Integrated proteogenomic characterization reveals an imbalanced hepatocellular carcinoma microenvironment after incomplete radiofrequency ablation.

BACKGROUND: Efforts to precisely assess tumor-specific T-cell immune responses still face major challenges, and the potential molecular mechanisms mediating hepatocellular carcinoma (HCC) microenvironment imbalance after incomplete radiofrequency ablation (iRFA) are unclear. This study aimed to provide further insight into the integrated transcriptomic and proteogenomic landscape and identify a new target involved in HCC progression following iRFA. METHODS: Peripheral blood and matched tissue samples were collected from 10 RFA-treated HCC patients. Multiplex immunostaining and flow cytometry were used to assess local and systemic immune responses. Differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were explored via transcriptomic and proteogenomic analyses. Proteinase-3 (PRTN3) was identified in these analyses. And then, the ability of PRTN3 to predict overall survival (OS) was assessed in 70 HCC patients with early recurrence after RFA. In vitro CCK-8, wound healing and transwell assays were conducted to observe interactions between Kupffer cells (KCs) and HCC cells induced by PRTN3. The protein levels of multiple oncogenic factors and signaling pathway components were detected by western blotting. A xenograft mouse model was built to observe the tumorigenic effect of PRTN3 overexpression on HCC. RESULTS: Multiplex immunostaining revealed no immediate significant change in local immune cell counts in periablational tumor tissues after 30 min of iRFA. Flow cytometry showed significantly increased levels of CD4+ T cells, CD4+CD8+ T cells, and CD4+CD25+CD127- Tregs and significantly decreased the levels of CD16+CD56+ natural killer cells on day 5 after cRFA (p < 0.05). Transcriptomics and proteomics revealed 389 DEGs and 20 DEPs. Pathway analysis showed that the DEP-DEGs were mainly enriched in the immunoinflammatory response, cancer progression and metabolic processes. Among the DEP-DEGs, PRTN3 was persistently upregulated and closely associated with the OS of patients with early recurrent HCC following RFA. PRTN3 expressed in KCs may affect the migration and invasion of heat stress-treated HCC cells. PRTN3 promotes tumor growth via multiple oncogenic factors and the PI3K/AKT and P38/ERK signaling pathways. CONCLUSIONS: This study provides a comprehensive overview of the immune response and transcriptomic and proteogenomic landscapes of the HCC milieu induced by iRFA, revealing that PRTN3 promotes HCC progression after iRFA. TRIAL REGISTRATION: ChiCTR2200055606, http://www.chictr.org.cn/showproj.aspx?proj=32588 .

Eimeria infections of plateau pika altered the patterns of temporal alterations in gut bacterial communities.

Intestinal parasites, such as Eimeria, are common among plateau pika (Ochotona curzoniae). The gut microbiome is an essential driver of the host response to gastrointestinal parasites. However, the effects of intestinal protozoal parasites on the temporal variations in the gut microbiome and behavioral and physiological activities remain unknown. Our study conducted treatments involving experimental feeding of pika with Eimeria oocysts or anticoccidia under laboratory conditions to focus on the parasite-associated alterations in gut bacterial communities, host behavioral activity, physiology, and host-bacteria relationships. The results showed insignificant differences in bacterial community structures among treatments on the basis of Bray-Curtis distance metrics, whereas the patterns of temporal alterations in the bacterial communities were changed by the treatments. Bacterial alpha diversities did not vary with the treatments, and experimental feeding with Eimeria slowed down the decrement rate of alpha diversity. Furthermore, few bacterial members were significantly changed by the treatments-only the genus Ruminococcus and the species Ruminococcus flavefaciens, which were associated with energy metabolism. Experimental feeding with Eimeria modified the temporal variations in the bacterial members, including a lower loss rate of the relative abundance of the dominant families Muribaculaceae and Ruminococcaceae in the group with Eimeria experimental feeding. Moreover, a shifting energy trade-off was suggested by the parasite-induced increments in thyroid hormones (triiodothyronine and tetraiodothyronine) and decrements in exploration behavior in the group with Eimeria feeding. However, we did not detect specific connections between gut bacterial communities and pika behaviors and physiology in terms of energy trade-offs. Further in-depth research is needed to examine the role of Eimeria-modified differences in the gut bacteria of plateau pika.

