Single-cell and bulk RNA sequencing reveal heterogeneity and diagnostic markers in papillary thyroid carcinoma lymph-node metastasis.

PURPOSE: Papillary thyroid carcinoma (PTC) is characterized by lymph-node metastasis (LNM), which affects recurrence and prognosis. This study analyzed PTC LNM by single-cell RNA sequencing (scRNA-seq) data and bulk RNA sequencing (RNA-seq) to find diagnostic markers and therapeutic targets. METHODS: ScRNA-seq data were clustered and malignant cells were identified. Differentially expressed genes (DEGs) were identified in malignant cells of scRNA-seq and bulk RNA-seq, respectively. PTC LNM diagnostic model was constructed based on intersecting DEGs using glmnet package. Next, PTC samples from 66 patients were used to validate the two most significant genes in the diagnostic model, S100A2 and type 2 deiodinase (DIO2) by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemical (IHC). Further, the inhibitory effect of DIO2 on PTC cells was verified by cell biology behavior, western blot, cell cycle analysis, 5-ethynyl-2'-deoxyuridine (EdU) assay, and xenograft tumors. RESULTS: Heterogeneity of PTC LNM was demonstrated by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis. A total of 19 differential genes were used to construct the diagnostic model. S100A2 and DIO2 differ significantly at the RNA (p < 0.01) and protein level in LNM patient tissues (p < 0.001). And differed in PTC tissues with different pathologic typing (p < 0.001). Further, EdU (p < 0.001) and cell biology behavior revealed that PTC cells overexpressed DIO2 had reduced proliferative capacity. Cell cycle proteins were reduced and cells are more likely to be stuck in G2/M phase (p < 0.001). CONCLUSIONS: This study explored the heterogeneity of PTC LNM using scRNA-seq. By combining with bulk RNA-seq data, diagnostic markers were explored and the model was established. Clinical diagnostic efficacy of S100A2 and DIO2 was validated and the treatment potential of DIO2 was discovered.

[Clinicopathological features of SMARCA4-deficient lung adenocarcinoma: a study of 42 cases].

Objective: To investigate the clinicopathological characteristics and genetic mutations of SMARCA4-deficient lung adenocarcinoma. Methods: From January 2021 to April 2023 in the First Affiliated Hospital of Zhengzhou University, 42 cases of SMARCA4-deficienct lung adenocarcinoma were diagnosed and now analyzed. All cases were retrospectively studied using hematoxylin-eosin staining and immunohistochemistry. The clinicopathological features were reviewed. Next-generation sequencing (NGS) was performed to investigate the mutations of related genes. Results: Among the 42 cases, there were 35 biopsy and 7 surgical specimens. There were 38 males and 4 females. The male to female ratio was 9.5∶1.0, with an age range from 42 to 78 years. Thirty-three patients were smokers. Overall, 4 cases (9.5%), 2 cases (4.7%), 18 cases (42.9%) and 18 cases (42.9%) were at stages Ⅰ, Ⅱ, Ⅲ, and Ⅳ, respectively. Microscopically, all the cases were non-mucinous adenocarcinoma, without lepidic pattern. The morphology was diverse. Rhabdomyoid cells, tumor giant cells and tumor necrosis were present. Most of the tumor cells had eosinophilic cytoplasm and occasionally clear cytoplasm. Defined cell borders and variable cytoplasmic hyaline secretory globules could be found. Inflammatory cells infiltrated the tumor stroma. Immunohistochemistry showed 29 cases (69.0%, 29/42) expressed TTF1, 10 cases (40.0%, 10/25) expressed Napsin A, and 20 cases (100.0%, 20/20) expressed INI1. Forty cases (95.2%, 40/42) showed BRG1 loss in all tumor cells, while 2 cases (4.8%, 2/42) had partial BRG1 loss. PD-L1 (22C3) was positive in 59.2% of the cases (16/27). NGS revealed mutations in EGFR, ROS1, MET, RET and KRAS. Six cases (6/8) showed SMARCA4 mutation, while some cases were accompanied by mutations of TP53 (7/15), STK11 (4/8), and KEAP1 (1/8). Driver gene mutations were more common in women (P<0.05). Patients were followed up for 1-25 months. Four patients died and 20 patients' diseases progressed. Conclusions: SMARCA4-deficient lung adenocarcinoma lacks characteristic morphology. Most of them express TTF1 and harbor driver gene mutations. It is necessary to identify this subset of lung adenocarcinoma by carrying out BRG1 stain routinely on lung adenocarcinoma. These patients can then be identified and benefit from targeted therapies.

[Clinical application value of modified Lee grading system to evaluate the degree of foraminal stenosis in patients with intervertebral foraminal lumbar disc herniation].

Objective: To investigate the clinical application value of the modified Lee grading system (abbreviated as the modified system) in evaluating the degree of intervertebral foraminal stenosis(IFS) in patients with foraminal lumbar disc herniations(FLDH). Methods: MRI data of 83 patients with FLDH-IFS (34 patients in the operation group and 49 patients in the conservative group) in Yantai Affiliated Hospital of Binzhou Medical University and Yantai Yantaishan Hospital from March 2018 to February 2021 were retrospectively collected. There were 43 males and 40 females, ranged from 34 to 82 years old, with an average of (61±10) years. MRI images of selected patients were independently evaluated and recorded by two radiologists in a blind method, using both the Lee grading system (abbreviated as Lee system) and the modified system, respectively and each method was evaluated twice. The difference between the evaluation level of the two systems, and the agreement of observer assessments of the two systems were compared, and the correlation between the evaluation level of the two grading systems and the clinical treatment modalities was analyzed. Results: The percentage of nongrade 3 (grade 0-2) patients with effective conservative treatment according to the two grading systems was 94.6 % (139/147) and 64.2 % (170/265), respectively. The percentage of grade 3 patients requiring surgical treatment according to the two grading systems was 69.2 % (128/185) and 61.2 % (41/67), respectively. There was a statistically significant difference between the evaluation levels of the modified system and the Lee system (Z=-5.16, P=0.001). In the Lee system, the intra-observer observation consistency Kappa values of the two radiologists were 0.735 and 0.542, respectively, which were highly and moderately consistent; and the inter-observer observation consistency Kappa values were 0.426-0.521, which were moderate consistency. In the modified system, the intra-observer consistency Kappa values of the two radiologists were 0.900 and 0.921, respectively, and the consistency was almost completely consistent; and the inter-observer consistency Kappa values were 0.783-0.861, which were highly consistent or almost completely consistent. Lee system and clinical treatment modalities was correlative (rs=0.39,P<0.001), and modified system and clinical treatment modalities was correlative (rs=0.61,P<0.001). Conclusion: According to FLDH-IFS, the modified system can comprehensively and accurately grade, with high reliability and reproducibility. The evaluation level has a more significant correlation with clinical treatment modalities.

