminocarbonylation of aromatic alkenes to branched amides under relatively mild reaction conditions. With hydroxylamine hydrochloride as an additive, both aliphatic and aromatic amines could be used as coupling partners for the present reaction, leading to production of branched amides in high yields with excellent regioselectivities.

Mitochondrial haplotypes may modulate the phenotypic manifestation of the LHON-associated ND1 G3460A mutation in Chinese families.

To investigate the pathophysiology of Leber's hereditary optic neuropathy (LHON), a cohort of 1164 Han Chinese subjects with LHON were screened for ND1 G3460A mutation. A total of 295 subjects from 16 Han Chinese families carrying the G3460A mutation underwent a clinical and genetic evaluation and molecular analysis of mitochondrial (mt)DNA. The incidence of G3460A mutation was 1.4% in this cohort of Chinese subjects with LHON. Twenty-seven (20 males/7 females) of 109 matrilineal relatives among 10 Chinese pedigrees carrying this mutation exhibited a wide range of severity and age-at-onset in visual impairment. Penetrances of optic neuropathy ranged from 7.1% to 50%, with the average of 24.5%. The age-at-onset of 27 affected matrilineal relatives varied from 10 to 40 years, with the average of 22 years. Molecular analysis identified the homoplasmic G3460A mutation and distinct sets of variants belonging to eight haplogroups. Haplogroup M with G3460A mutation was of higher frequency than those in controls. The penetrances of visual loss in families carrying mitochondrial DNA haplogroups A, B and M were higher than those in other families. Furthermore, haplogroup-specific variants tRNA(Ser(AGY)) A12223G, tRNA(Thr) G15927A and tRNA(Glu) A14693G may enhance the penetrance of visual loss in these families. The G3460A mutation occurred through recurrent origins and founder events in Chinese population. Mitochondrial modifiers may modulate the penetrance and expressivity of optic neuropathy among Chinese pedigrees carrying the G3460A mutation. Thus, our findings may provide new insights into the understanding of pathophysiology and valuable information on the management of LHON.

[The analysis of mitochondrial DNA haplogroups and variants for Leber's hereditary optic neuropathy in Chinese families carrying the m.14484T >C mutation].

The m.14484T>C mutation in mitochondrial ND6 gene (MT-ND6) is a primary mutation underlying the development of Leber's hereditary optic neuropathy (LHON) , but by itself not enough to cause visual loss. To explore the role of mitochondrial haplogroups on the expression of LHON for the people carrying the m.14484T>C mutation, we performed systematic and extended mutational screening of MT-ND6 gene in a cohort of 1177 Han Chinese patients with LHON. A total of 67 affected subjects carried the homoplasmic m.14484T>C mutation, accounting for 5.7% of this LHON population. The penetrances of optic neuropathy among 51 pedigrees carrying the m.14484T>C mutation ranged from 5.6% to 100.0%, with the average of 21.5%. The sequence analysis of entire mitochondrial genomes of 51 probands exhibited distinct sets of polymorphisms belonging to 18 Eastern Asian haplogroups. The frequencies of haplogroup A and haplogroup F were sig-nificantly less in the LHON mtDNA samples than those in 106 Chinese controls. On the other hand, the haplogroup M10a accounted for 9.8% of the patient's mtDNA samples but was absent in 106 Chinese controls. Strikingly, the average pene-trance (46.13%) of optic neuropathy for the pedigrees carrying mitochondrial haplogroup M10a was higher than those car-rying other mtDNA haplogroups. These observations indicated that mitochondrial haplogroup M10a may increase the risk of visual loss.

Prenatal detection of chromosome 7q deletion with duplication: A case report and literature review.

