[Preliminary analysis of neuroprotective effects of capillary fascia preservation recurrent laryngeal nerve anatomical method in right level â ¥ dissection].

Objective: To investigate the application and effect of capillary fascia preservation between the recurrent laryngeal nerve (RLN) and common carotid artery (fascia preservation method) in nerve protection when dissecting right level Ⅵ lymph nodes for patients with papillary thyroid carcinoma. Methods: A retrospective cohort study enrolling 195 patients with papillary thyroid carcinoma undergoing right level Ⅵ lymph node dissection in Beijing Tongren Hospital from March 2021 to August 2022 was carried out. The RLN was dissected by fascia preservation method in study group and by routine method in control group. The intraoperative electrical signal amplitude of the RLN, the number of dissected lymph nodes, and the postoperative complications were recorded and analyzed. Results: A total of 195 patients (study group: 94 cases, control group: 101 cases) were collected. There were 71 males and 124 females, with the median age of 32 (39, 51) years. In the study group, the total number of right level Ⅵ lymph nodes was significantly larger than the number of right Ⅵa level lymph nodes [8 (6, 11) vs 6 (4, 8), P<0.001]. There were no significant differences between the two groups in the number of level Ⅵa or level Ⅵb lymph nodes [Ⅵa: 6 (4, 8) vs 5 (3, 7), P=0.373; Ⅵb: 3 (1, 4) vs 2 (1, 4), P=0.337] and metastasis rate [Ⅵa: 51.1% (48/94) vs 52.5% (53/101), P=0.844; Ⅵb: 12.8% (12/94) vs 15.8% (16/101), P=0.541]. The ratio of electromyography (EMG) amplitude R2 in lower level Ⅵ and entry into larynx (grouped as>90%, 50%~90%,<50%) in the study group was significantly higher than that in the control group (P<0.001). No significant differences were detected between the two groups in temporary RLN paralysis [1.1% (1/94) vs 2.0% (2/101), P=1.000]. Conclusions: Fascia preservation method can decrease the stimulus and traction to RLN and preserve the capillary network serving RLN. It can thoroughly dissect lymph nodes and decrease the injury of RLN.

A combination of mRNA expression profile and miRNA expression profile identifies detection biomarkers in different tumor stages of laryngeal squamous cell carcinoma.

OBJECTIVE: This study was conducted to investigate microRNA (miRNA) target regulations during the disease progression in laryngeal squamous cell carcinoma (LSCC) and identify biomarkers in different tumor stages. MATERIALS AND METHODS: The mRNA dataset GSE59102 and miRNA dataset GSE70289 were used in this study. After pretreatment, differentially expressed genes/miRNAs (DEGs/DEMs) in different tumor stages (beginning vs. margin, advanced vs. margin, and beginning vs. advanced) were selected on the basis of their limma package. Then, the enrichment analysis for these DEGs was conducted using ClueGO. Protein-protein interaction (PPI) network analysis was performed on the basis of the BioGRID database. After prediction of target genes of DEMs according to three validated miRNA databases, an integrated miRNA target network and its pathways were drawn using the multiMiR package. RESULTS: Numerous DEGs were identified in different tumor stages of LSCC (beginning vs. margin, advanced vs. margin, and beginning vs. advanced), and a set of 18 DEMs was identified. Cell cycle was the most significantly enriched pathway of the DEGs. Four hub nodes (MCM2, EGFR, CDK2, and CDK1) were highlighted in the PPI network. In the integrated miRNA target network, 2 miRNAs were predominant: hsa-miR-331-3p (2 predicted targets, E2F1 and TNFRSF10B) and has-miR-375 (1 predicted target, TNNI3). These genes were tied up with cell cycle or apoptosis pathway. CONCLUSIONS: Several genes and miRNAs might be used as markers for LSCC in different tumor stages (e.g., MCM2, EGFR, CDK1, CDK2, hsa-miR-331-3p, hsa-miR-375). They might function through the involvement of the cell cycle pathway.

