[Diagnostic values of nuclear score combined with cyclin D1 immunocytochemistry in indeterminate thyroid follicular nodules in preoperative fine needle aspiration].

Objective: To assess the feasibility of nuclear score combined with cyclin D1 immunocytochemistry in classifying indeterminate thyroid nodules with fine-needle aspiration (FNA) cytological diagnosis of Bethesda category Ⅲ-Ⅴ. Methods: A consecutive cohort of 118 thyroid FNA specimens with indeterminate diagnosis (TBSRTC category Ⅲ-Ⅴ) and available histopathologic follow-up data were collected between December 2018 and April 2022 at the Department of Pathology, Beijing Hospital, China. These cases were subjected to cytological evaluation and cyclin D1 immunocytochemistry. The optimal cut-off points of a simplified nuclear score and the percentage of cyclin D1-positive cells for the diagnosis of malignancy or low-risk neoplasm were determined using the receiver operating characteristic (ROC) curves and area under the ROC curve (AUC). The specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) of nuclear score and cyclin D1 immunostaining were evaluated from the crosstabs based on cut-off points. The diagnostic accuracy of simplified nuclear score combined with cyclin D1 immunostaining was estimated using ROC curve analysis. Results: Nuclear grooves, intra-nuclear inclusions and chromatin clearing were more commonly found in malignancy/low-risk neoplasms than benign lesions (P=0.001, P=0.012 and P=0.001 respectively). A cut-off point of≥2 for the simplified nuclear score was sensitive for defining malignancy/low-risk neoplasm, and its PPV, NPV, sensitivity and specificity were 93.6%, 87.5%, 99.0% and 50.0% respectively. A positive cut-off point of 10% positive thyroid cells in cyclin D1 immunostaining demonstrated sensitivity of 88.5%, specificity of 100%, PPV of 100% and NPV of 53.8% for correctly detecting thyroid malignancy or low-risk neoplasm. The sensitivity and PPV of simplified nuclear score combined with cyclin D1 immunostaining were 93.3% and 100%, respectively. Both specificity and NPV were maintained at high levels (100% and 66.7%, respectively). The diagnostic accuracy of simplified nuclear score combined with cyclin D1 immunostaining in detecting thyroid malignancy/low-risk neoplasm was increased to 94.1% compared to using either of them alone. Conclusions: Combing simplified nuclear score and cyclin D1 immunostaining on FNA cytology specimens can increase the diagnostic accuracy in classifying thyroid nodules of indeterminate cytological categories. Thus, this supplementary approach provides a simple, accurate, and convenient diagnostic method for cytopathologists so that may reduce unnecessary thyroidectomies.

[Diagnostic values of cyclin D1 immunocytochemistry and molecular testing in preoperative fine needle aspiration of undeterminate thyroid nodules].

Objective: To assess the value of cyclin D1 immunocytochemistry combined with a small panel molecular analysis in indeterminate cytological diagnosis of Bethesda category Ⅲ-Ⅴ. Methods: A consecutive cohort of 96 thyroid FNA specimens with indeterminate diagnosis (TBSRTC category Ⅲ-Ⅴ) and available histopathologic follow-up data were collected between December 2018 and December 2021 in Department of Pathology, Beijing Hospital. The cases were evaluated by cyclin D1 immunocytochemistry and molecular testing of BRAFV600E or a small panel of markers (BRAF, N-RAS, H-RAS, K-RAS and TERT) in the FNA specimens. The identification of the optimal cut-off point of cyclin D1 for the diagnosis of malignancy was evaluated using the receiver operating characteristic (ROC) curves and the assessment of the area under the ROC curve (AUC). The specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) of all these markers were evaluated with the crosstabs and significance was calculated. Results: Ninty-six patients with 96 thyroid nodules were enrolled, including 42 cases of TBSRTC-III, 10 cases of TBSRTC-IV and 44 cases of TBSRTC-V. There were 79 females and 17 males with a median age of 47 years (range, 25 to 75 years). A 7.5% cut-off value for positive cyclin D1 nuclear immunostaining in thyroid cells demonstrated 100% PPV, 57.1% NPV, 81.0% sensitivity and 100% specificity for thyroid malignancy diagnosis. The sensitivity of the BRAFV600E mutation test or combined with a small panel test alone for thyroid malignancy diagnosis were 65.5% and 69.0% respectively. The sensitivity for thyroid malignancy diagnosis increased to 94.0% and 95.2% respectively when combining the cyclin D1 immunocytochemistry with the molecular test, and the specificities remained 100% and 91.7% respectively.The accuracy of cyclin D1 immunocytochemistry combined with a small panel of molecular test in detecting thyroid malignancy increased to 94.8% compared to using these markers alone. Conclusions: The addition of cyclin D1 immunocytochemistry and a small panel of molecular testing to FNA cytology can increase the sensitivity and NPV of cytology in indeterminate categories, and this supplementary approach provides a simple, accurate and convenient diagnostic method for reducing unnecessary thyroidectomies.