RATIONALE: With advances in prenatal diagnostic techniques, chromosomal microdeletions and microduplications have become the focus of prenatal diagnosis. 7q partial monosomy or trisomy due to a deletion or duplication of the 7q end is relatively rare and usually originates from parents carrying a balanced translocation. PATIENT CONCERNS: Noninvasive prenatal screening (NIPT) showed a fetus with partial deletion and duplication of chromosome 7q. It was not possible to determine whether the fetus was normal. DIAGNOSES: Conventional chromosome G-banding and chromosome microarray analysis (CMA) were performed on fetal amniotic fluid samples and parental peripheral blood samples. INTERVENTIONS: The pregnant women were given detailed genetic counseling by clinicians. OUTCOMES: The fetal karyotype was 46, XY on conventional G-banding analysis. The CMA test results showed a deletion of approximately 7.8 Mb in the 7q36.1q36.3 region and a duplication of 6.6Mb in the 7q35q36.1 region. The parents' karyotype analysis and CMA results were normal, indicating a new mutation. LESSONS: CMA molecular diagnostic analysis can effectively detect chromosomal microdeletions or microduplications, clarify the relationship between fetal genotype and clinical phenotype, and provide a reference for prenatal diagnosis of chromosomal microdeletion-duplication syndrome.

Gene mutation analysis and genetic counseling for patients with non-syndromic hearing loss in Linyi region.

Through gene mutation analysis of patients with non-syndromic hearing loss (NSHL) correct genetic counseling for patients with NSHL and their family members were provided. A total of 116 patients suffering from NSHL were selected, and Sanger sequencing was applied to analyze 31 mutation sites in four deafness genes [gap junction ß-2 (GJB2), solute carrier family 26, member 4 (SLC26A4), GJB3 and mitochondria 12S ribosomal ribonucleic acid (12SrRNA)]. Based on detection results, for the families with reproductive needs, amniotic fluid was extracted from pregnant women during proper gestational weeks to identify fetal genotypes and predict hearing state. Among 116 patients with NSHL, 51 patients carrying definite pathogenic mutation were found, including 35 patients with GJB2 mutations, 14 patients with SLC26A4 gene mutations and 2 patients with mitochondrial deoxyribonucleic acid 12SrRNA (mtDNA 12SrRNA) mutations. No GJB3 gene mutation site was detected. In addition, prenatal diagnosis to 17 pregnant women who had given birth to babies with deafness was performed, and results suggested that genotypes of 6 fetuses were consistent with those of probands, genotypes of 8 fetuses were consistent with those of their parents, and no mutation was found in the other 3 fetuses. Gene mutation analysis of patients with NSHL can identify the etiology and provide appropriate genetic counseling and birth guiding for patients with NSHL and their family members. In addition, prenatal diagnosis to the families who plan to give birth again can avoid the natality of fetuses with hearing loss.

Pneumocystis-mediated IL-8 release by macrophages requires coexpression of mannose receptors and TLR2.

Interaction with the unique fungus Pneumocystis (Pc) promotes IL-8 release by human alveolar macrophages (AM), although the receptor(s) mediating IL-8 release have not been identified. TLR2 recognizes fungal components and mediates release of host defense cytokines and chemokines, although whether TLR2 mediates signaling in response to Pc is not known. In the current study, Pc induced IL-8 release by human AM, and AM pretreatment with anti-TLR2 neutralizing antibody reduced IL-8 release. However, in nonphagocytic human embryonic kidney (HEK)293 cells transfected with human TLR2 cDNA, incubation with Pc did not induce IL-8 release, whereas these same cells released IL-8 in response to the TLR2 agonist lipoteichoic acid. Targeted gene silencing of AM mannose receptors (MR; phagocytic receptors for Pc) using small interfering RNA also reduced Pc-mediated IL-8 release in human AM. However, HEK293 cells transfected with human MR cDNA alone did not release IL-8 in response to Pc. In contrast, HEK293 cells cotransfected with human TLR2 and human MR cDNA released IL-8 in response to Pc. In human AM, Pc promoted direct interaction of MR and TLR2, IL-8 release was reduced markedly upon simultaneous blocking of TLR2 and gene silencing of MR, and IL-8 release was dependent in part on transcription factor NF-kappaB and ERK1/2 and JNK MAPKs. These studies demonstrate that Pc-mediated IL-8 release by human AM requires the coexpression of MR and TLR2 and further supports the concept that combinatorial interactions of macrophage innate receptors provide specificity of host defense cell responses to infectious challenge.

