Efficacy and safety of camrelizumab combined with TACE for hepatocellular carcinoma: a systematic review and meta-analysis.

OBJECTIVE: Hepatocellular carcinoma (HCC) represents a highly lethal and recurrent neoplasm, with limited effective treatment regimens available. Camrelizumab, as a novel PD1 inhibitor combined with transcatheter arterial chemoembolization (TACE), has been widely used in the treatment of HCC. However, there remains a contentious debate regarding the clinical value of the TACE and camrelizumab combination. This study seeks to investigate the efficacy and safety of this combination treatment regimen in patients with HCC. MATERIALS AND METHODS: The related studies were retrieved from four online databases, including Pubmed, Cochrane Library, EMBASE, and Web of Science, up to June 1, 2023. The selection of studies was based on screening of titles, abstracts, and full-texts. The primary efficacy outcomes included complete response (CR), objective response rate (ORR), and disease control rate (DCR), while safety outcomes evaluated all treatment-related adverse events (AEs). Additionally, secondary outcomes such as overall (OS) and progression-free survival (PFS) were extracted for further survival analysis. The quality of the included trials was assessed using the MINORS tool. Publication bias was evaluated through funnel plot and Egger's test. RESULTS: A total of 17 publications involving 1,377 cases were included. The pooled CR rate, ORR, and DCR of the patients treated with TACE plus camrelizumab had a pooled CR rate of 8% (95% CI: 0.01-0.15, p=0.03), ORR of 47% (95% CI: 0.42-0.52, p<0.00001) and DCR of 82% (95% CI: 0.77-0.88, p<0.00001), respectively. Compared with a control group that did not receive TACE or camrelizumab, the pooled RR of CR rate, ORR, and DCR were 1.61 (95% CI: 1.27-2.04, p<0.0001), 1.56 (95% CI: 1.19-2.05, p=0.001) and 1.55 (95% CI: 1.19-2.03, p=0.001), respectively. Besides, the combination regimen can prolong the OS (HR=2.60, 95% CI: 2.25-3.02, p<0.00001) and PFS (HR=4.90, 95% CI: 1.94-12.38, p=0.0008). However, the incidence of treatment-related AEs was relatively high (77%), with 29% for grade 3 AEs. The most common AEs observed were pain (47%), fever (46%), hepatic function abnormalities (44%), hypoalbuminemia (39%), and hypertension (37%). The combination treatment did not increase the incidence of AEs compared to the control group, except for the hand-foot skin reaction (RR=0.85, 0.74-0.97, p=0.01), hepatic encephalopathy (RR=4.29, 2.51-7.35, p<0.00001) and nausea (RR=1.35, 1.13-1.61, p=0.001). CONCLUSIONS: Combination therapy of TACE plus camrelizumab has shown notable clinical benefits, improved survival, and a manageable safety profile in patients with HCC, but it is essential to monitor and manage the specific toxicities, especially for the camrelizumab-related AEs.

[Analysis of clinicopathological features and prognosis of sporadic synchronous multiple primary colorectal cancers].

