An efficient and user-friendly method for cytohistological analysis of organoids.

Organoid culture is a recently developed in vitro three-dimensional (3D) cell culture technology. It has wide applications in tissue engineering studies. However, histological analysis of organoid is quite complex and tedious for researchers. This study proposes a user-friendly, affordable and efficient method for making formalin-fixed paraffin embedded (FFPE) organoid blocks and Optimal Cutting Temperature compound (OCT) embedded frozen organoid blocks. This method implements a key pre-embedding step for preparing paraffin embedded organoid blocks, which could concentrate organoid together without damaging or loss of samples. This method could be used to process even a small number of organoids with high efficiency. In addition, with minor modifications, the method is readily applied for OCT embedded organoid blocks. The slides generated were ready for H&E staining, immunohistochemistry staining and immunofluorescent staining. The method described in this study can be easily used for routine histological analysis of organoid, and could be performed in general pathology labs and requires no dedicated equipment and reagent.

The Anti-Tumor Effect of Nab-Paclitaxel Proven by Patient-Derived Organoids.

BACKGROUND: Nab-paclitaxel has been widely used in treating breast cancer and pancreatic patients for its low toxicity and high efficiency. However, its role in gastric cancer (GC) remains ambiguous. The aim of our study was to test the anti-tumor activity of nab-paclitaxel using GC patient-derived organoids. METHODS: By using the organoid culture system, we describe the establishment of human gastric cancer organoid lines from surgical samples of three patients with gastric cancer. The consistency of these organoids with original cancer tissues was evaluated by histopathological examination. The characteristics of the cancer organoids were tested using immunofluorescence (IF) staining. Using organoids, the anti-tumor efficiencies of nab-paclitaxel, 5-Fu and epirubicin were compared by CCK8 assay and Annexin V-FITC/PI staining. RESULTS: Three organoids were successfully established and passaged. The morphology of the established GC organoids was consistent with original cancer tissues. The IC50 of nab-paclitaxel was 3.68 µmol/L in hGCO1, 2.41 µmol/L in hGCO2 and 2.91 µmol/L in hGCO3, which was significantly lower than those of 5-FU (72.99 µmol/L in hGCO1, 28.32 µmol/L in hGCO2 and 2.91 µmol/L in hGCO3) and epirubicin (25.85µmol/L in hGCO1, 15.15 µmol/L in hGCO2 and 7.60 µmol/L in hGCO3). When each organoid lines were treated with nab-paclitaxel for increasing period of time, the percentage of the apoptotic cells in each organoid increased accordingly. CONCLUSION: Nab-paclitaxel showed strong anti-tumor activity and had the potential to become front-line drug for treating GC patients. Gastric cancer organoid may be a good tool to predict in vivo response to drugs.

Immune-infiltration based signature as a novel prognostic biomarker in gastrointestinal stromal tumour.

BACKGROUND: Accumulating evidence indicates that tumour-infiltrating lymphocytes (TILs) are the primary determinant of survival outcomes in various tumours. Thus, we sought to investigate the TIL distribution and density in gastrointestinal stromal tumours (GISTs) and to develop an immune infiltration (II)-based signature to predict prognosis. METHODS: The expression of 8 immune features in the tumour centre (TC) and tumour margin (TM) and PD-L1 in 435 GIST patients was investigated by immunohistochemistry. Then, a 4-feature-based II-GIST signature integrating the CD3+ TC, CD3+ TM, CD8+ TM and CD45RO+ TM parameters was developed using a LASSO Cox regression model in the training cohort and was validated in two separate validation cohorts. FINDINGS: High CD3+ TC, CD3+ TM, CD8+ TC, CD8+ TM, CD45RO+ TM, NKp46+ TM and CD20+ TM correlated with improved survival. Patients with high II-GIST scores have better RFS and OS outcomes than those with low II-GIST scores. Multivariable analyses demonstrated that the II-GIST signature is an independent prognostic factor. The receiver operating characteristic (ROC) curve demonstrated that the prognostic accuracy of the II-GIST signature is superior to that of the NIH risk criteria. Further analysis showed that moderate- and high-risk GIST patients with high II-GIST scores could gain survival benefits from adjuvant imatinib therapy. INTERPRETATION: The novel II-GIST signature accurately predicted the survival outcomes of GIST patients. In addition, the II-GIST signature was a useful predictor of survival benefit from imatinib therapy amongst moderate- and high-risk patients with GIST. FUNDING: This project was supported by National Natural Science Foundation of China (81702325), Natural Science Foundation of Guangdong Province (2017A030310565), and 3&3 Project of the First Affiliated Hospital of Sun Yat-sen University.

