Clinical significance of rapid detection and diagnosis of Listeria infection in blood with mass spectrometry.

OBJECTIVE: Listeriosis is caused by the bacterium, Listeria monocytogenes, and is a significant health concern because of high hospitalization and mortality rates. This study reports seven cases of pregnancy-associated listeriosis diagnosed with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry performed on infection-positive blood culture. METHODS: Blood culture-positive samples of seven patients whose pregnancy was complicated by Listeria infection and treated at Xuanwu Hospital of Capital Medical University between January 2016 and December 2021 were analyzed retrospectively. Strains identified by MALDI-TOF mass spectrometry were compared with colony identification results. Identification accuracy and consistency were assessed. RESULTS: A total of seven strains of Listeria were collected from seven pregnant women presented with fever (37.6-39.9°C). Clinical abnormalities included abnormal liver function, emaciation, hypoalbuminemia, hypocalcemia, hypokalemia, hyponatremia, ketosis, mild to moderate anemia, leukopenia, and thrombocytopenia. Compared with the traditional culture method, MALDI-TOF mass spectrometry led to much earlier identification (4-6 h vs. 3-4 days) with 100% identification accuracy. Of the seven pregnancies complicated by Listeria, only two led to live births. Of the five fetal deaths, three occurred in the second trimester. CONCLUSION: In this series of pregnancy-associated listeriosis cases, the fetal mortality rate was 71%. MALDI-TOF mass spectrometry is a valuable method that can identify Listeria from blood culture rapidly and accurately.

Voltage-Gated Membranes Incorporating Cucurbit[n]uril Molecular Containers for Molecular Nanofiltration.

Smart voltage-gated nanofiltration membranes have enormous potential for on-demand and precise separation of similar molecules, which is an essential element of sustainable water purification and resource recovery. However, the existing voltage-gated membranes are hampered by limited selectivity, stability, and scalability due to electroactive monomer dimerization. Here, for the first time, the host-guest recognition properties of cucurbit[7]uril (CB[7]) are used to protect the viologen derivatives and promote their assembly into the membrane by interfacial polymerization. Viologen functions as a voltage switch, whereas CB[7] complexation prevents its dimerization and improves its redox stability. The inhibited diffusion of the CB[7]-viologen complex enables the precise patterning of the surface structure. The resultant voltage-gated membrane displays 80% improved rejection performance, excellent recovery accuracy for similar molecules, and anti-fouling properties. This work not only provides an innovative strategy for the preparation of voltage-gated smart nanofiltration membranes but also opens up new avenues for ion-selective transmission in water treatment, bionic ion channels, and energy conversion.

Clinical and microbiological characteristics of Cryptococcus gattii isolated from 7 hospitals in China.

BACKGROUND: Infection, even outbreak, caused by Cryptococcus gattii (C. gattii) has been reported in Canada and the United States, but there were sparsely-reported cases of C. gattii in China. Our interest in occurrence, clinical manifestation, laboratory identification and molecular characterization of Chinese C. gattii strains leads us to this research. RESULTS: Out of 254 clinical isolates, initially identified as Cryptococcus neoformans (C. neoformans), eight strains were re-identified as C. gattii. Multi-locus sequence typing (MLST) showed genotype VGI accounted for the most (6 / 8), the other two strains were genotype VGII (VGIIa and VGIIb respectively) with 3 specific spectra of molecular weight about 4342, 8686, 9611 Da by MALDI-TOF MS. The minimal inhibitory concentrations (MICs) of Fluconazole with Yeast one was 2~4 times higher than that with ATB fungus 3 and MICs of antifungal agents against VGII strains were higher than against VGI strains. Comparative proteome analysis showed that 329 and 180 proteins were highly expressed by C. gattii VGI and VGII respectively. The enrichment of differentially expressed proteins was directed to Golgi complex. CONCLUSIONS: Infection by C. gattii in China occurred sparsely. Genotype VGI was predominant but VGII was more resistant to antifungal agents. There was significant difference in protein expression profile between isolates of VGI and VGII C. gattii.

ROCK inhibitors 2. Improving potency, selectivity and solubility through the application of rationally designed solubilizing groups.

