Linker Editing of Pneumococcal Lysin ClyJ Conveys Improved Bactericidal Activity.

Streptococcus pneumoniae is a leading human pathogen uniquely characterized by choline moieties on the bacterial surface. Our previous work reported a pneumococcus-specific chimeric lysin, ClyJ, which combines the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) enzymatically active domain (EAD) from the PlyC lysin and the cell wall binding domain (CBD) from the phage SPSL1 lysin, which imparts choline binding specificity. Here, we demonstrate that the lytic activity of ClyJ can be further improved by editing the linker sequence adjoining the EAD and CBD. Keeping the net charge of the linker constant, we constructed three ClyJ variants containing different lengths of linker sequence. Circular dichroism showed that linker editing has only minor effects on the folding of the EAD and CBD. However, thermodynamic examination combined with biochemical analysis demonstrated that one variant, ClyJ-3, with the shortest linker, displayed improved thermal stability and bactericidal activity, as well as reduced cytotoxicity. In a pneumococcal mouse infection model, ClyJ-3 showed significant protective efficacy compared to that of the ClyJ parental lysin or the Cpl-1 lysin, with 100% survival at a single ClyJ-3 intraperitoneal dose of 100 µg/mouse. Moreover, a ClyJ-3 dose of 2 µg/mouse had the same efficacy as a ClyJ dose of 40 µg/mouse, suggesting a 20-fold improvement in vivo Taking these results together, the present study not only describes a promising pneumococcal lysin with improved potency, i.e., ClyJ-3, but also implies for the first time that the linker sequence plays an important role in determining the activity of a chimeric lysin, providing insight for future lysin engineering studies.

First case of infective endocarditis caused by Methylobacterium radiotolerans.

Methylobacterium radiotolerans has only been identified in blood samples from end-stage renal failure or leukaemia patients in clinic. Here, we report a case of infective endocarditis (IE) caused by M. radiotolerans. 16S rRNA sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to identify the bacteria isolated from cardiac vegetation. A drug sensitivity test was conducted by disk diffusion on blood Mueller-Hinton agar. This isolate was identified as M. radiotolerans, which was susceptible to aminoglycosides and ciprofloxacin. Our findings also suggest that M. radiotolerans can cause infection in a patient with normal immune function.

[Genetic analysis of 90 families affected with spinal muscular atrophy].

OBJECTIVE: To carry out genetic testing and prenatal diagnosis for 90 families affected with spinal muscular atrophy (SMA), and discuss the necessity for carrier screening. METHODS: All families were subjected to multiplex ligation-dependent probe amplification (MLPA) analysis. Combined MLPA and allele-specific PCR (AS-PCR) was used for prenatal diagnosis of the pregnant women. RESULTS: Among the 90 couples, 84 (93%) had a negative family history, 85 (94%) had given birth to an affected child before. Eighty-five husbands and 88 wives carried heterozygous deletion of exon 7 of the SMN1 gene. Two wives had homozygous deletion of exon 7 of the SMN1 gene and were affected. Prenatal diagnosis showed that 19 fetuses were SMA patients, 48 fetuses were carriers, and 23 fetuses were normal. Of note, eighteen affected fetuses were conceived by couples without a family history, which accounted for 20% of all pregnancies and 95% of all affected fetuses. CONCLUSION: To screen SMA carriers using MLPA and carry out prenatal diagnosis using combined MLPA and AS-PCR can ensure accurate diagnosis, which has a significant value for the prevention of SMA affected births.

Identification of differentially expressed genes in primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease that affects exocrine glands. To study the molecular mechanism and identify crucial genes/pathways in pSS pathogenesis, the microarray-based whole-genome gene expression profiles from salivary glands of patients with pSS and non-sicca controls were retrieved. After normalization and subsequent batch effect adjustment, significance analysis of microarrays method was applied to five available datasets, and 379 differentially expressed genes (DEGs) were identified. The 300 upregulated DEGs were enriched in Gene Ontology terms of immune and inflammatory responses, including antigen processing and presentation, interferon-mediated signaling pathway, and chemotaxis. Previously reported pSS-associated genes, including HLA-DRA, TAP2, PRDM1, and IFI16, were found to be significantly upregulated. The downregulated DEGs were enriched in pathways of salivary secretion, carbohydrate digestion and absorption, and starch and sucrose metabolism, implying dysfunction of salivary glands during pathogenesis. Next, a protein-protein interaction network was constructed, and B2M, an upregulated DEG, was shown to be a hub, suggesting its potential involvement in pSS development. In summary, we found the activation of pSS-associated genes in pathogenesis, and provide clues for salivary glands dysfunction. Experimental investigation on the identified DEGs in this study will deepen our understanding on pSS.

c-Myc-Regulated lncRNA-IGFBP4 Suppresses Autophagy in Cervical Cancer-Originated HeLa Cells.