[Clinicopathological features and prognosis of SMARCA4-deficient non-small cell lung carcinoma: an analysis of 127 cases].

Objective: To investigate and elucidate the clinicopathological and prognostic characteristics of SMARCA4-deficient non-small cell lung cancer. Methods: The clinicopathological and prognostic data were collected in 127 patients with SMARCA4-deficient non-small cell lung cancer diagnosed in Shanghai Pulmonary Hospital, Shanghai, China from January 2020 to March 2022. The variation and expression of biomarkers related to treatment were retrospectively reviewed. Results: One hundred and twenty-seven patients were eligible for enrollment. Among them 120 patients (94.5%) were male and 7 cases (5.5%) were female, while the average age was 63 years (range 42-80 years). There were 41 cases (32.3%) of stage Ⅰ cancer, 23 cases (18.1%) of stage Ⅱ, 31 cases (24.4%) of stage Ⅲ and 32 cases (25.2%) of stage Ⅳ. SMARCA4 expression detected by immunohistochemistry was completely absent in 117 cases (92.1%) and partially absent in 10 cases (7.9%). PD-L1 immunohistochemical analyses were performed on 107 cases. PD-L1 was negative, weakly positive and strongly positive in 49.5% (53/107), 26.2% (28/107) and 24.3% (26/107) of the cases, respectively. Twenty-one cases showed gene alterations (21/104, 20.2%). The KRAS gene alternation (n=10) was most common. Mutant-type SMARCA4-deficient non-small cell lung cancer was more commonly detected in females, and was associated with positive lymph nodes and advanced clinical stage (P<0.01). Univariate survival analysis showed that advanced clinical stage was a poor prognosis factor, and vascular invasion was a poor predictor of progression-free survival in patients with surgical resection. Conclusions: SMARCA4-deficient non-small cell lung cancer is a rare tumor with poor prognosis, and often occurs in elderly male patients. However, SMARCA4-deficient non-small cell lung cancers with gene mutations are often seen in female patients. Vascular invasion is a prognostic factor for disease progression or recurrence in patients with resectable tumor. Early detection and access to treatment are important for improving patient survivals.

[MiR-340 mediates the involvement of high mobility group box 1 in the pathogenesis of liver fibrosis].

Objective: To explore the pathogenic mechanism of the miR-340/high mobility group box 1 (HMGB1) axis in the formation of liver fibrosis. Methods: A rat liver fibrosis model was established by injecting CCl(4) intraperitoneally. miRNAs targeting and validating HMGB1 were selected with gene microarrays after screening the differentially expressed miRNAs in rats with normal and hepatic fibrosis. The effect of miRNA expressional changes on HMGB1 levels was detected by qPCR. Dual luciferase gene reporter assays (LUC) was used to verify the targeting relationship between miR-340 and HMGB1. The proliferative activity of the hepatic stellate cell line HSC-T6 was detected by thiazolyl blue tetrazolium bromide (MTT) assay after co-transfection of miRNA mimics and HMGB1 overexpression vector, and the expression of extracellular matrix (ECM) proteins type I collagen and α-smooth muscle actin (SMA) was detected by western blot. Statistical analysis was performed by analysis of variance and the LSD-t test. Results: Hematoxylin-eosin and Masson staining results showed that the rat model of liver fibrosis was successfully established. Gene microarray analysis and bioinformatics prediction had detected eight miRNAs possibly targeting HMGB1, and animal model validation had detected miR-340. qPCR detection results showed that miR-340 had inhibited the expression of HMGB1, and a luciferase complementation assay suggested that miR-340 had targeted HMGB1. Functional experiments results showed that HMGB1 overexpression had enhanced cell proliferation activity and the expression of type I collagen and α-SMA, while miR-340 mimics had not only inhibited cell proliferation activity and the expression of HMGB1, type I collagen, and α-SMA, but also partially reversed the promoting effect of HMGB1 on cell proliferation and ECM synthesis. Conclusion: miR-340 targets HMGB1 to inhibit the proliferation and ECM deposition in hepatic stellate cells and plays a protective role during the process of liver fibrosis.

[One case of adult-onset dystonia presenting with chronic progressive external ophthalmoplegia].

We report a case of adult-onset dystonia presenting with chronic progressive external ophthalmoplegia. The patient had ptosis in both eyes, particularly the left eye, for no obvious reason since the age of 10, which was progressively aggravated. The clinical diagnosis was chronic progressive external ophthalmoplegia. However, whole gene sequencing revealed the mitochondrial A3796G missense mutation, so the patient was clearly diagnosed as adult-onset dystonia and given treatment to reduce blood glucose and improve muscle metabolism. The A3796G mutation in the ND1 subunit of the mitochondrial complex leading to ophthalmoplegia is relatively rare, requiring a combination with genetic testing for confirmation of diagnosis.

