Photobiomodulation by diffusing optical fiber on spinal cord: A feasibility study in piglet model

Previous studies on spinal cord injury (SCI) have confirmed that percutaneous photobiomodulation (PBM) therapy can ameliorate immunoinflammatory responses at sites of injury, accelerate nerve regeneration, suppress glial scar formation and promote the subsequent recovery of locomotor function. The current study was performed to evaluate a large‐animal model employing implanted optical fibers to accurately irradiate targeted spinal segments. The method's feasibility and irradiation parameters that do not cause phototoxic reaction were determined, and the methodology of irradiating the spinal cord with near‐infrared light was investigated in detail. A diffusing optical fiber was implanted above the T9 spinal cord of Bama miniature pigs and used to transfer near‐infrared light (810 nm) onto the spinal cord surface. After daily irradiation with 200, 300, 500 or 1000 mW for 14 days, both sides of the irradiated area of the spinal cord were assessed for temperature changes. The condition of the spinal cord and the position of optical fiber were investigated by magnetic resonance imaging (MRI), and different parameters indicating temperature increases or phototoxicity were measured on the normal spinal cord surface due to light irradiation (ie, heat shock responses, inflammatory reactions and neuronal apoptosis), and the animals' lower‐limb neurological function and gait were assessed during the irradiation process. The implanted device was stable inside the freely moving animals, and light energy could be directly projected onto the spinal cord surface. The screening of different irradiation parameters preliminary showed that direct irradiation onto the spinal cord surface at 200 and 300 mW did not significantly increase the temperature, stress responses, inflammatory reactions and neural apoptosis, whereas irradiation at 500 mW slightly increased these parameters, and irradiation at 1000 mW induced a significant temperature increase, heat shock, inflammation and apoptosis responses. HE staining of spinal cord tissue sections did not reveal any significant structural changes of the tissues compared to the control group, and the neurological function and gait of all irradiated animals were normal. In this study, we established an in‐vivo optical fiber implantation method, which might be safe and stable and could be used to directly project light energy onto the spinal cord surface. This study might provide a new perspective for clinical applications of PBM in acute SCI.     

Development of gabaergic connections in vitro: Increasing efficacy of synaptic transmission is not accompanied by changes in miniature currents

Development of inhibitory synaptic transmission was studied using a dissociated cell culture from the superior colliculus of neonatal rat. Patch-clamp recordings in the whole-cell configuration were performed to measure evoked (single-cell-activated) inhibitory postsynaptic currents (IPSCs), miniature IPSCs and current responses to maximal concentrations of exogenous γ-aminobutyric acid (GABA). Over a period of 3 weeks in vitro (DIV3-24), the fraction of synaptically coupled neurons raised from 0% to 76%. Evoked IPSCs were first observed at DIV5. They had an average amplitude of 33.9 pA during the first week (n = 13) and 129.7 pA during the fourth week (n = 48). This increase by a factor of 3.8 represents a significant rise in the efficacy of GABAergic transmission during in vitro development. However, no developmental change has been observed in the average amplitudes of miniature somatic IPSCs. The latter remained at an average level of about 9 pA (symmetrical chloride concentration and a driving force of 68 mV). No increase was found also in whole-cell current densities induced by saturating concentrations of exogenous GABA. Our results suggest that under the given conditions, synapse maturation was primarily the result of presynaptic sprouting. This conclusion is further supported by bouton counts in immunostained collicular cultures, where the number of axosomatic and axodendritic GABAergic contacts per neuron increased from 0.54 and 0.37, respectively, at DIV3, to 13.84 and >23.1, at DIV24. The overall density of GABAergic neurons decreased during this period from about 41,000/cm² to 15,600 cm², indicating that a growing number of contacts is formed by a declining number of presynaptic neurons. © 1992 John Wiley & Sons, Inc.     