Objective: To investigate the impact of relative locations of multiple foci and microsatellite status of sporadic, synchronous, multiple, primary, colorectal carcinomas on clinicopathological features and prognosis. Methods: The clinicopathologic and prognostic data of 278 patients with sporadic, synchronous, multiple, primary, colorectal carcinomas who had been admitted to the Department of Colorectal Surgery at Zhejiang Cancer Hospital from January 2008 to July 2022 were retrospectively collected. The patients were categorized into three groups based on the relative locations of their multiple cancer foci: (1) a right-sided group that comprised patients with multiple cancer foci in the cecum, ascending colon, hepatic flexure of the colon, and transverse colon; (2) a left-sided group that comprised patients with multiple cancer foci in the splenic flexure of the colon, descending colon, sigmoid colon, and rectum; and (3) a left- and right-sided group that comprised patients with multiple cancer foci in the right half of the colon and left half of the colon/rectum. Additionally, the patients were further divided into two groups based on microsatellite status: a high microsatellite instability (MSI-H) and a low MSI/stable MSI (MSI/L&MSS) group. We compared differences in clinical characteristics and prognostic indicators between these groups. The χ2 test was utilized to compare selected clinical characteristics, whereas Kaplan-Meier survival analyses and log-rank tests were performed to compare their effects on prognosis. Result: Among 278 patients with SSCRC, 256 (92.1%) presented with two cancer foci and 22 (7.9%) with more than two foci. Additionally, 255 patients (91.7%) had adenocarcinomas, whereas the remaining 23 (8.3%) had mucinous adenocarcinomas. Lymph node metastases were identified in 136 patients (48.9%); the cancer foci had infiltrated beyond the muscular layer in 238 (85.6%); and 147 patients (52.9%) were diagnosed with TNM Stage III-IV disease. There were 155 patients (55.8%) in the left-sided group, 55 (19.8%) in the right-sided group, and 68 (24.5%) in the left- and right-sided group. Immunohistochemical examination of all four mismatch repair proteins were performed in 199 cases, revealing that 166 of these patients had MSI/L&MSS and 33 MSI-H disease. In the left-sided, left- and right-sided, and right-sided groups, the proportion of women was 16.8% (26/155), 26.5% (18/68), and 49.1% (27/55), respectively; these differences are statistically significant (χ2=22.335, P<0.001). The proportions of patients with more than three cancer foci were 5.2% (8/155), 16.2% (11/68), and 5.5% (3/55), respectively; these differences are statistically significant (χ2=8.438, P=0.015). The proportions of mucinous adenocarcinomas were 4.5% (7/155), 8.8% (6/68), and 18.2% (10/55), respectively; these differences are statistically significant (χ2=10.026, P=0.007). The proportions of patients with lymph node metastases were 55.5% (86/155), 48.5% (33/68), and 30.9% (17/55); these differences are statistically significant (χ2=9.817, P=0.007). The proportions of patients with Stage T3 & T4 disease in each group according to location were 81.3% (126/155), 88.2% (60/68), and 94.5% (52/55), respectively; these differences are statistically significant (χ2=6.293,P=0.043). The proportions of TNM Stage III-IV tumors were 59.4% (92/155), 54.4% (37/68), and 32.7% (18/55), respectively; these differences are statistically significant (χ2=11.637, P=0.003). Age, size of cancer foci, presence of distant metastasis, adenoma, nerve invasion, and vascular invasion did not differ significantly between the three groups (all P>0.05). Compared with those with MSI-H, patients with MSI/L&MSS disease were more likely to be aged >65 years and male (50.6% [84/166] vs. 15.2% [5/33], χ2=13.994,P<0.001; 80.7% [134/166] vs. 54.5% [18/33], χ2=10.457,P=0.001), more likely to be in the left-sided group (63.3% [105/166] vs. 24.2% [8/33], χ2=18.232, P<0.001), had a higher proportion of cancer foci of diameter <4 cm (54.8% [91/166] vs. 33.3% [11/33], χ2=5.086,P=0.024), and a lower proportion of mucinous adenocarcinomas (4.2% [7/166] vs. 27.3% [9/33], χ2=19.791,P<0.001), more likely to develop distant metastases (22.3% [37/166] vs. 6.1% [2/33], χ2=4.601,P=0.032), more likely to have lymph node metastases (57.2% [95/166) vs. 24.2% [8/33], χ2=11.996,P<0.001) and nerve invasion (28.9% [48/166] vs. 6.1% [2/33], χ2=7.643, P=0.006), had a higher proportion of TNM Stage III-IV disease (60.2% [100/166] vs. 24.2% [8/33], χ2=14.374, P<0.001), and a smaller proportion of family history of tumors (28.9% [48/166] vs. 60.6% [20/33], χ2=12.228, P<0.001). All the above-listed differences are statistically significant (all P<0.05). The differences in number of cancer foci, depth of infiltration, presence or absence of adenomas, and vascular invasion were not statistically significant (all P>0.05). In the 33 patients with MSI-H status and mismatch repair protein loss, the highest frequency of deletion was found in PMS-2 (66.7%, 22/33), followed by MLH-1 (57.6%, 19/33), whereas the proportions of MSH-2 (33.3%, 11/33) and MSH-6 (24.2%, 8/33) deletions were relatively low. There were statistically significant differences in the 3-year overall survival rates among the groups according to relative locations of cancer foci. The 3-year overall survival rates were 96.8%, 79.6%, and 88.5% in the right-sided, left- and right-sided, and left-sided groups, respectively (P=0.021). As to microsatellite status, the 3-year overall survival rate of patients with MSI-H disease was 93.8%, which is significantly better than the 78.4% for those with MSI/L & MSS (P=0.026). Conclusions: Among sporadic, synchronous, multiple, primary, colorectal carcinomas, those with right-sided disease had the deepest local infiltration, whereas those with left-sided disease had the greatest number of lymph node metastases, most advanced clinical TNM stage, lowest percentage of MSI-H disease, and the poorest prognosis.

