HtrAs are essential for the survival of the haloarchaeon Natrinema gari J7-2 in response to heat, high salinity, and toxic substances.

Bacterial and eukaryotic HtrAs can act as an extracytoplasmic protein quality control (PQC) system to help cells survive in stress conditions, but the functions of archaeal HtrAs remain unknown. Particularly, haloarchaea route most secretory proteins to the Tat pathway, enabling them to fold properly in well-controlled cytoplasm with cytosolic PQC systems before secretion. It is unclear whether HtrAs are required for haloarchaeal survival and stress response. The haloarchaeon Natrinema gari J7-2 encodes three Tat signal peptide-bearing HtrAs (NgHtrA, NgHtrB, and NgHtrC), and the signal peptides of NgHtrA and NgHtrC contain a lipobox. Here, the in vitro analysis reveals that the three HtrAs show different profiles of temperature-, salinity-, and metal ion-dependent proteolytic activities and could exhibit chaperone-like activities to prevent the aggregation of reduced lysozyme when their proteolytic activities are inhibited at low temperatures or the active site is disrupted. The gene deletion and complementation assays reveal that NgHtrA and NgHtrC are essential for the survival of strain J7-2 at elevated temperature and/or high salinity and contribute to the resistance of this haloarchaeon to zinc and inhibitory substances generated from tryptone. Mutational analysis shows that the lipobox mediates membrane anchoring of NgHtrA or NgHtrC, and both the membrane-anchored and free extracellular forms of the two enzymes are involved in the stress resistance of strain J7-2, depending on the stress conditions. Deletion of the gene encoding NgHtrB in strain J7-2 causes no obvious growth defect, but NgHtrB can functionally substitute for NgHtrA or NgHtrC under some conditions.IMPORTANCEHtrA-mediated protein quality control plays an important role in the removal of aberrant proteins in the extracytoplasmic space of living cells, and the action mechanisms of HtrAs have been extensively studied in bacteria and eukaryotes; however, information about the function of archaeal HtrAs is scarce. Our results demonstrate that three HtrAs of the haloarchaeon Natrinema gari J7-2 possess both proteolytic and chaperone-like activities, confirming that the bifunctional nature of HtrAs is conserved across all three domains of life. Moreover, we found that NgHtrA and NgHtrC are essential for the survival of strain J7-2 under stress conditions, while NgHtrB can serve as a substitute for the other two HtrAs under certain circumstances. This study provides the first biochemical and genetic evidence of the importance of HtrAs for the survival of haloarchaea in response to stresses.

Lateral hypothalamic orexin neurons mediate electroacupuncture-induced anxiolytic effects in a rat model of post-traumatic stress disorder.

The lateral hypothalamus' orexinergic system has been associated with anxiety-related behaviors, and electroacupuncture (EA) modifies orexin neurons to control the anti-anxiety process. However, in a rat model of post-traumatic stress disorder (PTSD), the important role of LH orexin neurons (OXNs) in the anxiolytic effects induced by EA has not been explored. In this study, rats underwent modified single prolonged stress (MSPS) for seven days before developing EA. The rats were then subjected to elevated plus maze (EPM) and open field (OFT) tests, and western blot and c-Fos/orexin double labeling investigations were carried out to determine the functional activation of LH orexinergic neurons. Compared to MSPS model rats, it has been demonstrated that EA stimulation enhanced the amount of time spent in the central zone (TSCZ) in OFT and the amount of time spent in the open arm (TSOA) in EPM in MSPS model rats (P < 0.01). After behavioral testing, MSPS model rats had decreased activated c-Fos positive OXNs. Still, EA in SPS rats increased that number and elevated orexin type 1 receptors (OXR1) protein expression in the LH. Furthermore, after administering SB334867 (an OXR1 antagonist) to MSPS model rats, the effects of EA therapy on anxiety-like behaviors (ALBs) were significantly diminished. Additionally, when low-dose orexin-A (LORXA) was administered intracerebroventricularly together with EA stimulation in MSPS rats, the anxiolytic effects of the stimulation were substantially enhanced (P < 0.05). The results of this study reveal the mechanisms by which acupuncture may reduce PTSD and advance our understanding of the function of LH orexin signaling in EA's anxiolytic effects.