[Analysis of prognostic factors in Chinese patients with post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis].

Objective: To evaluate the performances of the prognostic scoring systems devised for primary myelofibrosis(PMF)and the new developed MYSEC- PM(Mysec Prognostic Model)and investigate the risk factors in Chinese patients with post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis(post- PV/ET MF). The most widely used prognostic scoring systems in PMF included the International Prognostic Scoring System(IPSS), dynamic International Prognostic Scoring System(DIPSS), refined DIPSS(DIPSS plus), modified IPSS for Chinese(IPSS-Chinese), and modified DIPSS for Chinese(DIPSS- Chinese). Methods: The clinical and hematologic information of 55 consecutive patients diagnosed with post- PV/ET MF from March 1984 to December 2013 were retrospectively collected. All post-PV/ET MF patients were categorized according to IPSS, DIPSS, DIPSS plus, IPSS-Chinese, DIPSS-Chinese and MYSEC-PM, and the possible prognostic factors were statistically analyzed. Results: Fifty five patients diagnosed with post-PV MF(n=32)or post-ET MF(n=23)were analyzed with a median age of 59(range: 20- 88)years old, including 20 males and 35 females. Median time from original diagnosis to myelofibrosis was 7.8(range: 1.1- 23.4)years. With a median follow up from post-PV/ET MF diagnosis of 37(range: 1-156)months, 44(80.0%)patients were censored alive, 11(20.0%)patients died. Median survival was 110(95% CI 87.5-132.8)months. Using IPSS, DIPSS, DIPSS plus, IPSS- Chinese and MYSEC- PM criteria, there were no statistically significances in survival among different risk groups(P>0.05). In univariate analyses HGB<100 g/L(P=0.003)was the only factor associated with poorer overall survival. The prognosis in subjects with HGB≥100 g/L was significantly better than that with HGB<100 g/L(median OS: not reached vs 47 months, P=0.003). Conclusion: IPSS, DIPSS, DIPSS plus, IPSS- Chinese and MYSEC- PM did not accurately discriminate different risk categories in post PV/ET MF patients. HGB< 100 g/L was associated with poor outcome in post-PV/ET MF patients.

[Characterization of N-linked glycosylation sites on envelope proteins of simian/human immunodeficiency virus in peripheral blood of Chinese rhesus macaques during acute infection].

Objective: To investigate the number and distribution of N-linked glycosylation sites of simian/human immunodeficiency virus envelope proteins(SHIVSF162P3)and SHIV transmission. Methods: Two female adult Chinese rhesus macaques(4 years old)were intravenously inoculated with 300 TCID50 SHIVSF162P3. The macaques weighed 4 and 5 kg and were identified as Rh1 and Rh2. We collected plasma samples at days 3, 7, 10, 14, 17, 21, 24, 28, 35, 42, 49, 56, 63, 70 and 77 post-challenge. Subsequently, we monitored plasma viral load by real-time PCR after viral RNA isolation and cDNA synthesis. We amplified the full-length envelope gene by single genome amplification(SGA)at days 7, 14, 28 and 77. BioEdit, MEGA, and the HIV Databases were used to analyze envelope sequences. Sequence diversity and N-linked glycosylation sites were compared between virus stock and plasma viruses of the two macaques. Results: A total of 55 env sequences were obtained from virus stock and their average pairwise distances were(0.166 6± 0.096 3)%. Viral loads peaked at 7.68 and 7.49 log10 copies/ml at day 10 and reached the set point at day 42(4.27 and 4.81 log10 copies/ml). The percentages of envelope sequences containing 25 potential N-linked glycosylation sites(PNGSs)were 83%(20/24)and 94%(29/31)in Rh1 and Rh2, respectively, at day 7; these were significantly higher than the proportion in SHIVSF162P3 stock(49%(27/55)). Viral diversity after infection increased with time whereas the proportion of sequences containing 25 PNGSs decreased and sequences containing 27 PNGSs gradually increased. In Rh1, the percentage of sequences containing 27 PNGSs increased to 29% at day 28 and reached 35% at day 77 in Rh2. By analyzing the number of PNGSs in the V1-V5 regions, we found that PNGS variation mainly occurred in the V4 loop. Compared with sequences containing 27 PNGSs, a seven amino acid(TWNNTIG)deletion was found in the V4 loop, which resulted in a loss of two PNGSs at positions 392 and 396. Conclusion: Low glycosylation of the SHIVSF162P3 V4 loop may facilitate spread of the SHIV virus whereas viruses with highly glycosylated V4 loops showed replication advantages after infection.