Next-generation sequencing yields the complete chloroplast genome of Abies kawakamii.

Abies kawakamii is endemic to the island of Taiwan and has been listed as a threatened species in the Red List. In present study, we reported the complete chloroplast genome of A. kawakamii. The chloroplast genome is 121,290 bp in size. It was composed of 114 genes and they were 68 peptide-encoding genes, 35 tRNA genes, four rRNA genes, six open reading frames and one pseudogene. Loss of ndh genes was identified in the genome of A. kawakamii. Inverted repeat sequences include trnS-psaM-ycf12-trnG and trnG-ycf12-psaM-trnS were recognized in 52-kb inversion points. The phylogenetic analysis confirms that the Abies species are strongly supported as monophyletic. The complete plastome of A. kawakamii will provide potential genetic resources for further conservation and management strategies.

The complete chloroplast genome sequence of Abies chensiensis (Pinaceae).

Abies chensiensis is listed as a threatened species in the Red List and categorized as key protected wild plants in China. Here, we determined the complete chloroplast genome of A. chensiensis using the Illumina MiSeq platform. The genome was 121,795 bp in length, comprising a large single copy (LSC) region of 67,160 bp, a small single copy (SSC) region of 54,107 bp, and two inverted repeat regions (IRa and IRb) of 264 bp each. It was composed of 114 genes, including 68 peptide-encoding genes, 35 transfer RNAs (tRNAs), four ribosomal RNAs (rRNAs), six open reading frames and one pseudogene. Phylogenetic analysis revealed that A. chensiensis was most closely related to A. beshanzuensis, with high bootstrap values. The present research will provide potential genetic resources for further conservation and management strategies.

Effects of Hemp seed soft capsule on colonic ion transport in rats.

AIM: To investigate the effect of Hemp seed soft capsule (HSCC) on colonic ion transport and its related mechanisms in constipation rats. METHODS: Sprague-Dawley male rats were randomly divided into three groups: normal group, constipation group and HSSC group. Rats in the constipation and HSSC groups were administrated loperamide 3 mg/kg per day orally for 12 d to induce the constipation model. Then, the HSSC group was given HSSC 0.126 g/kg per day by gavage for 7 d. The normal and constipation groups were treated with distilled water. After the treatment, the fecal wet weight and water content were measured. The basal short-circuit current (Isc) and resistance were measured by an Ussing Chamber. Besides the in vivo drug delivery experiment above, an in vitro drug application experiment was also conducted. The accumulative concentrations of HSSC (0.1 mg/mL, 0.5 mg/mL, 1.0 mg/mL, 2.5 mg/mL, 5.0 mg/mL, 10.0 mg/mL and 25.0 mg/mL) were added to the normal isolated colonic mucosa and the Isc was recorded. Further, after the application of either ion (Cl- or HCO3-) substitution, ion channel-related inhibitor (N-phenylanthranilic acid, glybenclamide, 4,4-diisothiocyano-2,2-stilbenedisulfonic acid or bumetanide) or neural pathway inhibitor [tetrodotoxin (TTX), atropine, or hexamethonium], the Isc induced by HSSC was also measured. RESULTS: In the constipation group, the fecal wet weight and the water content were decreased in comparison with the normal group (P < 0.01). After the treatment with HSSC, the fecal wet weight and the water content in the HSSC group were increased, compared with the constipation group (P < 0.01). In the constipation group, the basal Isc was decreased and resistance was increased, in comparison with the normal group (P < 0.01). After the treatment with HSSC, the basal Isc was increased (P < 0.05) and resistance was decreased (P < 0.01) in the HSSC group compared with the constipation group. In the in vitro experiment, beginning with the concentration of 1.0 mg/mL, differences in Isc were found between the experimental mucosa (with HSSC added) and control mucosa. The Isc of experimental mucosa was higher than that of control mucosa under the same concentration (1.0 mg/mL, P < 0.05; 2.5-25 mg/mL, P < 0.01). After the Cl- or HCO3- removal and pretreated with different inhibitors (cAMP-dependent and Ca2+-dependent Cl- channels, Na+-K+-2Cl- cotransporter (NKCC), Na+-HCO3- cotransporter or Cl-/HCO3- exchanger inhibitor), there were differences between experimental mucosa and control mucosa; the Isc of experimental mucosa was lower than that of control mucosa under the same concentration (P < 0.05). Meanwhile, after pretreatment with neural pathway inhibitor (TTX, atropine, or hexamethonium), there were no differences between experimental mucosa and control mucosa under the same concentration (P > 0.05). CONCLUSION: HSSC ameliorates constipation by increasing colonic secretion, which is mediated via the coaction of cAMP-dependent and Ca2+-dependent Cl- channels, NKCC, Na+-HCO3- cotransporter or Cl-/HCO3- exchanger.

