Diversity of the T cell receptor ß chain complementarity-determining region 3 in peripheral blood of neonates with sepsis: an analysis based on immune repertoire sequencing. / å ç–«ç»„åº“æµ‹åºåˆ†æžæ–°ç”Ÿå„¿è„“æ¯’ç—‡æ‚£è€ å¤–å‘¨è¡€T细胞受体ß链CDR3çš„å¤šæ ·æ€§.

OBJECTIVES: To investigate the diversity of peripheral blood T cell receptor (TCR) ß chain complementarity-determining region 3 (CDR3) based on immune repertoire sequencing in neonates with sepsis and the possible pathogenesis of neonatal sepsis. METHODS: A total of 12 neonates with sepsis were enrolled as the case group, and 9 healthy full-term infants, matched for gestational age, birth weight, and age, were enrolled as the control group. Omega nucleic acid purification kit (SQ blood DNA Kit II) was used to extract DNA from peripheral blood samples, TCR ß chain CDR3 was amplified by multiplex PCR, and then high-throughput sequencing was performed for the products to analyze the diversity of TCR ß chain CDR3 and the difference in expression. RESULTS: The length and type of TCR ß chain CDR3 were similar between the case and control groups, and Gaussian distribution was observed in both groups. With D50 and Shannon-Wiener index as the evaluation indices for diversity, the case group had a significantly lower diversity of TCR ß chain CDR3 than the control group (P<0.05). The frequency of 48 genes in TCR ß chain V segment was compared, and the results showed that compared with the control group, the case group had significantly higher frequencies of TRBV10-3, TRBV2, and TRBV20-1 (P<0.05). The frequency of 13 genes in TCR ß chain J segment were compared, and the results showed that compared with the control group, the case group had significantly higher frequencies of TRBJ2-3, TRBJ2-5, and TRBJ2-7 (P<0.05). CONCLUSIONS: There is a significant change in the diversity of TCR ß chain CDR3 in the peripheral blood of neonates with sepsis, suggesting that it might be associated with the immune pathogenesis of neonatal sepsis.

Value of near-infrared spectroscopy in monitoring intestinal tissue oxygen saturation in preterm infants with hemodynamically significant patent ductus arteriosus: a prospective research. / è¿‘çº¢å¤–å ‰è°±æŠ€æœ¯å¯¹æœ‰è¡€æµåŠ¨åŠ›å­¦æ„ä¹‰çš„åŠ¨è„‰å¯¼ç®¡æœªé—­æ—©äº§å„¿è‚ é“ç»„ç»‡æ°§é¥±å’Œåº¦ç›‘æµ‹ä»·å€¼çš„å‰çž»æ€§ç ”ç©¶.

OBJECTIVES: To study the change in regional oxygen saturation (rSO2) of intestinal tissue in preterm infants with hemodynamically significant patent ductus arteriosus (hsPDA) by near-infrared spectroscopy, and the clinical significance of the change in intestinal oxygen level in preterm infants with hsPDA. METHODS: The preterm infants with patent ductus arteriosus (PDA) who had gestational age <32 weeks and/or birth weight <1 500 g were prospectively enrolled, who were admitted to the Department of Neonatology, Shenzhen Longgang Central Hospital from October 2017 to October 2020.According to the diagnostic criteria for hsPDA, the preterm infants with patent ductus arteriosus (PDA) were divided into two groups: hsPDA and non-hsPDA. According to closure of the ductus arteriosus after oral administration of ibuprofen, the preterm infants in the hsPDA group were subdivided into two groups: hsPDA closure and hsPDA non-closure. Hemodynamic parameters were measured at diagnosis of PDA and after treatment, and the level of intestinal tissue rSO2 was monitored continuously to analyze its change. RESULTS: A total of 241 preterm infants with PDA were enrolled, with 55 infants (22.8%) in the hsPDA group and 186 infants (77.2%) in the non-hsPDA group. There were 36 infants (65%) in the hsPDA closure group and 19 infants (35%) in the hsPDA non-closure group. Compared with the non-hsPDA group, the hsPDA group had a significantly higher left atrial diameter/aortic root diameter ratio and significantly lower left ventricular ejection fraction and fractional shortening (P<0.05). At each time point within 6 hours after diagnosis (1, 2, 4, and 6 hours), the hsPDA group had significantly lower intestinal tissue rSO2 than the non-hsPDA group (P<0.05), and intestinal tissue rSO2 gradually decreased over time in the hsPDA group (P<0.05), with the lowest level of 0.448±0.014 at 6 hours. Compared with the hsPDA non-closure group, the hsPDA closure group had a significantly lower left atrial diameter/aortic root diameter ratio and significantly higher left ventricular ejection fraction and fractional shortening (P<0.05). At each time point within 48-96 hours after treatment (48, 72, and 96 hours), the hsPDA closure group had significantly higher intestinal tissue rSO2 than the hsPDA non-closure group (P<0.05), and intestinal tissue rSO2 gradually increased since 24 hours after treatment in the hsPDA closure group (P<0.05), with the highest level of 0.578±0.031 at 96 hours. CONCLUSIONS: hsPDA has an impact on intestinal tissue oxygenation in preterm infants, and continuous monitoring of intestinal tissue rSO2 by near-infrared spectroscopy can help to guide the clinical management of hsPDA in preterm infants.