Efficacy and safety of PD1/PDL1 inhibitors combined with radiotherapy and antiangiogenic drugs for hepatocellular carcinoma: a systematic review and meta-analysis.

OBJECTIVE: The triplet regimen based on the programmed cell death 1 (PD1)/ programmed cell death ligand 1 (PDL1) inhibitors combined radiotherapy and antiangiogenic drugs is a novel therapeutic strategy for hepatocellular carcinoma. We conducted a meta-analysis to evaluate the efficacy and safety of the triplet therapeutic regimen in the treatment of hepatocellular carcinoma. MATERIALS AND METHODS: We searched scientific literature databases and clinical trial databases through October 31, 2022, for required studies. The pooled hazard ratio (HR) was used to analyze the overall survival (OS), progression-free survival (PFS), and the pooled relative risk (RR) was used to analyze the objective response rate (ORR), disease control rate (DCR), mortality rate (MR), and adverse events (AEs) through random or fixed effects model, 95% confidence interval (CI) was determined for all outcomes. Qualities of the included literature were assessed by MINORS Critical appraisal checklist. Funnel plot was used to assess publication bias in the included studies. RESULTS: Five studies (3 single-arm and 2 non-randomized comparative trials), including 358 cases, were enrolled. Meta-analysis showed that the pooled ORR, DCR, and MR were 51% (95% CI: 34%-68%), 86% (95% CI: 69-102%), and 38% (95% CI: 18-59%), respectively. Compared with triplet regimen, the single or dual-combination treatments had shorter OS (HR=0.53, 95%: 0.34-0.83 via univariate analysis; HR=0.49, 95%: 0.31-0.78 via multivariable analysis) and PFS (HR=0.52, 95%: 0.35-0.77 via univariate analysis; HR=0.54, 95%: 0.36-0.80 via multivariable analysis). Common AEs to triplet regimens included skin reaction (17%), nausea/vomiting (27%), fatigue (23%), while severe AEs (10%), fever (18%), diarrhea (15%), and hypertension (5%) without statistically significant differences. CONCLUSIONS: In the treatment of hepatocellular carcinoma, PD1/PDL1 inhibitors combined radiotherapy and antiangiogenic drugs achieved better survival benefits than alone or dual-combination regimens. In addition, the triple-combination therapy has tolerable safety.

[Effects of ultrasonic bone curettes combined with high-speed drills in posterior laminectomy and decompression procedure for severe thoracic spinal stenosis].