Common swine leucocyte antigen (SLA) haplotypes in NIH and Sinclair miniature swine have similar effects on the expression of an inherited melanoma

Mixed lymphocyte culture and serological typing of NIH and Sinclair miniature swine indicate that the two herds share a common SLA haplotype. NIH haplotype a (International Haplotype H10) appears identical to Sinclair haplotype B, which has significant effects on the penetrance of Sinclair swine cutaneous malignant melanoma (SSCM). Offspring of crosses between melanoma-bearing Sinclair swine homozygous for the B haplotype and non-melanoma NIH aa swine have tumour incidence identical to Sinclair melanoma BB× Sinclair non-melanoma BB offspring. Our results provide further support for the involvement of the swine leucocyte antigen (SLA) complex in the inheritance of SSCM, and identify a new source of non-tumour animals that have all of the genes for SSCM except those at the tumour-initiator locus.     

Transposable elements and human cancer: A causal relationship?

Transposable elements are present in almost all genomes including that of humans. These mobile DNA sequences are capable of invading genomes and their impact on genome evolution is substantial as they contribute to the genetic diversity of organisms. The mobility of transposable elements can cause deleterious mutations, gene disruption and chromosome rearrangements that may lead to several pathologies including cancer. This mini-review aims to give a brief overview of the relationship that transposons and retrotransposons may have in the genetic cause of human cancer onset, or conversely creating protection against cancer. Finally, the cause of TE mobility may also be the cancer cell environment itself.     

The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction

Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. In the present study, we analyzed the role of Gef26 in synaptic development and function. We identified significant decreases in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, our data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a βPS integrin-mediated signaling pathway. Taken together, our findings define a novel role of Gef26 in regulating NMJ development and function.     

猪内源性反转录病毒在中国实验小型猪中的存在与表达

目的对中国实验小型猪中内源性反转录病毒的存在与mRNA的表达进行检测,摸清中国实验小型猪中内源性反转录病毒的携带情况.方法根据已发表的PERV的序列设计并合成了三对引物,分别用于检测PERV核心蛋白基因(gag)、多聚酶基因(pol)及囊膜基因(env)的存在与表达;同时,根据目前通用的env基因分型方法合成了三对用于分型检测的引物env-A、env-B、env-C.应用PCR、RT-PCR扩增的方法,对来自于中国实验小型猪外周血淋巴细胞的DNA和RNA样品进行了检测.结果在6个被检DNA样品中均检出了PERV特异性DNA的存在;同样,在被检RNA样品中均有PERV特异性RNA的表达,且所表达的PERV均为A型和B型,在所有样品中均未检出C型PERV的表达.结论初步表明中国实验小型猪中存在内源性反转录病毒序列,且能以mRNA的形式表达,这一结果为我国特有小型猪的开发、利用及其病毒安全性评价奠定了基础.     

Heat stress upregulation of Toll-like receptors 2/4 and acute inflammatory cytokines in peripheral blood mononuclear cell (PBMC) of Bama miniature pigs: an in vivo and in vitro study

Global warming is a challenge to animal health, because of increased heat stress, with subsequent induction of immunosuppression and increased susceptibility to disease. Toll-like receptors (TLR) are pattern recognition receptors that act as sentinels of pathogen invasion and tissue damage. Ligation of TLRs results in a signaling cascade and production of inflammatory cytokines, which eradicate pathogens and maintain the health of the host. We hypothesized that the TLR signaling pathway plays a role in immunosuppression in heat-stressed pigs. We explored the changes in the expression of TLR2, TLR4 and the concentration of acute inflammatory cytokines, such as IL-2, IL-8, IL-12 and IFN-γ in Bama miniature pigs subjected to 21 consecutive days of heat stress, both in vitro and in vivo models. The results showed that heat stress induced the upregulation of cortisol in the plasma of pigs (P<0.05); TLR4 mRNA was elevated, but IL-2 was reduced in peripheral blood mononuclear cells (PBMC, P<0.05). The white blood cell count and the percentage of granulocytes (eosinophilic+basophilic) decreased significantly in heat-stressed pigs (P<0.05). In the in vitro model (PBMC heat shocked for 1 h followed by a 9 h recovery period), TLR2 and TLR4 mRNA expression also increased, as did the concentration of IL-12 in supernatants. However, IFN-γ was significantly reduced in PBMC culture supernatants (P<0.05). We concluded that a consecutive heat stress period elevated the expression of TLR2 and TLR4 in PBMC and increased the plasma levels of inflammatory cytokines. These data indicate that TLR activation and dysregulation of cytokine expression in response to prolonged heat stress may be associated with immunosuppression and increased susceptibility to antigenic challenge in Bama miniature pigs.     