[Factors associated with long-term survival in critically ill patients following surgery for solid tumors complicated with paraneoplastic pemphigus].

OBJECTIVE: Critically ill patients with solid tumors complicated with paraneoplastic pemphigus are usually treated in intensive care units (ICU) for perioperative management after surgical treatment. In this study, the clinical characteristics and predictors of long-term prognosis of these critically ill patients were analyzed. METHODS: the clinical and laboratory data of 63 patients with solid tumors complicated with paraneoplastic pemphigus admitted to ICU from 2005 to 2020 were retrospectively analyzed, and the survival status of the patients were followed up. RESULTS: Among the 63 patients, 79.4% had Castleman disease as the primary tumor, and 20.6% with other pathological types; 69.8% had severe-extensive skin lesions, and 30.2% had other skin lesions; the patients with bronchiolitis obliterans accounted for 44.4%, and 55.6% were not merged. Postoperative fungal infection occurred in 23.8% of the patients, and 76.2% without fungal infection. The median follow-up time was 95 months, and 25 patients died during the study period. The 1-year, 3-year and 5-year survival rates were 74.6% (95%CI 63.8%-85.4%), 67.4% (95%CI 55.6%-79.2%) and 55.1% (95%CI 47.9%-62.3%), respectively. The log-rank univariate analysis showed that the patients had age>40 years (P=0.042), preoperative weight loss>5 kg (P=0.002), preoperative albumin < 30 g/L (P < 0.001), paraneoplastic pemphigus complicated with bronchiolitis obliterans (P=0.002), and perioperative fungal infection (P < 0.001) had increased mortality. Cox univariate analysis showed that preoperative weight loss >5 kg (P=0.005), preoperative albumin < 30 g/L (P < 0.001), paraneoplastic pemphigus complicated with bronchiolitis obliterans (P=0.009), preoperative bacterial pulmonary infection (P=0.007), prolonged surgical time (P=0.048), postoperative oxygenation index (P=0.012) and low albumin (P=0.010) and hemoglobin concentration (P=0.035) in ICU, acute physiology and chronic health evaluation (APACHE Ⅱ) score (P=0.001); sequential organ failure assessment (SOFA) score (P=0.010), and postoperative fungal infection (P < 0.001) were risk factors for long-term survival. Cox regression model for multivariate analysis showed that preoperative weight loss > 5 kg (HR 4.44; 95%CI 1.47-13.38; P=0.008), and preoperative albumin < 30 g/L (HR 4.38; 95%CI 1.72-11.12; P=0.002), bronchiolitis obliterans (HR 2.69; 95%CI 1.12-6.50; P=0.027), and postoperative fungal infection (HR 4.85; 95%CI 2.01-11.72; P < 0.001) were independent risk factors for postoperative mortality. CONCLUSION: The 5-year survival rate of critically ill patients undergoing surgery for paraneoplastic pemphigus combined with solid tumors is approximately 55.1%, with preoperative weight loss > 5 kg, albumin < 30 g/L, bronchiolitis obliterans and postoperative fungal infection were associated with an increased risk of near- and long-term postoperative mortality.

[Spectral CT multi-parameter imaging in preoperatively evaluation the status of lymphovascular and perineural invasion of gastric cancer].

Objective: To explore the application value of spectral CT multi-parameter imaging in preoperative assessment the status of lymphovascular invasion (LVI) and perineural invasion (PNI) in patients with gastric cancer. Methods: A total of 62 patients who underwent energy spectral CT and with pathology confirmed gastric cancer in Lanzhou Uiversity Second Hospital from September 2020 to September 2021 were retrospectively collected, which including 46 males and 16 females, aged from 36 to 71 (57.5±9.1) years. According to the presence or absence of LVI/PNI in postoperative pathological results, they were divided into the positive group (42 cases) and the negative group (20 cases). The CT values of 40 keV and 70 keV (CT40 keV, CT70 keV), iodine concentration (IC), normalized iodine concentration (NIC) were measured in the arterial phase, the venous phase, and the delay phase, and the spectral curve slope of 40 keV to 70 keV (K(40-70)) and the combined parameters (the arterial phase: AP~all, the venous phase: VP~all, the delay phase: DP~all) were calculated. Spectral parameters between the positive and negative groups were compared, and the receiver operating characteristic curve (ROC) with the area under the curve (AUC), sensitivity, specificity, and optimal threshold were calculated for evaluating the diagnostic performance of each parameter. Results: The CT40 keV, CT70 keV, K(40-70), IC, and NIC in the arterial phase and the venous phase and the CT70 keV and NIC in the delay phase of the LVI/PNI-positive group were all higher than those of the negative group [the representative parameters: the arterial phase NIC 0.14±0.04 vs 0.12±0.04, the venous phase NIC 0.5(0.5, 0.6) vs 0.4(0.4, 0.5), the delay phase NIC 0.6±0.1 vs 0.5±0.1, all P<0.05]. ROC curve analysis showed that the diagnostic efficacy of the parameters of the venous phase is better than that of the arterial phase and the delay phase, and the diagnostic efficiency of the combined parameters is better than that of the individual parameters. The AUC value, sensitivity, and specificity of the most optimal parameter VP~all of the venous phase were 0.931(95%CI:0.872-0.990), 80.95%, and 95.00%, respectively. Conclusions: In the preoperative evaluation the status of the LVI and PNI in gastric cancer, the diagnostic efficacy in the venous phase parameters is better than that in the arterial phase and delay phase, and the diagnostic efficacy of combined parameters is better than that of individual parameters.

[Application of ultra-short echo time-T2* component analysis technology in monitoring morphological and biochemical changes of achilles tendon in amateur marathon athletes].