Solubilizing groups have been frequently appended to kinase inhibitor drug molecules when solubility is insufficient for pharmaceutical development. Such groups are usually located at substitution sites that have minimal impact on target activity. In this report we describe the incorporation of solubilizing groups in a class of Rho kinase (ROCK) inhibitors that not only confer improved solubility, but also enhance target potency and selectivity against a closely related kinase, PKA.

ROCK inhibitors 3: Design, synthesis and structure-activity relationships of 7-azaindole-based Rho kinase (ROCK) inhibitors.

Rho kinase (ROCK) inhibitors are potential therapeutic agents for the treatment of a variety of disorders including hypertension, glaucoma and erectile dysfunction. Here we disclose a series of potent and selective ROCK inhibitors based on a substituted 7-azaindole scaffold. Substitution of the 3-position of 7-azaindole led to compounds such as 37, which possess excellent ROCK inhibitory potency and high selectivity against the closely related kinase PKA.

Reference intervals for thyroid hormones for the elderly population and their influence on the diagnosis of subclinical hypothyroidism.

Background: This study aims to establish reference intervals (RIs) for thyroid hormones in the elderly population and analyze their influence on the prevalence of subclinical hypothyroidism. Methods: Thyroid hormone records of subjects who underwent routine health checkup at our hospital between 2018 and 2020 were analyzed. Thyroid stimulating hormone (TSH), total triiodothyronine, total thyroxine, free triiodothyronine (FT3), and free thyroxine (FT4) levels were compared between young and elderly subjects. Thresholds of these thyroid hormones were established for elderly subjects. Results: A total of 22,207 subjects were included. Of them, 2,254 (10.15%) were aged ≥ 65 years. Elderly subjects had higher TSH, and lower FT3 and FT4 levels when compared with young subjects. In the elderly group, the RIs for TSH, FT3 and FT4 were 0.55-5.14 mIU/L, 3.68-5.47 pmol/L, and 12.00-19.87 pmol/L, respectively. The age and sex specific RIs for TSH were 0.56-5.07 mIU/L for men and 0.51-5.25 mIU/L for women. With whole-group RIs and age and sex-specific RIs for elderly people, the prevalence of subclinical hypothyroidism was 9.83% and 6.29% (p < 0.001), respectively. Conclusions: Elderly individuals had higher TSH levels than young individuals. Our study indicated that establishing specific RIs for elderly individuals is needed. This has implications for the diagnosis and management of subclinical hypothyroidism in the elderly population.

Clinical Efficacy, Antibiotic Resistance Genes, Virulence Factors and Outcome of Hospital-Acquired Pneumonia Induced by Klebsiella pneumoniae Carbapenemase 2-Producing with Tigecycline Treatment in the ICU.

Purpose: Tigecycline is an agent for carbapenemase-producing Klebsiella pneumonia (KPC-KP), given its penetration into lung tissues. Our study focused on the molecular and clinical efficacy of tigecycline for hospital-acquired pneumonia (HAP) in the ICU. Patients and Methods: A retrospective cohort study of 52 adult KPC-KP HAP patients by searching hospital medical records from January 2018 to December 2020 was established to investigate the epidemiology of KPC-KP infections for tigecycline treatment and the associated clinical efficacy of tigecycline. The KPC-KP isolates underwent multilocus sequence typing. Molecular typing, antimicrobial resistance, and virulence profiling were also analyzed by whole-genome sequencing of KPC-KP. Results: Among 52 patients with KPC-KP, the ICU mortality rate was 14/52 (27%), and there was no significant statistical difference in mortality between the effective group and failure group (p = 0.754). However, the duration of tigecycline was statistically different between the two groups of patients (14.4 vs 10 days, p=0.046). The total bacterial clearance rate was 6/52 (11.5%). There was no significant statistical difference in both groups (p=0.416). Antibiotic resistance genes (aac3iia) and virulence gene (AREO-iutA, Capsule-wzc) were negatively correlated with clinical efficacy (p = 0.011, OR = 1.237). Conclusions: Blakpc was the main carbapenemase in all K. pneumoniae strains. ST11-KL64 KPC-KP was the most common virulence factors in KPC-KP isolates. This study suggested that antibiotic resistance genes (aac3iia) and virulence gene (AREO-iutA, Capsule-wzc) were independent mortality risk factors for patients with Klebsiella pneumoniae carbapenemase-2 producing K. pneumoniae infections, when during the tigecycline treatment. Molecular analysis of K. pneumoniae may provide an option when choosing the antimicrobial treatment.

