Diagnostic accuracy of a real-time PCR assay for detection of Helicobacter pylori and resistance to clarithromycin and levofloxacin directly from stool.

OBJECTIVE: The non-invasive detection of Helicobacter pylori (H. pylori) and its resistance to clarithromycin and levofloxacin significantly improves the management of infected patients by enabling tailored eradication treatments without the need for endoscopic procedures. This study aimed to assess the effectiveness of real-time PCR (RT-PCR) assays in identifying H. pylori infection and antibiotic resistance in stool and gastric biopsy specimens. PATIENTS AND METHODS: Stool and gastric biopsy samples were collected from patients within three days of post-hospitalization. A total of 115 samples were analyzed for H. pylori infection, and an additional 115 samples were evaluated for resistance to clarithromycin and levofloxacin using an RT-PCR-based molecular test. Statistical analyses were performed using (SPSS 26.0 IBM Corp., Armonk, NY, USA). RESULTS: Among 115 patients (53 males, average age 50.8±13.2 years), H. pylori was detected in 93.1% of stool samples and 93.9% of gastric biopsies. The RT-PCR assay demonstrated a sensitivity of 99.1% and a specificity of 100%, with an overall diagnostic accuracy of 99.1%. Clarithromycin resistance was found in 37.3% of stool and 46.9% of gastric biopsy specimens, with the assay showing 79.6% sensitivity and 98.4% specificity. Levofloxacin resistance was identified in 32.1% of stool samples and 31.3% of gastric biopsies, with 86.3% sensitivity and 91.1% specificity of the molecular test. CONCLUSIONS: The RT-PCR-based detection of H. pylori and its resistance to clarithromycin and levofloxacin in stool samples represents a promising approach to enhance eradication therapy outcomes, potentially improving treatment efficacy. Chictr.org.cn: ChiCTR2300070267.

Down-regulation of miR-155 promotes apoptosis of nasopharyngeal carcinoma CNE-1 cells by targeting PI3K/AKT-FOXO3a signaling.

OBJECTIVE: Nasopharyngeal carcinoma is one of the common malignant tumors of the ear, nose, and throat in China. Cell apoptosis is expected to be closely related to prognosis. In recent years, with the development of non-coding RNA function research, it is proposed that miRNA plays an important role in the pathogenesis of nasopharyngeal carcinoma. This study aimed to investigate the role of miR-155 in the apoptosis of nasopharyngeal carcinoma cells. PATIENTS AND METHODS: Cell transfection was performed to knockdown or overexpress the level of miR-155 or knockdown of the level of FOXO3a. The expression of the related protein was detected by immunoblotting. The Real Time quantitative-PCR was used to detect miR-155 expression. Cell proliferation was assessed by MTT assay. The changes of cell apoptosis were observed by flow cytometry using AV-PI staining and TUNEL staining. RESULTS: The miR-155 inhibitor and mimics were successfully capable of knocking down or overexpressing miR-155 levels. After knocking down miR-155 level, cell proliferation was significantly attenuated, and apoptosis was significantly increased compared with the sham group (p < 0.05). After overexpression of miR-155, opposite results were observed. In addition, in the cells with the knockdown of miR-155 level, further knockdown of FOXO3a level significantly reduced the inhibitory effect of miR-155 on cell apoptosis compared with the control group (p < 0.05). CONCLUSIONS: In nasopharyngeal carcinoma CNE-1 cell line, miR-155 can inhibit the proliferation and promote apoptosis of nasopharyngeal carcinoma cells by targeting PI3K/AKT-FOXO3a signaling. MiR-155 may be a novel target for the treatment of nasopharyngeal carcinoma.

Diagnostic accuracy of adenosine deaminase for tuberculous pericarditis: a meta-analysis.