Primary Esophageal Extranasal NK/T Cell Lymphoma With Biphasic Morphology: A Case Report and Literature Review.

We report a case of esophageal extranasal NK/T cell lymphoma with biphasic morphologic features revealed by a deep large piecemeal biopsy. A 40-year-old man present with pharyngalgia, dysphagia, recurrent fever, and 5-kg weight loss for 8 months. Endoscopy demonstrated progressing longitudinal ulcers and mucosal bridges along the esophagus. The first and second biopsies obtained superficial mucosa with scattered bland-looking small lymphocytes. A subsequent large piecemeal snare abscission for biopsy showed atypical lymphoid cells infiltrating into the deep lamina propria and muscularis mucosae, whereas the superficial lamina propria was highly edematous with scant small lymphocytes. Immunohistochemical studies confirmed that both underlying atypical cells and superficial small lymphocytes were neoplastic, sharing an identical immunophenotype: positive for CD2, CD3, CD43, CD8, CD56, TIA-1 and granzyme B. Epstein-Barr virus-encoded small RNAs were found in both cells. The histologic findings were diagnostic of primary esophageal extranasal NK/T cell lymphoma. However, the patient developed bone marrow depression during chemotherapy and died of massive cerebral hemorrhage after the first cycle of chemotherapy. Primary esophageal extranodal NK/T cell lymphoma nasal type is extremely rare. We show the biphasic morphology of this disease, which highlights the importance of deep biopsy for accurate diagnosis.

TMEM16A overexpression contributes to tumor invasion and poor prognosis of human gastric cancer through TGF-ß signaling.

TMEM16A is a newly identified calcium activated chloride channel, and has been reported to be overexpressed by various solid malignant cancers to promote proliferation and invasion, yet little is known about its role in gastric cancer(GC). Therefore, we investigated the role of TMEM16A in GC and its clinical significance by a retrospective analysis of 367 GC patients, and in vitro study was performed for validation and underlying molecular mechanism.TMEM16A was significantly upregulated and amplified in GC tissues, and its overexpression was positively correlated with disease stage, negatively with patient survival and identified as an independent prognostic factor for patient outcome. A negative correlation between TMEM16A and E-cadherin was found in 367 GC specimens. TMEM16A silencing significantly decreased calcium activated chloride currents, impaired TGF-ß secretion, reduced E-cadherin expression, and inhibited the migration and invasion without affecting proliferation of GC cells (AGS and BGC-823). Supplement of TGF-ß reverted the effects of TMEM16A silencing on E-cadherin expression, cell migration and invasion.In conclusion, TMEM16A promotes invasion and metastasis in GC, and might be a novel prognostic biomarker and potential therapeutic target in the treatment of GC.