[Diagnosis of lung biopsy employing the 2015 WHO criteria and detection of related oncogenic driver mutations].

Objective: The diagnostic criteria of lung biopsy specimens by 2015 WHO lung tumor classification were used to evaluate lung biopsy specimens along with detection of genetic alterations of major tumor driving genes including epidermal growth factor receptor (EGFR). Methods: The clinical data, histological slides, immunohistochemical stains and special stains of 806 lung biopsy specimens at Beijing Hospital from July 2015 to July 2018 were retrospectively analyzed. Diagnosis of lung cancer was reclassified according to the 2015 WHO lung tumor classification and related gene mutation data were analyzed. Results: During a three-year period, the total number of lung cancer diagnosis was 483 cases, including 221 female and 262 male patients with age ranging from 37 to 85 years (median age of 65 years). There were 40 cases(8.28%) of small cell carcinoma,11 cases (2.28%) of large cell neuroendocrine carcinoma, 3 cases (0.62%) of combined neuroendocrine carcinoma, 2 cases(0.41%) of atypical carcinoid, 208 cases (43.06%) of adenocarcinoma, 92 cases(19.05%) of non-small cell carcinoma, favor adenocarcinoma, 66 cases (13.66%) of squamous cell carcinoma, 42 cases(8.70%) of non-small cell carcinoma, favor squamous cell carcinoma, 16 cases(3.31%) of non-small cell carcinoma, not otherwise specified, and 3 cases (0.62%) of non-small cell carcinoma, possible adenosquamous carcinoma. Among 202 cases tested, 107 cases (52.97%) showed EGFR mutations, including 86 of 133 cases (64.66%) of adenocarcinoma and 18 of 52 cases (34.62%) of non-small cell carcinoma, favor adenocarcinoma. Twenty two cases were found to have T790M mutation among 27 patients after EGFR TKI targeted drug therapy. Immunohistochemical staining of ALK (D5F3) was positive in 3 of 354 cases of non-small cell lung cancer, confirmed by EML4-ALK fusion gene fluorescence PCR. ROS1 gene fusion was found in 1 of 38 cases. Splicing mutations in exon 14 of MET gene were seen in one case of non-small cell carcinoma with spindle cell differentiation. Conclusion: The new diagnostic criteria by the 2015 WHO lung tumor classification is better suited for diagnosing lung biopsy specimens and providing accurate treatment guidance and improving the patient outcome.

[Histologic subtyping of poorly-differentiated solid lung cancer with molecular testing for epidermal growth factor receptor mutation and ALK gene rearrangement: an analyses of 167 cases].

Objective: To study the histological subtyping of poorly differentiated solid lung cancer by using immunohistochemistry and mucin staining along with analysis of epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) gene rearrangement. Methods: Among 827 cases of non-small cell lung cancer at Beijing Hospital from April 2014 to April 2017, 167 cases of solid poorly differentiated lung cancer were identified and histopathologically subtyped by mucin staining (D-PAS) and immunohistochemistry using 10 antibodies (CK7, vimentin, Ki-67, CK5/6, p40, TTF1, Napsin A, CD56, chromogranin A, and synaptophysin). Paraffin embedded tumor samples were subjected to mutation analysis of exons 18, 19, 20 and 21 of the EGFR gene by amplification refractory mutation system (ARMS) method. Immunohistochemistry (Ventana D5F3) for ALK gene rearrangement was performed followed by ALK fluorescence in situ hybridization (FISH) verification. Results: There were 79 females and 88 males in the study cohort. The patient's age ranged from 35 to 77 years (mean 62 years). Cases with solid growth pattern (at least >10%) and without typical histological features of adenocarcinoma, squamous cell carcinoma or neuroendocrine carcinoma were further divided based on immunohistochemistry and mucin stain into 64 cases(38.32%)of adenocarcinoma, 34 cases(20.35%) squamous cell carcinoma, 21 cases(12.57%)large cell neuroendocrine carcinoma, 5 cases(2.99%)combined large cell neuroendocrine carcinoma, 2 cases(1.20%)adenosquamous carcinoma and 41 cases(24.55%)large cell carcinoma. The Ki-67 positive rate ranged from 5% to 65%. Mutations of EGFR were detected in 5 cases (2.99%, 5/167) of adenocarcinoma(19del in 3 cases and L858R in 2 cases). Two cases(1.20%, 2/167) with ALK-rearranged were identified by immunohistochemistry (Ventana D5F3) and confirmed by ALK FISH. Conclusions: Poorly differentiated solid lung cancer without distinct morphological features can be further histologically subtyped by mucin staining and immunohistochemistry. Molecular testing should be performed for accurate molecular target therapy to improve the prognosis.