OBJECTIVE: Many studies suggest that adenosine deaminase is a marker for tuberculous pericarditis, while controversy exists as to its diagnostic utility. This study aims to summarize the overall diagnostic performance of adenosine deaminase for tuberculous pericarditis through a meta-analysis. MATERIALS AND METHODS: Literatures published before May 2015 were searched in PubMed and EMBASE. The data were retrieved and the sensitivity, specificity, positive/negative likelihood ratio (PLR/NLR), diagnostic odds ratio (DOR) of adenosine deaminase for diagnosing tuberculous pericarditis were pooled, and the summary receiver operating characteristic (SROC) curves were used to examine the overall performance of adenosine deaminase. RESULTS: In total, 11 studies with 938 subjects were included in the meta-analysis. The summary estimates of adenosine deaminase for diagnosing tuberculous pericarditis were listed as follows: sensitivity of 0.90 (95% CI: 0.86-0.93), specificity of 0.86 (95% CI: 0.83-0.89), PLR of 5.90 (95% CI: 4.46-7.82), NLR of 0.15 (95% CI: 0.09-0.26), and DOR of 42.55 (95% CI: 21.51-84.18). The area under the SROC curve was 0.92, and the Q value was 0.85. No publication bias was identified. CONCLUSIONS: Adenosine deaminase is a valuable marker with both high sensitivity and specificity in the diagnosis of tuberculous pericarditis. Nevertheless, the results of adenosine deaminase assays should be interpreted in combination with other test results and clinical characteristics of patients.

Neurofollicular hamartoma with strong diffuse S-100 positivity: a case report.

Neurofollicular hamartoma is a recently described lesion with a distinct pilosebaceous and spindle cell proliferation. Neurofollicular hamartoma is composed of spindle cells haphazardly arranged in a fibromyxoid stroma closely associated with an abnormal hyperplasia of folliculosebaceous units. Although this histologic pattern has been classified as "neurofollicular," all cases reported thus far have had only scattered spindle cells with S-100 positivity. We present a case of neurofollicular hamartoma with strong and diffusely positive staining of spindled cells for S-100 protein. This lesion also shows scattered positivity of spindle cells for monoclonal neuron specific enolase and synaptophysin. We interpret the results of immunostains of this lesion as evidence for neural differentiation. This case validates the concept of "neurofollicular" hamartoma.

Sebaceous neoplasm with reticulated and cribriform features: a rare variant of sebaceoma.

Troy and Ackerman defined the term sebaceoma (Am J Dermatopathol 1984: 6: 7-13) as benign neoplasm of basaloid cells with varying numbers of mature sebocytes. Steffen and Ackerman (Neoplasms with sebaceous differentiation. Philadelphia: Lee and Febiger, 1994: 401-425) illustrated many examples of sebaceoma, two of which had a reticulated and cribriform pattern. We report a case of sebaceoma from the scalp of a 52-year-old white female. Histologically, it displayed reticulated and cribriform basaloid epithelial islands. This is the third reported case of sebaceoma, to our knowledge, with these unusual features.

Ovarian mucinous cystadenoma associated with mural leiomyomatous nodule and massive ovarian edema.

Mural nodules associated with mucinous and serous tumors of the ovary may represent a reactive process, a benign tumor, or a malignant neoplasm. Mural leiomyomatous nodule in mucinous cystadenoma is extremely rare. Two such cases had been described previously. In this case a 43-year-old white female presented with 24-h history of left quadrant pain and a left adenexal cystic mass on ultrasound examination. An exploratory laparotomy revealed a left ovarian mass with torsion on its pedicle. Frozen section of the cystic mass showed a mucinous cystadenoma with mural smooth muscle proliferation. A total abdominal hysterectomy and bilateral salpingo-oophorectomy were performed. Histologic examination of the mass revealed a mucinous cystadenoma with a mural leiomyomatous nodule and an enlarged ovary with massive stromal edema. This is the first case of a mural leiomyomatous nodule in association with a mucinous cystadenoma in an ovary with massive edema. This case broadens the histologic spectrum in which a mural leiomyomatous nodule may be encountered.

