Clinical Characteristics of Chlamydia psittaci Pneumonia Infection in Central South China.

INTRODUCTION: Chlamydia psittaci pneumonia has been a global public health hotspot in recent years. Although some scattered cases of C. psittaci pneumonia have been reported, there is a lack of large case studies worldwide. METHODS: In this multicenter, observational study, we recruited all consecutive patients with confirmed C. psittaci pneumonia from October 4, 2018, to October 23, 2020, in nine tertiary general hospitals in Central-South China. Epidemiologic and clinical data from patients' electronic medical records were collected and analyzed. RESULTS: One hundred and sixteen patients with C. psittaci pneumonia were included in the study. The mean age was 59.7 years. Fever (96.6%) and cough (65.5%) were the most common clinical symptoms. Most patients presented with an increase in the proportion of neutrophils, neutrophil to lymphocyte ratio, LDH, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and a significant decrease in lymphocytes. The main CT lung findings were consolidation (81%) and pleural effusion (35.3%), and bilateral lung consolidation was mainly found in severe patients. Chlamydia psittaci DNA was detected in BALF (bronchoalveolar lavage fluid) or blood samples by metagenomic next-generation sequencing (mNGS) in all patients. Use of quinolone was associated with shorter length of hospital stay and fever duration after antibiotic use. Multivariate logistic regression analysis indicated that respiratory support was associated with both severe pneumonia and in-hospital death. CONCLUSIONS: The clinical phenotype of C. psittaci pneumonia is complex and variable. mNGS is helpful in the diagnosis and treatment of C. psittaci pneumonia, and early treatment with quinolone may benefit patients.

Survivors of COVID-19 exhibit altered amplitudes of low frequency fluctuation in the brain: a resting-state functional magnetic resonance imaging study at 1-year follow-up.

Although some short-term follow-up studies have found that individuals recovering from coronavirus disease 2019 (COVID-19) exhibit anxiety, depression, and altered brain microstructure, their long-term physical problems, neuropsychiatric sequelae, and changes in brain function remain unknown. This observational cohort study collected 1-year follow-up data from 22 patients who had been hospitalized with COVID-19 (8 males and 11 females, aged 54.2 ± 8.7 years). Fatigue and myalgia were persistent symptoms at the 1-year follow-up. The resting state functional magnetic resonance imaging revealed that compared with 29 healthy controls (7 males and 18 females, aged 50.5 ± 11.6 years), COVID-19 survivors had greatly increased amplitude of low-frequency fluctuation (ALFF) values in the left precentral gyrus, middle frontal gyrus, inferior frontal gyrus of operculum, inferior frontal gyrus of triangle, insula, hippocampus, parahippocampal gyrus, fusiform gyrus, postcentral gyrus, inferior parietal angular gyrus, supramarginal gyrus, angular gyrus, thalamus, middle temporal gyrus, inferior temporal gyrus, caudate, and putamen. ALFF values in the left caudate of the COVID-19 survivors were positively correlated with their Athens Insomnia Scale scores, and those in the left precentral gyrus were positively correlated with neutrophil count during hospitalization. The long-term follow-up results suggest that the ALFF in brain regions related to mood and sleep regulation were altered in COVID-19 survivors. This can help us understand the neurobiological mechanisms of COVID-19-related neuropsychiatric sequelae. This study was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University (approval No. 2020S004) on March 19, 2020.

Bi-allelic BRWD1 variants cause male infertility with asthenoteratozoospermia and likely primary ciliary dyskinesia.

Genetics-associated asthenoteratozoospermia is often seen in patients with multiple morphological abnormalities of the sperm flagella (MMAF). Although 24 causative genes have been identified, these explain only approximately half of patients with MMAF. Since sperm flagella and motile cilia (especially respiratory cilia) have similar axonemal structures, many patients with MMAF also exhibit respiratory symptoms, such as recurrent airway infection, chronic sinusitis, and bronchiectasis, which are frequently associated with primary ciliary dyskinesia (PCD), another recessive disorder. Here, exome sequencing was conducted to evaluate the genetic cause in 53 patients with MMAF and classic PCD/PCD-like symptoms. Two homozygous missense variants and a compound-heterozygous variant in the BRWD1 gene were identified in three unrelated individuals. BRWD1 staining was detected in the whole flagella and respiratory cilia of normal controls but was absent in BRWD1-mutated individuals. Transmission electron microscopy and immunostaining demonstrated that BRWD1 deficiency in human affected respiratory cilia and sperm flagella differently, as the absence of outer and inner dynein arms in sperm flagellum and respiratory cilia, while with a decreased number and outer doublet microtubule defects of respiratory cilia. To our knowledge, this is the first report of a BRWD1-variant-related disease in humans, manifesting as an autosomal recessive form of MMAF and PCD/PCD-like symptoms. Our data provide a basis for further exploring the molecular mechanism of BRWD1 gene during spermatogenesis and ciliogenesis.