Cdc42 and RhoB activation are required for mannose receptor-mediated phagocytosis by human alveolar macrophages.

Human alveolar macrophages (AMs) phagocytose Pneumocystis (Pc) organisms predominantly through mannose receptors, although the molecular mechanism mediating this opsonin-independent process is not known. In this study, using AMs from healthy individuals, Pc phagocytosis was associated with focal F-actin polymerization and Cdc42, Rac1, and Rho activation in a time-dependent manner. Phagocytosis was primarily dependent on Cdc42 and RhoB activation (as determined by AM transfection with Cdc42 and RhoB dominant-negative alleles) and mediated predominantly through mannose receptors (as determined by siRNA gene silencing of AM mannose receptors). Pc also promoted PAK-1 phosphorylation, which was also dependent on RhoGTPase activation. HIV infection of AMs (as a model for reduced mannose receptor expression and function) was associated with impaired F-actin polymerization, reduced Cdc42 and Rho activation, and markedly reduced PAK-1 phosphorylation in response to Pc organisms. In healthy AMs, Pc phagocytosis was partially dependent on PAK activation, but dependent on the Rho effector molecule ROCK. These data provide a molecular mechanism for AM mannose receptor-mediated phagocytosis of unopsonized Pc organisms that appears distinct from opsonin-dependent phagocytic receptors. Reduced AM mannose receptor-mediated Cdc42 and Rho activation in the context of HIV infection may represent a mechanism that contributes to the pathogenesis of opportunistic pneumonia.

Triterpenoids with antiplatelet aggregation activity from Ilex rotunda.

Phytochemical studies on the barks of Ilex rotunda Thunb. had resulted in the isolation of seven previously undescribed triterpenoids, rotundinosides E-K, along with sixteen known ones. The structures of previously undescribed compounds were elucidated on the basis of extensive spectroscopic analysis and the sugar moieties were further identified by HPLC and GC after acid hydrolysis. Among the isolates, rotundinoside F featured a rare triterpene-phenylpropanoid hybrid structure and rotundinoside H was an uncommon triterpene saponin with α-linked glucopyranosyl moiety at C-3. The antiplatelet aggregation of all compounds were evaluated against ADP induced rat platelet aggregation in vitro, and five compounds exhibited moderate inhibitory effects with IC50 values ranging from 22.4 to 32.8 µM.

Community-based stroke system of care improves patient outcomes in Chinese rural areas.

BACKGROUND: Building effective and efficient stroke care systems is a key step in improving prevention, treatment and rehabilitation of stroke. The aim of this study was to evaluate the effectiveness of this stroke system of care on stroke management during a 2-year follow-up. METHODS: A stroke system of care was developed from November 2009 to November 2010 in three townships in Ganyu County. Additional three matched townships were invited as controls. We first investigated the stroke incidence of these populations. Subsequently, this stroke system of care and an educational campaign in the three intervention townships were implemented and the effectiveness of the system was evaluated in the next 2 years. RESULTS: At postintervention, more patients in the intervention communities obtained stroke knowledge and then the proportion of patients with stroke who were admitted within 3 hours of onset markedly increased in 2012 (12.0% vs 8.1%, p=0.044) and in 2013 (15.2% vs 9.7%, p=0.008) compared with those in the control communities. In the intervention communities, this proportion of patients with acute ischaemic stroke who received thrombolytic treatment was markedly raised from 2.1% in 2012 to 3.0% in 2013. More importantly, the fatality rate substantially decreased in 2013 in the intervention communities compared with that in the control communities (6.1% vs 9.7%, p=0.032). Similarly, the disability rate significantly decreased in 2013 (45.3% vs 51.5%, p=0.045). CONCLUSIONS: The community-based stroke system of care was effective and practical for optimising stroke treatments and improving patient outcomes. TRIAL REGISTRATION NUMBER: ChiCTR-RCH-13003408, Post-results.