Crosstalk between acetylation and the tyrosination/detyrosination cycle of α-tubulin in Alzheimer's disease.

Microtubules (MTs) support a variety of neuronal functions, such as maintenance of cell structure, transport, and synaptic plasticity. Neuronal MTs are highly heterogeneous due to several tubulin isotypes and the presence of multiple post-translational modifications, such as detyrosination and acetylation. The tubulin tyrosination/detyrosination cycle is a key player in the maintenance of MT dynamics, as tyrosinated tubulin is associated with more dynamic MTs, while detyrosinated tubulin is linked to longer lived, more stable MTs. Dysfunction of tubulin re-tyrosination was recently correlated to Alzheimer's disease progression. The implication of tubulin acetylation in Alzheimer's disease has, however, remained controversial. Here, we demonstrate that tubulin acetylation accumulates in post-mortem brain tissues from Alzheimer's disease patients and human neurons harboring the Alzheimer's familial APP-V717I mutation. We further show that tubulin re-tyrosination, which is defective in Alzheimer's disease, can control acetylated tubulin in primary neurons irrespective of the levels of the enzymes regulating tubulin acetylation, suggesting that reduced MT dynamics associated with impaired tubulin re-tyrosination might contribute to the accumulation of tubulin acetylation that we detected in Alzheimer's disease.

Lateral Hypothalamic Orexin Neurons Mediate the Reward Effects of Pain Relief Induced by Electroacupuncture.

The reward of pain relief caused by acupuncture has been found to be clinically significant. However, the molecular mechanisms underlying acupuncture-induced reward of pain relief in chronic pain remain unclear and have not been analyzed in suitable preclinical models. Here, we investigated whether acupuncture could potentially induce the reward of pain relief and orexin neuronal signaling in the lateral hypothalamus (LH) and exhibit a possible role in electroacupuncture (EA)-induced reward in spared nerve injury (SNI) rats. Therefore, by using conditioned place preference (CPP) paradigm, we noticed that EA induced the preference for cues associated with EA-induced pain relief in the early, but not late, phase of chronic pain. These observations were different from the immediate antihyperalgesic effects of EA. c-Fos/orexin double labeling revealed that EA stimulation on 14 days but not on 28 days after SNI modeling activated greater numbers of c-Fos positive orexin neurons in the LH after the CPP test. Moreover, the administration of an orexin-A antagonist in the LH significantly blocked the reward effects of pain relief induced by EA. Furthermore, by using cholera toxin b subunit combined with c-Fos detection, we found that the orexin circuit from the LH to the nucleus accumbens (NAc) shell was significantly activated after EA induced CPP. Microinjection of the orexin antagonist into the NAc shell substantially attenuated the CPP induced by EA. Intravenous injection of low-dose orexin-A together with EA resulted in significantly greater antihyperalgesia effects and CPP scores. Together, these findings clearly demonstrated that LH orexin signaling could potentially play a critical role in the reward effects of pain relief induced by acupuncture. The observations of the present study extended our understanding of orexin signaling in the LH and its role in EA-induced reward, providing new insights into the mechanisms of acupuncture analgesia.

Tubulin tyrosination regulates synaptic function and is disrupted in Alzheimer's disease.

Microtubules play fundamental roles in the maintenance of neuronal processes and in synaptic function and plasticity. While dynamic microtubules are mainly composed of tyrosinated tubulin, long-lived microtubules contain detyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination cycle is a key player in the maintenance of microtubule dynamics and neuronal homeostasis, conditions that go awry in neurodegenerative diseases. In the tyrosination/detyrosination cycle, the C-terminal tyrosine of α-tubulin is removed by tubulin carboxypeptidases and re-added by tubulin tyrosine ligase (TTL). Here we show that TTL heterozygous mice exhibit decreased tyrosinated microtubules, reduced dendritic spine density and both synaptic plasticity and memory deficits. We further report decreased TTL expression in sporadic and familial Alzheimer's disease, and reduced microtubule dynamics in human neurons harbouring the familial APP-V717I mutation. Finally, we show that synapses visited by dynamic microtubules are more resistant to oligomeric amyloid-ß peptide toxicity and that expression of TTL, by restoring microtubule entry into spines, suppresses the loss of synapses induced by amyloid-ß peptide. Together, our results demonstrate that a balanced tyrosination/detyrosination tubulin cycle is necessary for the maintenance of synaptic plasticity, is protective against amyloid-ß peptide-induced synaptic damage and that this balance is lost in Alzheimer's disease, providing evidence that defective tubulin retyrosination may contribute to circuit dysfunction during neurodegeneration in Alzheimer's disease.