Objective: To explore the value of ultra-short echo time (UTE)-T2* component analysis techniques in dynamic monitoring the morphological and biochemical changes in amateur marathon athletes' achilles tendon before and after the marathon. Methods: Twenty-nine amateur marathon runners were recruited between October 2020 and March 2021 in Zhuhai City, Guangdong Province, including 25 males and 4 females, aged from 24 to 50 (40±6) years old. All volunteers underwent bilateral achilles tendon MRI examination 1 week before the marathon, 48 hours after the race, and 1 month after the race. The shape and signal of the achilles tendon were evaluated by routine T1-weighted, proton density weighted with fat saturation sequence and different echo time (TE) UTE sequence, and the changes of achilles tendon after running was quantitatively analyzed by UTE-T2* sequence. The values of single-component analysis (T2*M), short T2* components (T2*S), and long T2* components (T2*L) and Fraction values were obtained using UTE-T2* sequence. The value of the whole achilles tendon was measured on the sagittal images of achilles tendon, and the Achilles tendon was equally divided into three subregions [muscle-tendon junction (MTJ), middle (MID), and insertion (INS)]. The region of interest was delineated by two radiologists independently. The intra-group correlation coefficient (ICC) was used to evaluate the consistency of the data measured by two radiologists. Nonparametric Friedman M test was used to compare the differences of T2*M, T2*S, T2*L and Fraction values in different time points and different subregions. Wilcoxon rank-sum test was used to compare the difference between 48 h post-race and pre-race T2*S values (ΔT2*S) of different distance, different running posture, different pace and different amount of training, in which ΔT2*S equals the T2*S value of 48 h post-race minus the T2*S value of pre-race. Results: On the sequence of short TE (TE≤0.6 ms), achilles tendinopathy can manifest as scattered punctate hypointensity in areas of high signal intensity. The two radiologists showed a good consistency in measuring the T2*M, T2*S, T2*L and Fraction values of the achilles tendon, and the ICC values ​​were 0.96, 0.94, 0.83 and 0.94, respectively. The T2*s values was significantly higher in the whole Achilles tendon, MTJ and MID segment at 48 h post-exercise compared to pre-exercise, and decreased after 1 month of exercise, [0.49 (0.45, 0.59) vs 0.54 (0.49, 0.59) vs 0.53 (0.49, 0.57), 0.48 (0.44, 0.54) vs 0.53 (0.47, 0.58) vs 0.50 (0.46, 0.57), 0.48 (0.43, 0.58) vs 0.54 (0.47, 0.59) vs 0.52 (0.46, 0.57); respectively, all P<0.05]. The changes in T2*M, T2*L and Fraction values are not statistically significant (all P>0.05). In different running gestures, the ΔT2*S of achilles tendon who using the postures of front-middle feet is higher than that using the postures of back feet (0.03(-0.05, 0.07) vs -0.03(-0.17, 0.11), P=0.001). Conclusion: The Bi-component analysis of UTE-T2* technology is superior to single component analysis in monitoring the dynamic changes of achilles tendon before and after exercise, and T2*S is a more sensitive sequence to evaluate the subtle changes in the chemical composition of achilles tendon.

[Effects of postoperative urine protein levels on predicting acute kidney injury in critically ill patients undergoing non-cardiac surgery].

Objective: To investigate the predictive value of postoperative urine protein level in critically ill patients undergoing non-cardiac surgery with acute kidney injury (AKI). Methods: A total of 661 critically ill patients undergoing non-cardiac surgery, who visited the Department of Critical Care Medicine of Peking University First Hospital from May 20, 2019 to November 24, 2020, were enrolled in this prospective study. The clinical data of the patient's age, gender, body mass index, laboratory examination, surgical status, etc. were collected. AKI diagnostic criteria of the 2012 KDIGO guidelines were used to diagnose the occurrence of AKI after surgery. The independent predictors of AKI were determined by multivariate logistic regression. Results: The age of this patient cohort was (69±15) years. The prevalence of AKI was 45.4% (300/661). Multivariate logistic regression showed that urine protein semi-quantitative ≥2+(OR=2.62, 95%CI: 1.05-6.56, P=0.039) was independent factor for postoperative AKI in critically ill patients undergoing non-cardiac surgery, other independent factors include higher age (OR=1.04, 95%CI: 1.02-1.06, P=0.001), higher body mass index (BMI) (OR=1.12, 95%CI: 1.04-1.21, P=0.004), lower plasma hemoglobin level (OR=0.98, 95%CI: 0.97-1.00, P=0.019), lower central venous pressure (OR=0.89, 95%CI: 0.83-0.97, P=0.005) and lower total hypotension time (OR=1.01, 95%CI: 1.00-1.01, P=0.041). Conclusions: Urine protein semi-quantitative ≥2+after surgery is an independent predictive factor for the occurrence of postoperative AKI in critically ill patients undergoing non-cardiac surgery. It is important to check urine routine immediately after surgery to detect and deal with high-risk patients.

[Primary lung salivary gland-type duct carcinoma: a clinicopathological analysis of two cases and review of literature].

Objective: To investigate the clinicopathological features, diagnostic criteria and differential diagnosis of primary salivary gland-type duct carcinoma of lung(LSDC). Methods: Two patients with LSDC after surgical resection in Shanghai Pulmonary Hospital from 2020 to 2021 were included; their clinical parameters as well as pathological, immunohistochemical and molecular characteristics of the tumors were analyzed. The relevant literature was also reviewed. Results: Both patients were male, aged 49(case 1) and 64(case 2) years, respectively, and with a history of smoking. The chest computed tomography scan showed both lesions to be centrally located. Gross examination showed the maximum diameters were 16 mm and 35 mm, respectively. The histomorphology of LSDC resembled ductal carcinoma of breast, with intraductal islands of neoplastic cells, which also formed solid nests, papillary, micropapillary and cribriform structures. There was frequent accompanying comedo-like necrosis. The neoplasm cells were markedly heteromorphic, possessing large irregular nuclei with prominent nucleoli, abundant eosinophilic or clear cytoplasm, and mitotic figures were common. Both cases of LSDC were immunoreactive for CKpan, CK7, AR, HER2 staining was (2+) and were negative for TTF1, Napsin A, p40, GATA3, mammaglobin, GCDFP15, SOX10, PSA, P504S, ER, PR, vimentin, S-100, SMA, CK5/6 and p63. The tumor showed double-layer cell structure of the duct, and some basal cells/myoepithelial cells expressed p40 and CK5/6. Case 1 had no gene mutation while case 2 harbored TP53 and KMT2A gene mutation detected by next generation sequencing. Conclusions: LSDC is a very rare and highly aggressive salivary-type malignant tumor. The postoperative diagnosis mainly depends on histopathology and immunohistochemistry, attention should be paid to differential diagnosis to prevent missed diagnosis.