The clue of a possible etiology about spontaneous regression of hepatocellular carcinoma: a perspective on pathology.

Spontaneous regression of hepatocellular carcinoma (HCC) is a rare event. However, only a few of the causes of cases of HCC spontaneous regression are clear. More cases are ambiguous. We report on a patient who had a spontaneous regression of HCC as detected by histological and immunohistochemical exam, and compared this case to 20 cases of non-specific HCC. In our case, we found that the odd phenomenon is that CD163(+) macrophages are overactivated in surviving HCC, which is spontaneously regressing. Concomitantly, we cannot find a similar phenomenon in peritumoral liver tissue or non-specific HCC. According to our microscopical morphology and immunohistochemical study, we considered that a clue of a possible etiology about HCC spontaneous regression is that CD163(+) macrophages are overactivated.

Echocardiographic diagnosis of sinus of Valsalva aneurysm: a 17-year (1995-2012) experience of 212 surgically treated patients from one single medical center in China.

OBJECTIVE: To evaluate the value of echocardiography in the diagnosis of different pathological patterns of sinus of Valsalva aneurysms (SVAs). METHODS: Echocardiographic features and surgical findings of 212 consecutive patients with SVAs treated in the last 17 years (1995-2012) at the Union Hospital of Huazhong University of Science and Technology were compared and analyzed retrospectively. RESULTS: 212 Chinese patients with SVAs underwent surgical repairs from January 1995 to May 2012 in our hospital. The aneurysms originated from the right, non-, multiple and left coronary sinus in 77.8%, 19.3%, 2.4% and 0.5%, respectively. 71.7% were ruptured, most commonly into the right ventricle (67.9%) followed by the right atrium (27.4%). Other rare entry sites of rupture included the left atrium, the left ventricle, the interatrial septum, the interventricular septum and the pulmonary artery (0.5%-1.9%). 164 SVAs arising from the right coronary sinus were classified by the Sakakibara method: 47.6% type I, 33.5% type II, 6.1% type IIIv and 12.8% type IIIa. 41 aneurysms of the non-coronary sinus were classified by the Guo method: 61.0% type I, 34.1% type IIa and 4.9% type IIv. The three most common associated cardiovascular lesions were ventricular septal defect (VSD) (53.3%), stenosis of right ventricular outflow tract (RVOTS) (7.5%) and aortic valvular malformations (5.2%). Compared with surgical results, the sensitivity, specificity and accuracy of echocardiographic diagnosis of SVAs were 93.9%, 99.9% and 99.8%, respectively. Of the 13 SVAs that were missed on echocardiography, 77% were small aneurysms of the right coronary sinus extending into the right ventricle across a VSD. Of the 199 cases diagnosed by echocardiography prior to surgery, the diagnostic accuracy of aneurismal origination, termination and whether ruptured or not was 99.0%, 99.0% and 97.5%, respectively. Echocardiography also diagnosed accurately all of the complications of the SVAs with the exception of aneurismal vegetations. The sensitivity, specificity and accuracy of echocardiography in diagnosing the associated cardiovascular lesions were 89.2%, 99.9% and 99.0%, respectively. The most common misdiagnosis and misdiagnosed associated lesions were the RVOTS and the types of VSD, respectively. CONCLUSIONS: Echocardiography has a specific value in the diagnosis of different pathological patterns of the SVAs with distinguishing ultrasonic features. To the best of our knowledge, this is the largest series of patients with SVAs surgically treated in a single medical center.

Evaluation of left atrial function in patients with coronary artery disease by two-dimensional strain and strain rate imaging.