Isovitexin alleviates acute gouty arthritis in rats by inhibiting inflammation via the TLR4/MyD88/NF-κB pathway.

CONTEXT: The prevalence of gout has greatly increased, and it has become the most common inflammatory arthritis in men. Isovitexin possesses anti-inflammatory and antioxidant properties. OBJECTIVE: We explored the effects of isovitexin on rats with acute gouty arthritis (GA). MATERIALS AND METHODS: Fifty-four Sprague-Dawley rats were assigned to five groups: sham, model, positive (colchicine, 0.3 mg/kg), isovitexin (100 mg/kg), TLR4 inhibitor (TAK-242, 3 mg/kg) and isovitexin + TAK-242. The gait of rats and the ankle joint swelling index were monitored. The levels of tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6, and pathological changes in the synovial tissues were determined. RESULTS: Isovitexin significantly reduced the ankle joint swelling index at day 7 compared to that in the model group (4.39 ± 1.01 vs. 6.09 ± 1.31). Moreover, isovitexin alleviated the infiltration of inflammatory cells and ameliorated the proliferation of synovial cells. The levels of TNF-α (93.42 ± 5.02 pg/mL), IL-1ß (25.46 ± 1.91 pg/mL) and IL-6 (194.71 ± 7.92 pg/mL) in the isovitexin group were significantly lower than in the model group (129.39 ± 5.43, 39.60 ± 2.71 and 223.77 ± 5.35 pg/mL). The expression of TLR4, MyD88 and p-NF-κB-p65 was remarkably decreased after isovitexin and colchicine treatment. The effect of isovitexin was similar to that colchicine. Furthermore, the combination of isovitexin and TAK-242 had better effect, and there was no significantly difference with colchicine treatment. DISCUSSION AND CONCLUSIONS: Isovitexin ameliorates joint inflammation in acute GA via the TLR4/MyD88/NF-κB pathway. Isovitexin may be a potential substitute medicine for GA.

Electroacupuncture Attenuates Anxiety-Like Behaviors in a Rat Model of Post-traumatic Stress Disorder: The Role of the Ventromedial Prefrontal Cortex.

Electroacupuncture (EA) is a promising clinical approach to treating posttraumatic stress disorder (PTSD), yet the mechanisms whereby EA can alleviate anxiety and other PTSD symptoms have yet to be clarified. In the present report, rats underwent EA for 14 consecutive days following modified single prolonged stress (MSPS) exposure. These animals were then evaluated in open field and elevated plus maze tests (OFT and EPM), while Fos immunohistochemical staining was performed to assess ventromedial prefrontal cortex (vmPFC) functional activation. In addition, an extracellular recording and stimulation system was used to analyze vmPFC inputs into the ventral tegmental area (VTA) in these rats. Temporary vmPFC inactivation was further performed to assess whether this was sufficient to reverse the anxiolytic effects of EA. Overall, rats that underwent EA treatment spent more time in the central region (OFT) and the open arm (EPM) relative to MSPS model animals (P < 0.05). These MSPS model animals also exhibited significantly fewer activated Fos-positive nuclei in the vmPFC following behavioral testing, while EA was associated with a significant relative increase in c-Fos expression in this region. The transient inactivation of the vmPFC was sufficient to reverse the effects of EA treatment on anxiety-like behaviors in MSPS model rats. MSPS and SEA rats exhibiting no differences in bursting activity between baseline and vmPFC stimulation, whereas bursting activity rose relative to baseline upon ventral mPFC stimulation in EA treated and control rats. Together, these findings indicate that the vmPFC and its inputs into the VTA are functionally linked to the anxiolytic activity of EA, implicating this pathway in the EA-mediated treatment of PTSD.