Atractylenolide­1 alleviates gastroparesis in diabetic rats by activating the stem cell factor/c­kit signaling pathway.

Diabetic gastroparesis (DGP), also known as delayed gastric emptying, is a common complication of diabetes mellitus. There are numerous clinical symptoms associated with DGP, as well as high treatment costs and markedly reduced patient quality of life. However, the pathogenesis of DGP is not clear, thus effective treatment methods are yet to be established. In the present study, a DGP rat model was established in Sprague­Dawley rats by the intraperitoneal injection of streptozotocin (STZ). DGP model rats were treated with different doses of atractylenolide­1 to detect alterations in gastrointestinal function, including gastroparesis, gastric emptying, gastric motility, gastric peristalsis and gastric blood flow. Compared with the DGP group, atractylenolide­1 treatment significantly reduced glycaemia and the level of glycated hemoglobin, as well as restoring gastrointestinal function. Gastroparesis, gastric emptying, gastric motility, gastric peristalsis and gastric blood flow were significantly impaired in the STZ­induced group compared with the vehicle control group. Moreover, the STZ­induced group displayed downregulated expression levels of the DGP indicator KIT proto­oncogene, receptor tyrosine kinase (c­kit), as investigated by immunohistochemistry, and stem cell factor (SCF) protein, as assessed using ELISA, significantly enhanced rat interstitial cells of Cajal (ICC) apoptosis, and significantly altered levels of oxidative stress­related markers (malondialdehyde and superoxide dismutase) in the serum and gastric tissues compared with the vehicle control group. By contrast, treatment with atractylenolide­1 significantly counteracted the effects of DGP on peristalsis, inhibited apoptosis and suppressed oxidative stress by regulating the expression of heme oxygenase 1 in STZ­induced DGP model rats. Further research indicated that atractylenolide­1 regulated oxidative stress reactions and improved gastric function by activating the SCF/c­kit signaling pathway. Collectively, the results of the present study suggested that atractylenolide­1 promoted ICC survival and preserved the structure of the gastric tissue network in a DGP rat model via the SCF/c­kit signaling pathway, providing novel insights for the treatment of DGP.

[Ningmitai Capsules combined with doxycycline hydrochloride for Ureaplasma urealyticumpositive chronic prostatitis].

OBJECTIVE: To investigate the effects of Ningmitai Capsules (NMT) combined with doxycycline hydrochloride (DH) on chronic prostatitis induced by Ureaplasma urealyticum (Uu). METHODS: This randomized controlled trial included 240 male patients with Uupositive chronic prostatitis, treated orally with NMT at 4 capsules tid (n= 35), DH at 100 mg bid (n = 78), and NMT+DH at the corresponding doses (n = 127), respectively, all for 2 successive weeks. At 1 week after drug withdrawl, we conducted routine urine analysis, EPS examination, and drug sensitivity test of the cultured Uu. RESULTS: The positivetonegative rate of Uu was significantly higher in the NMT+DH group than in the NMT and DH groups (89.0% ［113/127］ vs 54.3% ［19/35］ and 71.8% ［56/78］, P< 0.05), so were the cure rate (25.2% vs 20.0% and 20.5%, P< 0.05) and total effectiveness rate (89.0% vs 54.3% and 71.8%, P< 0.05). CONCLUSIONS: The combination of Ningmitai Capsules and doxycycline hydrochloride is more effective than either Ningmitai Capsules or doxycycline hydrochloride used alone in the treatment of Uupositive chronic prostatitis.

Peripheral lymphocyte subset variation predicts prostate cancer carbon ion radiotherapy outcomes.