Objective: To evaluate the safety and efficacy of the combined use of ultrasonic bone curette with the high-speed drill in posterior laminectomy and decompression procedure for severe thoracic spinal stenosis, and propose the optimal cutting position for ultrasonic bone curette during the laminectomy. Methods: By observing and measuring the parameters of thoracic pedicle, lamina, inner wall of the vertebral canal and their relation with the surrounding structures on cadavers, we provided a morphological marker for laminectomy by an ultrasonic bone curette. Data of 19 patients with severe thoracic spinal stenosis treated by posterior laminectomy and decompression were collected from June 2017 to June 2018 in Shanghai Changzheng Hospital. There were 11 males and 8 females, aged (50±6) years. The patients received laminectomy with the combined use of ultrasonic bone curette and the high-speed drill (Group A, n=10) or the use of ultrasonic bone curette alone (Group B, n=9). Operational time of decompressive laminectomy, blood loss, as well as perioperative complications such as durotomy, cerebrospinal fluid leak, injury of the nerve root and spinal cord were recorded in these two groups. The improvement of symptoms and the decompression width of the spinal canal were evaluated after operation. Two independent samples t-test was used for the comparison of two sets of continuous normal distribution data. Results: We had done the measurement in 6 cadavers. The mean distance between the boundary of cancellous-cortical bone of lamina and the inner wall of spinal canal was (0.9±0.4) mm, and the distance between the boundary of cancellous-cortical bone of pedicle and the inner wall of the spinal canal was (1.2±0.6) mm. For the surgeries in groups A, the mean laminectomy time for each segment was (4.4±0.5) min, the mean width of posterior laminectomy was (21.8±0.5) mm; and for the surgeries in group B, the mean laminectomy time for each segment was (5.0±0.5) min, the mean width of posterior laminectomy was (19.9±1.0) mm; there were significant differences in laminectomy time for each segment and the width of posterior laminectomy between the two groups (t=-2.391, 3.491, both P<0.05). There was one case of dura injury and one case of thoracic nerve root injury during the operation in group B. Conclusions: It is safer and more reliable for the combined use of ultrasonic bone curette with the high-speed drill in posterior laminectomy and decompression procedure for the severe thoracic spinal stenosis. The interface between the cortical bone and the medial edge of cancellous bone of the pedicle could be identified as the cutting mark for ultrasonic bone curette in this procedure.

[The value of multi-gene testing in benign or malign diagnosis of thyroid nodules with indeterminate cytology].

Objective: To study the diagnostic value of a multi-gene molecular testing in cytologically indeterminate thyroid nodules. Methods: From February 2018 to September 2018, patients with thyroid nodules who underwent fine needle aspiration(FNA) at Peking University Cancer Hospital were enrolled. Three hundred and sixty patients were included, consisting of 86 men and 274 women, with a mean age of 45.8 years (between 13 and 89 years old). Among 391 nodules, 141 were cytologically inderminate and 75 were resected. FNA samples underwent prospective testing using a next-generation sequencing (NGS) assay, which included 16 genes for point mutations and 26 types of gene fusions. The testing results of indeterminate nodules were compared with surgical outcomes, to determine the diagnostic performance. The results were compared with the BRAF V600E single gene mutation analysis by χ(2) test. Results: The multi-gene testing showed a sensitivity of 73.2%, specificity of 96.8%, positive predictive value of 96.8%, and negative predictive value of 73.2%. The diagnostic accuracy of multi-gene testing was significantly higher than the BRAF V600E mutation test (83.3% vs 73.6%, χ(2)=31.588, P<0.01). Conclusion: Multi-gene testing in FNA samples is an effective method to diagnose cytologically indeterminate thyroid nodules, which has a higher accuracy than BRAF V600E mutation detection.

Expression of CD63 in Lung Tissue of Guinea Pigs Dying of Anaphylactic Shock.

ABSTRACT: Objective To study the protein expression of cluster of differentiation 63 （CD63） in lung tissues of guinea pigs that died of anaphylactic shock and discuss the diagnostic value of CD63 for death from anaphylactic shock. Methods Twenty guinea pigs were randomly divided into control group, anaphylactic shock immediate death group, cold storage group （4 ℃ for 48 h） and frozen group （-20 ℃ for 7 d）. The animal model of guinea pigs that died of anaphylactic shock was established with human mixed serum injection. The expression changes of CD63 protein and CD63 mRNA in lung tissues were detected by hematoxylin-eosin （HE） staining, immunohistochemical staining, Western blotting, enzyme-linked immunosorbent assay （ELISA） and real-time RT-PCR. Results HE staining results showed congestion, and edema of lung tissues, and eosinophil infiltration in the anaphylactic shock groups. Western blotting analysis results showed that the expression of CD63 protein in the lung tissues of guinea pigs that died of anaphylactic shock was significantly higher than that in the control group （P<0.05）. Comparison between the anaphylactic shock groups was made, and the differences had no statistical significance. The results of immunohistochemical staining and real-time RT-PCR were consistent with that of Western blotting. ELISA results showed that CD63 protein expression in the immediate death group was higher than that in the control group （P<0.05）. Conclusion The expression of CD63 protein and CD63 mRNA in the lung tissues of guinea pigs that died of anaphylactic shock is significantly enhanced. Animal carcasses which were put in cold storage for 48 h and frozen for 7 d do not affect the examination of the above indicators. CD63 protein is expected to become an auxiliary diagnostic indicator of death from anaphylactic shock.

