Granzyme B+ B cells detected by single-cell sequencing are associated with prognosis in patients with intrahepatic cholangiocarcinoma following liver transplantation.

B cells possess anti-tumor functions mediated by granzyme B, in addition to their role in antigen presentation and antibody production. However, the variations in granzyme B+ B cells between tumor and non-tumor tissues have been largely unexplored. Therefore, we integrated 25 samples from the Gene Expression Omnibus database and analyzed the tumor immune microenvironment. The findings uncovered significant inter- and intra-tumoral heterogeneity. Notably, single-cell data showed higher proportions of granzyme B+ B cells in tumor samples compared to control samples, and these levels were positively associated with disease-free survival. The elevated levels of granzyme B+ B cells in tumor samples resulted from tumor cell chemotaxis through the MIF- (CD74 + CXCR4) signaling pathway. Furthermore, the anti-tumor function of granzyme B+ B cells in tumor samples was adversely affected, potentially providing an explanation for tumor progression. These findings regarding granzyme B+ B cells were further validated in an independent clinic cohort of 40 liver transplant recipients with intrahepatic cholangiocarcinoma. Our study unveils an interaction between granzyme B+ B cells and intrahepatic cholangiocarcinoma, opening up potential avenues for the development of novel therapeutic strategies against this disease.

High prevalence of fluconazole resistant Candida tropicalis among candiduria samples in China: An ignored matter of concern.

Introduction: The rapid rise of azole resistance in Candida tropicalis causing invasive infections has become a public health concern; however, the prevalence of resistant isolates in urine samples was not well studied, because the clinical significance of candiduria was not unambiguous due to possible host colonization. Methods: We performed a 12-year laboratory-based surveillance study of C. tropicalis causing either invasive infection or candiduria and studied their susceptibility profiles to common antifungal drugs. The complete coding domain sequence of the ERG11 gene was amplified in all fluconazole resistant isolates, and aligned with the wild-type sequence to detect nucleotide mutations. Results: A total of 519 unique C. tropicalis strains isolates, 69.9% of which were isolated from urine samples and remaining 30.1% were invasive strains. Overall, 16.5% isolates were confirmed to be resistant to fluconazole, of which 91.9% were cross-resistant voriconazole. Of note, at the beginning of surveillance (2010-2011), the fluconazole resistance rates were low in both candiduria and invasive groups (6.8% and 5.9%, respectively). However, the resistant rate in the candiduria group significantly increased to 29.5% since 2012-2013 (p = 0.001) and stayed high since then, whilst the resistance rate in the invasive group only showed a gradually increasing trends till 2021 (p > 0.05). Sequence analysis of ERG11 from fluconazole-resistant strains revealed the prevalence of A395T/W mutations were relatively low (16.7%) in the beginning but reached 87.5-100% after 2014. Moreover, the A395W heterozygous mutation isolates became predominant (>60% of resistant strains) after 2016, and indeed isolates carrying corresponding amino acid substitution (Y132F) was highly resistant to fluconazole with MIC50 exceeded 256 µg/ml. Conclusion: Our study revealed high azole resistant rate in candiduria with its increasing trends observed much earlier than stains causing invasive infections. Given antimicrobial resistance as a critical "One Health" issue, the emergence of antifungal resistance in Candida species that are common commensal colonizers in the human body should be concerned.

Pneumocystis pneumonia in liver transplant recipients.

The clinical course of Pneumocystis pneumonia in liver transplant recipients has not been well investigated. Therefore, we collected and analyzed the clinical, epidemiological, and molecular data from patients with Pneumocystis pneumonia as well as paired controls (Chinese Clinical Trial Registry, ChiCTR2100046028; www.chictr.org.cn). There were a total of ten patients diagnosed with Pneumocystis pneumonia containing prospectively included six patients and retrospectively collected four patients, of which seven were transferred to the surgical intensive care unit and four died. The transmission map revealed that inter-patient transmission of Pneumocystis jirovecii was impossible; P. jirovecii detection was negative in all air samples. It was positive only in one sample from the twelve healthcare workers who had close contact with diseased patients. Five out of 79 liver transplant recipients during the outbreak were colonized with Pneumocystis jirovecii compared to 2 out of 94 after the outbreak upon admission (P>0.05). Liver transplant recipients with Pneumocystis pneumonia had totally different genotypes based on multilocus sequence typing. Additionally, we found an unreported mutation in the cytochrome b gene. The absolute CD19+ B-cell counts (odds ratio: 1.028; 95% confidence interval: 1.000-1.057; P=0.049) were defined to be the only significant independent risk factor. At a cut-off value of 117.16/µL, the sensitivity and specificity were 100% and 70%, respectively. Pneumocystis pneumonia is a severe complication following liver transplantation. The outbreak may not be caused by nosocomial transmission. A decrease in absolute CD19+ B-cell counts may be associated with the development of Pneumocystis pneumonia.