The synaptic life of microtubules.

In neurons, control of microtubule dynamics is required for multiple homeostatic and regulated activities. Over the past few decades, a great deal has been learned about the role of the microtubule cytoskeleton in axonal and dendritic transport, with a broad impact on neuronal health and disease. However, significantly less attention has been paid to the importance of microtubule dynamics in directly regulating synaptic function. Here, we review emerging literature demonstrating that microtubules enter synapses and control central aspects of synaptic activity, including neurotransmitter release and synaptic plasticity. The pleiotropic effects caused by a dysfunctional synaptic microtubule cytoskeleton may thus represent a key point of vulnerability for neurons and a primary driver of neurological disease.

Live imaging of microtubule dynamics at excitatory presynaptic boutons in primary hippocampal neurons and acute hippocampal slices.

Analyses of microtubule (MT) plus end dynamics at glutamatergic en passant boutons can be carried out in cultured primary neurons isolated from mouse or rat embryos or ex vivo in acute slices isolated from mice that had been electroporated in utero. Here, we describe a protocol for setting up and analyzing live image recordings of primary neurons and acute hippocampal slices expressing tagged versions of the MT plus end binding protein EB3 and the presynaptic vesicle markers vGlut1 or VAMP2. For complete information on the use and execution of this protocol, please refer to Qu et al. (2019).

Pathogenic role of delta 2 tubulin in bortezomib-induced peripheral neuropathy.

The pathogenesis of chemotherapy-induced peripheral neuropathy (CIPN) is poorly understood. Here, we report that the CIPN-causing drug bortezomib (Bort) promotes delta 2 tubulin (D2) accumulation while affecting microtubule stability and dynamics in sensory neurons in vitro and in vivo and that the accumulation of D2 is predominant in unmyelinated fibers and a hallmark of bortezomib-induced peripheral neuropathy (BIPN) in humans. Furthermore, while D2 overexpression was sufficient to cause axonopathy and inhibit mitochondria motility, reduction of D2 levels alleviated both axonal degeneration and the loss of mitochondria motility induced by Bort. Together, our data demonstrate that Bort, a compound structurally unrelated to tubulin poisons, affects the tubulin cytoskeleton in sensory neurons in vitro, in vivo, and in human tissue, indicating that the pathogenic mechanisms of seemingly unrelated CIPN drugs may converge on tubulin damage. The results reveal a previously unrecognized pathogenic role for D2 in BIPN that may occur through altered regulation of mitochondria motility.

Involvement of phospholipase PLA2 in production of cellulase and xylanase by Penicillium oxalicum.

Phospholipases play vital roles in immune and inflammatory responses in mammals and plants; however, knowledge of phospholipase functions in fungi is limited. In this study, we investigated the effects of deleting predicted phospholipase genes on cellulase and xylanase production, and morphological phenotype, in Penicillium oxalicum. Individual deletion of nine of the ten predicted phospholipase genes resulted in alteration of cellulase and xylanase production, and the morphological phenotypes, to various degrees. The mutant ∆POX07277 lost 22.5 to 82.8% of cellulase (i.e., filter paper cellulase, carboxymethylcellulase, and p-nitrophenyl-ß-cellobiosidase) and xylanase production, whereas p-nitrophenyl-ß-glucopyranosidase production increased by 5.8-127.8 fold. POX07277 (P. oxalicum gene No. 07277) was predicted to encode phospholipase A2 and was found to negatively affect the sporulation of P. oxalicum. Comparative transcriptomic and quantitative reverse transcription-PCR analysis indicated that POX07277 dynamically affected the expression of cellulase and xylanase genes and the regulatory genes for fungal sporulation, under micro-crystalline cellulose induction. POX07277 was required for the expression of the known regulatory gene PoxCxrB (cellulolytic and xylanolytic regulator B in P. oxalicum), which is involved in cellulase and xylanase gene expression in P. oxalicum. Conversely, POX07277 expression was regulated by PoxCxrB. These findings will aid the understanding of phospholipase functions and provide novel insights into the mechanism of fungal cellulase and xylanase gene expression. KEY POINTS : • The roles of phospholipases were investigated in Penicillium oxalicum. • POX07277 (PLA2) is required for the expression of cellulase and xylanase genes. • PoxCxrB dynamically regulated POX07277 expression.