[The effect of Hashimoto's thyroiditis on the diagnostic efficacy of ultrasound-guided fine needle aspiration cytology for thyroid nodules ≥ 1 cm].

Objective:To explore the diagnostic efficacy of ultrasound-guided fine needle aspiration cytology（US-FNAC） for thyroid nodules ≥1 cm, and the effect of Hashimoto's thyroiditis（HT） on it. Methods:The clinical data of 1027 cases of thyroid nodules ≥ 1 cm were retrospectively analyzed. Two-dimensional ultrasound, US-FNAC and BRAFV600E gene testing were performed. The postoperative pathological results were used as the criterion. The two dimensional ultrasound examination, clinical characteristics, follow-up results, and BRAFV600E were used to diagnosis for unoperated patients. The diagnostic efficiency of US-FNAC in HT（+） group and HT（-） group was compared, and the factors affecting the diagnostic efficiency were analyzed. Results:Of the 1027 nodules, the cytological results were nondiagnostic/unsatisfactory in 73 nodules（7.1%）, benign in 282（27.5%）, atypia of undetermined significance/follicular lesion of undetermined significance in 230（22.4%）, follicular neoplasm/suspicious for a follicular neoplasm in 20（1.9%）, suspicious for malignancy in 120（11.7%）, and malignant in 302（29.4%）. 515 cases underwent surgery. Among them, 495 were malignant and 20 were benign. 512 cases continued to be followed up without surgery, and the BRAFV600E of them were wild type. Combined with the two dimensional ultrasound examination, clinical features, and follow-up results, they were judged to be benign. The accuracy, sensitivity, specificity, positive predictive value, negative predictive value, false positive rate and the false negative rate the of US-FNAC were 98.7%, 98.4%, 99.3%, 99.5%, 97.5%, 0.7% and 1.6%, respectively. The accuracy, sensitivity and negative predictive value of the HT（+） group were 95.5%, 95.4% and 82.8%, respectively, which were lower than that of HT（-） group （99.5%, 99.4%, 99.2% ）（P=0.001, 0.018, P<0.001）. The false negative rate of the HT（+） group was 4.6%, higher than 0.6% of the HT（-） group（P=0.018）, and HT was an risk factor for increased FNR（OR=7.596, 95%CI: 1.452-39.740）. Conclusion:US-FNAC is an effective method for the diagnosis of thyroid nodules and it has high sensitivity and specificity in ≥ 1 cm nodules. However, the combination of HT reduces the diagnostic accuracy and HT is a risk factor for increased false negative rate.

Imaging-Guided Evaluation of the Novel Small-Molecule Benzosuberene Tubulin-Binding Agent KGP265 as a Potential Therapeutic Agent for Cancer Treatment.

The selective disruption of tumor-associated vasculature represents an attractive therapeutic approach. We have undertaken the first in vivo evaluation of KGP265, a water-soluble prodrug of a benzosuberene-based tubulin-binding agent, and found promising vascular-disrupting activity in three distinct tumor types. Dose escalation in orthotopic MDA-MB-231-luc breast tumor xenografts in mice indicated that higher doses produced more effective vascular shutdown, as revealed by dynamic bioluminescence imaging (BLI). In syngeneic orthotopic 4T1-luc breast and RENCA-luc kidney tumors, dynamic BLI and oxygen enhanced multispectral optoacoustic tomography (OE-MSOT) were used to compare vascular shutdown following the administration of KGP265 (7.5 mg/kg). The BLI signal and vascular oxygenation response (ΔsO2) to a gas breathing challenge were both significantly reduced within 2 h, indicating vascular disruption, which continued over 24 h. A correlative histology confirmed increased necrosis and hemorrhage. Twice-weekly doses of KGP265 caused significant growth delay in both MDA-MB-231 and 4T1 breast tumors, with no obvious systemic toxicity. A combination with carboplatin produced significantly greater tumor growth delay than carboplatin alone, though significant carboplatin-associated toxicity was observed (whole-body weight loss). KGP265 was found to be effective at low concentrations, generating long-term vascular shutdown and tumor growth delay, thus providing strong rationale for further development, particularly in combination therapies.