[Combined resection of thoracic and abdominal organ clusters: a series of 50 cases].

Objective: To examine the technique and effect of combined thoracic and abdominal organ clusters resection. Methods: From February 2019 to August 2021, totally 50 cases of combined thoracoabdominal organ cluster resection were completed at Transplant Medical Center, the Second Affiliated Hospital of Guangxi Medical University from donation after brain death donors. There were 47 males and 3 females, aging (34.8±12.3) years (range: 5 to 55 years). The length of hospital stay(M(IQR)) was 4(4) days (range: 2 to 43 days), the length of tube time was 4(2) days (range: 1 to 43 days). Through the midsternal incision and the abdominal grand cross incision, the cold perfusion was performing simultaneously when the perfusion lines of each target organ was established respectively. The combined resection was performed with the diaphragm as the boundary and the organ cluster as the unit. The heart and lung were separated on site and sent to the transplant hospital, and the abdominal organ cluster was directly preserved and returned to our hospital for further separation and repair. Results: Totaly 21 hearts, 47 pairs of lungs, 49 livers, 47 pairs of kidneys and 11 pancreas were harvested by this surgical treatment. The resection time was (32.6±6.5) minutes (range: 19 to 50 minutes), with no hot ischemia time. There was no accidental injury that affected organ quality and function. Heart transplantation was performed in 17 cases, combined heart-kidney transplantation in 2 cases, double lung transplantation in 43 cases, single lung transplantation in 6 cases, liver transplantation in 41 cases, combined liver-pancreas-duodenal cluster transplantation in 1 case, combined liver-kidney transplantation in 3 cases, combined pancreas-kidney transplantation in 9 cases, and kidney transplantation in 74 cases. Conclusion: Simultaneous perfusion and combined resection of thoracic and abdominal organ clusters for donation after brain death donors are feasible and effective.

Tributyrin supplementation in pasteurized waste milk: Effects on growth performance, health, and blood parameters of dairy calves.

This study evaluated the effects of incremental tributyrin supplementation in pasteurized waste milk on growth performance, health, and blood metabolism of dairy calves before and after weaning. Forty-eight newborn female Holstein dairy calves (39.6 ± 2.75 kg; mean ± standard deviation) were blocked by age and randomly assigned to 3 treatments: pasteurized waste milk (1) without supplementation, (2) with 1 g/L of tributyrin products (unprotected solid powder; containing 35% tributyrin), or (3) with 2 g/L of tributyrin products. The calves were weaned on d 56 and were raised until d 77. Data were analyzed for the preweaning, postweaning, and overall periods. The results showed that starter intake and hay intake were not different among treatments in any period of the trial, but the crude protein intake tended to increase linearly with tributyrin supplementation during the overall period. Although tributyrin supplementation had no effects on body weight during preweaning and overall periods, body weight increased linearly with tributyrin supplementation postweaning. The average daily gain tended to increase linearly during postweaning and overall periods. No effects were observed on feed efficiency in any period. A positive linear relationship between body length and tributyrin supplementation was observed during the postweaning period, but no differences were found for the other body structural measurements in any period. The results of diarrhea showed that tributyrin concentration had a negative linear relationship with diarrhea frequency during preweaning and overall periods. The rectal temperature did not differ among treatments in any period, but a treatment × week effect for rectal body temperature was observed. For blood metabolism, tributyrin supplementation had no effects on insulin, growth hormone, total protein, albumin, or globulin. No differences were found in serum amyloid A concentration in any of the periods, yet haptoglobin concentration decreased linearly with increasing tributyrin concentration during postweaning and overall periods. Endothelin concentration showed a tendency to decrease linearly during preweaning and postweaning periods and decreased linearly with tributyrin supplementation during the overall period. An increasing tributyrin concentration was associated with a negative linear relationship with IL-1ß concentration during the preweaning period, and no differences were found in the other periods. The concentration of IL-6 and tumor necrosis factor α were not different among treatments in any of the periods. These data suggest that increasing the concentration of tributyrin in pasteurized waste milk could increase growth performance and health of dairy calves, and incremental tributyrin supplementation could linearly reduce haptoglobin, endothelin, and IL-1ß concentrations, indicating a positive effect of tributyrin on alleviating oxidative stress and inflammatory status of dairy calves. Calves fed pasteurized waste milk supplemented with tributyrin products (containing 35% tributyrin) at 2 g/L compared with 1 g/L of milk had more improved growth and health.

[Clinical characteristics and outcome of malignant hydronephrosis associated with colorectal carcinoma].