[Effects of PI3Kδ-RhoA pathway on phagocytosis defect of alveolar macrophages in a mouse model of chronic obstructive pulmonary disease].

Objective: To investigate effects of Phosphoinositide3-Kinases (PI3Kδ)-Ras homolog gene family member A(RhoA) pathway on phagocytosis deficiency of alveolar macrophages (AMs) in a mouse model of chronic obstructive pulmonary disease (COPD). Methods: Twenty mice were exposed to cigarette smoking to establish the COPD model, with 20 mice as the control group. AMs were isolated from lung tissue by discontinuous density gradient centrifugation and then divided into a healthy control group, a COPD group, a healthy IC87114 group and a COPD IC87114 group. The culture of IC87114 group was mixed with a final concentration of 1 nmol/L IC87114 for 24 hours. Mean fluorescence intensity (MFI) and the positive percent of AMs engulfing flurescein isothiocyanate-labeled Escherichina coli (FITC-E.coli) (AM%) were detected by flow cytometry. Real-Time PCR(RT-PCR)and Western blot were applied to detect mRNA and protein. G-LISA RhoA Kit was used to detect the activity of RhoA, and laser scanning confocal microscopy was used to observe the cytoskeleton structure of AMs. Results: Phagocytosis of AM: MFI and AM %in the COPD group [(4 512±517), (32.2±4.6)%] were decreased than those in the healthy control group [(9 857±1 042), (68.0±4.0)%, all P<0.01]. Compared with the COPD group, MFI and AM% in the COPD IC87114 group [(6 894±472), (50.6±2.1)%] were increased (all P<0.01). The expressions of mRNA and protein of PI3Kδ in the COPD group (3.14±0.54, 0.84±0.08) were increased than those in the healthy control group (1.00±0.00, 0.57±0.07) (all P<0.01). Compared with the COPD group, the expressions of mRNA and protein of PI3Kδ in the COPD IC87114 group (1.52±0.28, 0.66±0.13) were decreased (all P<0.01). The RhoA mRNA, protein and activity in the COPD group (0.70±0.07, 0.41±0.10, 0.70±0.06) were decreased compared to those in the healthy control group (1.00±0.00, 0.56±0.09, 1.19±0.09) (all P<0.01). Compared with the COPD group, the expression of mRNA, protein and activity of RhoA in the COPD IC87114 group(0.91±0.08, 0.48±0.06, 0.86±0.06) were increased (P<0.01, P<0.05). Cytoskeleton of AM: The pseudopods of the healthy control group and the healthy IC87114 group extended well, and the ability of phagocytosing FITC-E.coli was intact, but there were some defects in the COPD group. Compared with the COPD group, the COPD IC87114 group was better, both in phagocytosing and extending of pseudopods. Negative correlations existed between the mRNA, protein of PI3Kδ with mRNA, protein and activity of RhoA. Negative correlations also existed between the mRNA, protein of PI3Kδ with MFI, but positive correlations between RhoA and MFI were observed in all groups. Conclusion: The phagocytosis of AMs in COPD mice was defective, with abnormal rearrangement of the cytoskeleton. PI3Kδ negatively regulated RhoA, while PI3Kδ over activation resulted in decreasing activity of RhoA and then induced abnormal cytoskeleton rearrangement in AMs, which led to phagocytosis deficiency.IC87114 inhibited PI3Kδ activation, improved the activity of RhoA and partly recovered phagocytosis of AMs.

[Accurate grading of pancreatic neuroendocrine tumors with Ki-67 index in fine-needle aspiration specimens: a comparative cytologic and histologic study].