LncRNAs are known to regulate a plethora of key events of cellular processes; however, little is known about the function of lncRNAs in autophagy. Here in the current study, we report lncRNA-IGFBP4 which has previously been known to regulate the proliferation and reprogramming of cancer cells, but its role in autophagy is not yet known. We found that serum starvation provokes autophagy-induced downregulation of lncRNA-IGFBP4 levels. Next, we determined that c-Myc can negatively regulate lncRNA-IGFBP4 in HeLa cells. Phenotypically, we found that upon depletion of lncRNA-IGFBP4, the HeLa cells undergo autophagy through ULK1/Beclin1 signaling. Furthermore, through TCGA data analysis, we found lncRNA-IGFB4 overexpressed in most cancers including cervical cancer. Based on these findings, we conclude that c-Myc maintains cellular homeostasis through negatively regulating lncRNA-IGFBP4 in cervical cancer cells.

Characterization of a blaNDM-1-Bearing IncHI5-Like Plasmid From Klebsiella pneumoniae of Infant Origin.

The spread of plasmid-mediated carbapenem-resistant clinical isolates is a serious threat to global health. In this study, an emerging NDM-encoding IncHI5-like plasmid from Klebsiella pneumoniae of infant patient origin was characterized, and the plasmid was compared to the available IncHI5-like plasmids to better understand the genetic composition and evolution of this emerging plasmid. Clinical isolate C39 was identified as K. pneumoniae and belonged to the ST37 and KL15 serotype. Whole genome sequencing (WGS) and analysis revealed that it harbored two plasmids, one of which was a large IncHI5-like plasmid pC39-334kb encoding a wide variety of antimicrobial resistance genes clustered in a single multidrug resistance (MDR) region. The blaNDM-1 gene was located on a ΔISAba125-blaNDM-1-bleMBL-trpF-dsbC structure. Comparative genomic analysis showed that it shared a similar backbone with four IncHI5-like plasmids and the IncHI5 plasmid pNDM-1-EC12, and these six plasmids differed from typical IncHI5 plasmids. The replication genes of IncHI5-like plasmids shared 97.06% (repHI5B) and 97.99% (repFIB-like) nucleotide identity with those of IncHI5 plasmids. Given that pNDM-1-EC12 and all IncHI5-like plasmids are closely related genetically, the occurrence of IncHI5-like plasmid is likely associated with the mutation of the replication genes of pNDM-1-EC12-like IncHI5 plasmids. All available IncHI5-like plasmids harbored 262 core genes encoding replication and maintenance functions and carried distinct MDR regions. Furthermore, 80% of them (4/5) were found in K. pneumoniae from Chinese nosocomial settings. To conclude, this study expands our knowledge of the evolution history of IncHI5-like plasmids, and more attention should be paid to track the evolution pathway of them among clinical, animal, and environmental settings.

Deciphering the Epidemiological Characteristics and Molecular Features of bla KPC-2- or bla NDM-1-Positive Klebsiella pneumoniae Isolates in a Newly Established Hospital.

The emergence of hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) was regarded as an emerging threat in clinical settings. Here, we investigated the prevalence of CRKP strains among inpatients in a new hospital over 1 year since its inception with various techniques, and carried out a WGS-based phylogenetic study to dissect the genomic background of these isolates. The genomes of three representative bla NDM-1-positive strains and the plasmids of four bla KPC-2-positive strains were selected for Nanopore long-read sequencing to resolve the complicated MDR structures. Thirty-five CRKP strains were identified from 193 K. pneumoniae isolates, among which 30 strains (85.7%) harbored bla KPC-2, whereas the remaining five strains (14.3%) were positive for bla NDM-1. The antimicrobial resistance profiles of bla NDM-1-positive isolates were narrower than that of bla KPC-2-positive isolates. Five isolates including two bla NDM-1-positive isolates and three bla KPC-2-positive strains could successfully transfer the carbapenem resistance phenotype by conjugation. All CRKP strains were categorized into six known multilocus sequence types, with ST11 being the most prevalent type. Phylogenetic analysis demonstrated that the clonal spread of ST11 bla KPC-2-positive isolates and local polyclonal spread of bla NDM-1-positive isolates have existed in the hospital. The bla NDM-1 gene was located on IncX3, IncFIB/IncHI1B, and IncHI5-like plasmids, of which IncFIB/IncHI1B plasmid has a novel structure. By contrast, all ST11 isolates shared the similar bla KPC-2-bearing plasmid backbone, and 11 of them possessed pLVPK-like plasmids. In addition, in silico virulome analysis, Galleria mellonella larvae infection assay, and siderophore secretion revealed the hypervirulence potential of most bla KPC-2-positive strains. Given that these isolates also had remarkable environmental adaptability, targeted measures should be implemented to prevent the grave consequences caused by hv-CRKP strains in nosocomial settings.