[Prognostic value of JAK2, MPL and CALR mutations in Chinese patients with primary myelofibrosis].

OBJECTIVE: To evaluate the prognostic value of JAK2, MPL and CALR mutations in Chinese patients with primary myelofibrosis (PMF). METHODS: Four hundred and two Chinese patients with PMF were retrospectively analyzed. The Kaplan-Meier method, the Log-rank test, the likelihood ratio test and the Cox proportional hazards regression model were used to evaluate the prognostic scoring system. RESULTS: This cohort of patients included 209 males and 193 females with a median age of 55 years (range: 15- 89). JAK2V617F mutations were detected in 189 subjects (47.0% ), MPLW515 mutations in 13 (3.2%) and CALR mutations in 81 (20.1%) [There were 30 (37.0%) type-1, 48 (59.3%) type-2 and 3 (3.7%) less common CALR mutations], respectively. 119 subjects (29.6%) had no detectable mutation in JAK2, MPL or CALR. Univariate analysis indicated that patients with CALR type-2 mutations or no detectable mutations had inferior survival compared to those with JAK2, MPL or CALR type- 1 or other less common CALR mutations (the median survival was 74vs 168 months, respectively [HR 2.990 (95% CI 1.935-4.619),P<0.001]. Therefore, patients were categorized into the high-risk with CALR type- 2 mutations or no detectable driver mutations and the low- risk without aforementioned mutations status. The DIPSS-Chinese molecular prognostic model was proposed by adopting mutation categories and DIPSS-Chinese risk group. The median survival of patients classified in low risk (132 subjects, 32.8% ), intermediate- 1 risk (143 subjects, 35.6%), intermediate- 2 risk (106 subjects, 26.4%) and high risk (21 subjects, 5.2%) were not reached, 156 (95% CI 117- 194), 60 (95% CI 28- 91) and 22 (95% CI 10- 33) months, respectively, and there was a statistically significant difference in overall survival among the four risk groups (P<0.001). There was significantly higher predictive power for survival according to the DIPSS-Chinese molecular prognostic model compared with the DIPSS-Chinese model (P=0.005, -2 log-likelihood ratios of 855.6 and 869.7, respectively). CONCLUSION: The impact of the CALR type- 2 mutations or no detectable driver mutation on survival was independent of current prognostic scoring systems. The DIPSS- Chinese molecular prognostic model based on the molecular features of Chinese patients was proposed and worked well for prognostic indication.

[Lycopene protects against hypoxia/reoxygenation injury in mouse cardiomyocytes by inhibiting endoplasmic reticulum stress induced apoptosis].

