Microglia-containing human brain organoids for the study of brain development and pathology.

Microglia are resident immune cells in the central nervous system, playing critical roles in brain development and homeostasis. Increasing evidence has implicated microglia dysfunction in the pathogenesis of various brain disorders ranging from psychiatric disorders to neurodegenerative diseases. Using a human cell-based model to illuminate the functional mechanisms of microglia will promote pathological studies and drug development. The recently developed microglia-containing human brain organoids (MC-HBOs), in-vitro three-dimensional cell cultures that recapitulate key features of the human brain, have provided a new avenue to model brain development and pathology. However, MC-HBOs generated from different methods differ in the origin, proportion, and fidelity of microglia within the organoids, and may have produced inconsistent results. To help researchers to develop a robust and reproducible model that recapitulates in-vivo signatures of human microglia to study brain development and pathology, this review summarized the current methods used to generate MC-HBOs and provided opinions on the use of MC-HBOs for disease modeling and functional studies.

Construction and validation of prognostic models in critically Ill patients with sepsis-associated acute kidney injury: interpretable machine learning approach.

BACKGROUND: Acute kidney injury (AKI) is a common complication in critically ill patients with sepsis and is often associated with a poor prognosis. We aimed to construct and validate an interpretable prognostic prediction model for patients with sepsis-associated AKI (S-AKI) using machine learning (ML) methods. METHODS: Data on the training cohort were collected from the Medical Information Mart for Intensive Care IV database version 2.2 to build the model, and data of patients were extracted from Hangzhou First People's Hospital Affiliated to Zhejiang University School of Medicine for external validation of model. Predictors of mortality were identified using Recursive Feature Elimination (RFE). Then, random forest, extreme gradient boosting (XGBoost), multilayer perceptron classifier, support vector classifier, and logistic regression were used to establish a prognosis prediction model for 7, 14, and 28 days after intensive care unit (ICU) admission, respectively. Prediction performance was assessed using the receiver operating characteristic (ROC) curve and decision curve analysis (DCA). SHapley Additive exPlanations (SHAP) were used to interpret the ML models. RESULTS: In total, 2599 patients with S-AKI were included in the analysis. Forty variables were selected for the model development. According to the areas under the ROC curve (AUC) and DCA results for the training cohort, XGBoost model exhibited excellent performance with F1 Score of 0.847, 0.715, 0.765 and AUC (95% CI) of 0.91 (0.90, 0.92), 0.78 (0.76, 0.80), and 0.83 (0.81, 0.85) in 7 days, 14 days and 28 days group, respectively. It also demonstrated excellent discrimination in the external validation cohort. Its AUC (95% CI) was 0.81 (0.79, 0.83), 0.75 (0.73, 0.77), 0.79 (0.77, 0.81) in 7 days, 14 days and 28 days group, respectively. SHAP-based summary plot and force plot were used to interpret the XGBoost model globally and locally. CONCLUSIONS: ML is a reliable tool for predicting the prognosis of patients with S-AKI. SHAP methods were used to explain intrinsic information of the XGBoost model, which may prove clinically useful and help clinicians tailor precise management.

Taxonomy and SSU rRNA gene-based phylogeny of two new Euplotes species from China: E. chongmingensis n. sp. and E. paramieti n. sp. (Protista, Ciliophora).

BACKGROUND: The genus Euplotes Ehrenberg, 1830, one of the most complicated and confused taxa, contains about 160 nominal species. It was once proposed to be divided into four genera, two of which were proved to be non-monophyletic. At least 19 new species have been discovered in the past decade, implying that there is a large undiscovered diversity of this genus. RESULTS: The morphology of two new freshwater euplotid ciliates, Euplotes chongmingensis n. sp. and E. paramieti n. sp., isolated from Shanghai, China, were investigated using live observations, protargol staining, and Chatton-Lwoff silver staining method. Euplotes chongmingensis is characterized by its small size (40-50 × 25-35 µm), about 24 adoral membranelles, 10 frontoventral cirri, two marginal and two caudal cirri, eight dorsolateral kineties with 11-16 dikinetids in the mid-dorsolateral kinety and a double type of silverline system. Euplotes paramieti n. sp. is 180-220 × 110-155 µm in vivo and strongly resembles E. amieti but having a difference of 57 bp in their SSU rRNA gene sequences. Phylogenetic analyses based on SSU rRNA gene sequence data were used to determine the systematic positions of these new taxa. CONCLUSIONS: The description of two new freshwater taxa and their SSU rRNA gene sequences improve knowledge of biodiversity and enrich the database of euplotids. Furthermore, it offers a reliable reference for environmental monitoring and resource investigations.

miR-146a-5p Attenuates Allergic Airway Inflammation by Inhibiting the NLRP3 Inflammasome Activation in Macrophages.