Negative regulatory role of mannose receptors on human alveolar macrophage proinflammatory cytokine release in vitro.

Alveolar macrophages (AM) are critical components of lung innate immunity and contribute to an effective host response to Pneumocystis pneumonia. Recognition of unopsonized Pneumocystis organisms by human AM is mediated predominantly via mannose receptors and results in phagocytosis, release of reactive oxygen species, and activation of the nuclear transcription factor (NF)-kappaB. However, the AM host defense genes activated by Pneumocystis have not been defined. In the present study, incubation of AM with unopsonized Pneumocystis organisms was not associated with release of interleukin (IL)-1beta, IL-6, or tumor necrosis factor (TNF)-alpha (important cytokines in the host response to Pneumocystis) and did not induce IL-1beta, IL-6, or TNF-alpha mRNA transcripts. These findings were not attributed to Pneumocystis-induced cytopathic changes, as these same AM released IL-8 and matrix metalloproteinase-9 in response to Pneumocystis. NF-kappaB-mediated IL-8 release was independent of Pneumocystis phagocytosis. The observed response was specific, as IL-1beta, IL-6, and TNF-alpha release and mRNA induction were preserved in response to lipopolysaccharide or serum-opsonized Pneumocystis. The absence of IL-1beta, IL-6, and TNF-alpha release in response to Pneumocystis was predominately influenced by AM mannose receptors, as blocking mannose receptors or targeted mannose receptor small interfering RNA functional gene silencing resulted in TNF-alpha release in response to unopsonized Pneumocystis organisms. Furthermore, ligation of AM mannose receptors by unopsonized Pneumocystis organisms reduced Toll-like receptor 4-mediated TNF-alpha release. Taken together, these data suggest that mannose receptors on human AM may suppress select proinflammatory cytokine release and may serve to regulate the innate inflammatory responses to infectious challenge in the lungs.

Investigation on the differences of accumulating Escherichia coli in three types of shellfish species, involving in the environmental factors.

This study investigated accumulation of Escherichia coli and aerobic colony count in three types of shellfish species. The results indicated that the capability of accumulating E. coli and aerobic colony count for Sinonovacula constricta was stronger than that for Meretrix meretrix and Tegillarca granosa, and capability of accumulating E. coli for M. meretrix was slightly stronger than that for T. granosa. However, no significant difference was observed in the capability of accumulating aerobic colony count between M. meretrix and T. granosa. Moreover, accumulation of E. coli in S. constricta is affected by contaminated seawater and E. coli were accumulated much faster and more in S. constricta when the seawater contaminated more serious. Meanwhile, the results suggested that the populations of E. coli in S. constricta changed in accordance with the weather. This is the first study to investigate the differences of accumulating E. coli in three types of shellfish species.

New clerodane diterpenoids from Croton crassifolius.

Two new clerodane diterpenoids (1-2), one new clerodane diterpenoid alkaloid (3), as well as thirteen known compounds were isolated from Croton crassifolius. The structures of new compounds were established by a combination of spectroscopic methods, including HRMS, (1)H NMR, (13)C NMR, (1)H (1)H COSY, HSQC, HMBC, NOESY and X-ray crystallographic analysis. Compound 3 is firstly reported as the clerodane-type diterpenoid alkaloid in natural products. All of the compounds were evaluated for in vitro cytotoxic activities against CT26.WT cell using the MTT method.

Leber's hereditary optic neuropathy caused by the homoplasmic ND1 m.3635G>A mutation in nine Han Chinese families.

