Protective Effects of Mitochondrial Uncoupling Protein 2 against Aristolochic Acid I-Induced Toxicity in HK-2 Cells.

Aristolochic acid I (AA I) is one of the most abundant and toxic aristolochic acids that is reported to cause Aristolochic acid nephropathy (AAN). This paper was designed to assess whether mitochondrial Uncoupling Protein 2 (UCP2), which plays an antioxidative and antiapoptotic role, could protect human renal proximal tubular epithelial (HK-2) cells from toxicity induced by AA I. In this study, HK-2 cells were treated with different concentrations of AA I with or without UCP2 inhibitor (genipin). To upregulate the expression of UCP2 in HK-2 cells, UCP2-DNA transfection was performed. The cell viability was evaluated by colorimetric method using MTT. A series of related biological events such as Reactive Oxygen Species (ROS), Glutathione peroxidase (GSH-Px), and Malondialdehyde (MDA) were evaluated. The results showed that the cytotoxicity of AA I with genipin group was much higher than that of AA I alone. Genipin dramatically boosted oxidative stress and exacerbated AA I-induced apoptosis. Furthermore, the increased expression of UCP2 can reduce the toxicity of AA I on HK-2 cells and upregulation of UCP2 expression can reduce AA I-induced oxidative stress and apoptosis. In conclusion, UCP2 might be a potential target for alleviating AA I-induced nephrotoxicity.

The interaction of MYB, bHLH and WD40 transcription factors in red pear (Pyrus pyrifolia) peel.

KEY MESSAGE: Sunlight enhanced peel color and significantly up-regulated the expression of PyMYB10 and PybHLH genes. MYB-bHLH-WD40 transcriptional complex forms in the light and is involved in regulating anthocyanin accumulation in the peel. Anthocyanin is the major pigment in the peel of Yunnan red pear (Pyrus pyrifolia (Burm.) Nak.). A transcriptional activation protein complex, involving members of the transcription factor classes of MYB, bHLH and WD40, regulates anthocyanin biosynthesis. This complex was examined in the peel of red pear. In order to clarify the interaction of PyMYB10, PybHLH and PyWD40, fruit were bagged then peel samples collected 0, 3, 5, and 7 days after bag removal. Samples were used for Western blotting and protein interaction analysis. The results showed that sunlight enhanced peel color and significantly up-regulated the expression of both PyMYB10 and PybHLH genes. Co-immunoprecipitation (Co-IP) analysis showed that PybHLH interacted with PyMYB10 or PyWD40, and PyMYB10 interacted with PyWD40. Using onion cells as a model system, bimolecular fluorescence complementation (BiFC) confirmed these interactions and showed that the interaction localized to the nuclei. GST Pull down and Far-Western blotting assays demonstrated that PybHLH interacted with PyMYB10 or PyWD40, respectively, and PyMYB10 interacted with PyWD40 in vitro. In addition, EMSA assay showed that PyMYB10 can directly bind to the promoter of the gene encoding the anthocyanin biosynthesis enzyme anthocyanidin synthase (PyANS). Taken together, these results showed that the ternary complex of PyMYB10, PybHLH and PyWD40 transcription factors forms to regulate anthocyanin biosynthesis and accumulation in Yunnan red pear.

Behavioral Consequences of a Bifacial Map in the Mouse Somatosensory Cortex.

