Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background

The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells.

Methods

A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses.

Results

Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles.

Conclusion

We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.     

Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis

The vectorial competence of the tick Rhipicephalus sanguineus is discussed in relation to the epidemiology of canine visceral leishmaniasis, taking into account its strict association with dogs and the low indices of natural infection presented by its known vector, the phlebotomine sand fly Lutzomyia longipalpis. In order to evaluate natural infection by Leishmania chagasi and the infectivity of these parasites in the tick, 39 specimens (6 females, 11 males and 22 nymphs) of R. sanguineus were removed from 21 dogs showing diverse symptoms of zoonotic visceral leishmaniasis (ZVL). Six ticks (15.4%) gave positive results for the genus Leishmania using the PCR technique. To determine the infectivity of the parasites, 36 hamsters were inoculated orally and peritoneally with macerates of ticks removed from nine dogs symptomatic for visceral leishmaniasis. After 6 months the hamsters were sacrificed and necropsied. Serum was removed for IFAT, as well as spleen and liver fragments to make imprint smears and for PCR. Eight (88.9%) of these dogs presented ticks that were infective for 14 hamsters (41.2%), 12 (85.7%) of them infected peritoneally and two (14.3%) orally. PCR revealed 27 smears (40.9%) to be positive, 20 (62.5%) of them infected peritoneally and seven (20.6%) orally. IFAT showed 14 positive animals (41.2%). Based on these findings, we suggest that the vectorial capacity of R. sanguineus for L. chagasi should be evaluated further, opening new perspectives in the epidemiology of ZVL.     

Milk production and composition,food consumption,and energy balance of postpartum crossbred Holstein-Gir dairy cows fed two diets of different energy levels

The objective of this study was to evaluate the production, consumption, and energy balance parameters of primiparous 3/4 and 7/8 Holstein × Gir (HG) dairy cows fed two diets of differing energy levels during the postpartum period. At the beginning of the study, 28 days prepartum, the average weight of both genetic groups was 498?±?12 kg and body condition score (BCS) was 3.5?±?0.05. At the end of the study, 61 days postpartum, the 3/4 HG cows had higher weight and body condition scores than the 7/8 HG (456?±?8 and 429?±?8 kg and 3.13?±?0.03 and 2.94?±?0.03 BCS for 3/4 HG and 7/8 HG, respectively). Milk from cows fed the high-energy diet had higher percentages of fat, protein, lactose, and total dry extract than cows fed the low-energy diet. Cows fed the high-energy diet had higher net energy intake (95.3?±?1.9 vs. 88.1?±?2.1 MJ/day) and higher energy balance (3.64?±?2.13 vs ??6.02?±?2.30 MJ/day). The 3/4 HG cows displayed higher energy for maintenance (33.1?±?0.4 MJ/day) than the 7/8 HG (31.5?±?0.5 MJ /day). In conclusion, although the primiparous 3/4 HG were heavier than the 7/8 HG and had a higher body condition score, no differences in milk produced up to 60 days postpartum were observed. The higher energy diet during the postpartum period increased energy balance, resulting in higher production of milk fat, protein, and lactose.

     

Effects of ketamine constant rate infusions on cardiac biomarkers and cardiac function in dogs

Objective

To evaluate the effects of ketamine continuous rate infusions (CRI) at two dose rates on cardiovascular function and serum creatine kinase MB isoenzyme (CK-MB) and troponin I in healthy conscious dogs.

Characterization of the LH peak after short and long fixed‐time artificial insemination protocols in sheep raised in the tropics

The aim of this study was to evaluate the peak in luteinizing hormone (LH) and the pregnancy rate of sheep (Texel × Santa Inês) in the tropics using short‐ (6 days) and long‐term (12 days) progesterone protocols followed by artificial insemination (AI) both in and out of the breeding season. Experiment 1 was conducted within (IN) the breeding season (autumn, n = 36), and experiment 2 was conducted outside (OUT) of the breeding season (spring, n = 43). In each experiment, the sheep were divided into two groups (6 or 12 days) according to the duration of treatment with a single‐use progesterone release vaginal device (CIDR^®, Pfizer, São Paulo, SP, Brazil), and blood samples were collected from 10 animals per group every 4 hr to measure the LH and progesterone concentrations. In the spring, the characteristics of the LH peak did not differ between groups; but in the autumn, there were differences between groups at the beginning (G‐6 IN: 36.44 ± 5.46 hr; G‐12 IN: 26.57 ± 4.99 hr) and end of the LH peak (G‐6 IN: 46.22 ± 7.51 hr; G‐12 IN: 34.86 ± 8.86 hr). The results showed alterations in the LH peak during the breeding season only in the sheep undergoing the short‐term protocol.     