Objective: To explore the clinical characteristics and outcome of hydronephrosis associated with advanced or metastatic colorectal carcinoma. Methods: Clinical data of 311 patients with locally advanced or metastatic colorectal carcinoma between June 2017 and March 2020 in National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital were retrospectively collected. Thirty-nine patients with hydronephrosis diagnosed by CT scan were analyzed. Kaplan-Meier method was used for survival analysis, Log rank method was used for comparison of survival between the two groups with or without hydronephrosis, and univariate and multivariate analyses was performed by Cox proportional risk regression model. Results: The incidence rate of malignant hydronephrosis associated with metastatic colorectal carcinoma was 12.5% (39/311), 26 were male, and 13 were female. The median age was 43 years (23-74 years). Among the 39 patients, 29 had unilateral hydronephrosis and 10 had bilateral hydronephrosis. Eleven patients with hydronephrosis at the initiate diagnosis, 28 patients with hydronephrosis at relapse or advanced course, and the median time to hydronephrosis was 17 months (4-62 months). The disease control rate (DCR, 77.8% and 84.6%, respectively) and progression free survival (PFS were 6 and 7 months) were not significantly different between patients with hydronephrosis and without hydronephrosis received the first-line chemotherapy (P>0.05). The median overall survival (OS) after presence of hydronephrosis was 26 months (95%CI: 8.3, 43.7). Multivariate analyses showed that the blood vessel invasion (LVSI) was an independent risk factor for OS (P<0.05). Conclusions: Malignant hydronephrosis had no effect on the efficacy of the first-line chemotherapy and PFS of patients with colorectal carcinoma received the first-line chemotherapy. LVSI was the independent prognostic factor for OS of patients with malignant hydronephrosis.

[Establishment and validation of prognostic risk score model for esophageal squamous cell carcinoma based on immune related genes].

Objective: To find the biomarkers that accurately predict the survival of patients with esophageal squamous cell carcinoma (ESCC). Methods: The immune related genes that were significantly related to the overall survival (OS) of patients with ESCC were screened from The Cancer Genome Atlas (TCGA) database to construct a prognostic risk score model. The prognoses of the high-risk and low-risk groups were compared by Kaplan-Meier method. The accuracy of the model was evaluated by the receiver operating characteristic (ROC) curve. Tumor tissue samples of 83 patients with pathological diagnosis of ESCC were collected from Anyang Cancer Hospital for external verification. Cox regression analysis was used to comprehensively evaluate the effects of prognostic risk score and various clinical characteristics on OS of patients with ESCC. Results: Seven immune-related genes that were significantly related to survival prognosis were selected from the TCGA database and included in the prognostic risk score model, which were S100A12, SLC40A1, FABP9, TNFSF10, IGHA2, IL1F10, and STC2. The 1- and 2-year survival rates of the low-risk group (40 cases) were 94.3% and 82.5%, respectively, while those of the high-risk group (40 cases) were 75.9% and 32.9%, respectively.The prognosis of the high-risk group was worse than that of the low-risk group (P<0.001). The 83 external validation samples obtained consistent results by using the prognostic risk score model. The prognostic risk score was positively correlated with the content of CD4(+) T lymphocytes in ESCC (r(s)=0.259, P=0.020), but not correlated with the content of B lymphocytes, CD8(+) T lymphocytes, neutrophils, macrophages or dendritic cells (P>0.05). Conclusions: S100A12, SLC40A1, FABP9, TNFSF10, IGHA2, IL1F10, and STC2 were risk genes significantly associated with OS of patients with ESCC. The prognostic risk score was an independent prognostic factor for the OS of patients with ESCC, and it was correlated with the content of CD4(+) T lymphocytes in ESCC tissue.

[The suppressive effects of microRNA-139-5p on proliferation and invasion of esophageal squamous cell carcinoma].

Objective: To investigate the role of microRNA-139-5p (miR-139-5p) in the occurrence and development of esophageal squamous cell carcinoma (ESCC) and its effects on cell proliferation and invasion of ESCC cells and its molecular mechanisms. Methods: Seventy-five cases of ESCC tissues and paired normal tissues were obtained from thoracic surgery of the First Affiliated Hospital of Zhengzhou University from February 2017 to March 2018. Experiment was divided into two group: ESCC (n=75) and normal esophageal tissues (n=75).GEO datasets and real-time quantitative PCR (qRT-PCR) were used to detect the expression of miR-139-5p in ESCC tissues and cells. miR-139-5p inhibitor, miR-139-5p mimic, negative control, control siRNA, T-box transcliption factor 1(TBX1) siRNA, pcDNA3.1 and pcDNA3.1-TBX1 were transfected into ESCC Eca109 and TE1 cells. qRT-PCR was used to detect the expressions of miR-139-5p and TBX1 in transfected ESCC cells. Cell counting kit 8 (CCK-8) and Transwell chamber were employed to detect cell proliferation and invasion of ESCC cells, respectively. Dual-Luciferase Reporter assay was used to analyze the interaction between miR-139-5p with TBX1. qRT-PCR, Western blot and immunohistochemistry were utilized to detect the expression of TBX1 in ESCC tissues. Western blot was used to detect the expressions of E-cadherin, N-cadherin and Vimentin after transfection. Results: The level of miR-139-5p in ESCC tissues was significantly lower than that in normal tissues (1.17±0.43 vs 5.16±3.62,P<0.001). Log-rank test showed that the survival rate of ESCC patients with high miR-139-5p level (n = 43) was significantly higher than that with low miR-139-5p level (n=32) (67.44% vs 25.00%, P = 0.005). The expression level of miR-139-5p in ESCC cells was significantly lower than that of normal esophageal epithelial cell Het-1A (all P<0.001). The proliferation and invasion ability of ECA109 and TE1 cells with high expression of miR-139-5p were significantly lower than those transfected with negative control (NC) (all P<0.05). Dual-Luciferase Reporter assay showed that miR-139-5p could bind to the 3'-untranslated region of TBX1. miR-139-5p mimic or inhibitor suppressed or promoted the expression of TBX1 protein in Eca109 and TE1 cells, respectively (all P<0.05). Downregulation of TBX1 significantly suppressed proliferation and invasion of ECA109 and TE1 cells, while overexpression of TBX1 significantly promoted proliferation and invasion of ECA109 and TE1 cells (all P<0.05). In addition, pcDNA3.1-TBX1 partially reversed the inhibition of miR-139-5p-mediated invasion ability (all P<0.05), while TBX1 siRNA partially reversed the enhancement of miR-139-5p inhibitor-mediated invasion ability (all P<0.05). Conclusion: miR-139-5p suppressed ESCC cell proliferation and invasion by targeting TBX1.