The whisker system is an important sensory organ with extensive neural representations in the brain of the mouse. Patterned neural modules (barrelettes) in the ipsilateral principal sensory nucleus of the trigeminal nerve (PrV) correspond to the whiskers. Axons of the PrV barrelette neurons cross the midline and confer the whisker-related patterning to the contralateral ventroposteromedial nucleus of the thalamus, and subsequently to the cortex. In this way, specific neural modules called barreloids and barrels in the contralateral thalamus and cortex represent each whisker. Partial midline crossing of the PrV axons, in a conditional Robo3 mutant (Robo3R3-5cKO) mouse line, leads to the formation of bilateral whisker maps in the ventroposteromedial, as well as the barrel cortex. We used voltage-sensitive dye optical imaging and somatosensory and motor behavioral tests to characterize the consequences of bifacial maps in the thalamocortical system. Voltage-sensitive dye optical imaging verified functional, bilateral whisker representation in the barrel cortex and activation of distinct cortical loci following ipsilateral and contralateral stimulation of the specific whiskers. The mutant animals were comparable with the control animals in sensorimotor tests. However, they showed noticeable deficits in all of the whisker-dependent or -related tests, including Y-maze exploration, horizontal surface approach, bridge crossing, gap crossing, texture discrimination, floating in water, and whisking laterality. Our results indicate that bifacial maps along the thalamocortical system do not offer a functional advantage. Instead, they lead to impairments, possibly due to the smaller size of the whisker-related modules and interference between the ipsilateral and contralateral whisker representations in the same thalamus and cortex.SIGNIFICANCE STATEMENT The whisker sensory system plays a quintessentially important role in exploratory behavior of mice and other nocturnal rodents. Here, we studied a novel mutant mouse line, in which the projections from the brainstem to the thalamus are disrupted. This led to formation of bilateral whisker maps in both the thalamus and the cortex. The two whisker maps crowd in a space normally devoted to the contralateral map alone and in a nonoverlapping fashion. Stimulation of the whiskers on either side activates the corresponding region of the map. Mice with bilateral whisker maps perform well in general sensorimotor tasks but show poor performance in specific tests that require whisker-dependent tactile discrimination. These observations indicate that contralateral, instead of bilateral, representation of the sensory space plays a critical role in acuity and fine discrimination during somesthesis.

A Novel Corynebacterium glutamicum l-Glutamate Exporter.

Besides metabolic pathways and regulatory networks, transport systems are also pivotal for cellular metabolism and hyperproduction of biochemicals using microbial cell factories. The identification and characterization of transporters are therefore of great significance for the understanding and engineering of transport reactions. Herein, a novel l-glutamate exporter, MscCG2, which exists extensively in Corynebacterium glutamicum strains but is distinct from the only known l-glutamate exporter, MscCG, was discovered in an industrial l-glutamate-producing C. glutamicum strain. MscCG2 was predicted to possess three transmembrane helices in the N-terminal region and located in the cytoplasmic membrane, which are typical structural characteristics of the mechanosensitive channel of small conductance. MscCG2 has a low amino acid sequence identity (23%) to MscCG and evolved separately from MscCG with four transmembrane helices. Despite the considerable differences between MscCG2 and MscCG in sequence and structure, gene deletion and complementation confirmed that MscCG2 also functioned as an l-glutamate exporter and an osmotic safety valve in C. glutamicum Besides, transcriptional analysis showed that MscCG2 and MscCG genes were transcribed in similar patterns and not induced by l-glutamate-producing conditions. It was also demonstrated that MscCG2-mediated l-glutamate excretion was activated by biotin limitation or penicillin treatment and that constitutive l-glutamate excretion was triggered by a gain-of-function mutation of MscCG2 (A151V). Discovery of MscCG2 will enrich the understanding of bacterial amino acid transport and provide additional targets for exporter engineering.IMPORTANCE The exchange of matter, energy, and information with surroundings is fundamental for cellular metabolism. Therefore, studying transport systems that are essential for these processes is of great significance. Besides, transport systems of bacterial cells are usually related to product excretion as well as product reuptake, making transporter engineering a useful strategy for strain improvement. The significance of our research is in identifying and characterizing a novel l-glutamate exporter from the industrial workhorse Corynebacterium glutamicum, which will enrich the understanding of l-glutamate excretion and provide a new target for studying bacterial amino acid transport and engineering transport reactions.

Thalamic NMDA receptor function is necessary for patterning of the thalamocortical somatosensory map and for sensorimotor behaviors.