Evaluation of the biomechanical properties of the posterior eye using Shear Wave Elastography in patients with increased intracranial pressure.

AIMS: This study aims to investigate the biomechanical properties of the posterior eye using the shear wave elastography (SWE) in increased intracranial pressure (ICP) patients and healthy subjects. MATERIAL AND METHODS: This study enrolled 54 healthy subjects and 54 patients with increased ICP, including 29 patients with space-occupying lesions (SOLs) and 25 patients with intracranial haemorrhage (ICH). Optic nerve sheath diameter and the stiffness of the optic nerve head, peripapil-lary sclera and optic nerve were evaluated. RESULTS: Patients with increased ICP had a significantly higher optic nerve sheath diameter, Young's modulus of optic nerve head, and Young's modulus of peripapillary sclera than healthy subjects (p<0.01 for all). The optic nerve sheath diameter, Young's modulus of optic nerve head and Young's modulus of the peripapillary sclera were similar between the SOLs group and ICH group (p>0.05 for all). Young's modulus of the optic nerve in the ICH group was similar to the control group (p>0.05), whereas the SOLs group had higher Young's modulus of optic nerve comparing the control group (p<0.05). CONCLUSION: The stiffness of the optic nerve head and peripapillary sclera were higher in patients with increased ICP than healthy subjects, and the optic nerve stiffness of SOLs patients was the highest in all subjects. The SWE could provide a quantitative analysis of the posterior eye's biomechanical properties and help the clinic diagnose the degree of visual impairments.

A multimodal imaging-guided nanoreactor for cooperative combination of tumor starvation and multiple mechanism-enhanced mild temperature phototherapy.

Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), has shown great promise for cancer treatment in many preclinical studies. This study reports a nanoreactor designed for an enhanced mild temperature phototherapy which utilizes multiple mechanisms including simultaneous glucose consumption, oxygen supply, glutathione (GSH) depletion and heat-resistance relief. The nanoreactor is prepared using an Fe-doped polydiaminopyridine (Fe-PDAP) nanozyme with an intrinsic catalase-like activity coloaded with glucose oxidase (GOx) and indocyanine green (ICG). Evidence shows that glucose plays a vital role in tumor progression. Initiated by the breakdown of glucose into gluconic acid and H2O2 by GOx, Fe-PDAP promotes reoxygenation by catalyzing the reaction-supplied and tumor cell-supplied H2O2 into O2, which then enhances the O2-dependent PDT. Moreover, Fe-PDAP depletes GSH in tumor cells for more efficient reactive oxygen species (ROS) production. Meanwhile, the heat resistance of tumor cells is relieved by GOx-induced glucose exhaustion and heat shock protein (HSP) reduction, improving the efficiency of PTT. In particular, the nanoreactor also serves as a contrast agent for fluorescence, photoacoustic, and magnetic resonance multimodal imaging. Consequently, this nanoreactor efficiently inhibits tumor growth through mild temperature phototherapy under multimodal imaging guidance, resulting in successful tumor ablation with minimal systemic toxicity.

Artificial Nanotargeted Cells with Stable Photothermal Performance for Multimodal Imaging-Guided Tumor-Specific Therapy.

Organic-inorganic hybrid materials have drawn increasing attention as photothermal agents in tumor therapy due to the advantages of green synthesis, high loading efficiency of hydrophobic drugs, facile incorporation of theranostic iron, and excellent photothermal efficiency without inert components or additives. Herein, we proposed a strategy for biomimetic engineering-mediated enhancement of photothermal performance in the tumor microenvironment (TME). This strategy is based on the specific characteristics of organic-inorganic hybrid materials and endows these materials with homologous targeting ability and photothermal stability in the TME. The hybrid materials perform the functions of cancer cells to target homolytic tumors (acting as "artificial nanotargeted cells (ANTC)"). Inspired by the pH-dependent disassembly behaviors of tannic acid (TA) and ferric ion (FeIII) and subsequent attenuation of photothermal performance, cancer cell membranes were self-deposited onto the surfaces of protoporphyrin-encapsulated TA and FeIII nanoparticles to achieve ANTC with TME-stable photothermal performance and tumor-specific phototherapy. The resulting ANTC can be used as contrast agents for concurrent photoacoustic imaging, magnetic resonance imaging, and photothermal imaging to guide the treatment. Importantly, the high loading efficiency of protoporphyrin enables the initiation of photodynamic therapy to enhance photothermal therapeutic efficiency, providing antitumor function with minimal side effects.