Crystal structure of desheptapeptide(B24-B30)insulin at 1.6 A resolution: implications for receptor binding.

The crystal structure of desheptapeptide (B24-B30) insulin (DHPI), a virtually inactive analog of insulin, was determined at 1.6 A resolution. In the refined structure model, DHPI retains three alpha-helices (A1-A8, A12-A18, and B9-B19) as its structural framework, while great conformational changes occur in the N and C termini of B-chain. The beta-turn, which lies in B20-B30 in insulin and insulin analogs with high potency, no longer exists in DHPI. Relative motion is observed among the three alpha-helices, each as a rigid functional group. In contrast, a region covering B5-B6 and A6-A11 exhibits a relatively stable conformation. We interpret our results as identifying: (i) the importance of beta-turn in determining the receptor-binding potency of insulin and (ii) a leading role of PheB24 in maintaining the beta-turn structure.

Stable photobleaching of P840 in Chlorobium reaction center preparations: presence of the 42-kDa bacteriochlorophyll a protein and a 17-kDa polypeptide.

Simple procedures for the anaerobic preparation of photoactive and stable P840 reaction centers from Chlorobium tepidum and Chlorobium limicola in good yield are presented and quantitated. The subunit composition was tested by cosedimentation in sucrose density gradients. For C. limicola, it minimally comprises four subunits: the P840 reaction center protein PscA, the BChla antenna protein FMO, the FeS protein PscB with centers A and B, and a positively charged 17-kDa protein denoted PscD. The preparation from Chlorobium tepidum additionally contained PscC, a cytochrome c-551. The BChla absorption peak of the purified complexes was at 810 nm, with a shoulder at 835 nm. The ratio of the shoulder to the peak was 0.25, which corresponds to 1 reaction center per 70 BChla molecules if a uniform extinction coefficient of BChla is assumed. However, bleaching at 610 nm in continuous light corresponded up to 1 photoactive reaction center per 50 BChla molecules. Therefore, either the extinction coefficient of BChla in the reaction center is overestimated or the one for photobleaching is underestimated. In any case, the major portion of the reaction center was photoactive in the preparations. A P840 reaction center subcomplex, lacking PscD and deficient in FMO and PscB, but retaining the cytochrome c subunit, was obtained as a side product. It was photoinactive and had an absorption peak at 814 nm and a 835/814 absorbance ratio of 0.42. FMO and PscB show the tendency to form a complementary subcomplex. FMO and PscD are apparently required to stabilize the photoactive reaction center, while the cytochrome c subunit is not.

Cartilage chondrolysis by fibronectin fragments is associated with release of several proteinases: stromelysin plays a major role in chondrolysis.

We have reported that three different Fn fragments (Fn-f) added to bovine articular cartilage cultured in serum-free DMEM cause marked elevation of proteoglycan (PG) degradation and release into the culture media. We report here that the PG release required the continual presence of Fn-f, that PG release still occurred when serum-free cultures were switched to bovine synovial fluid media, and that addition of recombinant IGF-1, TGF-beta, and recombinant interferon gamma to cultures did not affect Fn-f-mediated PG release. The Fn-f caused a 25-fold enhanced release of stromelysin-1 protein from cartilage by Day 1 and up to 120-fold by Day 3. The stromelysin form released was 43 kDa, the activated form of pro-stromelysin-1. This stromelysin form apparently played a major role in Fn-f-mediated PG release, since addition of Sepharose-bound anti-stromelysin-1 to cartilage cultures greatly slowed rates of PG release. Potential activators of pro-stromelysin-1, plasmin, and u-PA (urinary plasminogen activator), were also detected in conditioned media of Fn-f-treated cartilage. u-PA levels were increased in the presence of the Fn-f but by only a few fold. Addition of alpha-1-antiproteinase inhibitor, which can block enzymatic activity of u-PA, was found to inhibit about half the PG-releasing activity of the Fn-f. Levels of TIMP-1, the 30-kDa tissue inhibitor of metalloproteinases, which can inhibit stromelysin, doubled within 24 h when a Fn-f was added to culture. These data suggest that stromelysin-1 may be a major mediator of Fn-f-mediated PG release from cartilage.