Case Report: A Case of Sarocladium strictum Meningoencephalitis in an Immunocompetent Patient After Invasive Operation.

As an opportunistic phytopathogen, Sarocladium strictum has only been shown to cause neurological disease in immunocompromised patients, where antifungal therapy was not effective. We report a case of Sarocladium strictum meningoencephalitis in an apparently immunocompetent young woman who presented with severe headache and slight fever after undergoing transnasal endoscopic repair of cerebrospinal fluid rhinorrhea. Chronic sinusitis and suspicious intracranial fungal lesions were observed on enhanced magnetic resonance imaging (MRI). Both culture and metagenomic next-generation sequencing of her cerebrospinal fluid were positive for Sarocladium strictum. After local debridement, treatment with amphotericin B plus voriconazole and Ommaya reservoir implantation, the patient improved significantly. Unfortunately, her symptoms worsened again despite plenty of antifungal therapy for a month.

Discovery of a Novel Series of Potent and Selective Alkynylthiazole-Derived PI3Kγ Inhibitors.

Phosphoinositide 3-kinases (PI3Ks) are a family of enzymes that control a wide variety of cellular functions such as cell growth, proliferation, differentiation, motility, survival, and intracellular trafficking. PI3Kγ plays a critical role in mediating leukocyte chemotaxis as well as mast cell degranulation, making it a potentially interesting target for autoimmune and inflammatory diseases. We previously disclosed a novel series of PI3Kγ inhibitors derived from a benzothiazole core. The truncation of the benzothiazole core led to the discovery of a structurally diverse alkynyl thiazole series which displayed high PI3Kγ potency and subtype selectivity. Further medicinal chemistry optimization of the alkynyl thiazole series led to identification of compounds such as 14 and 32, highly potent, subtype selective, and CNS penetrant PI3Kγ inhibitors. Compound 14 showed robust inhibition of PI3Kγ mediated neutrophil migration in vivo.

Discovery of Novel, Orally Bioavailable Pyrimidine Ether-Based Inhibitors of ELOVL1.

In our efforts to identify novel small molecule inhibitors for the treatment of adrenoleukodystrophy (ALD), we conducted a high-throughput radiometric screen for inhibitors of elongation of very long chain fatty acid 1 (ELOVL1) enzyme. We developed a series of highly potent, central nervous system (CNS)-penetrant pyrimidine ether-based compounds with favorable pharmacokinetics culminating in compound 22. Compound 22 is a selective inhibitor of ELOVL1, reducing C26:0 VLCFA synthesis in ALD patient fibroblasts and lymphocytes in vitro. Compound 22 reduced C26:0 lysophosphatidyl choline (LPC), a subtype of VLCFA, in the blood of ATP binding cassette transporter D1 (ABCD1) KO mice, a murine model of ALD to near wild-type levels. Compound 22 is a low-molecular-weight, potent ELOVL1 inhibitor that may serve as a useful tool for exploring therapeutic approaches to the treatment of ALD.

Vaginal microbiome analysis of healthy women during different periods of gestation.