Efficacy and Safety of an Aclidinium Bromide Treatment for 12 Weeks or Longer in Patients with Moderate-To-Severe COPD: A Meta-Analysis.

BACKGROUND: Bronchodilators are commonly used to manage patients with stable chronic obstructive pulmonary disease (COPD). This meta-analysis assessed the efficacy of aclidinium bromide with respect to clinical events, health-related quality of life and symptom scales, pulmonary function, and safety among patients with stable COPD. METHODS: A comprehensive search of MEDLINE, EMBASE, CINAHL and the Cochrane Library databases was undertaken to identify randomized controlled trials (RCTs) that compared aclidinium with placebo, treatment over at least 12 weeks. Trials were measured using either odds ratios (OR) or mean differences (MD) with 95% confidence intervals (CI). RESULTS: Seven studies, containing 7001 patients, were included in this meta-analysis. Compared with placebo, aclidinium bromide reduced the incidence of exacerbation-related hospitalizations (OR 0.64; 95% CI 0.47 to 0.89) and improved quality of life as measured by a lower total George's Respiratory Questionnaire [SGRQ] score (MD -2.34; 95% CI -3.18 to -1.51) and attenuated dyspnea symptom as assessed by changes in the Transitional Dyspnea Index [TDI] (MD 0.76; 95% CI 0.43 to 1.10). Similar changes were observed with regard to trough FEV1 and FVC and peak FEV1 and FVC. No significant differences were observed with regard to all-cause mortality, COPD exacerbations, non-fatal serious adverse events or cardiac adverse events. CONCLUSIONS: Aclidinium reduced the incidence of exacerbation-related hospitalizations and improved quality of life, COPD symptoms and pulmonary function. In addition, aclidinium did not increase the incidence of non-fatal serious adverse events, cardiac adverse events, or COPD exacerbations and was not associated with increased mortality.

Pathogenicity and complete genome sequence of a fowl adenovirus serotype 8 isolate.

In this study we determined and analyzed the complete nucleotide sequence of the genome of a fowl adenovirus serotype 8 (FAdV-8) isolate and examined its pathogenicity in chickens. The full genome of FAdV-8 was 44,055 nucleotides in length with a similar organization to that of FAdV-1 and FAdV-9 genomes. No regions homologous to early regions E1, E3 and E4 of mastadenoviruses were recognized. Along with FAdV-9, FAdV-8 has only one fiber gene and with regard to sequence composition and genome organization, FAdV-8 is closer to FAdV-9 than to FAdV-1. Moreover, our findings suggest that FAdV-1 of species Fowl adenovirus A as the current type species despite its historical priority is not representative of the genus Aviadenovirus, and that FAdV-8 or FAdV-9 in species Fowl adenovirus E and Fowl adenovirus D, respectively, would be more suitable for that designation. Additionally, pathogenicity of FAdV-8 was studied in specific pathogen free chickens following oral and intramuscular inoculations. Despite lack of clinical signs and pathological changes virus was found in tissues and cloacal swabs of all birds with the highest viral copy numbers present in the cecal tonsils. The highest virus titers in the feces for orally and intramuscularly inoculated chickens were recorded at days 10 and 3 post-infection, respectively.

Performance characteristics of a microfluidic Lab-on-Chip electrophoresis system for high-density lipoprotein (HDL) subfraction separation and measurement.

BACKGROUND: High-density lipoprotein (HDL) is believed to be protective against coronary heart disease (CHD). HDL is comprised of different subfractions. Of these, HDL 2b is believed to be the most important in preventing CHD. Current methods for HDL subfraction measurements are not standardized and often exhibit poor analytical performance, which can limit their usefulness in clinical practice. METHODS: We have developed a microfluidic Lab-on-Chip electrophoresis system for serum HDL subfraction measurements. Linear polymers are used as a sieving buffer for HDL particle separation. Nine samples and two controls can be analyzed in 30 min. The percent HDL 2b that comprises total high-density lipoprotein cholesterol (HDL-C) is reported. RESULTS: Samples with HDL-C of 0.259-2.072 mmol/L could be evaluated for percent HDL 2b with coefficient of variations (%CVs) below 8%. Total precision was typically below 1.5% and linearity was observed between 8% and 32% HDL 2b. Measurements were not affected by many therapeutic and biological compounds. Consistent results were obtained by three laboratories and showed r(2) values between 0.93 and 0.95. CONCLUSIONS: The assay allows for easy and reproducible measurement of percent HDL 2b. The experimental procedure and small size of the instrumentation needed for measurement make it viable in clinical settings.