Objective: To study the cytomorphologic features and determine whether pancreatic neuroendocrine tumors (PanNET) sampled by fine-needle aspiration (FNA) can be accurately graded based on the Ki-67 index when compared to surgical samples. Methods: Corresponding intraoperative (19 cases) or endoscopic ultrasound-guided (3 cases) FNA cytology and surgical tissue specimens were obtained from 22 tumors, which were reviewed and stained for Ki-67 proliferation marker. The cytological samples included more than 200 tumor cells. Samples were graded by scoring the Ki-67 positive index in accordance with the 2010 WHO criteria. The grading scores assigned to the FNA cytology samples were compared with the scores assigned to the corresponding histological samples. Concordance was achieved by using 5% (instead of 2%) as a cut-off value for defining G2 tumors. One cytological sample included less than 500 tumor cells was excluded in the concordance calculation. Results: The cytological smears consisted of uniform, monotonous and isolated cells, loose cellular aggregates and rosette-like formations. Some tumor cells clustered around segments of capillaries. The cells demonstrated distinct cytoplasmic and nuclear features. Mitoses and necrosis were rarely seen. When traditional 2% Ki-67 index cut-off value were used to classify G2 tumors, the majority (86.4%, κ=0.812, P<0.01) of FNA cytology samples and corresponding surgical tissue specimens demonstrated concordance. When a 5% cut-off value was adopted, the concordance rate was 95.5% (21/22, κ=1.000, P<0.01). Similar concordance rates between the cytological and histological grades were achieved with threshold value of cytological assessment material set at more than 500 or 200 cells. Conclusions: The cytological Ki-67 index in adequate material (>200 tumor cells) is useful in grading pancreatic neuroendocrine tumors, and a cut-off value of 5% showed better predictive value compared with that of 2%. Accurate grading of PanNET is critical for predicting tumor biology, patient prognosis, and making informed decisions regarding patient management and treatment.

[Mechanisms of cytoskeleton and PI3Kδ-RhoA in fine particulate matter deteriorating phagocytosis defect of alveolar macrophage in mice with chronic obstructive pulmonary disease].

Objective: To explore the mechanism of cytoskeleton and PI3Kδ-RhoA in fine particulate matter deteriorating phagocytosis defect of alveolar macrophage (AM) in chronic obstructive pulmonary disease (COPD) mice. Methods: Forty mice were randomly divided into four groups: health control group, COPD group, health PM2.5 group, COPD PM2.5 group and with ten in each group. A mouse model of COPD was established by cigarette smoke exposure, and health PM2.5 group and COPD PM2.5 group mice were given PM2.5 (588 µg/m(3)) aerosol inhalation for 90 days. AM were isolated from lung tissue by discontinuous density gradient centrifugation. Mean fluorescence intensity (MFI) and the percent of alveolar macrophage engulfing flurescein isothiocyanate-labeled Escherichia coli (FITC-E.coli) AM (AM%) were detected by flow cytometry. The mRNA and protein expression were measured by real time polymerase chain reaction (RT-PCR) and Western blot. The activity of RhoA was measured by GTPase linked immunosorbent assay (G-LISA) Kit. Cytoskeleton was observed by laser scanning confocal microscopy. Results: The MFI and the AM% in COPD group [4 512±517, (32.19±4.57)%] and health PM2.5 group [7 631±585, (50.78±4.58)%] were significantly lower than those in health control group [9 857±1 042, (68.53±2.88)%], while those in COPD PM2.5 group [3 121±393, (21.90±2.58)%] were lower than those in COPD group (all P<0.01). The mRNA and protein of PI3Kδ in COPD group (3.41±0.54, 0.84±0.08)and health PM2.5 group (1.52±0.35, 0.71±0.11) were higher than those in health control group (1.00±0.00, 0.57±0.07) (all P<0.05), and in COPD PM2.5 group (5.53±0.42, 1.17±0.25), the above parameters were remarkably increased as compared to those in COPD group (all P<0.01). The mRNA, protein and activity of RhoA in COPD group (0.70±0.07, 0.41±0.10, 0.70±0.06) and health PM2.5 group (0.84±0.06, 0.46±0.11, 0.87±0.07) were lower than those in health control group (1.00±0.00, 0.56±0.09, 1.19±0.09) (all P<0.05), and above parameters of COPD PM2.5 group (0.42±0.05, 0.31±0.06, 0.44±0.04) were significantly lower than COPD group (all P<0.01). Cytoskeleton of AM: long and dense filopodia and membrane fold could been seen clearly around the AM of health control group; in COPD group and health PM2.5 group, short and sparse filopodia and slightly deformed AM can been seen. Filopodia remarkably decreased and rigid cells with impaired capacity of engulfing FITC-E.coli can be generally observed in COPD PM2.5 group. Negative correlations were existed between PI3Kδ mRNA, protein and RhoA mRNA, protein, activity in all groups (all P<0.01). Negative correlations were existed between PI3Kδ mRNA, protein and MFI, and positive correlations were existed between RhoA mRNA, protein, activity and MFI in all groups (all P<0.05). Conclusion: Fine particulate matter (PM2.5) can deteriorate the phagocytosis of AM from COPD mice through over activating PI3Kδ and inhibiting the activity of RhoA then causing cytoskeleton abnormal rearrangement.