The immune system plays a complementary role in the cytotoxic activity of radiotherapy. Here, we examined changes in immune cell subsets after heavy ion therapy for prostate cancer. The lymphocyte counts were compared with acute radiotherapy-related toxicity, defined according to the Common Terminology Criteria for Adverse Events, and short-term local efficacy, defined based on prostate-specific antigen concentrations. Confirmed prostate cancer patients who had not received previous radiotherapy were administered carbon ion radiotherapy (CIR) in daily fractions of 2.74 GyE with a total dose of 63-66 GyE. Lymphocyte subset counts were investigated before, during and after radiotherapy, and at a 1 month follow-up. Most notable among our findings, the CD4/CD8 ratio and CD19+ cell counts were consistently higher in patients with a complete response (CR) or partial response (PR) to CIR than in those classified in the stable disease (SD) group (P<0.05 for both). But CD3+ and CD8+ cell counts were lower in the CR and PR groups than in the SD group. These results indicate that variations in peripheral lymphocyte subpopulations are predictive of outcome after CIR for prostate cancer.

Haemocyte apoptosis of the tiger shrimp Penaeus monodon exposed to cadmium.

This study investigated the effect of ambient Cadmium (Cd) on haemocyte apoptosis of the shrimp, Penaeus monodon. Cellular response was determined in Cd-exposed (0, 0.05, 0.5 and 5 mg L(-1)) shrimp. Results showed that 0.05 mg L(-1) Cd(2+) had no significant effect on the haemocyte parameters during the 48 h exposure. Cadmium at doses of 0.5 and 5 mg L(-1) depressed the total haemocyte count (THC), and increased reactive oxygen species (ROS) production and apoptosis ratio in haemocytes. Esterase activity increased in shrimp exposed to 0.5 mg L(-1) Cd(2+) for 6 h, and decreased to the initial level later. Depressed esterase activity could be observed in shrimp after 24 and 48 h exposure to 5 mg L(-1) Cd(2+). These results demonstrated that Cd(2+) modified esterase activity and induced ROS generation, which led to haemocyte apoptosis and THC reduction. Oxidative stress is one of the induction mechanisms for Cd-caused apoptosis of shrimp haemocytes.

The potential effects of climate change on the distribution and productivity of Cunninghamia lanceolata in China.

Climate changes may have immediate implications for forest productivity and may produce dramatic shifts in tree species distributions in the future. Quantifying these implications is significant for both scientists and managers. Cunninghamia lanceolata is an important coniferous timber species due to its fast growth and wide distribution in China. This paper proposes a methodology aiming at enhancing the distribution and productivity of C. lanceolata against a background of climate change. First, we simulated the potential distributions and establishment probabilities of C. lanceolata based on a species distribution model. Second, a process-based model, the PnET-II model, was calibrated and its parameterization of water balance improved. Finally, the improved PnET-II model was used to simulate the net primary productivity (NPP) of C. lanceolata. The simulated NPP and potential distribution were combined to produce an integrated indicator, the estimated total NPP, which serves to comprehensively characterize the productivity of the forest under climate change. The results of the analysis showed that (1) the distribution of C. lanceolata will increase in central China, but the mean probability of establishment will decrease in the 2050s; (2) the PnET-II model was improved, calibrated, and successfully validated for the simulation of the NPP of C. lanceolata in China; and (3) all scenarios predicted a reduction in total NPP in the 2050s, with a markedly lower reduction under the a2 scenario than under the b2 scenario. The changes in NPP suggested that forest productivity will show a large decrease in southern China and a mild increase in central China. All of these findings could improve our understanding of the impact of climate change on forest ecosystem structure and function and could provide a basis for policy-makers to apply adaptive measures and overcome the unfavorable influences of climate change.

Trapping red blood cells in living animals using optical tweezers.

The recent development of non-invasive imaging techniques has enabled the visualization of molecular events underlying cellular processes in live cells. Although microscopic objects can be readily manipulated at the cellular level, additional physiological insight is likely to be gained by manipulation of cells in vivo, which has not been achieved so far. Here we use infrared optical tweezers to trap and manipulate red blood cells within subdermal capillaries in living mice. We realize a non-contact micro-operation that results in the clearing of a blocked microvessel. Furthermore, we estimate the optical trap stiffness in the capillary. Our work expands the application of optical tweezers to the study of live cell dynamics in animals.

Measurement of intracellular nitric oxide (NO) production in shrimp haemocytes by flow cytometry.