[Fine-needle aspiration for the diagnosis of lymph node metastasis in papillary thyroid carcinoma].

Objective: To investigate the diagnostic performance of fine-needle aspiration (FNA) cytology for the detection of lateral lymph node metastases in patients with papillary thyroid carcinoma (PTC). Methods: A total of 109 lateral lymph nodes with suspicious metastases from 85 patients were retrospectively subjected to FNAC, fine-needle aspiration thyroglobulin measurement (FNATg), and FNATg/SerumTg measurement. Lymph node pathological results after surgery were taken as the gold standard. Using Mann-Whitney U test, Pearson linear model and ROC curve were used for data analysis. Results: The sensitivity, specificity and accuracy of FNATg for the diagnosis of lateral neck lymph node metastasis were respectively 93.7%, 90.0% and 93.3% and those of FNATg/SerumTg were respectively 89.9%, 90.0% and 93.2% respectively, the threshold values for FNATg and FNATg/SerumTg were 0.925 ng/ml and 1.007, respectively. The sensitivity, specificity and accuracy of FNATg combined with FNAC were respectively 91.0%,93.5% and 94.4%. The existence of thyroid tissue and the expression of serum Tg did not affect the expression of lymph node FNATg. The FNATg cutoff value of 0.925 ng/ml showed the best diagnostic performance in patients with a thyroid gland, while the FNATg/SerumTg cutoff ratio of 14.95 showed the best diagnostic performance in patients without a thyroid gland. The serum TgAb significantly interfered with the expression of FNATg in the lateral neck metastatic lymph nodes (P=0.049). Conclusions: FNATg alone or the combination of FNATg with FNAC are highly reliable in the diagnosis of lateral neck lymph node metastases in patients with PTC. The expression of TgAb may interfere with the accuracy of the diagnostic performance of FNATg.

[Evaluation of the tear film function in diabetic patients using the Keratograph 5M].

Objective: To evaluate the tear film function of diabetic patients using the Keratograph 5M and to analyze its related factors. Methods: Case-control study. A total of 89 inpatients (174 eyes) diagnosed with diabetes in the Department of Endocrinology at our hospital were recruited. According to the fundus fluorescein angiography results, subjects were divided into two groups, diabetic retinopathy (DR) group and none-DR group. All subjects were examined for duration of diabetes, glycosylated hemoglobin, creatinine, 24-hour urinary protein, and 24-hour urinary microalbumin/urinary creatinine, and by electromyography. The Keratograph 5M was used to obtain the first and average tear film break-up time (BUTf and BUTav), tear meniscus height, the upper and lower meibomian gland loss scores. The data were analyzed by SPSS. Results: Compared to the non-DR group, the BUTf and the BUTav in the DR group were shorter [(8.42±4.71) s vs. (10.24±5.32) s, (11.20±5.25) s vs. (12.67±5.52) s; P=0.000]. There was no significant difference in the tear meniscus height between the three groups (P=0.067). The tear meniscus height were negatively related with glycosylated hemoglobin, and not related with duration of diabetes, 24-hour urinary microalbumin/urinary creatinine, creatinine, 24-h urinary protein, and electromyography results. As the duration of diabetic longer, the upper and lower meibomian gland loss scores added(Z=19.514, 15.342, P<0.05). Patients with EMG abnormal, the lower meibomian gland loss scores added(Z=-2.312, P<0.05). Conclusions: The Keratograph 5M can directly evaluate the tear film condition of diabetic patients.Diabetic patients with retinopathy and higher glycosylated hemoglobin tend to suffer tear film dysfunction. (Chin J Ophthalmol, 2018, 54:762-766).