猪胰岛素启动子调控EGFP表达载体的优化及体外验证

目的获得猪胰岛素启动子调控外源基因的高效表达载体,为制备转基因猪胰岛特异性表达目的基因奠定基础。方法利用猪胰岛素启动子（PIP,包含5＇调控区、第一外显子、第一内含子及第二外显子ATG前的序列共1.5 kb）构建表达载体,连接PIP和EGFP的酶切位点HindⅢ设计在起始密码子前,命名为PIP-Hind IIIEGFP。鉴于酶切位点的插入位置可能会影响外源基因的表达效率,对载体进行了优化：将Hind III酶切位点删除,实现PIP和EGFP无缝连接,载体命名PIP-EGFP;将第一内含子3＇端的内含子剪接受体位点（splicing acceptor site,SA）突变为Hind III内切酶识别位点,命名为PIP-SA（M）-EGFP。三种载体分别电转染小鼠胰岛素瘤β细胞株MIN-6细胞、猪耳成纤维细胞以及猪肾细胞,48 h后通过荧光强度、流式分析、RT-PCR及Western blot检测,验证载体的表达效率。结果转染细胞后,三种载体都仅在MIN-6胰岛β细胞表达绿色荧光。RT-PCR及其产物测序结果显示三种载体的胰岛素启动子第一内含子的剪切存在差异：载体PIP-Hind III-EGFP和PIP-EGFP的内含子存在剪切和不剪切两种情况,剪切不稳定;载体PIP-SA（M）-EGFP的SA位点突变后内含子不剪切,流式细胞及Western blot检测显示,与另外两种载体相比,该载体目的蛋白的表达量最高。结论通过突变胰岛素启动子第一内含子的3＇端的剪接受体位点,成功获得了胰岛β细胞的高效表达载体,可用于制备胰岛特异表达外源基因的转基因猪。     

Evidence that microgynes of Myrmica rubra ants are social parasites that attack old host colonies

Ant microgynes are miniaturized queen forms found together with normal queens (macrogynes) in species occurring across the ant phylogeny. Their role is not yet fully understood: in some cases, they seem to be nonparasitic alternative reproductive morphs, in others incipient social parasites, and thus potential models for studying the evolution of social parasitism. Whether they are regarded as parasitic or not has traditionally been based on genetic differentiation from syntopic macrogynes and/or the queen/worker ratio of their offspring rather than measuring fitness traits. We confirmed previously reported genetic differentiation between microgynes and macrogynes of Myrmica rubra in a population studied for the first time. Further, we measured virulence and infectivity of M. rubra microgynes in a controlled laboratory experiment. Nests headed only by macrogynes (controls), only by microgynes, and naturally and artificially mixed nests were kept under identical conditions. We found reduction in worker numbers of both naturally and artificially mixed macrogyne/microgyne nests compared with controls, and strong reduction but also surprising variation in fitness of nests headed only by microgynes. Microgyne nests produced workers, males and microgynes. Microgynes did not themselves reproduce in artificially mixed nests, but reproduced most in naturally mixed nests that had lost their macrogyne queen. This, together with higher mortality of field‐collected macrogyne queens from naturally infested colonies and greater estimated relative age of macrogyne queens in naturally infected nests, suggests that they preferentially exploit older host colonies. We conclude that M. rubra microgynes are intraspecific social parasites specialized on exploiting old host colonies.