NMDARs play a major role in patterning of topographic sensory maps in the brain. Genetic knock-out of the essential subunit of NMDARs in excitatory cortical neurons prevents whisker-specific neural pattern formation in the barrel cortex. To determine the role of NMDARs en route to the cortex, we generated sensory thalamus-specific NR1 (Grin1)-null mice (ThNR1KO). A multipronged approach, using histology, electrophysiology, optical imaging, and behavioral testing revealed that, in these mice, whisker patterns develop in the trigeminal brainstem but do not develop in the somatosensory thalamus. Subsequently, there is no barrel formation in the neocortex yet a partial afferent patterning develops. Whisker stimulation evokes weak cortical activity and presynaptic neurotransmitter release probability is also affected. We found several behavioral deficits in tasks, ranging from sensorimotor to social and cognitive. Collectively, these results show that thalamic NMDARs play a critical role in the patterning of the somatosensory thalamic and cortical maps and their impairment may lead to pronounced behavioral defects.

The Intrinsic Dynamics and Unfolding Process of an Antibody Fab Fragment Revealed by Elastic Network Model.

Antibodies have been increasingly used as pharmaceuticals in clinical treatment. Thermal stability and unfolding process are important properties that must be considered in antibody design. In this paper, the structure-encoded dynamical properties and the unfolding process of the Fab fragment of the phosphocholine-binding antibody McPC603 are investigated by use of the normal mode analysis of Gaussian network model (GNM). Firstly, the temperature factors for the residues of the protein were calculated with GNM and then compared with the experimental measurements. A good result was obtained, which provides the validity for the use of GNM to study the dynamical properties of the protein. Then, with this approach, the mean-square fluctuation (MSF) of the residues, as well as the MSF in the internal distance (MSFID) between all pairwise residues, was calculated to investigate the mobility and flexibility of the protein, respectively. It is found that the mobility and flexibility of the constant regions are higher than those of the variable regions, and the six complementarity-determining regions (CDRs) in the variable regions also exhibit relative large mobility and flexibility. The large amplitude motions of the CDRs are considered to be associated with the immune function of the antibody. In addition, the unfolding process of the protein was simulated by iterative use of the GNM. In our method, only the topology of protein native structure is taken into account, and the protein unfolding process is simulated through breaking the native contacts one by one according to the MSFID values between the residues. It is found that the flexible regions tend to unfold earlier. The sequence of the unfolding events obtained by our method is consistent with the hydrogen-deuterium exchange experimental results. Our studies imply that the unfolding behavior of the Fab fragment of antibody McPc603 is largely determined by the intrinsic dynamics of the protein.

Conformational Motions and Functionally Key Residues for Vitamin B12 Transporter BtuCD-BtuF Revealed by Elastic Network Model with a Function-Related Internal Coordinate.

BtuCD-BtuF from Escherichia coli is a binding protein-dependent adenosine triphosphate (ATP)-binding cassette (ABC) transporter system that uses the energy of ATP hydrolysis to transmit vitamin B12 across cellular membranes. Experimental studies have showed that during the transport cycle, the transporter undergoes conformational transitions between the "inward-facing" and "outward-facing" states, which results in the open-closed motions of the cytoplasmic gate of the transport channel. The opening-closing of the channel gate play critical roles for the function of the transporter, which enables the substrate vitamin B12 to be translocated into the cell. In the present work, the extent of opening of the cytoplasmic gate was chosen as a function-related internal coordinate. Then the mean-square fluctuation of the internal coordinate, as well as the cross-correlation between the displacement of the internal coordinate and the movement of each residue in the protein, were calculated based on the normal mode analysis of the elastic network model to analyze the function-related motions encoded in the structure of the system. In addition, the key residues important for the functional motions of the transporter were predicted by using a perturbation method. In order to facilitate the calculations, the internal coordinate was introduced as one of the axes of the coordinate space and the conventional Cartesian coordinate space was transformed into the internal/Cartesian space with linear approximation. All the calculations were carried out in this internal/Cartesian space. Our method can successfully identify the functional motions and key residues for the transporter BtuCD-BtuF, which are well consistent with the experimental observations.