Facilitating the measurement of circulatory hydrogen sulfide with fluorescence probe-coated microplates.

OBJECTIVE: The hydrogen sulfide (H2S) role in pathogenesis of various diseases were wildly addressed in recent decade. The circulatory (plasma or serum) and biological fluid H2S measurement is still an enormous issues due to the technical limitation. This paper aimed to develop a novel measurement method based on fluorescence probe. METHODS: Firstly, 20 µL ethanol was used to dissolve 100 pmol fluorescence probe, then added in a 96-well plate. An equal volume of ethanol was also added to the blank well of the plate. The plate was placed in a dark room for about 1 h until the fluorescence probe was evenly coated in the 96-well microplate and dried. The plate was frozen at -20 °C for later use. Secondly, the plasma or serum sample was added with saturated ammonium sulfate buffer (pH 7.8) and then centrifuged to remove the proteins. The equal volume supernatant liquid was added to the probe-coated well and the probe-uncoated well. The plate was incubated in a dark environment at 37 °C for 2 h. Finally, after incubation, the fluorescence density was acquired at ΛEx/ΛEm 340/445 nm in a microplate reader. The differences of the fluorescence density values between the probe-coated well and probe-uncoated well were counted and H2S concentration of plasma/serum was calculated by standard curve with NaHS. RESULTS: The method had high sensitivity (from 0.3 to 100 µmol/L) and specificity for measuring H2S as compared with other biologically relevant reactive sulfur species and sulfur-containing amino acid. Serum H2S concentrations were assayed in 188 health volunteers using this method [(12.1±3.5) µmol/L, 95%CI: 4.6-19.8 µmol/L], and the frequency distribution showed a normal tendency(one-sample Kolmogorov-Smirnov test, P>0.1). The serum H2S concentrations in 30 hypertension patients were decreased compared with 22 age- and gender-matched health individuals (paired-samples t test, t=9.937, P<0.001). There were no differences of H2S concentration in serum [(19.66±2.32) µmol/L] or plasma [(18.67±2.07) µmol/L], between the samples acquired from artery [(19.34±0.51) µmol/L] or vein [(18.99±0.50) µmol/L] of male Wistar rats (repeated measurement of ANOVA, P=0.38). One week frozen samples did not affect the detection. The values of the repeated measurement did not differ (two-way ANOVA, P>0.05). CONCLUSION: The present method is easily performed with high sensitivity, specificity and repeatability for circulatory H2S. It is also quick and may apply for large samples.

Comparing effects of cervical anterior approach and laminoplasty in surgical management of cervical ossification of posterior longitudinal ligament by a prospective nonrandomized controlled study.

INTRODUCTION: The laminoplasty has been the most widely used surgical method for OPLL. In recent years, increasing attention has been drawn to the anterior operative approaches for surgical treatment of cervical OPLL. However, which method is the optimum selection for therapy of cervical OPLL is still obscure. Therefore, we performed this prospective nonrandomized clinical study in patients with multilevel cervical myelopathy due to OPLL and compare the therapeutic efficiency of laminoplasty and anterior approach (cervical discectomy and/or cervical corpectomy) in the management of multilevel cervical OPLL. HYPOTHESIS: There is no difference in clinical effects between anterior cervical spine surgery and laminoplasty in the treatment of multilevel cervical OPLL. MATERIAL AND METHODS: A total of 150 consecutive patients with multilevels of cervical OPLL underwent anterior approaches (ACDF, ACCF and HDF) from July 2010 to June 2014, which were enrolled in this study. During the same period, one hundred and two patients receiving the laminoplasty were enrolled in the study. The clinical effects, alignment and range of motion (ROM) of cervical spine in patients of the anterior group and posterior group were assessed, respectively. The effects of high signals in T2 weighed MRI scans and percentage of spinal canal stenosis in these patients were also evaluated. Finally, postoperative complications regarding each group were analyzed. RESULTS: Although significant differences in types of OPLL and preoperative sagittal alignment of cervical spine occurred in the two groups (P<0.05), clinical effects of the two groups were similar (P>0.05). The cervical curvature in laminoplasty group showed significant decrease at final follow-up (P<0.05). For ROM of cervical spine, no significant alteration was observed in both groups. The high T2 weighed signals and rate of spinal canal stenosis can influence clinical effects of both anterior group and laminoplasty group. In addition, significantly higher complication rate was observed in laminoplasty group compared with anterior group (P<0.05). DISCUSSION: Both anterior and laminoplasty approaches can be considered effective and safe procedures in the treatment of the multilevel OPLL. However, the anterior approach with relatively lower incidence of postoperative complications is a better choice for cases with poor cervical curvature and serious spinal canal stenosis. TYPE OF STUDY AND LEVEL OF PROOF: Level 3 nonrandomized, controlled clinical trials.