A transcription unit for the Rieske FeS-protein and cytochrome b in Chlorobium limicola.

A transcription unit petCB from Chlorobium limicola is described. The leading gene petC codes for a Rieske FeS-protein of 19.04 kDa with 181 amino acid residues. The following gene petB codes for a cytochrome b of 47.48 kDa with 428 amino acid residues. The transcription unit lacks a third gene pet-A for cytochrome c 1 or-f, which is found in the fbc-operons of gram-negative bacteria. In the derived amino acid sequence for the Rieske FeS-protein the four cysteines and the 2 histidines are conserved in the peptides binding the 2Fe2S-cluster, although the redox potential of the cluster is about 150 mV more negative in Chlorobium. The gene for cytochrome b includes the coding region for an N-terminal, positively charged extension which is typical for Chlorobium. The gene is not split into two parts for cytochrome b 6 and subunit IV. However, a fourteenth amino acid between the two histidines in the fourth, putative transmembrane helix, and the lack of an eighth transmembrane helix at the C-terminus, among other features, clearly resemble the cytochrome b 6 f-complexes. Therefore, the separation into b 6 f- and bc 1-type complexes during evolution must have occurred before the split of the gene.

Targeted inactivation of the gene psaL encoding a subunit of photosystem I of the cyanobacterium Synechocystis sp. PCC 6803.

Photosystem I is a multisubunit pigment-protein complex that functions as a light-driven plastocyanin-ferredoxin oxidoreductase in thylakoid membranes of cyanobacteria and higher plants. A 16-kDa protein subunit of photosystem I complex was isolated from the cyanobacterium Synechocystis sp. PCC 6803. The sequence of its NH2-terminal residues was determined and a corresponding oligonucleotide probe was used to isolate the gene encoding this subunit. The gene, designated as psaL, codes for a protein of 16,605 Da. The deduced amino acid sequence is homologous to the subunit PsaL of barley photosystem I. There are two conserved hydrophobic regions in the subunit PsaL that may cross or interact with thylakoid membranes. The gene psaL exists as a single copy in the genome and is expressed as a monocistronic RNA. Stable mutant strains in which the gene psaL was interrupted by a gene conferring resistance to chloramphenicol, were generated by targeted mutagenesis. Growth and photosynthetic characteristics of a selected mutant strain under photoautotrophic conditions were similar to those of the wild type, suggesting that the function of PsaL is dispensable for photosynthesis in Synechocystis sp. PCC 6803. Western analysis and subunit composition of purified photosystem I revealed that the mutant strain contained other subunits of photosystem I in thylakoid membranes and in the assembled complex. When photosystem II activity was inhibited and glucose was supplied in the medium, mutant strains grew faster than the wild type. Under these conditions of growth, re-reduction of P700 in the mutant cells, but not in the wild type cells, showed a component with an uncharacteristically rapid half-time.

The atp2 operon of the green bacterium Chlorobium limicola.

The operon (atp2) encoding the beta and epsilon subunits of F-ATPase from Chlorobium limicola was cloned and sequenced. In contrast with purple bacteria these genes are arranged in a separate operon similar to the cyanobacteria. The operon terminates with a pronounced stem-loop structure. About 0.8 kb upstream of the beta subunit a gene encoding the enzyme phospho enol pyruvate carboxykinase was identified. This gene is transcribed in the opposite direction of the atp2 operon and also ends with a stem-loop structure. These genes of green bacteria are among the first to be sequenced, and therefore the genetic distance between these genes and corresponding genes from other bacteria and eukaryotes was studied. Even though the operon structure resembles that of cyanobacteria, the evolutionary tree compiled from these data places the chlorobium gene close to purple bacteria. Chlorobium limicola beta and epsilon subunits complemented Escherichia coli mutants defective in the corresponding subunits, indicating that the hybrid enzyme formed from subunits of the two bacteria is active in ATP synthesis.