Neonatal infraorbital nerve crush-induced CNS synaptic plasticity and functional recovery.

Infraorbital nerve (ION) transection in neonatal rats leads to disruption of whisker-specific neural patterns (barrelettes), conversion of functional synapses into silent synapses, and reactive gliosis in the brain stem trigeminal principal nucleus (PrV). Here we tested the hypothesis that neonatal peripheral nerve crush injuries permit better functional recovery of associated central nervous system (CNS) synaptic circuitry compared with nerve transection. We developed an in vitro whisker pad-trigeminal ganglion (TG)-brain stem preparation in neonatal rats and tested functional recovery in the PrV following ION crush. Intracellular recordings revealed that 68% of TG cells innervate the whisker pad. We used the proportion of whisker pad-innervating TG cells as an index of ION function. The ION function was blocked by ∼64%, immediately after mechanical crush, then it recovered beginning after 3 days postinjury and was complete by 7 days. We used this reversible nerve-injury model to study peripheral nerve injury-induced CNS synaptic plasticity. In the PrV, the incidence of silent synapses increased to ∼3.5 times of control value by 2-3 days postinjury and decreased to control levels by 5-7 days postinjury. Peripheral nerve injury-induced reaction of astrocytes and microglia in the PrV was also reversible. Neonatal ION crush disrupted barrelette formation, and functional recovery was not accompanied by de novo barrelette formation, most likely due to occurrence of recovery postcritical period (P3) for pattern formation. Our results suggest that nerve crush is more permissive for successful regeneration and reconnection (collectively referred to as "recovery" here) of the sensory inputs between the periphery and the brain stem.

Secondary Conformational Checkpoint in CRISPR-Cas9.

A specific checkpoint between target DNA binding and cleavage primarily governs the precision of Cas9 gene editing. Although various CRISPR-Cas9 variants have been developed to improve DNA cleavage accuracy, we still lack a comprehensive understanding of how they work at the molecular level. Herein, we have focused on studying the late-stage conformational transitions of Cas9 and an evolved Cas9 mutant (evoCas9) that start from the precleavage state. Our submilliseconds of dynamic simulations reveal that the presence of base mismatches leads the HNH nuclease domain of Cas9 to alter its principal functional modes of motion, thereby impairing its conformational activation. This observation suggests the existence of a secondary conformational checkpoint that fine-tunes the final DNA cleavage activation. Remarkably, evoCas9 is prone to deviating from the normal activation pathway with base mismatches. This is characterized by a noticeable shift in the positioning of the HNH domain and a significantly perturbed allosteric communication network within the enzyme. Therefore, the mutations evolved in evoCas9 also reinforce the secondary checkpoint in addition to the previously identified primary checkpoint, collectively ensuring this variant's high gene-editing accuracy. This mechanism should also apply to other Cas9-guide RNA variants with enhanced fidelity.

Gemological characteristics and inclusions of green rutilated quartz from Huanggangliang, Inner Mongolia.

Normally, various minerals exist in quartz as inclusions. In this study, methods such as gem microscopy, polarizing microscopy, Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and electron probe microanalysis (EPMA) were used to systematically study the gemological characteristics and inclusions in green rutilated quartz from Inner Mongolia. Results show that the sample appears green due to the chaotic distribution of green inclusions in the shape of hair filaments. Combined with the chemical composition, the inclusions are Ca-Fe-rich amphiboles with compositions very close to those of the end-member ferro-actinolite. According to the principle of amphibole nomenclature, the inclusions are named ferro-actinolite in the subclass of calc-alkaline amphiboles with a few named ferro-hornblende. Results suggested that the inclusions in green rutilated quartz were formed during the late stage of quartz crystallization. This work provides a new theoretical basis for the study of green rutilated quartz in Huanggangliang, Inner Mongolia.