OBJECTIVE: Asthma is a common inflammatory respiratory disease with increasing incidence worldwide. This study aimed to investigate the mechanism of miR-146a-5p in reducing allergic airway inflammation by inhibiting NLRP3 inflammasome activation in macrophages. METHODS: Allergic mouse models were established by ovalbumin stimulation, and mice were treated with miR-146a-5p agomir and oe-TIRAP 3 h before OVA stimulation. The pathological changes of lung tissues were observed by hematoxylin-eosin staining. The airway hyperresponsiveness of mice were examined. The miR-146a-5p level was detected by RT-qPCR. The inflammatory cytokines (IL-18/TNF-α) and anti-inflammatory cytokine IL-10 levels in bronchoalveolar lavage fluid and serum IgE levels were examined by ELISA. Airway inflammation in mice was detected after miR-146a-5p overexpression. The levels of NLRP3/ASC/caspase1 proteins and macrophage M1/M2 surface markers in mouse lung tissues were examined using immunohistochemistry, Western blot, and flow cytometry. The targeting relationship between miR-146a-5p and TIRAP was verified by dual-luciferase assay. The p65 levels in the cytoplasm/nucleus of mouse lung tissue were measured. RESULTS: miR-146a-5p was downregulated in the lung tissues of allergic mice, and miR-146a-5p overexpression alleviated airway inflammation in asthmatic mice. miR-146a-5p suppressed NLRP3 inflammasome activation in macrophages of allergic mice, reduced NLRP3/ASC/caspase1 protein levels in lung tissues, blocked M1 polarization, and promoted M2 polarization. miR-146a-5p targeted TIRAP. TIRAP overexpression partially reversed the promoting effect of miR-146a-5p on M2 polarization. miR-146a-5p can inhibit the activation of the TIRAP/NF-κB pathway. CONCLUSION: miR-146a-5p inhibited NLRP3 inflammasome activation in macrophages in the lung tissue of allergic mice, prevented pro-inflammatory phenotype M1 polarization, and promoted anti-inflammatory phenotype M2 polarization by targeting the TIRAP/NF-κB pathway, thus alleviating airway inflammation in allergic asthma.

Taxonomy and phylogeny of the freshwater tintinnid Tintinnopsis tubuformis Chiang, 1956 (Ciliophora, Oligotrichea) and a proposed synonymization of T. longa nom. corr. Chiang, 1956.

Tintinnid ciliates are traditionally identified by their loricae; however, increasing evidence indicates that some lorica features (e.g. its length, spiraled structures) are not reliable. The vast majority of tintinnids inhabit the marine pelagial; merely, about thirty species live in freshwater. In the present study, two morphotypes with similar lorica shapes and opening diameters but deviating lorica lengths were isolated from freshwater samples collected at different water temperatures near Chongming Island in the Yangtze Estuary, China. The specimens were studied in vivo and after protargol staining, and their phylogenetic placement was inferred from three ribosomal RNA markers; further, cell division was investigated in the short morphotype. Based on the original descriptions, the longer morphotype is identified as Tintinnopsis longa nom. corr. Chiang, 1956, and the shorter one as Tintinnopsis tubuformis Chiang, 1956. Despite distinct differences in the lorica lengths, the identity of the three molecular markers in both morphotypes suggests conspecificity, which is supported by overlapping ranges in the lorica opening diameters and the length-independent features of the somatic ciliary pattern (e.g. number of kineties). Hence, we synonymized T. longa nom. corr. with T. tubuformis and neotypified the later species.

Hydrogen sulfide prevents arecoline-induced neurotoxicity via promoting leptin/leptin receptor signaling pathway.

Arecoline, a major alkaloid of the areca nut, has potential toxicity to the nervous system. Our previous study reveals that the neurotoxicity of arecoline involves in inhibited endogenous hydrogen sulfide (H2 S) generation. Therefore, the present study investigated whether exogenous H2 S protects against arecoline-induced neurotoxicity and further explore the underlying mechanisms focusing on leptin/leptin receptor signaling pathway. The cell viability was measured by CCK-8 kit. The apoptosis were detected by Hoechst 33258 and Annexin V/PI (propidium iodide) staining. The protein expressions were determined by Western blot analysis. Our results demonstrated that NaHS, an exogenous H2 S donor, significantly increases the cell viability, decreases apoptosis ratio, and reduces caspase-3 activity as well as Bax/Bcl-2 ratio in PC12 cells exposed to arecoline, indicating the protection of H2 S against arecoline-induced cytotoxicity and apoptosis. Also, NaHS attenuated arecoline-induced endoplasmic reticulum (ER) stress, as evidenced by the decreases in the expressions of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and Cleaved caspase-12. Meanwhile, NaHS promoted leptin/leptin receptor signaling pathway in arecoline-exposed PC12 cells, as illustrated by upregulations of leptin and leptin receptor expressions. Furthermore, leptin tA, an antagonist of leptin receptor, obviously abolished the inhibitory effects of NaHS on arecoline-induced cytotoxicity, apoptosis, and ER stress in arecoline-exposed PC12 cells. Taken together, these results suggested that H2 S prevents arecoline-induced neurotoxicity via enhancing the leptin/leptin receptor signaling pathway.