Release of elastase from monocytes adherent to a fibronectin-gelatin surface.

Fibronectin (Fn) is a circulating and extracellular matrix glycoprotein that may serve to facilitate phagocytosis because of its ability to bind many inflammatory ligands and to a monocyte receptor. Fn fragments have been shown in many systems to have augmented properties over those of native Fn. We show in this report that although Fn fragments did not cause elastase release from monocytes in suspension, fragments did cause elastase release from monocytes that were first bound to Fn-gelatin surfaces. An amino-terminal 29-Kd and a 140-Kd integrin-binding fragment were half-maximally active at 100 nmol/L, whereas the Arg-Gly-Asp-Ser integrin-recognition peptide was half-maximally active at 100 mumol/L. Fluid-phase Fn was ineffective yet blocked the activity of the Fn fragments. Complexing of Fn with gelatin or with heparin partially removed the blocking effect of Fn. Similar results were obtained with U-937 cells. Substitution of the Fn-gelatin surface with bovine articular cartilage also promoted elastase release. Therefore, in conditions in vivo in which monocytes bind to tissue surface, a high ratio of Fn fragments to native Fn may upregulate certain monocyte activities such as protease release.

Identification of the subunit carrying FeS-centers A and B in the P840-reaction center preparation of Chlorobium limicola.

The product of the second gene in a transcription unit for the P840-reaction center of Chlorobium limicola f.sp. thiosulfatophilum, which codes for a protein of 23.87 kDa with 232 amino acids, was identified as the subunit migrating in SDS-PAGE at the apparent molecular weight of 32 kDa in reaction center preparations, by Western blotting and N-terminal sequencing. This protein corresponds to PsaC, a 8 kDa-subunit of Photosystem 1 which carries the FeS-centers A and B.

Photosynthetic reaction center genes in green sulfur bacteria and in photosystem 1 are related.

Oxygenic photosynthesis of chloroplasts and cyanobacteria involves two photosystems, which originate from different prokaryotic ancestors. The reaction center of photo-system 2 (PS2) is related to the well-characterized reaction center of purple bacteria, while the reaction center of photosystem 1 (PS1) is related to the green sulfur bacteria, as is convincingly documented here. An operon encoding the P840 reaction center of Chlorobium limicola f.sp. thiosulfatophilum has been cloned and sequenced. It contains two structural genes, coding for proteins of 730 and 232 amino acids. The first protein resembles the large subunits of the PS1 reaction center. Putative binding elements for the primary donor, P840 in Chlorobium and P700 in PS1, and for the acceptors A0, A1, and FeS center X are conserved. The second protein is related to the PS1 subunit carrying the FeS centers A and B. An adjacent third gene, not belonging to the reaction center, encodes a protein related to dolichyl-phosphate-D-mannose synthase from yeast. The different origins of PS1 and PS2 are discussed.

Fibronectin fragments in osteoarthritic synovial fluid.

Fibronectin is an adhesive multifunctional glycoprotein found in the extracellular matrix of most types of cells and that exerts growth factor, differentiative and chemotactic activities toward many types of cells, including those cells found in knee joint tissue. Since fibronectin levels in the synovial fluid (SF) and on the cartilage surface of patients with osteoarthritis (OA) have been shown to be greatly increased over normal levels and since protease levels are also enhanced in diseased cartilage, we have investigated the presence of fibronectin fragments in the SF of patients with OA. We report that concentrations of at least 1 microM of 100 to 200 kDa fragments were found in all OA fluids examined. Since we have recently shown that fibronectin fragments can cause cartilage to release metalloproteinases, resulting in severe proteoglycan depletion, and others have shown that fragments also enhance metalloproteinase expression in synovial fibroblasts, the presence of these fragments suggests pathologic consequences in arthritis.