Structural dynamics of Populus euphratica forests in different stages in the upper reaches of the Tarim River in China.

We selected four Populus euphratica Oliv. forest plots (100 m × 100 m) in the upper reaches of the Tarim River in the Xinjiang Uygur Autonomous Region of China. Each of the four forest plots was chosen to represent a different growth and death stage of P. euphratica forest: juvenile forest, mature forest, dying forest, and dead forest. In each plot, we measured the coordinates, DBH, height, and status of all P. euphratica individuals. We used (1) spatial pattern analysis to explore spatial distribution patterns and associations of live trees and dead trees, (2) a random mortality model to test whether the tree death was random or non-random, and (3) a generalized linear mixed-effect model (GLMM) to analyse factors related to tree survival (or death). In the juvenile plot, live trees were significantly aggregated at all scales (p < 0.05); while in the mature and dying plots, live trees were more aggregated at small scales and randomly distributed at larger scales. Live trees and dead trees showed a significantly positive association at all scales in the juvenile plot (p < 0.05). While in the mature and dying plots, live trees and dead trees only showed a significantly positive association at scales of 0-3 m (p < 0.05). There was significant density-dependent mortality in the juvenile plot; while mortality was spatially random at all scales in the mature and dying plots. The distance from the river showed significantly negative correlations with tree survival (p < 0.01). DBH and height had significantly positive associations with tree survival in the juvenile, mature, and dying plots (p < 0.05). In extreme drought, dying trees appeared to be shape-shifting into more shrub-like forms with clumps of root sprouts replacing the high canopies. The shift under extreme drought stress to more shrub-like forms of P. euphratica may extend their time to wait for a favourable change.

Perfluorohexane-encapsulated fullerene nanospheres for dual-modality US/CT imaging and synergistic high-intensity focused ultrasound ablation.

PURPOSE: The objective of this study was to develop a multifunctional contrast agent for bioimaging and synergistic high-intensity focused ultrasound (HIFU) therapy to achieve theranostic. MATERIALS AND METHODS: A novel type of perfluorohexane-encapsulated fullerene (PFH-C60) nanosphere was successfully developed via a vacuum ultrasonic emulsification and centrifugation method and subsequently used in ultrasound/computed tomography (CT) dual-modality and HIFU ablation of dissected bovine livers. In addition, transmission electron microscopic examination was employed to detect structural changes in the target tissue for HIFU ablation. RESULTS: The use of composite nanospheres effectively enhanced ultrasound and CT imaging. Moreover, the HIFU ablation of dissected bovine livers was also significantly enhanced. CONCLUSION: Composite nanospheres demonstrate potential theranostic application as a multifunctional contrast agent for dual-modality biological imaging and highly efficient synergistic imaging-guided HIFU ablation.

Phase-shift, targeted nanoparticles for ultrasound molecular imaging by low intensity focused ultrasound irradiation.