[The clinical characteristics, treatment and outcome of macrolide-resistant Mycoplasma pneumoniae pneumonia in children].

OBJECTIVE: To investigate the drug resistance of Mycoplasma pneumoniae among children with community-acquired pneumonia (CAP), and to explore the clinical and radiological characteristics of and the role of azithromycin in the treatment of of macrolide-resistant (MR) Mycoplasma pneumoniae pneumonia. METHODS: Cases of CAP in children (n = 179) were prospectively enrolled in the Pediatric ward of Beijing Chaoyang Hospital from 1st September, 2010 to 31st August 2011. Pharyngeal swabs were collected for detection of Mycoplasma pneumoniae DNA. Mycoplasma pneumoniae culture and in vitro susceptibility testing were also performed. RESULTS: Eighty-three cases met the diagnostic criteria of mycoplasma pneumonia, accounting for 46% of the CAP patients. Mycoplasma pneumoniae culture was positive in 45 cases, including 44 highly resistant to macrolides (MR) in vitro, and 1 sensitive. The 44 cases caused by MR pathogen presented with fever for (8 ± 3) d and cough for (17 ± 5) d, with higher fever (39.5 ± 0.7) °C and more irritating dry cough. In most of the children, peripheral blood leukocytes (8 ± 4)×10(9)/L were normal, with normal or elevated (0.60 ± 0.94) neutrophils, normal or slightly elevated erythrocyte sedimentation rate [(24 ± 14) mm/1 h] and CRP (12.8 mg/L). Chest X-ray showed lobar consolidation in 10 cases (23%, 10/44), among them 3 in the lower left lung, 2 in the left lung, 3 in the right lower lung, 2 in the right upper lung. Pleural effusion (small amount), combined with right lower lung consolidation, was found in 1 case. Patchy shadows were found in 27 cases, and interstitial lung infiltrate in 7 cases. Of the 44 cases caused by MR Mycoplasma pneumoniae, 19 had lung computed tomography (CT) scanning, among them 13 had lobar or segmental consolidation. Azithromycin therapy started in an average of 4.0 days after onset of illness, with duration of therapy averaging (9 ± 4) d. Cephalosporin or penicillin (n = 1) was the initial antibiotic choice in 12 of them, while combination therapy with azithromycin and cephalosporin or penicillin antibiotics was given in 41 of them. The duration of fever averaged (6 ± 3) d after treatment of azithromycin and duration of cough averaged (17 ± 5) d after treatment. Among patients with MR Mycoplasma pneumonia, those with lobar consolidation had longer duration of fever after treatment with azithromycin, compared with those without consolidation (P < 0.05). CONCLUSIONS: The macrolide resistance rate was 98% (44/45) in our patients. Fever and duration of therapy with azithromycin in MR infection was longer in patients with lobar consolidation. The 44 children with MR Mycoplasma pneumonia recovered with no serious complications.

Tyrosine sulfation in N-terminal domain of human C5a receptor is necessary for binding of chemotaxis inhibitory protein of Staphylococcus aureus.

AIM: Staphylococcus aureus evades host defense through releasing several virulence proteins, such as chemotaxis inhibitory protein of staphylococcus aureus (CHIPS). It has been shown that extracellular N terminus of C5a receptor (C5aR) forms the binding domain for CHIPS, and tyrosine sulfation is emerging as a key factor in determining protein-protein interaction. The aim of this study was to evaluate the role of tyrosine sulfation of N-terminal of C5aR in its binding with CHIPS. METHODS: Expression plasmids encoding C5aR and its mutants were prepared using PCR and site-directed mutagenesis and were used to transfect HEK 293T cells using calcium phosphate. Recombinant CHIPS protein was purified. Western blotting was used to examine the binding efficiency of CHIPS to C5aR or its mutants. RESULTS: CHIPS exclusively binds to C5aR, but not to C5L2 or C3aR. A nonspecific sulfation inhibitor, sodium chlorate (50 nmol/L), diminishes the binding ability of C5aR with CHIPS. Blocking sulfation by mutation of tyrosine to phenylalanine at positions 11 and 14 of C5aR N terminus, which blocked sulfation, completely abrogates CHIPS binding. When tyrosine 14 alone was mutated to phenylalanine, the binding efficiency of recombinant CHIPS was substantially decreased. CONCLUSION: The results demonstrate a structural basis of C5aR-CHIPS association, in which tyrosine sulfation of N-terminal C5aR plays an important role. Our data may have potential significance in development of novel drugs for therapeutic intervention.

Anticancer effect and neurotoxicity of S-(+)-deoxytylophorinidine, a new phenanthroindolizidine alkaloid that interacts with nucleic acids.