A family study of congenital dysfibrinogenemia caused by a novel mutation in the FGA gene: A case report.

Congenital dysfibrinogenemia (CD) is a rare hereditary fibrinogen disorder characterized by normal fibrinogen antigen levels associated with lower functional activities. The aim of this study is to analyze the phenotype and genotype of a family of CD. Routine coagulation screening tests were performed on the proband, her parents, and her grandparents. Then, the purified genomic DNA extracted from peripheral blood was amplified by PCR, and Sanger sequencing was performed to further confirm the mutation. The prothrombin time and activated partial thromboplastin time of the proband were normal, thrombin time prolonged, and the activity of fibrinogen (Fg:Ac) decreased significantly, but fibrinogen antigen (Fg:Ag) level was normal. The coagulation function indices of the proband's father and grandfather were similar to her, and the indices of her mother and grandmother were normal. Sequencing results showed that the proband had a heterozygous missense mutation in FGA gene c.92G > A, which caused the mutation of amino acid 31 from glycine to glutamic acid (p.Gly31Glu). Her father had the same heterozygous mutation. In conclusion, the proband suffered from CD. The change of Gly31Glu in A chain due to the c.92G > A heterozygous missense mutation in the FGA gene is the cause of CD in the family. To the best of our knowledge, the mutation site is new and first reported so far.

Myocardial amyloidosis following multiple myeloma in a 38-year-old female patient: A case report.

Multiple myeloma (MM) is an immunoglobulin-producing tumor of plasma cells, which occurs commonly in the elderly. The incidence of myocardial amyloidosis with MM is extremely low and early clinical manifestations are nonspecific. The diversity of clinical manifestations and first episode symptoms often cause misdiagnosis in young patients with myocardial amyloidosis following MM. In this study, we analyzed the clinical data of a young woman with MM and impaired cardiac function combined with echocardiography, electrocardiography (ECG), laboratory data, cell Congo Red staining, and other manifestations to diagnose amyloidosis. Considering the rapid progression, short survival, and poor prognosis in most patients, a clear, definitive, and timely diagnosis is essential for the treatment of patients with MM complicated with myocardial amyloidosis.

Retracted Article: Exosomal miR-25-3p derived from hypoxia tumor mediates IL-6 secretion and stimulates cell viability and migration in breast cancer.

Hypoxia is a major hallmark of solid tumors and is associated with malignant phenotypes. Exosomal miRNAs derived from hypoxia tumor cells are implicated in the modulation of cancer progression, whereas, the mechanisms underlying the association between hypoxia and exosomal miR-25-3p during breast cancer progression remain to be further clarified. The present study aimed to investigate the role of exosomal miR-25-3p in regulating breast cancer progression. Herein, we found that miR-25-3p expression was increased in hypoxia tumor-derived exosomes a HIF-1α-dependent manner. Hypoxia exosomes markedly stimulated the viability and migration of normoxia breast cancer cells, which was reversed by miR-25-3p depletion. Inhibition of exosomes miR-25-3p lowered hypoxic-induced the expression of IL-6 and NF-κB from THP-1 and RAW264.7 cells in a TLR7/8-dependent way. Treatment of macrophage supernatant (MS) initially incubated with hypoxic-responsed exosomes accelerated the viability and migration of breast cancer cells, and miR-25-3p depletion relieved the stimulatory effects of hypoxic on cell viability and migration. Moreover, miR-25-3p knockdown dramatically suppressed HIF-1α-induced tumor growth in vivo via inactivation of IL-6/STAT3 signaling pathway, reflected by the abated abundances of IL-6 and p-STAT3. These data suggested that absence of exosomal miR-25-3p rescued breast cancer aggressiveness through inhibiting cell viability and migration by regulation of IL-6 secretion from macrophages, providing a potential biomarker for breast cancer treatment.

Honokiol Attenuates the Memory Impairments, Oxidative Stress, Neuroinflammation, and GSK-3ß Activation in Vascular Dementia Rats.

Vascular dementia (VaD) is caused by chronic decreases in brain blood flow and accounts for 15-20% of dementia cases worldwide. In contrast to Alzheimer's disease (AD), no effective drug treatments are currently available for VaD. Previous studies have suggested that oxidative stress and neuroinflammation in the brain play important roles in the pathogenesis of VaD. Honokiol (HKL) is a well-known bioactive and nutraceutical compound that can act as an antioxidant and anti-inflammatory molecule. HKL can protect against memory impairments in AD mouse models. In this study, we explored whether the application of HKL was also protective against the insult of chronic cerebral hypoperfusion (CCH) in rats. We found that HKL supplementation prevented the memory impairments in the inhibitory avoidance step-down and Morris water maze tasks in CCH rats. HKL also suppressed the levels of oxidative stress and inflammation in CCH rats. Moreover, HKL prevented dendritic spines abnormalities in CCH rats. We also found that HKL inhibited the activity of GSK-3ß, which may be critical for the neuroprotective activity of HKL. Thus, our study demonstrated the protective role of HKL in VaD.