Pregnancy influences expression of interferon-stimulated genes, progesterone receptor and progesterone-induced blocking factor in ovine thyroid.

OBJECTIVE: Embryonic interferon-tau (IFNT) and progesterone affect expression of interferonstimulated genes (ISGs), progesterone receptor (PGR) and progesterone-induced blocking factor (PIBF) in the ovine thyroid. METHODS: Thyroids of ewes were sampled at day 16 of nonpregnancy, days 13, 16, and 25 of pregnancy, and real-time quantitative polymerase chain reaction assay, western blot and immunohistochemistry were used to detect expression of ISGs, PGR, and PIBF. RESULTS: Free ISG15 protein was undetected, but ISG15 conjugated proteins upregulated at day 16 of pregnancy, and expression levels of ISG15 conjugated proteins, PGR isoform (70 kDa), PIBF, interferon-gamma-inducible protein 10 and myxovirusresistance protein 1 peaked, but expression level of signal transducer and activator of transcription 1 was the lowest at day 16 of pregnancy. In addition, the expression levels of PGR isoform (70 kDa) and signal transducer and activator of transcription 1 (STAT1) decreased, but levels of PGR isoform (43 kDa), 2',5'-oligoadenylate synthetase, IP-10 and MX1 increased at day 25 of pregnancy comparing with day 16 of the estrous cycle. CONCLUSION: Early pregnancy affects expression of ISGs, PGR, and PIBF in maternal thyroid through IFNT and progesterone, which may regulate thyroid autoimmunity and thyroid hormone secretion in ewes.

Bilateral Whisker Representations in the Primary Somatosensory Cortex in Robo3cKO Mice Are Reflected in the Primary Motor Cortex.

In Robo3cKO mice, midline crossing defects of the trigeminothalamic projections from the trigeminal principal sensory nucleus result in bilateral whisker maps in the somatosensory thalamus and consequently in the face representation area of the primary somatosensory (S1) cortex (Renier et al., 2017; Tsytsarev et al., 2017). We investigated whether this bilateral sensory representation in the whisker-barrel cortex is also reflected in the downstream projections from the S1 to the primary motor (M1) cortex. To label these projections, we injected anterograde viral axonal tracer in S1 cortex. Corticocortical projections from the S1 distribute to similar areas across the ipsilateral hemisphere in control and Robo3cKO mice. Namely, in both genotypes they extend to the M1, premotor/prefrontal cortex (PMPF), secondary somatosensory (S2) cortex. Next, we performed voltage-sensitive dye imaging (VSDi) in the left hemisphere following ipsilateral and contralateral single whisker stimulation. While controls showed only activation in the contralateral whisker barrel cortex and M1 cortex, the Robo3cKO mouse left hemisphere was activated bilaterally in both the barrel cortex and the M1 cortex. We conclude that the midline crossing defect of the trigeminothalamic projections leads to bilateral whisker representations not only in the thalamus and the S1 cortex but also downstream from the S1, in the M1 cortex.

Effect of altrenogest treatment before weaning on reproductive performance and production efficiency in primiparous and multiparous sows.