The photosystem I-like P840-reaction center of green S-bacteria is a homodimer.

An operon encoding the P840 reaction center of Chlorobium limicola f.sp.thiosulfatophilum has been cloned and sequenced. It contains two structural genes coding for proteins of 730 and 232 amino acids. The first protein resembles the large subunits of the Photosystem I (PS I) reaction center. Putative binding elements for the primary donor, P840 in Chlorobium and P700 in PS I and for the acceptors A(o), A(1) and FeS-center X are conserved. The second protein is related to the PS I subunit carrying the FeS-centers A and B. Since all our efforts to find a gene for a second, large subunit failed, the P840 reaction center probably is homodimeric.

Fibronectin fragments cause chondrolysis of bovine articular cartilage slices in culture.

Elevated fibronectin (Fn) and Fn fragment concentrations are found in the synovial fluid of osteoarthritic and rheumatoid arthritic patients. Fn has been shown to affect expression of chondrocytic matrix proteins, and Fn fragments have been shown to elevate gene expression of neutral proteinases in synoviocytes. For these reasons, we tested the effects of Fn fragments on protease release and resultant proteoglycan release from cartilage in serum-free bovine articular cartilage explant cultures. We have found that 1 microM amino-terminal 29- and 50-kDa gelatin-binding Fn fragments caused over a 50-fold enhancement of gelatinolytic and collagenolytic proteinase release with a 23-fold enhancement of proteoglycan (PG) release. Release was significant at fragment concentrations as low as 20 nM. An integrin-binding 140-kDa fragment mixture was the least active fragment, whereas native Fn had little activity. The relative activities of the fragments correlated with their relative abilities to bind to cartilage. The RGDS integrin-recognition peptide also caused release, although sequence mutants did not. PG release was blocked by actinomycin D, cycloheximide, and deoxyglucose. Fn fragment-mediated PG release was decreased in 10% serum by over 10-fold but was still 2-fold greater than in controls. In the presence of insulin-like growth factor-1, PG release was as great as without serum. We suggest that Fn fragments, as found in diseased synovial fluid, may contribute to protease-mediated damage to cartilage.

The amino-terminal 29- and 72-Kd fragments of fibronectin mediate selective monocyte recruitment.

Proteolytic fragments of fibronectin (Fn) can possess properties not inherent to intact Fn. Previously, only mixtures of low molecular weight Fn fragments, and the 120-Kd fibroblastic cell-binding segment, but not intact Fn, were shown to be selectively chemotactic for human monocytes (MOs). In order to determine if other structural domains of Fn were responsible, we tested six Fn fragments. The amino-terminal 72-Kd fragment at 1.5 microns was about 75% as potent as zymosan-activated serum (ZAS). Its amino-terminal 29-Kd degradation product at 1.0 micron was about one third as potent as ZAS. Checkerboard analysis confirmed chemotaxis. Complexing gelatin to 72-Kd fragments reduced MO chemotaxis by 28% to 30%. Reducing disulfide bonds in 29- and 72-Kd segments had no effect. A synthetic peptide containing the thrombin cleavage site between the 29- and 50-Kd segments of the 72-Kd fragment was chemotactic. The 50-, 190/170-, 35-, and 160/150/120-Kd fragments, and intact Fn were not chemotactic for MOs. The data suggest that the 72-Kd fragment and its 29-Kd subfragment are additional Fn fragments that mediate selective MO chemotaxis. We speculate that proteinases present at inflammatory sites can liberate such fragments that selectively recruit MOs.

[Ultraviolet spectrophotometric determination of cantharidin in Mylabris].

In this study the content of cantharidin in Mylabris was analyzed quantitatively by UV. Analytical results thus obtained agree well with those by the neutralization method set forth in ChP. The average recovery of cantharidin is 99.98% and the coefficient of variation is 0.719%.