[Cytology of basaloid squamous cell carcinoma and small cell carcinoma of lung: a comparative study].

OBJECTIVE: To evaluate the roles of cytomorphology and immunohistochemistry in distinguishing between basaloid squamous cell carcinoma (BSC) and small cell carcinoma (SCC) of lung. METHODS: The direct smears and/or liquid-based cytology preparation (ThinPrep) of bronchial brushing/washing and fine-needle aspiration (FNA) specimens from 17 cases of biopsy-proven BSC of lung were retrospectively reviewed and compared with those from 17 cases of SCC. The cytomorphologic parameters analyzed included proportion of cohesive cell clusters, cell palisades/rosettes, adenoid cystic features, crushing artifact, nuclear maximum diameter, nuclear molding, scantiness of cytoplasm,"salt-and-pepper"nuclei, distinct nucleoli, spindly configuration, individual cell keratinization, necrosis, hyaline material, apoptosis and mitotic activity. Immunocytochemical/immunohistochemical study of 25 cases was performed. Ten FNA samples of basaloid squamous cell carcinoma were also analyzed for epidermal growth factor receptor mutations in exons 18, 19, 20 and 21 using amplification refractory mutation system. RESULTS: Most of the 17 BSC cases (15/17) showed a predominance of tightly cohesive tumor cell clusters. The proportion of isolated tumor cells was high in SCC (more than 60% in 14 cases). The nuclear maximum diameter of BSC was slightly larger than that of SCC (9 to 11 µm in BSC versus 7 to 9 µm in SCC)."Salt-in-pepper"nuclei, nuclear molding and crushing artifact were detected in all SCC cases (15/17, 17/17 and 14/17, respectively). These features were only occasionally found in BSC group. Nucleoli were present in BSC and rarely (2/17) in SCC. Only 9 of 17 BSC cases showed individual cell keratinization. The differences in the above-mentioned cytomorphologic features were statistically significance (P<0.05). The results of immunohistochemistry performed on the cell block sections and immunocytochemistry performed on the ThinPrep slides were identical to that performed on the corresponding biopsy specimens. The tumor cells in BSC were consistently positive for CK5, p40 and p63. TTF1, chromogranin A, synaptophysin and CD56 were positive in most of SCC. One of SCC cases showed focal PAX5 expression. No EGFR mutations were detected in the 10 BSC cases studied. CONCLUSIONS: Selected cytomorphologic features, including presence of cohesive cell clusters, larger nuclear size, distinct nucleoli, lack of crushing artifact, absence of nuclear molding and presence of individual cell keratinization, are helpful in diagnosing BSC on cytology specimens. Immunohistochemistry using a panel of TTF1, CK5, p40/p63 and chromogranin A/synaptophysin/CD56 provides further clues in differential diagnosis between BSC and SCC. EGFR mutation study is often negative in lung BSC.

[Clinicopathological diagnosis of diffuse alveolar hemorrhage].

OBJECTIVE: To improve knowledge about the clinical and pathological features of diffuse alveolar hemorrhage (DAH). METHODS: Six cases DAH with intact clinical and pathological data were retrospectively analyzed during the period from May 1999 to May 2015 in Beijing Hospital. There were altogether 2 males and 4 females, with age ranging from 32 to 68 years (mean 58.8 years). Specimens were obtained by autopsy (3 cases), open lung biopsy (2 cases) and renal biopsy (2 cases), including 1 case of open lung biopsy in 2003, renal biopsy in 2012. RESULTS: Clinically, the patients presented with cough, shortness of breath and dyspnea, including 5 cases of hemoptysis, 4 cases of fever, 3 cases of skin and mucosa bleeding, 2 cases of gross hematuria, 2 cases of microscopic hematuria, 3 cases of renal functional impairment. A total of 5 cases had different levels of elevated erythrocyte sedimentation rate and C-reactive protein, 6 cases had moderate anemia, hypoxemia, diffuse infiltrates with alveolar filling in chest CT. Serum antineutrophil cytoplasmic antibody was positive in 3 cases, anti-glomerular basement membrane antibody was present in 1 case. Pathological diagnosis: 2 cases of Granulomatosis with polyangiitis (GPA), 2 cases of Microscopic polyangiitis (MPA), 1 case of Goodpasture syndrome, 1 case of pulmonary veno-occlusive disease (PVOD). PROGNOSIS: 3 cases died; 2 cases were discharged; 1 case received symptomatic treatment, follow-up after discharge. CONCLUSION: The mainly clinical characteristics of DAH are varied degree of dyspnea, anemia, hypoxemia, and extensive ground-glass opacification or consolidation in image, with or without haemoptysis; diffuse acute or chronic pulmonary hemorrhage in lung tissue is the main pathological feature.