BACKGROUND: Left ventricular (LV) dysfunction in patients with coronary artery disease is shown by strain and strain rate imaging. However, left atrium (LA) function in patients with coronary artery disease (CAD) has not been assessed by this method. METHODS AND RESULTS: In 34 CAD patients, including 17 patients with enlarged LA (LA diameter ≤ 4.0 cm) and 17 with normal-size LA (LA diameter ≤ 4.0 cm), two-dimensional strain echocardiographic imaging (2DSE) was performed. Twenty healthy subjects as a control group were included. Both conventional parameters and strain parameters, such as LA peak systolic strain (LAs S/SR), preatrial contraction strain (LAa S), peak systolic (LAs SR), early diastolic strain rate (LAe SR) and late diastolic strain rate (LAa SR), were measured. Conventional parameters were abnormal in CAD patients with enlarged LA (ELA), but there were no significant differences between CAD with normal-size left atrium (NLA) and control groups. LAs S/SR and LAe SR were lower in patients than in normal controls, and were even lower in CAD-ELA group (P < 0.05). LAa S/SR were lower in CAD patients with ELA (P < 0.05), but without a significant difference between CAD-NLA and control groups. A significant correlation was observed between LAs S/SR and LA emptying fraction (r = 0.85, P < 0.05; r = 0.72, P < 0.05, respectively). LAa S/SR related well to LA ejection fraction (r = 0.68, P < 0.05; r = 0.61, P < 0.05, respectively). LAs SR was most accurate in identifying both CAD patients with NLA from controls and CAD patients from controls (area under the curve: 0.91; 0.95, respectively). CONCLUSIONS: LA diastolic dysfunction occurs prior to LA systolic dysfunction in CAD patients, and LAs SR is the most accurate index in identifying patients with CAD.

Echocardiographic characteristics of the criss-cross heart.

OBJECTIVE: To assess the ultrasonic characteristics of the criss-cross heart, and explore the value of echocardiography in the diagnosis of this rare congenital cardiac defect. METHODS: We reviewed the echocardiographic findings in 4 patients having criss-crossed atrioventricular connections at our hospital, and compared the findings with observations at surgery in 3 of the patients. RESULTS: In all 4 patients, there was usual atrial arrangement, right hand ventricular topology, and concordant atrioventricular connections. The inlet components of the ventricular mass, however, crossed such that the apical component of the morphologically right ventricle was situated anteriorly and superiorly, and extended to the left relative to the apex of the morphologically left ventricle. The ventriculo-arterial connections were concordant in 1 patient, double outlet from the morphologically right ventricle in 2, and discordant in the other. In all 4 patients, it proved impossible to obtain the standard 4-chamber view showing simultaneously all four chambers and both atrioventricular valves. A series of apical 4-chamber or subcostal coronal views, obtained by tilting the transducer from posterior to anterior, demonstrate initially the connection of the left-sided left atrium and the right-sided left ventricle through the mitral valve. More anterior angulation of the transducer then showed the right-sided right atrium to be connected to the left-sided right ventricle through the tricuspid valve, confirming the presence of twisted atrioventricular connections. Color Doppler imaging displayed the crossing of the atrioventricular connections without mixing of the streams. Short-axis views across the ventricular mass confirmed that the right ventricle was superior, anterior, and to the left of the left ventricle, and demonstrated the horizontal position of the ventricular septum. When viewed subcostally, the distance between the tricuspid valve and the orifice of the inferior vena cava was significantly increased relative to normal findings. The echocardiographic findings were confirmed during surgical interventions in 3 patients, apart from the failure to diagnose one instance of persistent patency of the left superior vena cava. CONCLUSIONS: The failure to obtain a characteristic 4-chamber view in any cut was diagnostic for recognition of the criss-crossed atrioventricular junctions. Transthoracic echocardiography provides definitive images of this rare arrangement, and accurately defines the associated cardiac abnormalities.

Diagnosis of constrictive pericarditis by quantitative tissue Doppler imaging.