To assess the vaginal microbiome throughout full-term uncomplicated pregnancy, a longitudinal study was designed for 12 healthy women who had prepared to become pregnant and then delivered at term (38-42 weeks) without complications. The vaginal microbial community was studied at pre-pregnancy, 8-12, 24-28, 37-38 weeks of gestation, and puerperium, using hypervariable tag sequencing of the V3-V4 region of the 16S rRNA gene. Sequencing produced approximately 10 million reads on the Illumina MiSeq. Members of the Firmicutes phyla were prevailing before and during pregnancy periods, and the proportion was quite as Proteobacteria until puerperium. Lactobacillus genus was abundant before and during pregnancy, but post-delivery vaginal microflora variety turned diverse. The species-level analysis revealed that a healthy vaginal microbiome before or during pregnancy was prominently dominated by Lactobacillus crispatus. Furthermore, PCoA analysis revealed for differences in the bacterial community composition between the two levels of Lactobacillus species in pre-pregnancy and pregnancy period (PC1 contribution of 58.46%, PC3 contribution of 8.64%). Based on the taxonomic and PCoA analysis, we found that L. crispatus was dominant in the vaginal microflora of healthy women before or during pregnancy, but at the puerperium, the status changed leading to decreased abundance of protective Lactobacillus species that made vaginal micro-ecological barrier vulnerable to diseases. Additionally, vaginal pH was an important environmental property affecting the vaginal microbial community.

Structure-based design and parallel synthesis of N-benzyl isatin oximes as JNK3 MAP kinase inhibitors.

A series of N-benzylated isatin oximes were developed as inhibitors of the mitogen-activated kinase, JNK3. X-ray crystallographic structures aided in the design and synthesis of novel, selective compounds, that inhibit JNK3, but not p38 MAP kinase and provided key insights into understanding the behavior of gatekeeper residue methionine-146 in determining target selectivity for this series.

The preparation and clinical application of diagnostic DNA microarray for the detection of pathogens in intracranial bacterial and fungal infections.

The present study prepared 2 types of DNA diagnostic chips based on 16S ribosomal DNA (rDNA) and 18S-28S rDNA, and evaluated their values in the detection of pathogens in intracranial bacterial/fungal infections. A total of 14 probes of bacteria (Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Haemophilus influenza, Stenotrophomonas maltophilia, Neisseria meningitidis, Enterobacter spp., Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumonia and coagulase negative staphylococcus) and 4 probes of fungi (Candida albicans, Candida tropicalis, Candida glabrata and Cryptococcus neoformans), determined frequently in cerebrospinal fluid (CSF), were designed and used for preparation of microarrays. CSF samples from 88 patients with clinically suspected intracranial infection and standard strains were used to evaluate the chips. The same samples were also analyzed by culture and sequencing. The results demonstrated that the sensitivity, specificity and false-positive rate of the microarray assay compared with culture method were 100 vs. 68.3% (P<0.05), 97.1 vs. 100%, and 2.9 vs. 0%, respectively. The minimum concentration of detection with the chips was 10 cfu ml-1 for bacteria and 100 cfu ml-1 for fungi. The specificity of the probes was confirmed, and no cross-reaction was detected in the present study. Furthermore, 13 cases were positive in the microarray and negative in culture. However, 4 cases were not identified as clear pathogens and only positive in the 16S probe sites. The diagnostic DNA microarray for intracranial infections has proven to be more rapid and sensitive, and it may be a better option for clinical application than culture methods.

Design and Synthesis of a Novel Series of Orally Bioavailable, CNS-Penetrant, Isoform Selective Phosphoinositide 3-Kinase γ (PI3Kγ) Inhibitors with Potential for the Treatment of Multiple Sclerosis (MS).

The lipid kinase phosphoinositide 3-kinase γ (PI3Kγ) has attracted attention as a potential target to treat a variety of autoimmune disorders, including multiple sclerosis, due to its role in immune modulation and microglial activation. By minimizing the number of hydrogen bond donors while targeting a previously uncovered selectivity pocket adjacent to the ATP binding site of PI3Kγ, we discovered a series of azaisoindolinones as selective, brain penetrant inhibitors of PI3Kγ. This ultimately led to the discovery of 16, an orally bioavailable compound that showed efficacy in murine experimental autoimmune encephalomyelitis (EAE), a preclinical model of multiple sclerosis.

Flipped out: structure-guided design of selective pyrazolylpyrrole ERK inhibitors.

The Ras/Raf/MEK/ERK signal transduction is a key oncogenic pathway implicated in a variety of human cancers. We have identified a novel series of pyrazolylpyrroles as inhibitors of ERK. Aided by the discovery of two distinct binding modes for the pyrazolylpyrrole scaffold, structure-guided optimization culminated in the discovery of 6p, a potent and selective inhibitor of ERK.