In this report, we investigated the molecular mechanism underlying Leber's hereditary optic neuropathy (LHON)-associated mitochondrial m.3635G>A (p.S110N, ND1) mutation. A mutational screening of ND1 gene in a cohort of 1070 Han Chinese subjects LHON identified the m.3635G>A mutation in nine Chinese families with suggestively maternally transmitted LHON. Thirty-eight (22 males/16 females) of 162 matrilineal relatives in these families exhibited the variable severity and age-at-onset of optic neuropathy. Molecular analysis of their mitochondrial genomes identified the homoplasmic m.3635G>A mutation and distinct sets of polymorphisms belonging to the Asian haplogroups G2a1, R11a, D4, R11a, M7b2, G1a, F1a1, B4, and N9a3, respectively. Using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from one Chinese family into mtDNA-less (ρ(0)) cells, we showed ~27% decrease in the activity of NADH:ubiquinone oxidoreductase (complex I) in mutant cybrids carrying the m.3635G>A mutation, compared with control cybrids. The respiratory deficiency caused by the m.3635G>A mutation results in decreased efficiency of mitochondrial ATP synthesis. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cybrids. The data provide the direct evidence for the m.3635G>A mutation leading to LHON. Our findings may provide new insights into the understanding of pathophysiology of LHON.

Frequency and spectrum of mitochondrial ND6 mutations in 1218 Han Chinese subjects with Leber's hereditary optic neuropathy.

PURPOSE: To investigate the molecular pathogenesis of Leber's hereditary optic neuropathy (LHON) in Chinese families. METHODS: A cohort of 1218 Han Chinese subjects with LHON and 316 control subjects underwent the clinical and genetic evaluation and molecular analysis of mitochondrial (mt)DNA. RESULTS: The age at onset of optic neuropathy in these subjects ranged from 5 to 55 years, with the average of 18 years. Mutational analysis of ND6 gene identified 92 (73 known and 19 novel) variants in these subjects. These variants included 29 (9 novel and 20 known) missense mutations and 63 silence variants. A total of 94 subjects carrying one of the known T14484C, T14502C, and G14459A mutations accounted for 7.7% cases of this cohort, particularly 4.4% for T14484C mutation. Furthermore, eight putative LHON-associated ND6 mutations accounted for 1.1% case of this cohort. Thus, 106 subjects carrying one of ND6 mutations accounted for 8.7% cases of this cohort. Low penetrance of optic neuropathy in pedigrees carrying one of eight putative mutations indicated that the mutation(s) is necessary, but itself insufficient to produce a clinical phenotype. Mitochondrial DNAs in 98 probands carrying the ND6 mutation(s) were widely dispersed among 12 Eastern Asian subhaplogroups. In particular, the occurrences of haplogroups M9, M10, M11, and H2 in patients carrying the ND6 mutations were higher than those in controls. CONCLUSIONS: These data further support that the ND6 gene is the hot spot for mutations associated with LHON. Thus, our findings may provide valuable information for the further understanding of pathophysiology and management of LHON.

HIV impairs TNF-alpha mediated macrophage apoptotic response to Mycobacterium tuberculosis.

The factors that contribute to the exceptionally high incidence of Mycobacterium tuberculosis (MTb) disease in HIV(+) persons are poorly understood. Macrophage apoptosis represents a critical innate host cell response to control MTb infection and limit disease. In the current study, virulent live or irradiated MTb (iMTbRv) induced apoptosis of differentiated human U937 macrophages in vitro, in part dependent on TNF-alpha. In contrast, apoptosis of differentiated HIV(+) human U1 macrophages (HIV(+) U937 subclone) was markedly reduced in response to iMTbRv and associated with significantly reduced TNF-alpha release, whereas apoptosis and TNF-alpha release were intact to TLR-independent stimuli. Furthermore, reduced macrophage apoptosis and TNF-alpha release were independent of MTb phagocytosis. Whereas surface expression of macrophage TLR2 and TLR4 was preserved, IL-1 receptor associated kinase-1 phosphorylation and NF-kappaB nuclear translocation were reduced in HIV(+) U1 macrophages in response to iMTbRv. These findings were confirmed using clinically relevant human alveolar macrophages (AM) from healthy persons and asymptomatic HIV(+) persons at clinical risk for MTb infection. Furthermore, in vitro HIV infection of AM from healthy persons reduced both TNF-alpha release and AM apoptosis in response to iMTbRv. These data identify an intrinsic specific defect in a critical macrophage cellular response to MTb that may contribute to disease pathogenesis in HIV(+) persons.