OBJECTIVES: To observe the motion of pericardium and myocardium in patients with constrictive pericarditis (CP) and normal subjects using two-dimensional (2D) echocardiography and quantitative tissue Doppler imaging (QTDI), and to investigate the value of this echocardiographic approach in the diagnosis of pericardial adhesion in CP. BACKGROUND: The relationship of the motion of pericardium and myocardium in CP has not been investigated by QTDI. METHODS: The motions of pericardium and myocardium and the difference between them were investigated using 2D echocardiography combined with QTDI technique in 20 patients with CP and 20 age- and sex-matched normal subjects. Systolic peak displacements of pericardium (D(1)), outer-layer myocardium (D(2)) and inner-layer myocardium (D(3)) were measured from quantitative tissue displacement curves. The ratios of (D(3)-D(2))/(D(2)-D(1)) were then calculated. RESULTS: In normal subjects, the motion of myocardium was found to be stronger than that of pericardium, but the motions of outer-layer and inner-layer myocardium were virtually identical. However, in patients with CP, the motion of outer-layer myocardium was significantly reduced approaching that of pericardium, while the motion of inner-layer myocardium was stronger than that of outer-layer myocardium. The ratios of (D(3)-D(2))/(D(2)-D(1)) were significantly higher in patients with CP than those in normal subjects (5.0+/-4.7 vs 0.6+/-0.7, P<0.05). CONCLUSIONS: Obvious differences exist in the motion of pericardium and myocardium between normal subjects and patients with CP; observations of these differences using 2D echocardiography and QTDI provide a new and sensitive method in the diagnosis of pericardial adhesion in CP.

Echocardiographic diagnosis of anomalous origin of the left coronary artery from the pulmonary artery.

The objective of this study was to analyze echocardiographic characteristics of anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) and to explore the diagnostic value of transthoracic echocardiography. The echocardiographic characteristics of 8 patients hospitalized with ALCAPA from 2000 to 2005 were analyzed retrospectively, including the results of real time three-dimensional echocardiography in one case, and compared with angiographic results. Eight cases included 6 older type patients and 2 infant type patients. Echocardiography showed abnormal vessel inserting into pulmonary artery (PA), continuous shunt into PA and intercoronary collateral signals within the ventricular septum in all cases and bifurcate structure of the abnormal vessel with retrograde filling in 4 cases. The morphological and functional changes and valvular regurgitation induced by insufficient myocardial perfusion were also evaluated. In former 4 patients, 2 cases were misdiagnosed as right coronary artery-PA fistula and the other 2 cases were given an uncertain diagnosis of anomalous origin of the coronary artery because of the visualization of the echo-free linear structure which apparently arose from the aorta resembling a normal left coronary artery. The latter 4 patients were correctly diagnosed by excluding the aforementioned interference. The diagnosis of ALCAPA was confirmed by angiocardiography in all patients and by intraoperative findings in 4 patients. Based on the apprehension of ultrasonic features and the enhancement of diagnostic alertness, the echocardiography can evaluate ALCAPA accurately and give more information than angiography. It may be the first diagnostic choice.

Assessment of atrial septal defect area changes during cardiac cycle by live three-dimensional echocardiography.

OBJECTIVES: To investigate the accuracy of measurement of the atrial septal defect (ASD)area and dynamic change by live three-dimensional echocardiography (L3DE). METHODS: L3DE was performed in patients with ASD using a three-dimensional workstation to obtain the en face view of the ASD and measure its area at the peak of P-wave, the peak of R-wave, the initial and the destination point of T-wave, and the period of P-T. Parameters derived from L3DE were compared with intraoperative measurements. RESULTS: The ASD area changed significantly during cardiac cycles (mean change 46.1%, p < 0.0001; range 15.2-72.5%), with the maximal area at endsystole and the minimal area at enddiastole. There was excellent correlation between L3DE and intraoperative measurements for the area of ASD at the peak of P-wave (r = 0.92). There were good correlations between the two methods during the other phases of cardiac cycle (r = 0.81-0.86). CONCLUSIONS: L3DE provides accurate and feasible measurements of the ASD area. Investigation of the dynamic changes during the cardiac cycle may lead to an improved understanding of the hemodynamics of ASD.