[Analysis of the relationship between the anatomical location of intrapulmonary metastatic lymph nodes and relapse risk and survival in patients with N1 non-small cell lung cancer].

Objective: To evaluate the relationship between the anatomical location of intrapulmonary metastatic lymph nodes and relapse risk and survival in patients with N1 non-small cell lung cancer(NSCLC). Methods: A retrospective analysis of the clinical and pathological data of 138 patients with completely resected N1 NSCLC was conducted. There were 79 males and 59 females, aged from 26 to 81 years with an average of (59±10) years. All of them were treated in the Department of Thoracic Surgery Ⅱ of Peking University Cancer Hospital between January 2007 and December 2015. Patients were stratified based on the 8th edition of the American Joint Committee on Cancer (AJCC) N1 classification and the modified pathological N1 classification strategy, respectively. According to modified pathological N1 classification strategy, which was defined based on the anatomical location of intrapulmonary metastatic lymph nodes, N1 nodes were subcategorized into the hilar (stations 10-11, mN1b) (n=36) and peripheral (stations 12-14, mN1a) (n=102) zones. The Kaplan-Meier curves were plotted to compare the relapse risk and survival analysis, disease-free survival (DFS), and overall survival (OS) were compared between the two staging methods through univariate and multivariate analysis to evaluate the effectiveness of the two classifications in stratifying patients with distinct risks of disease relapse and survival. Results: According to the modified N1 classification, the differences in 5-year DFS and OS between the subgroups (mN1a vs mN1b) were statistically significant(59.5% vs 35.7%; 81.2% vs 56.0%; both P<0.05). However, following the 8th edition of the AJCC N1 classification, no significant differences were found in DFS and OS between the subgroups (both P>0.05). Multivariate analysis showed that the modified N1 classification was an independent prognostic factor to DFS (HR=1.814, 95%CI: 1.005-3.275) and OS (HR=3.919, 95%CI: 1.918-8.009) (all P<0.05). However, the 8th edition of the AJCC N1 classification was not an independent prognostic factor to DFS (HR=1.360, 95%CI:0.767-2.412) or OS (HR=1.620, 95%CI:0.839-3.131) (both P>0.05) as revealed by multivariate analysis. Conclusions: The relapse risk and survival could be assessed effectively using the modified pathological N1 classification, which was defined and subcategorized based on the anatomical location of intrapulmonary metastatic lymph nodes for N1 NSCLC patients. The modified pathological N1 classification is superior to the 8th edition of the AJCC classification.

[Cytological analysis of lymphoblastic lymphoma/acute lymphoblastic leukemia in serous effusion].

Objective: To study the diagnostic clues and significance in serous effusion cytology associated with lymphoblatic lymphoma/acute lymphoblastic leukemia (LBL/ALL). Methods: Forty-five serous effusion specimens with final diagnosis of LBL/ALL were collected from August 2011 to December 2019 at the First Affiliated Hospital of Zhengzhou University. All cases were reviewed for their clinical profiles, cytomorphologic features and ancillary studies. Cell blocks and immunocytochemistry were prepared in 22 cases; flow cytometric immunophenotyping was performed in three cases and gene rearrangement analysis (T-cell recepter, TCR and immunoglobulin, Ig) was performed in five cases. Results: Among the 45 cases, there were 35 males and 10 females with male to female ratio of 3.5∶1.0. The median age was 15 years. Mediastinal mass was the initial presentation in 39 patients (86.7%) and high LDL level were observed in 34 patients (75.6%). Microscopically, the majority of the specimens (86.7%) were hypercellular. The smears demonstrated dispersed lymphoblasts that were predominantly small to intermediate in size with scanty basophilic cytoplasm and irregular or convoluted nuclei with fine chromatin condensation and inconspicuous nucleoli. Mitoses were frequently observed. Karyorrhexis and apoptosis were seen in all cases. By immunophenotyping, TdT was expressed in 19 cases (86.4%) and CD99 in 20 cases (90.9%). Ki-67 expression varied from 65% to 95%. Flow cytometry in three cases demonstrated positivity for TdT, CD2, CD3 and CD7. Monoclonal TCR gene rearrangement was found in 4 of 5 cases, and both monoclonal TCR and Igκ gene were found in 1 case. Conclusions: In LBL/ALL, primary diagnosis could be made basing on clinical features (younger male patients with a mediastinum mass) and cytomorphology (monotonous, small to medium sized lymphoid cells with prominent irregular nuclei, fine chromatin and frequent mitoses, karyorrhexis and apoptosis). If immunocytochemistry and other ancillary studies are performed, the accuracy and reliability of the results could be improved.

[Biocompatibility of extracellular matrix hydrogel with human iPSCs differentiated cardiomyocytes].