The effects of laser stimulation at acupoint ST36 on anxiety-like behaviors and anterior cingulate cortex c-Fos expression in a rat post-traumatic stress disorder model.

Post-traumatic stress disorder (PTSD) is a mental disorder that is linked with the onset of multiple anxiety-like behaviors. This study was designed to assess how these behaviors and anterior cingulate cortex (ACC) c-Fos expression were impacted by 10.6-µm laser stimulation at acupoint ST36 a rat model of PTSD. A rat model of PTSD was prepared via prolonged exposure of animals to a stressor, followed by a 7-day period during which animals were allowed to rest undisturbed in their cages. Rats were randomized into four experimental groups (n = 12/group): the control, PTSD, LS, and sham LS groups. Control group animals were not subjected to SPS procedures prior to behavioral testing. LS and sham LS animals were administered LS treatment at bilateral ST36 acupoints or non-acupoints, respectively, for a 7-day period. Animals were then assessed for performance in elevated plus maze (EPM) tests and open-field tests (OFT), and their plasma corticosterone levels were measured. In addition, c-Fos-positive nuclei in the ACC were detected via immunohistochemical staining. Relative to sham LS treatment and PTSD model control rats, LS was associated with increased time spent in both open EPM test arms and in the central area in the OFT (P < 0.05). The PTSD model group exhibited a significant reduction in ACC c-Fox expression, while LS treatment significantly increased this expression (P < 0.001). In addition, a correlation was detected between anxiety-like behaviors and altered ACC neuronal activation. The results of this study indicate that LS at acupoint ST36 can have a previously unreported effect on anxiety-like behaviors in the context of PTSD, with ACC neuronal activation potentially being implicated as a driver of this effect.

Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton.

Small molecule inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biological models and not requiring genetic engineering. However, traditional chemical inhibitors cannot be experimentally applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesised photoswitchable paclitaxel-based microtubule stabilisers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biological response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilisers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiology.

Characterization of a GH3 halophilic ß-glucosidase from Pseudoalteromonas and its NaCl-induced activity toward isoflavones.

A novel ß-glucosidase gene was isolated from Pseudoalteromonas sp. GXQ-1 and heterologously expressed in Escherichia coli. The activity of the encoded enzyme, PABGL, toward p-nitrophenyl-ß-D-glucopyranoside was increased 8.74-fold by the presence of 3 M NaCl relative to the absence of added NaCl. PABGL hydrolyzed a variety of soy isoflavone substrates. For the conversion of daidzin to daidzein, the production rate was 1.44 mM/h. The addition of NaCl enhanced the hydrolytic activity of PABGL toward daidzin and genistein; the maximum activation by NaCl was 3.48- and 6.79-fold, respectively. This is the first report of a halophilic ß-glucosidase from Pseudoalteromonas spp., and represents the ß-glucosidase with the highest multiple of activation by NaCl. PABGL exhibits strong potential for applications in food processing and industrial production.

miR-296-5p inhibits IL-1ß-induced apoptosis and cartilage degradation in human chondrocytes by directly targeting TGF-ß1/CTGF/p38MAPK pathway.