HIV impairs TNF-alpha release in response to Toll-like receptor 4 stimulation in human macrophages in vitro.

The molecular mechanisms for increased risk of bacterial pneumonia in HIV+ persons remain incompletely understood. Recognizing the critical role of Toll-like receptor (TLR) signaling in host defense, this study showed that human U937 macrophage stimulation by the TLR4-specific ligand, lipid A (biologically active component of bacterial LPS), promoted TNF-alpha release through extracellular regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase phosphorylation. In contrast, HIV+ U1 macrophages had significantly reduced TNF-alpha release (despite preserved TLR4 expression) and reduced ERK1/2 phosphorylation, whereas TNF-alpha release was intact via a TLR4-independent pathway. In HIV+ U1 cells, reduced ERK1/2 phosphorylation was not due to reduced upstream MEK1/2 activation, but was associated with a reciprocal induction of MAP kinase phosphatase-1 (MKP-1). HIV nef protein was sufficient to reduce TNF-alpha release and induce MKP-1 in healthy macrophages. Pharmacologic inhibition of endogenous cellular phosphatases increased ERK1/2 phosphorylation and partially restored TLR4-mediated TNF-alpha release in HIV+ macrophages. Furthermore, targeted gene silencing of MKP-1 partially restored lipid A-mediated TNF-alpha release in HIV+ U1 cells. Similar results were observed using clinically relevant human alveolar macrophages, comparing healthy to asymptomatic HIV+ persons at clinical risk for bacterial pneumonia. Thus, reduced TLR4-mediated TNF-alpha release through altered ERK1/2 regulation by HIV may impair an effective innate immune response to bacterial challenge. Inhibition of cellular phosphatases may serve as a potential therapeutic target in the management of bacterial pneumonia in HIV+ persons.

Pneumocystis activates human alveolar macrophage NF-kappaB signaling through mannose receptors.

Alveolar macrophages (AM) represent important effector cells in the innate immune response to the AIDS-related pathogen Pneumocystis, but the early AM host defense signaling events are poorly defined. Using AM from healthy individuals, we showed in the present study that Pneumocystis organisms stimulate AM NF-kappaB p50 and p65 nuclear translocation in a time-dependent and multiplicity-of-infection-dependent manner as determined by electrophoretic mobility shift assay and immunofluorescence microscopy and that NF-kappaB nuclear translocation is associated with I-kappaB phosphorylation. Importantly, competitive inhibition of mannose receptor and targeted short interfering RNA-mediated gene suppression of mannose receptor mRNA and protein is associated with complete elimination of NF-kappaB nuclear translocation in response to Pneumocystis. Furthermore, human immunodeficiency virus (HIV) infection of AM (as a model human disease state of reduced AM mannose receptor expression and function) inhibits Pneumocystis-mediated NF-kappaB nuclear translocation and is associated with reduced I-kappaB phosphorylation and reduced interleukin-8 (IL-8) release. In contrast, NF-kappaB nuclear translocation and IL-8 release in response to lipopolysaccharide are intact in AM from both healthy and HIV-infected individuals, indicating that the observed impairment is not a global disturbance of the NF-kappaB pathway. Thus, in addition to phagocytic and endocytic effector functions, the present study identifies mannose receptors as pattern recognition receptors capable of NF-kappaB activation in response to infectious non-self challenge. AM mannose receptor-mediated NF-kappaB activation may represent an important mechanism of the host cell response to Pneumocystis, and altered NF-kappaB activation in the context of HIV infection may impair a critical innate immune signaling response and may contribute to pathogenesis of opportunistic lung infections.