Hydrogen sulphide attenuates neuronal apoptosis of substantia nigra by re-establishing autophagic flux via promoting leptin signalling in a 6-hydroxydopamine rat model of Parkinson's disease.

Previous studies reveal that hydrogen sulphide (H2 S) exerts neuroprotection against neurotoxin-induced Parkinson's disease (PD), but the underlying mechanism remains elusive. The present study was aimed to investigate whether H2 S inhibits neuronal apoptosis of substantia nigra with the involvement of autophagy via promoting leptin signalling in 6-hydroxydopamine (6-OHDA)-induced PD rats. In this study, neuronal apoptosis was analysed by TUNEL staining, the activity of caspase-3 was measured by Caspase-3 fluorometric assay kit, the expressions of Bax, Bcl-2, Beclin-1, LC3II, P62 and leptin were determined by Western blot analysis, and the numbers of autophagosomes and autolysosomes were assessed by transmission electron microscopy. Results showed that NaHS, a donor of exogenous H2 S, mitigates 6-OHDA-induced the increases in the numbers of TUNEL-positive cells, the activity of caspase-3 and the expression of Bax, and attenuates 6-OHDA-induced a decrease in the expression of Bcl-2 in substantia nigra of rats. In addition, 6-OHDA enhanced the expressions of Beclin-1, LC3-II and P62, increased the number of autophagosomes, and decreased the number of autolysosomes in the substantia nigra, which were also blocked by administration of NaHS. Furthermore, NaHS reversed 6-OHDA-induced the down-regulation of leptin expression in the substantia nigra, and treatment with leptin-OBR, a blocking antibody of leptin receptor, attenuated the inhibition of NaHS on neuronal apoptosis and the improvement of NaHS on the blocked autophagic flux in substantia nigra of 6-OHDA-treated rats. Taken together, these results demonstrated that H2 S attenuates neuronal apoptosis of substantia nigra depending on restoring impaired autophagic flux through up-regulating leptin signalling in PD.

SIRT1 Mediates H2S-Ameliorated Diabetes-Associated Cognitive Dysfunction in Rats: Possible Involvement of Inhibiting Hippocampal Endoplasmic Reticulum Stress and Synaptic Dysfunction.

Diabetes-associated cognitive dysfunction (DACD) characterized by hippocampal injury increases the risk of major cerebrovascular events and death. Endoplasmic reticulum (ER) stress and synaptic dysfunction play vital roles in the pathological process. At present, no specific treatment exists for the prevention and/or the therapy of DACD. We have recently reported that hydrogen sulfide (H2S) exhibits therapeutic potential for DACD, but the underlying mechanism has not been fully elucidated. Silent information regulator 1 (SIRT1) has been shown to play a role in regulating the progression of diabetes and is also indispensable for memory formation and cognitive performance. Hence, the present study was performed to explore whether SIRT1 mediates the protective effect of H2S on streptozotocin (STZ)-induced cognitive deficits, an in vivo rat model of DACD, via inhibiting hippocampal ER stress and synaptic dysfunction. The results showed that administration of NaHS (an exogenous H2S donor) increased the expression of SIRT1 in the hippocampus of STZ-induced diabetic rats. Then, results proved that sirtinol, a special blocker of SIRT1, abrogated the inhibition of NaHS on STZ-induced cognitive deficits, as appraised by Morris water maze test, Y-maze test, and Novel object recognition behavioral test. In addition, administration of NaHS eliminated STZ-induced ER stress as evidenced by the decreases in the expressions of ER stress-related proteins including glucose-regulated protein 78, C/EBP homologous protein, and cleaved caspase-12 in the hippocampus, while these effects of NaHS were also reverted by sirtinol. Furthermore, the NaHS-induced up-regulation of hippocampal synapse-related protein (synapsin-1, SYN1) expression in STZ-induced diabetic rats was also abolished by sirtinol. Taken together, these results demonstrated that SIRT1 mediates the protection of H2S against cognitive dysfunction in STZ-diabetic rats partly via inhibiting hippocampal ER stress and synaptic dysfunction.

Formaldehyde induces ferritinophagy to damage hippocampal neuronal cells.