Osteoarthritis (OA) is characterized by apoptosis of chondrocytes and an imbalance of extracellular matrix (ECM) synthesis and catabolism. Emerging evidence has demonstrated that miRNAs are involved in OA pathologies, but the role of miR-296-5p in OA remains unclear. The present study proposes to reveal the functions and mechanisms of miR-296-5p in a cell model of OA. In this study, human chondrocytes were treated with 5 ml interleukin-1 beta (IL-1ß) to induce apoptosis and cartilage degradation. Our results showed that miR-296-5p was downregulated in chondrocytes stimulated with IL-1ß. Overexpressed miR-296-5p enhanced cell proliferation and inhibited apoptosis and matrix degrading enzyme expression in response to IL-1ß stimulation, and knockdown of miR-296-5p showed the opposite effect. Further, we found that miR-296-5p directly targeted the 3'-untranslated region (3'-UTR) of TGF-ß1 mRNA, and miR-296-5p inactivated the TGF-ß1/CTGF/p38MAPK signaling pathway. Overexpression of TGF-ß1 alleviated the inhibition of miR-296-5p on chondrocyte apoptosis and cartilage degradation. In conclusion, miR-296-5p inhibited the progression of OA through the CTGF/p38MAPK pathway by directly targeting TGF-ß1.

Approach Behavior Induced by 10.6-µm Laser Stimulation at Acupoint ST36 in a Rat Model of Incisional Pain.

Objective: Laser stimulation (LS) at both the injury site and specific acupoints may induce analgesic effects. The purpose of this study is to investigate the effects of LS at injury site or acupoint on analgesic-associated approach behavior and determine whether opioid receptors in the anterior cingulate cortex (ACC) were involved. Methods: The left hindpaw incision was established in rats. LS (10.6 µm) was performed at the ipsilateral (left) acupoint ST36 (Zusanli) or locally to the incision site. Characteristic guarding pain behavior was measured to assess incision-induced pain. A two-chamber conditioned place preference (CPP) paradigm was used to measure approach behavior induced by pain relief. To inhibit opioid receptors, naloxone was microinjected into the ACC before LS. Results: A delayed analgesic effect (24 h after treatment) was induced in both the LS groups (ST36 and incision site) as compared with the sham control or model groups (p < 0.05). An immediate (30 min after the end of the LS) decrease in guarding pain (p < 0.001) and CPP for the LS chamber (p < 0.001) were observed only in the ST36 LS group. The administration of naloxone in ACC inhibited the LS-induced analgesic effect and CPP (p < 0.05). Conclusions: Our results highlight the novel approach behavior of pain relief induced by 10.6-µm LS at ST36 in a rat model of incisional pain, and implicate ACC opioid receptor signaling in these actions.

Laser photobiomodulation for cartilage defect in animal models of knee osteoarthritis: a systematic review and meta-analysis.

To review and assess the efficacy of laser photobiomodulation for cartilage defect in animal models of knee osteoarthritis (KOA). Medline, Web of Science, and EMBASE were searched. Studies were considered if the global quality score of cartilage were parallelly reported between laser and untreated control groups. The methodological quality of each study was assessed using a modified 10-item checklist. The effect size was estimated by standardized mean difference (SMD) and pooled based on the random-effects model. Stratified analysis and regression analysis were conducted to partition potential heterogeneity. An adjusted significant level of 0.01 was acceptable. Five hundred eight initial search recordings were identified, of which 14 studies (including 274 animals) were included for quantitative analysis. The global quality scores mostly weighted by the structural integrity and chondrocyte distribution were measured by different four scales including Histologic Histochemical Grading System (HHGS), Osteoarthritis Research Society International (OARSI), Pineda, and Huang. There were considerable variances on laser parameters and irradiation time among those included studies. Overall, a moderate level of methodological qualities was determined. The synthesis results indicated that the SMD effect size was significantly larger in HHGS (z = 2.61, P = 0.01) and Huang (z = 4.90, P < 0.01) groups. Stratified by irradiance, SMD of low (< 1 W/cm2) but not high (≥ 1 W/cm2) level estimated significant difference (z = 5.62, P < 0.01). Meta-regression identified a significant association for SMDs and irradiation time (P < 0.01). Yet, Egger's test detected small study effect (P < 0.01). No individual study with significant variance was found in homogeneity tests. The results demonstrated the positive effect of laser photobiomodulation for cartilage defect in animal models of KOA under proper irradiance and adequate irradiation time.

Activity-Dependent Nucleation of Dynamic Microtubules at Presynaptic Boutons Controls Neurotransmission.