A flow cytometric method to measure the production of intracellular nitric oxide (NO) was adapted for use with shrimp haemocytes. We applied fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) for NO detection in haemocytes from the tiger shrimp Penaeus monodon, and used flow cytometry to quantify fluorescence intensity in individual haemocyte. The optimized protocol for intracellular NO analysis consists to incubate haemocytes with DAF-FM DA at 10 µM for 60 min to determine the mean fluorescence intensity. Result showed that NO was also produced in the untreated shrimp haemocytes. NO level in granular cells and semigranular cells were much higher than that in hyaline cells. Defined by different characteristic of NO content, three subsets of haemocytes were observed. Zymosan A at dose of 10 or 100 particles per haemocyte triggered higher DAF-FM fluorescence intensity in granular and semigranular cells, than PMA that had no significant impact on all three cell types. These results indicate that granular and semigranular cells are the primary cells for NO generation. Cytochalasin B significantly inhibited the NO level induced by zymosan A. NG-Monomethyl-L-arginine (L-NMMA) and diphenylene iodonium chloride (DPI) significantly suppressed the DAF-FM fluorescence in haemocytes, but apocynin could not modulate it, indicating that the DAF-FM fluorescence was closely related to the activity of NO-synthase pathway. The NO donor sodium nitroprusside (SNP) improved the DAF-FM fluorescence in haemocytes, while the NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) significantly decreased the fluorescence, demonstrating that the fluorescence intensity of DAF-FM is mainly dependent on the intracellular NO level.

Real-time monitoring of rare circulating hepatocellular carcinoma cells in an orthotopic model by in vivo flow cytometry assesses resection on metastasis.

The fate of circulating tumor cells (CTC) is an important determinant of metastasis and recurrence, which leads to most deaths in hepatocellular carcinoma (HCC). Therefore, quantification of CTCs proves to be an emerging tool for diagnosing, stratifying, and monitoring patients with metastatic diseases. In vivo flow cytometry has the capability to monitor the dynamics of fluorescently labeled CTCs continuously and noninvasively. Here, we combine in vivo flow cytometry technique and a GFP-transfected HCC orthotopic metastatic tumor model to monitor CTC dynamics. Our in vivo flow cytometry has approximately 1.8-fold higher sensitivity than whole blood analysis by conventional flow cytometry. We found a significant difference in CTC dynamics between orthotopic and subcutaneous tumor models. We also investigated whether liver resection promotes or restricts hematogenous metastasis in advanced HCC. Our results show that the number of CTCs and early metastases decreases significantly after the resection. The resection prominently restricts hematogenous metastasis and distant metastases. CTC dynamics is correlated with tumor growth in our orthotopic tumor model. The number and size of distant metastases correspond to CTC dynamics. The novel in vivo flow cytometry technique combined with orthotopic tumor models might provide insights to tumor hematogenous metastasis and guidance to cancer therapy.

Biological evaluation of a novel copper-containing composite for contraception.

OBJECTIVE: To investigate the biocompatibility of a novel copper-containing composite to provide preclinical data for clinical application of intrauterine device (IUD) or intra-vas device (IVD). DESIGN: Prospective experimental study. SETTING: Good laboratory practices laboratories. ANIMALS: Twenty healthy adult mice (SPF grade Kunming white mice, animal code SCXK 2003-0005). INTERVENTION(S): Cytotoxicity tests in vitro were conducted to evaluate the influence of the materials on the morphology, growth, and proliferation of cultured L929 mouse fibroblasts. Acute systemic toxicity tests were conducted to investigate the acute systemic toxic reaction with mice, and then the materials were implanted into the spinal muscle of rabbits (n = 15). The rabbits were sacrificed for pathologic examination at 1, 4, and 12 weeks after surgery. MAIN OUTCOME MEASURE(S): Evaluation of cytotoxicity by MTT assay, cytotoxicity test by direct contact assay, acute systemic toxicity test, and material implantation test. RESULT(S): The cytotoxicity grade of the copper-containing composite was 0-1, suggesting that the material was free of cytotoxicity; no acute systemic toxicity was found in any mice; mild inflammatory reaction was observed in the surrounding tissues of the implanted material in the early implantation stage, which was similar to that of the sham-operated sides. Twelve weeks after implantation, the inflammatory reaction was completely disappeared in the implanted tissue, similarly to the sham-operated sides. The fibrosis membrane surrounding the material became stable gradually over time. CONCLUSION: The copper-containing composite has excellent biocompatibility, which is feasible and safe for the clinical application as a novel contraceptive material.

Initial studies on a novel filtering-type intra-vas device in male dogs.