Effect of sophoridine on Ca²âº induced Ca²âº release during heart failure.

Sophoridine is a type of alkaloid extract derived from the Chinese herb Sophora flavescens Ait (kushen) and possess a variety of pharmacological effects including anti-inflammation, anti-anaphylaxis, anti-cancer, anti-arrhythmic and so on. However, the effect of sophoridine on heart failure has not been known yet. In this study, the effect of sophoridine on heart failure was investigated using Sprague-Dawley (SD) rat model of chronic heart failure. Morphological results showed that in medium and high dose group, myofilaments were arranged orderly and closely, intermyofibrillar lysis disappeared and mitochondria contained tightly packed cristae compared with heart failure group. We investigated the Ca(2+) induced Ca(2+) transients and assessed the expression of ryanodine receptor (RyR2) and L-type Ca(2+) channel (dihydropyridine receptor, DHPR). We found that the cytosolic Ca(2+) transients were markedly increased in amplitude in medium (deltaF/F(0)=43.33+/-1.92) and high dose groups (deltaF/F(0)=47.21+/-1.25) compared with heart failure group (deltaF/F(0)=16.7+/-1.29, P<0.01), Moreover, we demonstrated that the expression of cardiac DHPR was significantly increased in medium- and high dose-group compared with heart failure rats. Our results suggest that sophoridine could improve heart failure by ameliorating cardiac Ca(2+) induced Ca(2+) transients, and that this amelioration is associated with upregulation of DHPR.

Defective Ca(2+) handling proteins regulation during heart failure.

Abnormal release of Ca(2+) from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction in heart failure (HF). We previously demonstrated that RyR2 macromolecular complexes from HF rat were significantly more depleted of FK506 binding protein (FKBP12.6). Here we assessed expression of key Ca(2+) handling proteins and measured SR Ca(2+) content in control and HF rat myocytes. Direct measurements of SR Ca(2+) content in permeabilized cardiac myocytes demonstrated that SR luminal [Ca(2+)] is markedly lowered in HF (HF: DeltaF/F(0) = 26.4+/-1.8, n=12; control: DeltaF/F(0) = 49.2+/-2.9, n=10; P<0.01). Furthermore, we demonstrated that the expression of RyR2 associated proteins (including calmodulin, sorcin, calsequestrin, protein phosphatase 1, protein phosphatase 2A), Ca(2+) ATPase (SERCA2a), PLB phosphorylation at Ser16 (PLB-S16), PLB phosphorylation at Thr17 (PLB-T17), L-type Ca(2+) channel (Cav1.2) and Na(+)- Ca(2+) exchanger (NCX) were significantly reduced in rat HF. Our results suggest that systolic SR reduced Ca(2+) release and diastolic SR Ca(2+) leak (due to defective protein-protein interaction between RyR2 and its associated proteins) along with reduced SR Ca(2+) uptake (due to down-regulation of SERCA2a, PLB-S16 and PLB-T17), abnormal Ca(2+) extrusion (due to down-regulation of NCX) and defective Ca(2+) -induced Ca(2+) release (due to down-regulation of Cav1.2) could contribute to HF.

Combining polymerase chain reaction restriction enzyme analysis with phenotypic characters for mycobacteria identification in Taiwan.

SETTING: Many hospitals use the fully-automated BACTEC 960 Mycobacteria Growth Indicator Tube (MGIT) system and acid-fast staining to detect acid-fast bacilli (AFB) in clinical specimens; however, labour-intensive biochemical methods are used for further mycobacterial species identification. OBJECTIVE: To develop a user-friendly algorithm for mycobacterial species identification from AFB smear-positive BACTEC tubes. DESIGN: AFB smear-positive BACTEC tubes were collected and mycobacteria were isolated and identified by biochemical methods. The tubes were subgrouped by rpoB duplex polymerase chain reaction restriction enzyme analysis (rpoB DPRA). The results were combined with key phenotypic characters of mycobacteria isolated from the tubes to develop a species identification algorithm with 16S rDNA sequencing of the isolate being used as the gold standard method. RESULTS: By rpoB DPRA, 441 AFB smear-positive BACTEC tubes were correctly subgrouped into 100 tubes containing Mycobacterium tuberculosis complex, 335 tubes containing non-tuberculous mycobacteria and six tubes containing both. A species identification algorithm was developed by combining the rpoB DPRA results of the tubes with growth rate, photoreactivity and two biochemical results of mycobacteria recovered from the tubes. CONCLUSION: This user-friendly algorithm can be used for mycobacterial species identification from AFB smear-positive BACTEC tubes.