Phenanthroindolizidine alkaloids are a family of plant-derived compounds with significant antineoplastic activity as well as other effects like antiamebicidal, antiviral, and anti-inflammatory activities. The specific biomolecular targets of these compounds have not yet been clearly identified. S-(+)-Deoxytylophorinidine (CAT) is a new phenanthroindolizidine alkaloid, originally extracted from the roots of Tylophora atrofolliculata and Tylophora ovata. Potent anticancer activity was observed in vitro and in vivo. Neurotoxicity of CAT was also studied and it was far less serious than that of vinblastine. Interactions between this compound and DNA had been studied in detail in our laboratory previously, and we further studied its interactions with RNA.

[Three different drug interaction mathematical models used to evaluate the cytotoxicity of SAHA and arsenic trioxide in combination].

To clarify the source of deviations of drug combination effects evaluated with different drug interaction mathematical models, the cytotoxicity of SAHA and arsenic trioxide and their combinations were observed in a series of human cancer cell lines and a normal cell line. The combined effects were evaluated with three drug interaction models: Loewe Additivity (LA), Bliss Independence (BI) and Chou's Median Effect Model. The evaluations with three different models were further compared with each other. We demonstrated that when dose-response curves were fitted with the same method, similar evaluated results for drug combinations would be derived with different models. The deviations of evaluated effects of drug combinations were attributed to different curve fitting methods used rather than the models themselves. The effects of drug combinations showed discrepancies on different cell lines, and at different combined drug concentrations on same cell line.

Chemokine receptor CXCR3 is important for lung tissue damage and airway remodeling induced by short-term exposure to cigarette smoking in mice.

AIM: To investigate the role of chemokine receptor CXCR3 in cigarette smoking (CS)-induced pulmonary damage. METHODS: CXCR3 knockout (CXCR3-/-) mice were used. Differences in airspace enlargement, mRNA expression of matrix metalloproteinases (MMPs), transforming growth factor (TGF) beta1, CXCL10 in lung homogenates, and CXCL10 content in bronchoalveolar lavage (BAL) fluids and homogenates were compared between CXCR3-/- mice and wild-type (WT) mice three days after three-day CS exposures. RESULTS: The linear intercept was significantly less in CXCR3-/- mice than in WT mice (30.1+/-0.9 microm vs 40.3+/-2.4 microm, P<0.01). Morphologically, collagen was deposited less around airways and vessels in CXCR3-/- mice. The lung hydroxyproline content was significantly lower in CXCR3-/- mice than in WT mice (6.0+/-1.0 microg/mL vs 12.0+/-1.6 microg/mL, P<0.05). Profoundly lower mRNA expression of MMP2, MMP12, TGF beta 1, and CXCL10 was seen in lung homogenates from CXCR3-/- mice. CXCL10 concentrations in BAL fluid and lung homogenates were significantly lower in CXCR3-/- mice than in WT mice (BAL fluid: 19.3+/-1.4 pg/mL vs 24.8+/-1.6 pg/mL, P<0.05; lung homogenates: 76.6+/-7.0 pg/mL vs 119.5+/-15.9 pg/mL, P<0.05). CONCLUSION: CXCR3 is important in mediating lung tissue damage and airway remodeling following a short-term CS insult, possibly through up-regulation of CXCL10 and inducement of mRNA expression of MMPs. Targeting CXCR3 may be helpful for prevention of CS-induced pulmonary pathology.

[Application of circular dichroism to the study of interactions between small molecular compounds and DNA].

Circular dichroism (CD) is an useful technique for monitoring DNA conformation changes resulting from changes in environmental conditions, such as temperature, ionic strength, and pH, and also for the study of the interaction between DNA and ligands (including small molecules and proteins). CD spectroscopy of DNA arises from the asymmetric backbone sugars and by the helical structures often adopted by nucleic acids. By the interpretation of induced circular dichroism (ICD) of ligand signals resulting from the coupling of electric transition moments of the ligand, DNA bases within the asymmetric DNA environment, ligand-DNA interactions, as well as the DNA-binding mode can be assessed. A number of important conclusions have been reported that related to the observed ICD signals resulting from the interactions between intercalators and groove binders with DNA. If short oligonucleotide sequences are used in the study, sequences-specific of binding also can be deduced. CD determination requires smaller amounts of sample, and not limited by the molecular weight or size and can be performed rapidly; though CD is of low resolution, but it's a complement to NMR and X-ray diffraction methods. This review will introduce the characters of the CD spectra of DNA, and its application to the studies of DNA with small molecules; some progress of the studies in our laboratory will also be discussed. CD is expected to be used as a screening method in seeking more DNA-targeted drugs, such as, antineoplastic, antimicrobial and antiviral drugs.