BACKGROUND: To relieve the side effects induced by the complete obstruction of the vas deferens, we created a filtering-type intra-vas device (IVD) which is made of materials composed of nano-SiO(2)-copper complex cross-linking polymer composites. STUDY DESIGN: Eight male beagle dogs were grouped into nonimplanted control group and IVD-implanted group. We tested the efficacy of the sperm filtering effect of the new IVD material for 12 months and examined the influence of the IVD materials on the cells of the vas deferens, epididymis and testis. RESULTS: The densities of sperm were reduced significantly after the IVD was implanted; no motile sperm were found after the third month. No obvious morphological changes were found in the cells of the vas deferens, epididymis and testis in the IVD-implanted group. CONCLUSIONS: The filtering-type nano-SiO(2)-copper complex/polymer composite IVD is able to filter the sperm of the male dogs, and the IVD material did not cause obvious damage to the cells of the male reproductive organs after 1 year of implantation.

Dihydroartemisinin (DHA) induces caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells.

BACKGROUND: Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, is recommended as the first-line anti-malarial drug with low toxicity. DHA has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways, although the molecular mechanisms are not well understood. METHODS: In this study, cell counting kit (CCK-8) assay was employed to evaluate the survival of DHA-treated ASTC-a-1 cells. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. Collapse of mitochondrial transmembrane potential (DeltaPsim) was measured by dynamic detection under a laser scanning confocal microscope and flow cytometry analysis using Rhodamine123. Caspase-3 activities measured with or without Z-VAD-fmk (a broad spectrum caspase inhibitor) pretreatment by FRET techniques, caspase-3 activity measurement, and western blotting analysis. RESULTS: Our results indicated that DHA induced apoptotic cell death in a dose- and time-dependent manner, which was accompanied by mitochondrial morphology changes, the loss of DeltaPsim and the activation of caspase-3. CONCLUSION: These results show for the first time that DHA can inhibit proliferation and induce apoptosis via caspase-3-dependent mitochondrial death pathway in ASTC-a-1 cells. Our work may provide evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of lung adenocarcinoma.

[Establishment of an animal model of azoospermia in male mice].

OBJECTIVE: To establish a stable animal model of azoospermia in male mice. METHODS: Two groups of mice, with 30 in each, were intervened respectively by chemotherapy (intraperitoneal injection of Busulfan and Cyclophosphamide) and subcutaneous injection of Estradiol. At different times after the injection, the microscopic structures of the seminiferous tubules of both groups were observed and compared. The recovery of the germ cells in the seminiferous tubules was also evaluated after the termination of the intervention. RESULTS: A stable animal model was established by chemotherapeutic method with Busulfan and cyclophosphamide, while the model constructed by muscle injection of Estradiol was not stable. CONCLUSION: Compared with estrogen injection, chemotherapeutic intervention is a reliable method for constructing an animal model of azoospermia in male mice.

Effect of sildenafil citrate on penile erection of rhesus macaques.

AIM: To examine the effect of sildenafil citrate on penile erection of male rhesus macaque. METHODS: Twenty Macaca mulatta were divided into the sildenafil treated and the control groups of 10 animals each. The penile size, the corpus cavernosal electromyogram (EMG) and the intra-corpus cavernosal pressure (ICP) were determined. RESULTS: The diameter of penis and the ICP were significantly increased and the corpus cavernosal EMG significantly reduced in the sildenafil group. CONCLUSION: Sildenafil citrate increases the penile size and ICP and reduces the corpus cavernosal EMG in male rhesus macaque.

[Effect of sildenafil ciltrate on the sexual activities of male rats].

OBJECTIVE: To obtain related pharmacodynamic data for the clinical experiment by observing the sexual activities of male rats after using sildenafil ciltrate through stomach irrigation. METHODS: Forty male Sprague-Dawley rats were distributed into 4 groups with different dosages (control with distilled water, low dosage: 0.08%, medium dosage: 0.24% and high dosage: 0.72%). After the male Sprague-Dawlay rats were mated with their female counterparts in pairs, the latent period of chasing, the frequencies of chasing in 60 minutes, the latent period of intercourse and the frequencies of intercourse in 60 minutes were recorded. RESULTS: Compared with the control, the frequencies of chasing were significantly increased and the latent periods of chasing were significantly shortened in both high dosage and medium dosage groups after using sildenafil (P < 0.01); The frequencies of intercourse in 60 minutes were significantly increased and the latent periods of intercourse were significantly shortened in all the groups after the use of sildenafil. CONCLUSIONS: The sexual activities of male rats treated with sildenafil were significantly activated.