PURPOSE: Ultrasound (US) molecular imaging provides a non-invasive way to visualize tumor tissues at molecular and cell levels and could improve diagnosis. One problem of using US molecular imaging is microbubbles challenges, including instability, short circulation time, and poor loading capacity and penetrability. It is urgent to design new acoustic contrast agents and new imaging methods to facilitate tumor-targeted imaging. In this study, phase-shift poly lactic-co-glycolic acid (PLGA) nanoparticles modified with folate as an efficient US molecular probe were designed and the long-term targeted imaging was achieved by low-intensity focused US (LIFU) irradiation. METHODS: A new 5-step method and purification procedure was carried out to obtain uniform folic acid polyethylene glycol PLGA (PLGA-PEG-FA), the structure of which was confirmed by 1H nuclear magnetic resonance spectroscopy and thin-layer chromatography. Perflenapent (PFP) was wrapped in PLGA-PEG-FA by a double emulsion solvent evaporation method to obtain PFP/PLGA-PEG-FA nanoparticles. The targeted ability of the resulting nanoparticles was tested in vivo and in vitro. LIFU irradiation can irritate nanoparticle phase-shift to enhance tumor imaging both in vivo and in vitro. RESULTS: PLGA-PEG-FA was a light yellow powder with a final purity of at least 98%, the structure of which was confirmed by 1H nuclear magnetic resonance spectroscopy and thin-layer chromatography. Highly dispersed PFP/PLGA-PEG-FA nanoparticles with spherical morphology have an average diameter of 280.9±33.5 nm, PFP load efficiency of 59.4%±7.1%, and shells, thickness of 28±8.63 nm. The nanoparticles can specifically bind to cells expressing high folate receptor both in vivo and in vitro. Ultrasonic imaging was significantly enhanced in vitro and in vivo by LIFU irradiation. The retention time was significantly prolonged in vivo. CONCLUSION: Phase-shift PFP/PLGA-PEG-FA nanoparticles induced by LIFU can significantly enhance ultrasonic imaging, specifically targeting tumors expressing folate receptor. As a potential targeting acoustic molecular probe, PFP/PLGA-PEG-FA nanoparticles can be used to achieve targeted localization imaging.

The vascular disrupting agent combretastatin A-4 phosphate causes prolonged elevation of proteins involved in heme flux and function in resistant tumor cells.

Vascular disrupting agents (VDAs) represent a promising class of anti-cancer drugs for solid tumor treatment. Here, we aim to better understand the mechanisms underlying tumor reccurrence and treatment resistance following the administration of a VDA, combretastatin A-4 phosphate (CA4P). Firstly, we used photoacoustic tomography to noninvasively map the effect of CA4P on blood oxygen levels throughout subcutaneous non-small cell lung cancer (NSCLC) tumors in mice. We found that the oxygenation of peripheral tumor vessels was significantly decreased at 1 and 3 hours post-CA4P treatment. The oxygenation of the tumor core reduced significantly at 1 and 3 hours, and reached anoxia after 24 hours. Secondly, we examined the effect of CA4P on the levels of proteins involved in heme flux and function, which are elevated in lung tumors. Using immunohistochemistry, we found that CA4P substantially enhanced the levels of enzymes involved in heme biosynthesis, uptake, and degradation, as well as oxygen-utilizing hemoproteins. Furthermore, measurements of markers of mitochondrial function suggest that CA4P did not diminish mitochondrial function in resistant tumor cells. These results suggest that elevated levels of heme flux and function contribute to tumor regrowth and treatment resistance post-VDA administration.

Development of EST-SSR markers for Taxillus nigrans (Loranthaceae) in southwestern China using next-generation sequencing.

PREMISE OF THE STUDY: We developed transcriptome microsatellite markers (simple sequence repeats) for Taxillus nigrans (Loranthaceae) to survey the genetic diversity and population structure of this species. METHODS AND RESULTS: We used Illumina HiSeq data to reconstruct the transcriptome of T. nigrans by de novo assembly and used the transcriptome to develop a set of simple sequence repeat markers. Overall, 40 primer pairs were designed and tested; 19 of them amplified successfully and demonstrated polymorphisms. Two loci that detected null alleles were eliminated, and the remaining 17, which were subjected to further analyses, yielded two to 21 alleles per locus. CONCLUSIONS: The markers will serve as a basis for studies to assess the extent and pattern of distribution of genetic variation in T. nigrans, and they may also be useful in conservation genetic, ecological, and evolutionary studies of the genus Taxillus, a group of plant species of importance in Chinese traditional medicine.

Hyperbaric oxygen inhibits venous neointimal hyperplasia following arteriovenous fistulization.