BACKGROUND: Most sows will experience negative energy balance during lactation resulting in impaired follicular development. This study aimed to treat 28-day lactating sows with altrenogest (ALT) to suppress follicle enlargement during lactation, and to assess the estrus and reproductive performance post-weaning. METHODS: In this study, we conducted two trials. In trial 1, we monitored the follicular development of lactating sows including 10 primiparous sows and 10 multiparous sows during the whole lactation to confirm the ALT administration time. In trial 2, a total of 42 primiparous and 111 multiparous sows were allocated to three treatments: Ctrl (control group, n = 51): no treatment; TAI (timed artificial insemination group, n = 51): sows were injected with equine chorionic gonadotropin (eCG) after weaning 24 h and gonadotropin-releasing hormone (GnRH) when they expressed estrus; and AT-TAI (ALT treatment-timed artificial insemination group, n = 51): base on the process of TAI group, the sows were fed with 20 mg ALT per day before weaning 10 days. All sows were artificially inseminated twice at 12 h and 36 h after estrus. The follicle size changes and serum hormone levels were explored in this process. RESULTS: Although the follicle size of multiparous sows was larger than primiparous sows during the whole lactation (P < 0.05), similar change trends of follicle size were observed in primiparous and multiparous sows. Meanwhile, the FSH, LH and E2 levels of multiparous sows were higher than primiparous sows. The ALT treatment significantly inhibits the increase in follicle size (P < 0.05) and reduces the serum levels of FSH, LH and E2 (P > 0.05). Additionally, ALT treatment increases estrus concentration and the preovulatory follicle size (P < 0.05), meanwhile, it delays the weaning-to-estrus interval (WEI, P < 0.001). However, the estrus rate, pregnancy rate, total pigs born and born alive did not differ between treatments (P > 0.05). CONCLUSIONS: There were significant differences in the size of follicles in the lactation between primiparous and multiparous sows. ALT treatment during the last ten days of lactation concentrated estrus expression leading to higher work efficiency of breeder in batch production, however, with no improvement in reproductive performance.

The Antioxidant Salidroside Ameliorates the Quality of Postovulatory Aged Oocyte and Embryo Development in Mice.

Postovulatory aging is known to impair the oocyte quality and embryo development due to oxidative stress in many different animal models, which reduces the success rate or pregnancy rate in human assisted reproductive technology (ART) and livestock timed artificial insemination (TAI), respectively. Salidroside (SAL), a phenylpropanoid glycoside, has been shown to exert antioxidant and antitumor effects. This study aimed to investigate whether SAL supplementation could delay the postovulatory oocyte aging process by alleviating oxidative stress. Here, we show that SAL supplementation decreases the malformation rate and recovers mitochondrial dysfunction including mitochondrial distribution, mitochondrial membrane potential (ΔΨ) and ATP content in aged oocytes. In addition, SAL treatment alleviates postovulatory aging-caused oxidative stress such as higher reactive oxygen species (ROS) level, lower glutathione (GSH) content and a reduced expression of antioxidant-related genes. Moreover, the cytoplasmic calcium ([Ca2+]c) and mitochondrial calcium ([Ca2+]mt) of SAL-treated oocytes return to normal levels. Notably, SAL suppresses the aging-induced DNA damage, early apoptosis and improves spindle assembly in aged oocytes, ultimately elevating the embryo developmental rates and embryo quality. Finally, the RNA-seq and confirmatory experience showed that SAL promotes protective autophagy in aged oocytes by activating the MAPK pathway. Taken together, our research suggests that supplementing SAL is an effective and feasible method for preventing postovulatory aging and preserving the oocyte quality, which potentially contributes to improving the successful rate of ART or TAI.

Amyloid Deposition and Dendritic Complexity of Corticocortical Projection Cells in Five Familial Alzheimer's Disease Mouse.

In Alzheimer's disease and related dementias, amyloid beta (Aß) and amyloid plaques can disrupt long-term synaptic plasticity, learning and memory and cognitive function. Plaque accumulation can disrupt corticocortical circuitry leading to abnormalities in sensory, motor, and cognitive processing. In this study, using 5xFAD (five Familial Alzheimer's Disease - FAD - mutations) mice, we evaluated amyloid plaque formation in different cortical areas, and whether differential amyloid accumulation across cortical fields correlates with changes in dendritic complexity of layer 3 corticocortical projection neurons and functional responses in the primary somatosensory cortex following whisker stimulation. We focused on three cortical areas: the primary somatosensory cortex (S1), the primary motor cortex (M1), and the prefrontal cortex (PFC including the anterior cingulate, prelimbic, and infralimbic subdivisions). We found that Aß and amyloid plaque accumulation is not uniform across 5xFAD cortical areas, while there is no expression in littermate controls. We also found that there are differential layer 3 pyramidal cell dendritic complexity changes across the three areas in 5xFAD mice, compared to same age controls, with no apparent relation to differential amyloid accumulation. We used voltage-sensitive dye imaging (VSDi) to visualize neural activity in S1, M1 and PFC following whisker activation. Control mice show normal physiological responses in all three cortical areas, whereas 5xFAD mice only display physiological responses in S1. Taken together our results show that 5xFAD mutation affects the overall dendritic morphology of layer 3 pyramidal cells across sensory-motor and association cortex irrespective of the density and distribution of the Aß amyloid proteins. Corticocortical circuitry between the sensory and motor/association areas is most likely disrupted in 5xFAD mice as cortical responses to whisker stimulation are altered.