Changes in the gut microbiome of the Chinese mitten crab (Eriocheir sinensis) in response to White spot syndrome virus (WSSV) infection.

Intestinal microorganisms play important roles in maintaining host health, but their functions in aquatic animal hosts have yet to be fully elucidated. The Chinese mitten crab, Eriocheir sinensis, is one such example. We attempted to identify the shift of gut microbiota that occurred in response to infection of white spot syndrome virus (WSSV), an emerging viral pathogen in the crab aquaculture industry. The microbiota may exert some control over aspects of the viral pathogenesis. We investigated the changes in composition and structure of the crab gut microbiome during various WSSV infection stages of 6 h post-infection (hpi) and 48 hpi, using a 16S rRNA approach on the MiSeq Illumina sequencing platform. Four phyla (Firmicutes, Proteobacteria, Tenericutes and Bacteroidetes) were most dominant in the gut of E. sinensis regardless of the WSSV infection stages. However, further analysis revealed that over 12 bacterial phyla, 44 orders and 68 families were significantly different in abundance at various states of WSSV infection. Several intriguing aspects of E. sinensis gut bacteria that had not been previously reported were also uncovered, such as class Mollicutes was dominant here, but absent in crabs from Yangtze River estuary and Chongming Islands. Overall, this study provided the first evidence that changes in gut microbiome were closely associated with the severity of WSSV infection and that indicator taxa could be used to evaluate the crab health status.

Glioma cells promote angiogenesis through the release of exosomes containing long non-coding RNA POU3F3.

OBJECTIVE: Angiogenesis is a key event in the progression of gliomas, and emerging evidence suggests that exosomes are signaling extracellular organelles that modulate the tumor microenvironment and promote angiogenesis and tumor progression. This study aimed to explore the mechanism by which glioma-derived exosomes affect angiogenesis. MATERIALS AND METHODS: qRT-PCR was used to determine the expression level of linc-POU3F3 in glioma tissue as well as glioma cell lines. Ultrafiltration combined with a purification method was used to isolate exosomes derived from A172 cells (A172-Exo) and linc-POU3F3 shRNA-treated A172 cells (shA172-Exo). Transmission electron microscopy, Western blot and tunable resistive pulse sensing (TRPS) were used to identify exosomes. In vitro migration, proliferation, and tube formation experiments, as well as in vivo CAM assays, were used to analyze the pro-angiogenesis ability of exosomes. qRT-PCR and Western blot were used to identify expression levels of angiogenesis-related genes and proteins in human brain microvascular endothelial cells (HBMECs) after being cultured with exosomes. RESULTS: The levels of linc-POU3F3 were upregulated in glioma tissue and significantly correlated with the advanced tumor stage. A172 cells exhibited the highest expression level. A172-Exo was similar to shA172-Exo (50-100 nm in diameter) and expressed Alix, Tsg101 and CD9, while the expression level of linc-POU3F3 in A172-Exo was significantly higher than that in shA172-Exo. HBMECs rapidly internalized A172-Exo and shA172-Exo, and the linc-POU3F3 expression level in HBMECs treated with A172-Exo was significantly higher than the level in HBMECs treated with shA172-Exo. A172-Exo exhibited better function in promoting HBMECs migration, proliferation, tubular-like structure formation in vitro and arteriole formation in vivo. The gene and protein expression level of bFGF, bFGFR, VEGFA, and Angio in HBMECs treated with A172-Exo was much higher than that of HBMECs treated with shA172-Exo. CONCLUSIONS: These results indicated that gliomas can induce angiogenesis by secreting exosomes enriched in linc-POU3F3. Exosomes and lncRNA-POU3F3 may, therefore, function as a putative therapeutic target in glioma.