OBJECTIVE: To investigate the effects of lycopene on primary cultured neonatal mouse cardiomyocytes with hypoxia/reoxgenation (H/R) injury and explore related mechanism. METHODS: Primary cultured neonatal mouse cardiomyocytes were randomly divided to control group (control); lycopene group (5 µmol/L, lyc); H/R group (4 hours hypoxia followed by 6 hours reoxgenation); lycopene+ H/R group (lyc+ H/R, the cardiomyocytes were incubated with 5 µmol/L lycopene for 4 hours before H/R treatment). The cell viability of cardiomyocytes was assessed by CCK-8 assay. The apoptotic rate of cardiomyocytes was evaluated by flow cytometry using AnnexinV-PI double staining. Western blot was used to determine the GRP78, CHOP, Bax and Bcl-2 protein expression in cardiomyocytes. The mRNA expressions of ATF6、eIF2α and sXbp-1 were detected by real-time PCR. The fluorescence intensity for reactive oxygen species (ROS) in cardiomyocytes was measured with Olympus fluorescence microscope. RESULTS: Compared to control group, the cell viability of cardiomyocytes was significantly reduced ((64.28±6.12)% vs. (100.00±4.98)%, P<0.01), the apoptotic rate ((24.42±1.76)% vs. (5.16±1.31)%, P<0.01) and ratio of Bax/Bcl-2 (2.33±0.20 vs. 1.00±0.09, P<0.01) significantly increased, the ATF6, eIF2α and sXbp-1 mRNA expression, the CHOP and GRP78 protein expression (1.98±0.15 vs. 1.00±0.12, 2.09±0.11 vs. 1.00±0.09) as well as fluorescence intensity for ROS ((262.13±22.03)% vs. (100.00±12.35)%) were markedly increased in H/R group (all P<0.01). Compared to the H/R group, pretreatment with lycopene markedly improved the cell viability of cardiomyocytes ((81.75±6.85)%, P<0.01), significantly decreased the apoptotic rate ((17.24±2.02)%, P<0.01) and ratio of Bax/Bcl-2(1.64±0.13, P<0.01), significantly down-regulated the mRNA expression levels of ATF6, eIF2α and sXbp-1, and the protein expression levels of CHOP (1.54±0.12) and GRP78 (1.53±0.12), significantly reduced the fluorescence intensity for ROS ((171.18±19.09)%, all P<0.01). CONCLUSIONS: Lycopene could attenuate hypoxia/reoxygenation-injury in primary cultured neonatal mouse cardiomyocytes, possibly through inhibiting the ER stress and alleviating the ER stress-induced apoptosis.

Intracerebroventricular injection of senktide-induced Fos expression in vasopressin-containing hypothalamic neurons in the rat.

Intracerebroventricular injection of senktide, a selective agonist for neurokinin B receptor (NK3), induced Fos expression in many neurons of the rat hypothalamus. Fos-positive neurons were predominantly present in the supraoptic and paraventricular hypothalamic nuclei, and some of them were seen in the lateral preoptic area, lateral hypothalamic area, arcuate nucleus, perifornical region, posterior hypothalamic area, circular nucleus, and along relatively large blood vessels (lateral hypothalamic perivascular nucleus) in the anterior hypothalamus. A double labeling study was performed to examine if vasopressin-containing neurons in the hypothalamus could be activated by the treatment. Neurons with both Fos-like immunoreactivity (-LI) and vasopressin-LI were found in the paraventricular nucleus, supraoptic nucleus, circular nucleus and lateral hypothalamic perivascular nucleus. In the supraoptic nucleus, about 87% of vasopressin-containing neurons exhibited Fos-LI, which corresponded to about 64% of Fos-positive neurons in the nucleus. In the paraventricular nucleus, about 80% of vasopressin-like immunoreactive neurons exhibited Fos-LI, which constituted about 51% of the total population of Fos-positive neurons in the region. The results suggest that NK3 receptor may be involved in the modulation of release of vasopressin from the hypothalamus in the rat.

Neurokinin B receptor (NK3)-containing neurons in the paraventricular and supraoptic nuclei of the rat hypothalamus synthesize vasopressin and express Fos following intravenous injection of hypertonic saline.