Regulation of IkappaB Protein Expression by Early Gestation in the Thymus of Ewes.

The thymus is an essential component of maternal immune systems that play key roles in recognizing the placenta as immunologically foreign. The inhibitor of the NF-κB (IκB) family has essential effects on the NF-κB pathway; however, it is unclear whether early pregnancy modulates the expression of the IκB family in the thymus. In this study, maternal thymuses were sampled on day 16 of nonpregnancy and different gestation stages in the ovine, and the expression of IκB proteins was analyzed. The data showed that B cell leukemia-3 and IκBß increased; however, IκBα, IκBε, and IKKγ deceased during gestation. Furthermore, there was an increase in IκBNS and IκBζ expression values on day 13 of pregnancy; however, this decreased on day 25 of gestation. In summary, the expression of the IκB family was modulated in the thymus during early gestation, suggesting that the maternal thymus can be associated with maternal immunologic tolerance and pregnancy establishment in ewes.

Combination therapy of Ulinastatin with Thrombomodulin alleviates endotoxin (LPS) - induced liver and kidney injury via inhibiting apoptosis, oxidative stress and HMGB1/TLR4/NF-κB pathway.

Sepsis is a type of systemic inflammation response syndrome that leads to organ function disorders. Currently, there is no specific medicine for sepsis in clinical practice. Lipopolysaccharide (LPS) is an important endotoxin that causes sepsis. Here, we report an effective two-drug combination therapy to treat LPS-induced liver and kidney injury in endotoxic rats. Ulinastatin (UTI) and Thrombomodulin (TM) are biological macromolecules extracted from urine. In our study, combination therapy significantly improved LPS-induced liver and kidney pathological structure and functional injury, and significantly improved the survival rate of endotoxic rats. Results of TUNEL staining and Western blot showed that UTI combined with TM inhibited the excessive apoptosis of liver and kidney cells caused by LPS. The drug combination also promoted the proliferation of liver and kidney cells, reduced the levels of pro-inflammatory factors interleukin (IL)-6, IL-1ß, tumor or necrosis factor (TNF)-α and nitric oxide, and down-regulated the expression of High Mobility Group Box 1 (HMGB1), Toll-like receptor (TLR) 4 and Nuclear Factor (NF)-κB phosphorylation to inhibit inflammation. In addition, the combination of UTI and TM also promoted the production of a variety of antioxidant enzymes in the tissues and inhibited the production of lipid peroxidation malondialdehyde (MDA) to enhance antioxidant defenses. Our experiments also proved that UTI combined with TM did not reduce the anticoagulant effect of TM. These results suggested that UTI combined with TM can improve endotoxin-induced liver and kidney damage and mortality by inhibiting liver and kidney cell apoptosis, promoting proliferation, and inhibiting inflammation and oxidative injury.

Layers 3 and 4 Neurons of the Bilateral Whisker-Barrel Cortex.