Design, Synthesis, and Structure-Activity Relationships of Pyridine-Based Rho Kinase (ROCK) Inhibitors.

The Rho kinases (ROCK1 and ROCK2) are highly homologous serine/threonine kinases that act on substrates associated with cellular motility, morphology, and contraction and are of therapeutic interest in diseases associated with cellular migration and contraction, such as hypertension, glaucoma, and erectile dysfunction. Beginning with compound 4, an inhibitor of ROCK1 identified through high-throughput screening, systematic exploration of SAR, and application of structure-based design, led to potent and selective ROCK inhibitors. Compound 37 represents significant improvements in inhibition potency, kinase selectivity, and CYP inhibition and possesses pharmacokinetics suitable for in vivo experimentation.

Susceptibility patterns and molecular epidemiology of multidrug-resistant Acinetobacter baumannii strains from three military hospitals in China.

To date, little has been reported on the susceptibility patterns and molecular characterisation of multidrug-resistant Acinetobacter baumannii (MDRAB) clinical isolates from different Chinese military hospitals. In this study, 49 MDRAB strains were collected from three military hospitals during 2007. The minimum inhibitory concentrations (MICs) of 13 antibiotics were determined for each strain. Genotyping and dendrogram analysis of MDRAB strains were performed using the repetitive sequence-based polymerase chain reaction (rep-PCR) DiversiLab Microbial Typing System. PCR screening was carried out to investigate the distribution of various genes contributing to each resistance phenotype in the main clonal types. The rates of resistance to the majority of antibiotics tested varied between 75.5% and 100%, with the exception of polymyxin B. Two DiversiLab rep-PCR clones (A and B) were widespread in three hospitals in different cities, one clone (D) existed only in two hospitals located in the same city (Beijing), and the other two clones (C and E) were present in only one hospital. In addition, this study shows a high distribution of intI1, ISAba1, bla(OXA-23), bla(ADC), adeB, adeJ, abeM and tet(B) genes, which mediate resistance to structurally unrelated antimicrobials in MDRAB isolates. These results suggest that all isolates were resistant to at least three classes of antibiotics. In addition, clonal dissemination among the three hospitals located in two different cities in China, previously documented in many regions of Europe and Asia-Pacific nations, emphasises the epidemic potential of these MDRAB isolates.

[Study on the molecular characteristics of multidrug-resistant Acinetobacter baumannii].

OBJECTIVE: To investigate antibiotic resistance, carbapenemase genotype and the molecular epidemiology of multidrug-resistant Acinetobacter baumannii (Aba) collected from 3 military hospitals in China. METHODS: The minimum inhibitory concentrations (MIC) were examined by ager dilution method. Genotypes of carbapenemases were amplified by multiplex PCR and its products were sequenced. PCR was used to detect per gene. Homology of the resistant isolates was analyzed by pulse-field gel electrophoresis (PFGE). RESULTS: Among the 64 MDRA strains, 78.1% (50) strains possessed bla(OXA-23) gene, 89.1% (57) carried Class 1 integrase gene, 39.1% (25) with bla(PER-1) gene, and 1 strain with bla(OXA-58-like) gene. PFGE showed that 13 (A, B, C, D, E genotype) different clones were identified in these strains. A, B, and U clones were the predominant clones in three hospitals, respectitively. CONCLUSION: Outbreaks of multidrug-resistant Aba occurred at 3 military hospitals with the most prevalent carbapenemase as OXA-23 enzyme. OXA-58 type of carbapenemase and per-1 in Aba were also isolated.

Structure-guided design of potent and selective pyrimidylpyrrole inhibitors of extracellular signal-regulated kinase (ERK) using conformational control.

The Ras/Raf/MEK/ERK signal transduction, an oncogenic pathway implicated in a variety of human cancers, is a key target in anticancer drug design. A novel series of pyrimidylpyrrole ERK inhibitors has been identified. Discovery of a conformational change for lead compound 2, when bound to ERK2 relative to antitarget GSK3, enabled structure-guided selectivity optimization, which led to the discovery of 11e, a potent, selective, and orally bioavailable inhibitor of ERK.