Development of In situ hybridization and real-time PCR assays for the detection of Hepatospora eriocheir, a microsporidian pathogen in the Chinese mitten crab Eriocheir sinensis.

A microsporidian parasite, Hepatospora eriocheir, is an emerging pathogen for the Chinese mitten crab Eriocheir sinensis. Currently, there is scant information about the way it transmits infection in the crustacean of commercial importance, including its pathogenesis, propagation and infection route in vivo. In this study, chromogenic in situ hybridization (ISH) and quantitative real-time PCR (qPCR) assays were developed to address this pressing need, and we provided an advance in the detection methods available. Pathogens can be seen in situ with associated lesions using ISH. Positive hybridization signals were noted inside the epithelial cells of the hepatopancreas, and putative free parasite spores were observed within the tubule lumen, which were associated with lesions detected by electron microscopy and haematoxylin and eosin (H&E) analysis. qPCR allows the determination of parasite loads in infected tissues, which is important for understanding disease progression and transmission. The hepatopancreas displayed the biggest statistical copy numbers among different tissues of infected crabs, confirming a tissue-specific pathogen infection characteristic. The qPCR assay also proved to be suitable for the diagnosis of asymptomatic carrier crabs. Combination of the two methods could facilitate the study of H. eriocheir infection mechanism in E. sinensis, enhance the early diagnosis of the pathogen and improve the management of microsporidian diseases in commercial crustaceans.

Fibulin-5: a novel biomarker for evaluating severity and predicting prognosis in patients with acute intracerebral haemorrhage.

BACKGROUND AND PURPOSE: The aim was to investigate relationships between serum fibulin-5 concentration and the severity or prognosis in patients with acute intracerebral haemorrhage (ICH). METHODS: Consecutive ICH patients and healthy controls were included and clinical data were collected. National Institute of Health Stroke Scale (NIHSS) and Glasgow Coma Scale (GCS) were assessed at admission time within 3 days after bleeding. Cerebral haemorrhage volume was calculated and serum fibulin-5 concentration was measured at the same time. Multivariate linear regression analyses were performed to determine risk factors for serum fibulin-5 concentration and Spearman correlation coefficients were obtained to explore the relationships between fibulin-5 concentration and NIHSS or GCS scores. Patients were followed up for 3 months and the modified Rankin Scale was evaluated for all survivors. Receiver operating characteristic (ROC) curves were obtained to explore fibulin-5 concentration in predicting prognosis. RESULTS: Serum fibulin-5 concentration had increased in ICH patients compared with healthy controls (65.86 ± 26.39 µg/l vs. 40.66 ± 5.03 µg/l, P = 0.00) and was mainly influenced by haemorrhage volume (ß = 0.905, P = 0.000) and extension to ventricles (ß = 10.173, P = 0.097). Serum fibulin-5 concentration was positively correlated with NIHSS score (r = 0.511, P = 0.000) but inversely correlated with GCS score (r = -0.585, P = 0.000). Based on the ROC curves, the optimal cut-off point was 80.68 µg/l for death, and the sensitivity and specificity values of serum fibulin-5 were 77.8% and 93.2%, whilst the optimal cut-off point was 48.45 µg/l for poor prognosis and the sensitivity and specificity values were 86.4% and 54.1%, respectively. CONCLUSIONS: Serum fibulin-5 concentration can be regarded as a biomarker for evaluating disease severity and predicting prognosis in ICH patients.