In Robo3R3-5cKO mouse brain, rhombomere 3-derived trigeminal principal nucleus (PrV) neurons project bilaterally to the somatosensory thalamus. As a consequence, whisker-specific neural modules (barreloids and barrels) representing whiskers on both sides of the face develop in the sensory thalamus and the primary somatosensory cortex. We examined the morphological complexity of layer 4 barrel cells, their postsynaptic partners in layer 3, and functional specificity of layer 3 pyramidal cells. Layer 4 spiny stellate cells form much smaller barrels and their dendritic fields are more focalized and less complex compared to controls, while layer 3 pyramidal cells did not show notable differences. Using in vivo 2-photon imaging of a genetically encoded fluorescent [Ca2+] sensor, we visualized neural activity in the normal and Robo3R3-5cKO barrel cortex in response to ipsi- and contralateral single whisker stimulation. Layer 3 neurons in control animals responded only to their contralateral whiskers, while in the mutant cortex layer 3 pyramidal neurons showed both ipsi- and contralateral whisker responses. These results indicate that bilateral whisker map inputs stimulate different but neighboring groups of layer 3 neurons which normally relay contralateral whisker-specific information to other cortical areas.

Modulation of Nod-like Receptor Expression in the Thymus during Early Pregnancy in Ewes.

Nucleotide-binding oligomerization domain receptors (NOD-like receptors, NLRs) are involved in modulating the innate immune responses of the trophoblast and the placenta in normal pregnancy. The thymus participates in regulation of innate and adaptive immune responses. However, it is unclear whether expression of NLR is modulated in the maternal thymus during early pregnancy. In this study, thymuses were sampled at day 16 of the estrous cycle, and at days 13, 16 and 25 of gestation (n = 6 for each group) from ewes after slaughter. Different stages were chosen because the maternal thymus was under the different effects of interferon-tau and/or progesterone or not. RT-qPCR, Western blot and immunohistochemistry analysis were used to analyze the expression of the NLR family, including NOD1; NOD2; major histocompatibility complex class II transactivator (CIITA); NLR family apoptosis inhibitory protein (NAIP); nucleotide-binding oligomerization domain and Leucine-rich repeat and Pyrin domain containing protein 1 (NLRP1), NLRP3 and NLRP7. The results showed that expression level of NOD1 was changed with the pregnancy stages, and expression levels of NOD2, CIITA, NAIP, NLRP1, NLRP3 and NLRP7 mRNA and proteins were peaked at day 13 of pregnancy. The levels of NOD2 and CIITA were increased during early pregnancy. The stainings for NOD2 and NLRP7 proteins were located in epithelial reticular cells, capillaries and thymic corpuscles. In summary, pregnancy stages changed expression of NLR family in the maternal thymus, which may be related to the modulation of maternal thymic immune responses, and beneficial for normal pregnancy in sheep.

Astrocytes promote peripheral nerve injury-induced reactive synaptogenesis in the neonatal CNS.

Neonatal damage to the trigeminal nerve leads to "reactive synaptogenesis" in the brain stem sensory trigeminal nuclei. In vitro models of brain injury-induced synaptogenesis have implicated an important role for astrocytes. In this study we tested the role of astrocyte function in reactive synaptogenesis in the trigeminal principal nucleus (PrV) of neonatal rats following unilateral transection of the infraorbital (IO) branch of the trigeminal nerve. We used electrophysiological multiple input index analysis (MII) to estimate the number of central trigeminal afferent fibers that converge onto single barrelette neurons. In the developing PrV, about 30% of afferent connections are eliminated within 2 postnatal weeks. After neonatal IO nerve damage, multiple trigeminal inputs (2.7 times that of the normal inputs) converge on single barrelette cells within 3-5 days; they remain stable up to the second postnatal week. Astrocyte proliferation and upregulation of astrocyte-specific proteins (GFAP and ALDH1L1) accompany reactive synaptogenesis in the IO nerve projection zone of the PrV. Pharmacological blockade of astrocyte function, purinergic receptors, and thrombospondins significantly reduced or eliminated reactive synaptogenesis without changing the MII in the intact PrV. GFAP immunohistochemistry further supported these electrophysiological results. We conclude that immature astrocytes, purinergic receptors, and thrombospondins play an important role in reactive synaptogenesis in the peripherally deafferented neonatal PrV.