Hypoxia following arteriovenous fistulization results in venous neointimal hyperplasia (VNH), potentially causing early arteriovenous fistula (AVF) dysfunction. In this study, we used hyperbaric oxygen (HBO) in a rabbit model of AVF to determine whether it could ameliorate early AVF failure. Chronic renal failure was induced by adenine in 96 adult rabbits randomly divided into 3 groups (n=32 in each group). The sham + HBO group underwent sham operation and received HBO. The AVF alone group underwent fistulization, but did not receive HBO. The AVF + HBO group underwent fistulization and received HBO. Each group was further divided into 4 subgroups of 8 rabbits each that were euthanized at 1, 7, 14 or 28 days post-operatively. At each time point, blood flow changes in the AVF venous segment were detected using a high-frequency duplex ultrasonography system. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and hematoxylin and eosin staining were performed to evaluate VNH. Western blot analysis was performed to confirm the expression of hypoxia-inducible factor (HIF)-1α. At 14 and 28 days following HBO treatment, blood flow in the AVF + HBO group was greater than that at day 0. The AVF + HBO group had a smaller ratio of intima to media area, a lower HIF-1α protein expression, and a smaller percentage of PCNA-positive cells in the proximal vein than did the AVF alone group. Our results thus suggest that continuous HBO treatment following AVF significantly inhibits VNH and promotes blood flow. Therefore, early AVF failure may be prevented by the use of HBO therapy.

Low-intensity focused ultrasound mediated localized drug delivery for liver tumors in rabbits.

OBJECTIVE: To explore the antitumor effects of low-intensity focused ultrasound (LIFU) mediated localized drug delivery of adriamycin-microbubble-PLGA nanoparticle complexes on rabbits VX2 liver tumor. METHODS: ADM-NMCs were prepared by covalent linking of ADM-PLGA nanoparticles (ADM-NPs) to the shell of the microbubbles. A fixed water bag filled with microbubbles was subjected to LIFU and non-focused ultrasound respectively, and the ultrasound images of which were recorded before and after ultrasonication. A total of 54 VX2 liver tumor-burdened rabbits were divided into six groups randomly, including control, ADM-NPs combined with LIFU, microbubbles combined with LIFU, ADM-NPs and microbubbles combined with LIFU, ADM-NMCs combined with LIFU and ADM-NMCs combined with Non-FUS. The tumor volume and volume inhibition rate (VIR) of tumor progression were calculated and compared. Apoptotic cells were labeled by terminal deoxyuridine nick end. Proliferating cell nuclear antigen was detected by immunohistochemistry. The median survival time of the animals were recorded and compared. RESULTS: ADM-NMCs were successfully prepared with an average diameter of 1721 nm. The highest VIR and apoptotic index (AI) were found in the group of ADM-NMCs combined with LIFU while the lowest proliferating index (PI) was simultaneously observed in this group. The median survival time of the rabbits in the ADM-NMCs combined with LIFU group was the longest (71days) among all groups. CONCLUSIONS: ADM-NMCs combined with LIFU could inhibit the rabbits VX2 liver tumor progress by delaying the tumor proliferation and accelerating apoptosis, which presents a novel process for liver tumor targeting chemotherapy.

Potential enhancement of intravenous nano-hydroxyapatite in high-intensity focused ultrasound ablation for treating hepatocellular carcinoma in a rabbit model.

The aim of the present study was to evaluate the safety and efficiency of an intravenously delivered nano-hydroxyapatite (Nano-HA) solution into a rabbit model (Oryctolagus cuniculus) to determine the potential enhancement of high-intensity focused ultrasound (HIFU) for the ablation of hepatocellular carcinoma (HCC) in liver tissue. The present study clearly indicated that the intravenous delivery of large quantities of Nano-HA into the body of the rabbit model over relatively short periods of time may be absorbed by the hepatic reticuloendothelial system. Subsequent HIFU treatment for HCC, as well as intravenous Nano-HA, produced a rapid increase in temperature and an enlargement of the coagulated necrotic area during ablation in the in vivo and ex vivo environments. In addition, it was found that the therapeutic doses of Nano-HA produced mild and transient abnormalities in the normal renal function and hepatic enzymes during the first 24 h following administration. The results of the current study indicated that the combination of Nano-HA and HIFU may provide a safe and effective alternative to conventional surgical procedures.

Analysis of variable sites between two complete South China tiger (Panthera tigris amoyensis) mitochondrial genomes.