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) interacts with dengue virus core protein.

Heterogeneous nuclear ribonucleoprotein K (hnRNP K), a component of hnRNP particles, is involved in several steps of gene expression regulation. Dengue (DEN) virus, a member of the Flaviviridae, is the primary cause of illnesses such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. In mature DEN virus particles, the core protein is a structural protein that forms a nucleocapsid complex with genomic RNA. Very little of its biologic functions is known. Here, using an in vitro binding assay and coimmunoprecipitation analysis, we report a protein-protein interaction between the DEN virus core protein and hnRNP K. The C-terminal hydrophilic region of the DEN virus core protein, spanning amino acid residues 73 to 100, is required for such interaction. Results of glutathione-S transferase binding assays indicated that the core protein-hnRNP K interaction might be abolished in the presence of hnRNP K cognate nucleic acids. Furthermore, in a cotransfection experiment, the repressive effect of hnRNP K on C/EBPbeta-mediated transcription activation could be reversed by full-length DEN virus core protein but not by a truncated form containing amino acids 1-72. Our results suggest that, on DEN virus infection, the multiple functions of cellular hnRNP K may be affected by the virus core protein.

Negative regulation of IS2 transposition by the cyclic AMP (cAMP)-cAMP receptor protein complex.

Three sequences similar to that of the consensus binding sequence of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex were found in the major IS2 promoter region. Experiments were performed to determine whether the cAMP-CRP complex plays a role in the regulation of IS2 transposition. In the gel retardation assay, the cAMP-CRP complex was found to be able to bind the major IS2 promoter. A DNA footprinting assay confirmed that the cAMP-CRP complex binds to the sequences mentioned above. With an IS2 promoter-luciferase gene fusion construct, the cAMP-CRP complex was shown to inhibit transcription from the major IS2 promoter. IS2 was found to transpose at a frequency approximately 200-fold higher in an Escherichia coli host defective for CRP or adenyl cyclase than in a wild-type host. These results suggest that the cAMP-CRP complex is a negative regulator of IS2 transposition.

Functional domains of the InsA protein of IS2.

The InsA protein is a transcriptional regulator. It binds to the promoter region of insA and insAB'. To understand the molecular mechanism for the interaction between InsA and its binding sequence, the functional domains of InsA were identified. The glutaraldehyde cross-linking method and the two-hybrid expression system were used to study the protein-protein interaction of InsA. The results of these experiments showed that InsA forms homodimers. Deletion of the last 44 amino acid residues at its C terminus, but not the first 12 or 57 residues at the N terminus, abolished the ability of InsA to form homodimers, indicating that the protein-protein interaction domain of InsA is located at its C terminus. Gel retardation assays revealed that deletion of the last 29 amino acid residues at its C terminus had no effect on the DNA binding ability of InsA. In contrast, deletion of the first N-terminal 12 residues abolished the DNA binding capability of InsA. These results indicate that the DNA binding domain of InsA is located at its N terminus.

Detection of an IS2-encoded 46-kilodalton protein capable of binding terminal repeats of IS2.

The genome of the transposable element IS2 contains five open reading frames that are capable of encoding proteins greater than 50 amino acids; however, only one IS2 protein of 14 kDa had been detected. By replacing the major IS2 promoter located in the right terminal repeat of IS2 with the T7 promoter to express IS2 genes, we have detected another IS2 protein of 46 kDa. This 46-kDa protein was designated InsAB'. Analyses of the InsAB' sequence revealed motifs that are characteristic of transposases of other transposable elements. InsAB' has the ability to bind both terminal repeat sequences of IS2. It was shown to bind a 27-bp sequence (5'-GTTAAGTGATAACAGATGTCTGGAAAT-3', positions 1316 to 1290 by our numbering system [16 to 42 by the previous numbering system]) located at the inner end of the right terminal repeat and a 31-bp sequence (5'-TTATTTAAGTGATATTGGTTGTCTGGAGATT-3', positions 46 to 16 [1286 to 1316]), including the last 27 bp of the inner end and the adjacent 4 bp of the left terminal repeat of IS2. This result suggests that InsAB' is a transposase of IS2. Since there is no open reading frame capable of encoding a 46-kDa protein in the entire IS2 genome, this 46-kDa protein is probably produced by a translational frameshifting mechanism.

Isolation and identification of influenza viruses from clinical materials in 1977-1993 at Veterans General Hospital-Taipei.