Subnuclear localization of neurokinin B receptor (NK3) in the paraventricular and supraoptic nuclei of the hypothalamus was immunohistochemically investigated in the rat. In the paraventricular hypothalamic nucleus, intense neurokinin B receptor-like immunoreactivity was found in the posterior magnocellular part, moderate to weak neurokinin B receptor-like immunoreactivity was seen in the other parts. In the supraoptic nucleus, intense neurokinin B receptor-like immunoreactivity was distributed in its principal part, and a few neurons with neurokinin B receptor-like immunoreactivity were found in the retrochiasmatic part. Co-localization of neurokinin B receptor-like immunoreactivity with vasopressin-like immunoreactivity was examined through serial adjacent sections. Neurons with both neurokinin B receptor-like immunoreactivity and vasopressin-like immunoreactivity were primarily found in the supraoptic nucleus and posterior magnocellular part of the pavaventricular nucleus. A small number of neurons with neurokinin B receptor-like immunoreactivity and vasopressin-like immunoreactivity were also seen in the circular nucleus and the region surrounding blood vessel in the anterior hypothalamus. Many neurokinin B receptor-containing neurons in the paraventricular and supraoptic nuclei, as well as in circular nucleus and the region surrounding the blood vessel, expressed Fos-like immunoreactivity after intravenous injection of hypertonic saline. The present study demonstrated that a large proportion of neurokinin B receptor-like immunoreactive neurons in the paraventricular hypothalamic and supraoptic nuclei contained vasopressin-like immunoreactivity, and expressed Fos-like immunoreactivity after intravenous administration of hypertonic saline. The results suggest that neurokinin B receptor in the two nuclei may be involved in modulation of the release of vasopressin when the internal environment is disturbed.

Polymerization of myosin on activation of rat anococcygeus smooth muscle.

The in vivo state of assembly of myosin in vertebrate smooth muscle is controversial. In vitro studies on purified smooth muscle myosin show that it is monomeric (10S) under relaxing conditions and filamentous under contraction conditions. Electron microscopic and antibody labelling studies of intact smooth muscles, on the other hand, suggest that myosin is filamentous in the relaxed as well as the contracting state and that 10S myosin occurs only in trace amounts. However, birefringence, conventional EM and X-ray diffraction evidence suggests that in certain smooth muscles in vivo (e.g. rat anococcygeus), while myosin filaments exist in the relaxed state, their number increases on contraction. Here, we have used low temperature electron microscopic techniques (rapid freezing followed by freeze-substitution), which preserve labile components in close to their in vivo state, to detect any change in filament number on contraction. The results from rat anococcygeus have been compared with those from guinea pig taenia coli, in which other techniques have revealed no change in filament number. In the anococcygeus, we find evidence for a 23% increase in filament density in transverse sections of contracting muscle compared with relaxed muscle. In the taenia coli we find no change. These results are in qualitative agreement with earlier findings. They provide evidence for polymerization of myosin in contracting rat anococcygeus, and suggest that this process is subtle and occurs only in some smooth muscles.

Myosin filament structure in vertebrate smooth muscle.

The in vivo structure of the myosin filaments in vertebrate smooth muscle is unknown. Evidence from purified smooth muscle myosin and from some studies of intact smooth muscle suggests that they may have a nonhelical, side-polar arrangement of crossbridges. However, the bipolar, helical structure characteristic of myosin filaments in striated muscle has not been disproved for smooth muscle. We have used EM to investigate this question in a functionally diverse group of smooth muscles (from the vascular, gastrointestinal, reproductive, and visual systems) from mammalian, amphibian, and avian species. Intact muscle under physiological conditions, rapidly frozen and then freeze substituted, shows many myosin filaments with a square backbone in transverse profile. Transverse sections of fixed, chemically skinned muscles also show square backbones and, in addition, reveal projections (crossbridges) on only two opposite sides of the square. Filaments gently isolated from skinned smooth muscles and observed by negative staining show crossbridges with a 14.5-nm repeat projecting in opposite directions on opposite sides of the filament. Such filaments subjected to low ionic strength conditions show bare filament ends and an antiparallel arrangement of myosin tails along the length of the filament. All of these observations are consistent with a side-polar structure and argue against a bipolar, helical crossbridge arrangement. We conclude that myosin filaments in all smooth muscles, regardless of function, are likely to be side-polar. Such a structure could be an important factor in the ability of smooth muscles to contract by large amounts.