Control of microtubule (MT) nucleation and dynamics is critical for neuronal function. Whether MT nucleation is regulated at presynaptic boutons and influences overall presynaptic activity remains unknown. By visualizing MT plus-end dynamics at individual excitatory en passant boutons in axons of cultured hippocampal neurons and in hippocampal slices expressing EB3-EGFP and vGlut1-mCherry, we found that dynamic MTs preferentially grow from presynaptic boutons, show biased directionality in that they are almost always oriented toward the distal tip of the axon, and can be induced by neuronal activity. Silencing of γ-tubulin expression reduced presynaptic MT nucleation, and depletion of either HAUS1 or HAUS7-augmin subunits increased the percentage of retrograde comets initiated at boutons, indicating that γ-tubulin and augmin are required for activity-dependent de novo nucleation of uniformly distally oriented dynamic MTs. We analyzed the dynamics of a wide range of axonal organelles as well as synaptic vesicles (SVs) relative to vGlut1+ stable presynaptic boutons in a time window during which MT nucleation at boutons is promoted upon induction of neuronal activity, and we found that γ-tubulin-dependent presynaptic MT nucleation controls bidirectional (SV) interbouton transport and regulates evoked SV exocytosis. Hence, en passant boutons act as hotspots for activity-dependent de novo MT nucleation, which controls neurotransmission by providing dynamic tracks for bidirectional delivery of SVs between sites of neurotransmitter release.

Temporal effect of electroacupuncture on anxiety-like behaviors and c-Fos expression in the anterior cingulate cortex in a rat model of post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a psychiatric disease which leads to a series of anxiety-like behaviors. In this study, we investigated the temporal effects of electroacupuncture (EA) at acupoint ST36 on anxiety-like behaviors and the expression of c-Fos in the anterior cingulate cortex (ACC) in a rat model of PTSD. PTSD was induced by a single prolonged stress procedure comprising three stages: restraint for 2 h, forced swim for 20 min, and pentobarbital sodium anesthesia. EA at acupoint ST36 was performed from 7:00-9:00 once a day for 7 consecutive days. Open field test (OFT) and elevated plus maze (EPM) test were used to assess the success of the model and evaluate anxiety-like behaviors. Immunohistochemistry was used to detect Fos-positive nuclei in the ACC. We observed that EA performed from 7:00-9:00 was associated with significantly more time spent in the center area during the OFT and in the open arm during the EPM, as well as lower corticosterone response compared with that of regular EA (P < 0.05). PTSD rats expressed significantly less c-Fos in the ACC. Timed EA significantly increased c-Fos expression in the ACC. The effect of timed EA acting on PTSD rats was linked to altered neuronal activation in the ACC. Compared to regular EA, timed EA exhibited superior therapeutic effects by attenuating anxiety-like behaviors in PTSD rats. These results emphasize the association between temporal parameters of EA manipulation and acupuncture effects. Timed acupuncture therapy may be a novel therapeutic application in the treatment of PTSD.

Highly specific sophorose ß-glucosidase from Sphingomonas elodea ATCC 31461 for the efficient conversion of stevioside to rubusoside.

Enzyme specificity and particularity is needed not only in enzymatic separation methods, but also in enzymatic determination methods for plant compound extraction. Stevioside, rubusoside, and rebaudioside A are natural sweet compounds from plants. These compounds have the same skeleton and only contain different side-chain glucosyl groups, making them difficult to separate. However, enzymes that target diterpenoid compounds and show specific activity for side-chain glucosyl groups are rare. Herein, we report the identification and characterization of an enzyme that can target both diterpenoid compounds and sophorose, namely, ß-glucosidase SPBGL1 from Sphingomonas elodea ATCC 31461. SPBGL1 displayed high specificity toward sophorose, and activity toward stevioside, but not rebaudioside A. The stevioside conversion rate was 98%. SPBGL1 also operated at high substrate concentrations, such as in 50% crude steviol glycoside extract. Glucose liberated from stevioside was easy to quantify using the glucose oxidase method, allowing the stevioside content to be determined.