From 1977 to 1993, 15,189 throat swab samples were received for isolation and identification of influenza virus in the Clinical Virology Laboratory, Veterans General Hospital-Taipei. Most of the samples came from the Pediatric Department. There were 634 identified strains of the influenza virus; the successful isolation rate was 4.17% in average/year. Among these isolates, 56.3% (357/634) were influenza B; 12.1% (77/634) were influenza A/H1N1 and 28.1% (178/634) were influenza A/H3N2. About 3.5% (22/634) were classified as flu-like agents because of no reaction with available monoclonal antibodies. In recent years, reverse transcriptase polymerase chain reaction (RT-PCR) was established here to re-evaluate these virus stocks. This method can provide rapid diagnosis method to identify influenza A/H1N1, A/H3N2 and B. Further, the RT-PCR method and sequencing of amplified DNA could be used to see the variation of virus isolates which were recirculated or which reappeared in the Taipei area.

Gene for cytochrome c oxidase subunit II in the mitochondrial DNA of Culex quinquefasciatus and Aedes aegypti (Diptera: Culicidae).

The cytochrome c oxidase subunit II (COII) gene between transfer RNA for Leu and Lys in the mitochondrial DNA of Culex quinquefasciatus Say and Aedes aegypti (L.) was amplified by the polymerase chain reaction (PCR) technique. Both the gene order and direction of transcription were identical to Anopheles and Drosophila. Nucleotide sequences of the PCR-amplified COII genes in these two mosquitoes exhibited 88% homology, and the frequency of transition was very close to that of transversion. The homology of deduced amino acid sequences of COII between these two mosquitoes was 95%. Two highly conserved segments of COII proteins were found in mosquitoes, fruit flies, locust, and honeybee. These segments contain the major amino acid residues of cytochrome c oxidase involved in electron transport and ligand binding. The amino acid residues are located at the positions similar to those of the mammalian enzymes. Two sets of the phylogenetic trees of a similar pattern were generated by comparing the divergences of nucleotide and amino acid sequences of COII. The branch lengths of the trees estimated by amino acid and nucleotide sequences showed different evolution rates of Aedes and Culex from their common ancestor.

Functional analysis of the 14 kDa protein of insertion sequence 2.

The IS2 sequence encodes five open reading frames (ORF1 to ORF5) that are greater than 150 nucleotides each. Only one protein of 14 kDa was detected when the expression of IS2 genes was examined in minicells. This 14 kDa protein was referred to as InsA in this study and was determined to be encoded by ORF1. A sixfold decrease in IS2 transposition frequency was observed when insA was overexpressed. DNA footprinting results indicated that InsA binds to the sequence 5'-TAAATAA-3' located at IS2 nucleotide numbers 1286 to 1292. (The IS2 right terminal repeat spans nucleotides 1290 to 1331.) This InsA binding sequence is situated 4 bp upstream from the putative "-10" sequence of the insA promoter that overlaps the right terminal repeat of IS2. The presence of a promoter located in this region was demonstrated by the ability of a DNA fragment containing the right terminal repeat to drive the expression of a promoterless lacZ gene. The transcription of insA was determined to start at the A residue located at nucleotide number 1268. With the same insA promoter-lacZ fusion construct, overexpression of insA in the same cell was found to decrease the beta-galactosidase activity. The results of this study suggest that InsA affects IS2 transposition by regulating the transcription of IS2 genes.

Carpal bone dislocations: an analysis of twenty cases with relative emphasis on the role of crushing mechanisms.

Twenty cases of carpal bone dislocation were encountered during a 7-year period, with an average of 27 months of follow-up. There were ten types of dislocation in this series; the most common type was transscaphoid perilunate dislocation which was seen in nine cases. In addition, there were two scaphoid subluxations; one volar lunate dislocation; one dorsal perilunate dislocation; one scaphoid perilunate dislocation; one hamate and pisiform dislocation; one transhamate pisiform dislocation; one trapezoid dislocation with dislocation of carpometacarpal joints two to five; one dislocation of the trapezium, trapezoid, and carpometacarpal joints two to four; and two trapezium periscapholunate dislocations. Methods of treatment included open reduction, closed reduction, proximal row carpectomy, total wrist arthrodesis, and excision of the lunate. In this series, the patterns of dislocation were different for crushing injuries and dorsiflexion injuries. The clinical results associated with the soft-tissue injuries of the ipsilateral hand were mostly caused by crushing forces. Although carpal instabilities were noted, there was no significant correlation between the clinical and roentgenographic results in some of our cases. Best results invariably relied on a stable anatomic reduction and an adequate period of immobilization. Poor results were demonstrated in the cases with incomplete initial reduction, secondary degenerative arthrosis, or nonunion.