Effects of suspended solids in the survival and haematological parameters of pacu juveniles (Piaractus mesopotamicus) in a biofloc technology culture system

This study determined the effects of total suspended solids (TSS) on pacu (Piaractus mesopotamicus) culture in a biofloc technology (BFT) system by performing two experiments. Experiment 1 evaluated the LC50-96 h of pacu at seven TSS levels (0, 1500, 3000, 4000, 5000, 6000 and 7000 mg L^?1). Experiment 2 evaluated haematological variables (glucose, pH, haematocrit, haemoglobin, erythrocytes and haematimetric indices) in juveniles exposed to five different sublethal TSS concentrations (0, 250, 500, 750 and 1000 mg L^?1) for 5 days. Treatments below 5000 mg L^?1 did not cause mortality, and the LC50-96 h of TSS was estimated at 5477 mg L^?1. The TSS concentrations that caused mortality in pacu juveniles were extremely high and rarely maintained in culture systems. Exposure to high TSS concentrations (mainly 750 and 1000 mg L^?1) increased blood glucose, pH, heamatocrit, erythrocytes, haemoglobin values and haematimetric indices on the first day. On the fifth day, most of the evaluated parameters stabilized at different TSS concentrations. In conclusion, pacu exposed to high TSS concentrations for short periods may undergo physiological changes, and TSS concentrations below 250 mg L^?1 are recommended for its culture in a BFT system.     

Early detection of Neophysopella tropicalis in grapevine leaves and on spore traps by qPCR

Neophysopella tropicalis, one of the causal agents of Asian grapevine leaf rust (AGLR), can cause severe epidemics in Brazil that lead to yield losses in commercial vineyards. An early detection of the pathogen by air sampling of urediniospores on spore traps or in symptomless leaves would be valuable to multiple studies, such as epidemics modelling, risk forecasting, monitoring of pathogen introductions in rust-free areas, and predicting the beginning of epidemics. This study developed a quantitative PCR (qPCR) protocol to quantify N. tropicalis urediniospores attached to adhesive tapes and in grapevine leaves before symptom appearance. A specific primer pair was designed based on the internal transcribed spacer (ITS) sequence region of the AGLR pathogen. Standard amplification curves using genomic DNA from urediniospores of N. tropicalis and from urediniospores attached to adhesive tapes were established. Grapevine leaves inoculated with N. tropicalis were collected at 2, 5, and 7 days postinoculation (dpi). One primer pair (580F/720R) amplified a 140 bp product in all AGLR isolates but did not amplify products of other rust genera, such as Phakopsora, Puccinia, Hemileia, Tranzschelia, Cerotelium, and Coleosporium. As little as 0.1 pg DNA and 10 urediniospores of N. tropicalis attached to adhesive tapes could be detected. qPCR enabled the detection of the pathogen as early as 2 dpi, before symptom appearance. This method can be used to monitor N. tropicalis inoculum in grapevine-growing areas and to quantify symptomless infections of the AGLR pathogen.     

接种内生菌对干旱胁迫下甘蔗的生理影响

探索内生菌对甘蔗抗旱能力的影响,以期为开发利用甘蔗内生菌抗旱性功能菌株提供理论依据和技术支撑.以课题组前期分离鉴定的6株甘蔗内生菌为供试菌株,甘蔗品种'ROC22'幼苗接种7天后进行干旱胁迫,然后取完全展开的第一叶叶片测定相关生理指标.结果 表明,E3、09和YC89均有较高的ACC脱氨酶活性,不同甘蔗内生菌菌株对甘蔗...     

割手密线粒体DNA的简易快速提取方法

高效快速地分离提取高质量的线粒体DNA(mtDNA)是研究植物线粒体基因及其起源进化的重要前提。为获得高质量的mtDNA进行割手密资源的线粒体基因序列扩增及测序分析,本研究以割手密黄化苗为材料,通过简单差速离心分离得到线粒体,经DNaseⅠ消化处理,去除核DNA杂质得到较纯的线粒体,然后经过5%SDS和蛋白酶K充分裂解线粒体,利用饱和的苯酚/氯仿(1:1)和氯仿两次抽提除去蛋白质,并经过RNase A的消化处理除去RNA,无水乙醇沉淀等一系列操作得到mtDNA。所提取的mtDNA样品经紫外吸收光度测定,琼脂糖凝胶电泳和PCR扩增分析其浓度和纯度,结果表明利用该方法提取的mtDNA纯度高,完全能满足后续PCR分析及分子克隆测序的要求。该方法不仅DNA提取纯度高,操作简单、快速经济,可为今后开展甘蔗及其各野生种的研究提供技术支持。     

咸宁职业技术学院园林绿化现状与改造对策

咸宁职业技术学院是由老校区和正在扩建的新校区所组成,建设成一所环境优雅、具有地方人文特色的高等职业院校是该院绿化的根本宗旨。因此,对新老校区园林植物的配置与改造显得尤为重要。笔者在对咸宁职业技术学院的园林绿化现状进行广泛调查分析基础上,提出了学院后期校园绿化和改造的几点建议。