Development of an oligonucleotide-based DNA microarray for transcriptional analysis of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) genes.

A modified oligonucleotide-based two-channel DNA microarray was developed for characterization of temporal expression profiles of select Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) ORFs including its 7 unique ORFs. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements as well as five host genes. Total RNA was isolated at different times post infection from Cf203 insect cells infected with CfMNPV. The cDNA was synthesized, fluorescent labelled with Cy3, and co-hybridized to the microarray chips along with Cy5-labelled viral genomic DNA, which served as equimolar reference standards for each probe. Transcription of the 7 CfMNPV unique ORFs was detected using DNA microarray analysis and their temporal expression profiles suggest that they are functional genes. The expression levels of three host genes varied throughout virus infection and therefore were unsuitable for normalization between microarrays. The DNA microarray results were compared to quantitative RT-PCR (qRT-PCR). Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene, was also detected by array analysis and confirmed by qRT-PCR. The polyhedrin antisense transcript, based on long-range RT-PCR analysis, appeared to be a read-through product of an adjacent ORF in the same orientation as the antisense transcript.

DNA versus protein immunisation for production of monoclonal antibodies against Choristoneura fumiferana ecdysone receptor (CfEcR).

The full-length ecdysone receptor cDNA of Choristoneura fumiferana (CfEcR-B) was cloned into bacterial expression systems and the recombinant protein was expressed either with a His-tag (His-EcR-B) or glutathione-S-transferase (GST) fusion (GST-EcR-B). The His-EcR-B was expressed mostly as insoluble aggregates, while the GST-EcR-B was partially soluble and could be purified using affinity chromatography. Mice were then immunised with the purified GST-EcR-B protein. Due to the time-consuming protein expression and purification procedures and the solubility problem of the recombinant protein, we also inserted the full-length CfEcR-B cDNA into the mammalian DNA vaccine expression vector, pVAC1-mcs for DNA immunisation. In vitro expression of CfEcR-B in mammalian cells transfected with the pVAC-EcR-B plasmid was confirmed prior to the delivery of the DNA vaccine into mice. The anti-CfEcR-B MAbs generated from both DNA and protein vaccines were characterised and shown to recognise native CfEcR-B protein induced by 20E in CF-203 insect cells. DNA immunisation was shown to overcome the solubility problem of the bacterial expressed EcR and created a more direct route for monoclonal antibody production for this receptor protein obviating the need for EcR expression and purification to generate the antigen.

Choristoneura fumiferana nucleopolyhedrovirus encodes a functional 3'-5' exonuclease.

The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III 3'-5' exonucleases. The CfMNPV v-trex ORF was cloned into the Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf21 cells with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified V-TREX confirmed that this viral protein is a functional 3'-5' exonuclease that cleaves oligonucleotides from the 3' end in a stepwise, distributive manner, suggesting a role in proofreading during viral DNA replication and DNA repair. Enhanced degradation of a 5'-digoxigenin- or 5'-(32)P-labelled oligo(dT)(30) substrate was observed at increasing incubation times or increased amounts of V-TREX. The 3'-excision activity of V-TREX was maximal at alkaline pH (9.5) in the presence of 5 mM MgCl(2), 2 mM dithiothreitol and 0.1 mg BSA ml(-1).

Overexpression, purification, and structural analysis of the hydrophobic E5 protein from human papillomavirus type 16.

The E5 proteins of human papillomavirus (HPV) are highly hydrophobic transmembrane proteins that display weak transforming activity. The HPV E5 proteins are localized largely to intracellular membranes, such as the Golgi apparatus and endoplasmic reticulum, but also appear in the plasma membrane. Infection with HPV16 is the cause of over 90% of human cervical cancers. HPV E5 is known to interact with growth factor receptors and gap junction proteins and is believed to play a role during the initiation of neoplasia. The structure of HPV E5 and the mechanism of its interactions with growth factor receptors remain largely unknown. In the present studies, the E5 protein of HPV16 was cloned into the pBAD/TOPO vector fused to an N-terminal thioredoxin leader and a C-terminal His-tag, and expressed in Escherichia coli. The identity of the protein was confirmed by immunoblotting using antibodies against a V5-epitope tag engineered into the protein. Due to formation of high molecular mass superaggregates of the protein, two chromatography steps were employed for its purification: (1) gel filtration chromatography to separate the superaggregated protein from other soluble proteins and (2) Ni-chelate affinity chromatography in the presence of detergent. The superaggregates of the E5-fusion protein were broken down to monomers and various oligomers by sonication in the presence of 0.2% SDS. The purified E5-fusion protein was then reconstituted into lipid vesicles and initial structural analysis of the protein was performed using circular dichroism spectroscopy.