In order to investigate the mitochondrial genome of Panthera tigris amoyensis, two South China tigers (P25 and P27) were analyzed following 15 cymt-specific primer sets. The entire mtDNA sequence was found to be 16,957 bp and 17,001 bp long for P25 and P27 respectively, and this difference in length between P25 and P27 occurred in the number of tandem repeats in the RS-3 segment of the control region. The structural characteristics of complete P. t. amoyensis mitochondrial genomes were also highly similar to those of P. uncia. Additionally, the rate of point mutation was only 0.3% and a total of 59 variable sites between P25 and P27 were found. Out of the 59 variable sites, 6 were located in 6 different tRNA genes, 6 in the 2 rRNA genes, 7 in non-coding regions (one located between tRNA-Asn and tRNA-Tyr and six in the D-loop), and 40 in 10 protein-coding genes. COI held the largest amount of variable sites (9 sites) and Cytb contained the highest variable rate (0.7%) in the complete sequences. Moreover, out of the 40 variable sites located in 10 protein-coding genes, 12 sites were nonsynonymous.

[Apoptosis and caspase-12 expression in progressive compressive spinal cord injury: experiment with rats].

OBJECTIVE: To investigate the apoptosis and caspase-12 expression in progressive compression of spinal cord (PCSC). METHODS: 120 adult Wistar rats were randomly divided into 2 equal groups, experimental group, undergoing operation so as to establish PCSC models, and control group, undergoing sham operation. 1, 3, 7, 14, 21, and 28 days after the operation, 5 rats from each group were anesthetized with their spinal cords taken out. TUNNEL method was used to observe the apoptosis of the neurons in the compressed segments. Another 5 rats from each group at different time points were anesthetized with their spinal cords as well. Immunohistochemistry was used to detect the mRNA expression of caspase-12 in the compressed segments. Western blotting was used to detect the protein expression of caspase-12. RESULTS: The apoptotic rates of neurons and gliocytes 1, 3, 7, 14, 21, and 28 days after were 12.5% +/- 2.3%, 13.0% +/- 3.6%, 17.2% +/- 4.3%, 29.4% +/- 4.4%, 36.1% +/- 6.5%, and 2.3% +/- 7.9% respectively, with significant differences among the values 14, 21, and 28 days and those at other time points after the operation (all P < 0.05). Immunohistochemistry showed that the number of caspase-12 positive neurons increased since 1 day after, and became remarkably high 14, 21, and 28 days after with significant differences among different time points (all P < 0.05). Western blotting showed that the protein expression of caspase-12 was low 1, 3, and 7 days after, and peaked 14 days after, and then gradually decreased, however, the expression levels 21 and 28 days after were still significantly higher then those 1, 3, and 7 days after (all P < 0.05). CONCLUSION: Caspase-12 is involved in the apoptosis of neurons in PCSC.

Responses of photosynthesis to NaCl in gametophytes of Acrostichum aureum.

Gametophytes of Acrostichum aureum were cultured in 0.0 to 1.0% NaCl solutions or in NaCl-free solution and then transferred to 1.0% NaCl solution. Photosynthetic light-response curves, efficiency of the primary photochemical reaction, relative electron transport rate, and photochemical and non-photochemical quenching at steady state were determined by photosynthetic O2 evolution and in vivo chlorophyll fluorescence. Results obtained showed that the chlorophyll fluorescence parameters, Fv /Fm and F'v /F'm and αO2 (the initial linear slope of the photosynthetic light-response curve) increased in gametophytes grown in NaCl. Linear electron transport rate was stimulated by NaCl. Based on the chlorophyll content, light-saturated photosynthesis in gametophytes grown in 0.2 to 0.7% NaCl increased slightly; it decreased in gametophytes grown in 1.0% NaCl. Photochemical quenching decreased in NaCl-grown gametophytes at all photosynthetic photon flux density (PPFD) levels measured, but there was no increase in non-photochemical quenching. The chlorophyll a/b ratio increased with increasing NaCl concentration in culture solutions. These results indicated that NaCl enhanced photochemical efficiency of photosystem II (PSII) and photosynthetic linear electron transport, thus resulting in the development of an excitation pressure in PSII. Such excitation pressure might act as a signal for photosynthetic acclimation to salt stress, thus allowing the gametophytes to grow in their natural habitats.