Objective: To observe the biocompatibility of porcine omental derived extracellular matrix (ECM) hydrogel with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and the feasibility of ECM hydrogel as a delivery vector of cell transplantation. Methods: A series of chemical, physical and enzymatic methods were applied to acellularize the porcine omentum. Subsequently, the extracted ECM was prepared into thermosensitive hydrogel. The biochemical composition of the hydrogel was identified by histological staining. The microstructure was observed by scanning electron microscopy. The hydrogel was then injected into the myocardium of mice to observe its in situ gelation ability. Differentiation of human induced pluripotent stem cells into cardiomyocytes was achieved by small molecule induction, and then the obtained hiPSC-CMs were cultured. hiPSC-CMs cultured onto the prepared hydrogel were defined as the hydrogel group, while conventionally cultured hiPSC-CMs were defined as the control group. Cardiomyocyte viability and growth patterns were detected using live/dead staining, CCK-8 and phalloidin staining. Immunofluorescence staining and Western blot of cardiomyocytes were used to determine the survival and phenotypic maintenance markers of cardiomyocytes in materials. Results: The results of HE staining, oil red O staining and DAPI fluorescence staining showed that there was no significant cell debris, nucleus and lipid residue in the prepared ECM hydrogel. The Sirius red staining and Alcian blue staining showed that the hydrogel retained collagen and glycolaminoglycan, which were the main components of ECM. The prepared hydrogel behaves as a viscous liquid at 4 ℃ and as a gel state at 37 ℃. Scanning electron microscope results showed that the microstructure of the hydrogel was composed of irregular fibers and pores of different sizes. Under the guidance of ultrasound, the prepared ECM hydrogel could be successfully injected into the myocardium of mice. Immediately after the injection, the hyperechoic signal could be observed under ultrasound, suggesting that the hydrogel remained in the myocardium. HE staining of myocardial tissue evidenced that there was lump of gel in the injection area. The differentiated hiPSC-CMs were co-cultured with the prepared ECM hydrogel, and the results of live/dead staining showed that most of the hiPSC-CMs in the hydrogel group and the control group were alive, dead cells were scanty. The results of CCK-8 test showed that the absorbance values of the two groups were similar (P>0.05). The results of phalloidin staining showed that hiPSC-CMs could extend normally when co-cultured with ECM hydrogel. The cell morphology of the hydrogel group was similar with that of the control group, and there was no statistically significant difference in the F-actin coverage area per cell between the two groups (P>0.05). Immunofluorescence staining of cardiomyocyte markers showed that there was no significant difference in the coverage area of α-actinin and connexin-43 (Cx-43) per field between the hydrogel group and the control group (both P>0.05), the quantitative results of DAPI staining showed that there was no statistically significant difference in the number of cells between the two groups (P>0.05). Meanwhile, the results of Western blot showed that the expression levels of α-actinin and Cx-43 in cardiomyocytes in the hydrogel group were similar as those in the control group (both P>0.05). Conclusions: These results show that preparation of the ECM hydrogel from porcine omentum is successful. The hydrogel has good biocompatibility and no obvious cytotoxicity. Besides, the hydrogel can support the survival of hiPSC-CMs in vitro and maintain its phenotype. These properties make it a promising injectable cardiac tissue engineering material.

Adjuvant chemotherapy for patients with gastric neuroendocrine carcinomas or mixed adenoneuroendocrine carcinomas.

BACKGROUND: The aim of this study was to evaluate whether adjuvant chemotherapy is associated with improved survival in patients with resectable gastric neuroendocrine carcinomas (G-NECs) or mixed adenoneuroendocrine carcinomas (G-MANECs). METHODS: The study included patients with G-NECs or G-MANECs who underwent surgery in one of 21 centres in China between 2004 and 2016. Propensity score matching analysis was used to reduce selection bias, and overall survival (OS) in different treatment groups was estimated by the Kaplan-Meier method. RESULTS: In total, 804 patients with resectable G-NECs or G-MANECs were included, of whom 490 (60·9 per cent) received adjuvant chemotherapy. After propensity score matching, OS in the chemotherapy group was similar to that in the no-chemotherapy group. Among patients with G-NECs, survival in the fluorouracil (5-FU)-based chemotherapy group and the non-5-FU-based chemotherapy group was similar to that in the no-chemotherapy group. Similarly, etoposide plus cisplatin or irinotecan plus cisplatin was not associated with better OS in patients with G-NECs. Among patients with G-MANECs, OS in the non-5-FU-based chemotherapy group was worse than that in the no-chemotherapy group. Patients with G-MANECs did not have better OS when platinum-based chemotherapy was used. CONCLUSION: There was no survival benefit in patients who received adjuvant chemotherapy for G-NECs or G-MANECs.

ANTECEDENTES: El objetivo de este estudio fue evaluar si la quimioterapia adyuvante mejoraba la supervivencia en pacientes con carcinomas gástricos resecables neuroendocrinos (gastric neuroendocrine carcinomas, G-NECs) y carcinomas adenoneuroendocrinos mixtos (mixed adenoneuroendocrine carcinomas, G-MANECs). MÉTODOS: Se incluyeron pacientes con G-NECs y G-MANECs tratados quirúrgicamente en 21 centros en China entre 2004 y 2016. Se utilizó un análisis de emparejamiento por puntaje de propensión para reducir el sesgo de selección y el método de Kaplan-Meier para estimar la supervivencia global (overall survival, OS) de los pacientes en los diferentes grupos de tratamiento. RESULTADOS: En total, se incluyeron en el estudio 804 pacientes con G-NECs y G-MANECs resecables y 490 pacientes (60,9%) recibieron quimioterapia adyuvante. Después del emparejamiento por puntaje de propensión, la OS del grupo con quimioterapia fue similar a la del grupo sin quimioterapia. En los pacientes con G-NECs, la supervivencia en los grupos con quimioterapia basada en 5-FU (fluorouracilo) y de quimioterapia sin 5-FU fue similar a la del grupo sin quimioterapia. Asimismo, la combinación de etopósido y cisplatino o de irinotecán y cisplatino no se asoció con una mejor OS en pacientes con G-NECs. En pacientes con G-MANECs, la OS del grupo con quimioterapia sin 5-FU fue peor que la del grupo sin quimioterapia. Los pacientes con G-MANECs no presentaron una mejor OS cuando se administró quimioterapia basada en platinos. CONCLUSIÓN: La administración de quimioterapia adyuvante en pacientes con G-NECs y G-MANECs no mejoró la supervivencia.