Increased serum aminotransferases associated with anti-mitochondrial antibodies in systemic lupus erythematosus patients with autoimmune liver disease.

BACKGROUND: Serum aminotransferase activities are increased in many liver diseases, but the causes for the elevation might be difficult to determine. Whether the elevation of aminotransferases correlates with anti-mitochondrial antibodies (AMA) in systemic lupus erythematosus (SLE) patients with autoimmune liver disease deserves further consideration. METHODS: A meticulous review was done in a large SLE cohort searching for laboratory features of the presence of AMA. Forty-eight hospitalized SLE patients with AMA and 60 randomly selected SLE patients without AMA as a matched case control were enrolled into the retrospective study. Laboratory data were collected, analyzed and compared in SLE patients with and without AMA. RESULTS: Serum activities of aminotransferases were significantly increased in the 48 SLE patients with AMA compared with the 60 subjects without AMA. Meanwhile, we found a positive correlation between serum AMA titers and serum aminotransferase activities. CONCLUSION: Although much remains to be learned about the pathogenesis of autoimmune liver disease associated with AMA, it is possible to suggest that AMA might contribute to the elevation of aminotransferases in SLE patients with the progressive disease.

[The analysis of hepatitis delta virus infective markers in the hepatitis is B virus infective people].

OBJECTIVE: To investigate the distribution of hepatitis delta virus (HDV) marker among hepatitis B virus (HBV) infected patients and to reveal its clinical significance. METHOD: To collect the clinical data and sera samples of HBV infected patients and to detect HDAg, Anti-HDV as well as HBV infection markers by means of enzyme-linked immunosorbnent assay. These data combined with clinical diagnostic results and biochemical index were then analyzed. RESULT: 462 samples of HBV infected patients were collected including 210 HBV carriers without symptom, 175 chronic HBV infections, 35 acute HBV infections and 42 liver fibrosis. The HDV infection rate was 4.8% overall. The highest infection rate of 9.5% was found in the group of liver fibrosis whereas the lower rate of 6.9% was found in HBV chronic carriers. HDV infection rate was 7.8% among the population of 40-60 years old, obviously higher than any other age groups. CONCLUSION: HDV infection was significantly higher in the chronic HBV patients and liver fibrosis patients. Because HDV infection was highly associated with the progress of liver disease, we suggest the screen of HDV markers among hepatitis patients and discriminate whether the patient was co-infected with HDV.

[A clinical study on combined serum hepatic fibrosis-related markers and ultrasound parameters to diagnose hepatic fibrosis].

OBJECTIVE: This study attempted to explore the value of combining serum hepatic fibrosis-related markers and ultrasound parameters together on diagnosis of hepatic fibrosis. METHODS: Six serum markers and 8 ultrasound parameters were measured from 100 patients with chronic hepatitis B or cirrhosis. The results of the serum hepatic fibrosis-related markers and ultrasound in disease group were analyzed and compared with the findings of hepatic pathology. RESULTS: By filtrating,the group of platelet derived growth factor-BB (PDGF-BB) plus hyaluronic acid (HA) plus echo characteristics of liver parenchyma (LPEC) plus length of spleen (SL) had the highest Se and Spe, which were 90.7% and 85.4% respectively. CONCLUSION: The advantageous combination of serum markers and ultrasound parameters can significantly improve Se and Spe, which is superior to any single serum index or ultrasound parameter. And it was a better non-invasive method for diagnosing hepatic fibrosis.

The presence of anti-mitochondrial antibodies in Chinese patients with liver involvement in systemic lupus erythematosus.

Sixty-six hospitalized patients with systemic lupus erythematosus (SLE) were enrolled into this study. The test for anti-mitochondrial antibodies (AMAs) was performed and biochemical parameters were determined. AMAs were detected in 15 of the 66 patients with SLE. Meanwhile, we compared enzymatic levels in AMA-positive and -negative patients and found that serum aminotransferase levels were significantly higher in AMA-positive patients than in AMA-negative individuals. Furthermore, we found a positive correlation between serum AMA titration and serum aminotransferase levels. This study suggests that AMAs might contribute to the elevation of aminotransferases. Although much remains to be learned about the pathogenesis of autoimmune liver disease associated with AMAs, this report might provide greater insight into the metabolic mechanisms of AMAs in AMA-positive patients.