Formaldehyde (FA) causes neurotoxicity and contributes to the occurrence of neurodegenerative diseases. However, the mechanism of FA-induced neurotoxicity has not been fully elucidated. Ferritinophagy, an autophagy process of ferritin mediated by the nuclear receptor coactivator 4 (NCOA4), is a potential mechanism of neurotoxicity. In this study, we explored whether ferritinophagy is associated with the neurotoxicity of FA. Our results showed that FA (50, 100, 200 µM; 24 h) exposure upregulated ferritinophagy in the mouse hippocampal neuronal HT22 cells, which was evidenced by the upregulated autophagic flux, the increased colocalizations of NCOA4 with ferritin heavy chain (FTH1) and NCOA4 with microtubule-associated protein 1 light chain-3B (LC3B), the augmented expression of NCOA4, and the reduced content of FTH1. We also found that FA (0.1, 1, and 10 µmol, i.c.v., 7d) administration boosted ferritinophagy in the hippocampus of Sprague-Dawley (SD) rats, which was demonstrated by the accumulated autophagosomes, the increased expressions of LC3II/I and NCOA4, and the decreased contents of p62 and FTH1 in the hippocampus. Further, we confirmed that inhibition of ferritinophagy by silencing the expression of NCOA4 decreased FA-induced toxic damage in HT22 cells. These results indicated that FA induces neurotoxicity by promoting ferritinophagy. Our findings suggest a potential mechanism insight into the FA-induced neurotoxicity, which in turn provides a new thought for the treatment of FA-related neurodegenerative diseases.

Description and Morphogenesis of a Chinese Population of Bakuella granulifera Foissner, Agatha and Berger, 2002 (Ciliophora, Hypotricha).

In this study, the morphology and morphogenesis of Bakuella granulifera Foissner, Agatha, and Berger, 2002, isolated from soil in the Chanba National Wetland Park, Xi'an, China, were studied based on in vivo observations and protargol staining. The Chinese population is distinctly smaller than the type population from Namibia. Details of the ontogenetic process are provided for the first time and are summarized as follows: (1) the parental adoral zone is partly retained; (2) the left of the endoral forms a rather wide anlage and the proximal adoral membranelles are renewed; (3) the parental midventral complex contributes to the formation of the FVT cirral anlagen; (4) the marginal rows anlagen and dorsal kineties anlagen develop intrakinetally; (5) no caudal cirri are produced; and (6) the macronuclear nodules fuse to a single mass. In combination with previous studies, these findings suggest that the genus Bakuella is ontogenetically diverse.

Morphology and Phylogeny of Two Novel Pleurostomatids (Ciliophora, Litostomatea), Establishing a New Genus.

Pleurostomatida Schewiakoff, 1896 is a cosmopolitan order of ciliates. In the present study, we investigated two new pleurostomatid species, Apolitonotus lynni gen. et sp. nov. and Protolitonotus clampi sp. nov., with state-of-the-art methods. Apolitonotus lynni lacks its oral extrusomes and its right kineties form an anterior semi-suture near the dorsal margin. Based on these two features, the new genus Apolitonotus was established within the Protolitonotidae Wu et al., 2017. Protolitonotus clampi differs from its congeners by its size of 80-130 × 15-30 µm, 4-6 left, and 9-11 right kineties, extrusomes arranged along the oral slit, and two macronuclear nodules. Because Litonotus antarcticus possesses an anterior semi-suture and oral extrusomes, it was transferred to the genus Protolitonotus, becoming P. antarctius comb. nov. (basionym Litonotus antarcticus Song and Wilbert, 2002). Phylogenetic analyses based on SSU rDNA sequences suggest a sister group relationship of P. clampi and the family Kentrophyllidae, and A. lynni is adelphotaxon to Litonotus gracilis, both within the order Pleurostomatida. Based on the new findings, an improved diagnosis for Protolitonotus was also provided.

Protective Effect of miR-340-5p against Brain Injury after Intracerebral Hemorrhage by Targeting PDCD4.

OBJECTIVE: Intracerebral hemorrhage (ICH) is a common cerebrovascular disease. Increasing evidence has documented the crucial role of microRNAs in ICH. The present study aimed to investigate the role and underlying mechanism of miR-340-5p in ICH. METHODS: The collagenase-induced ICH rat model was established. The neurological function of rats and the cerebral water content of rat brain tissue were measured to assess the brain injury. BV-2 cells were recruited and treated by LPS to mimic ICH-induced inflammatory response. qRT-PCR was used for the measurement of miR-340-5p. The protein levels of TNF-α, IL-6, and IL-1ß were detected using ELISA. Luciferase reporter gene assay was performed to confirm the target gene. RESULTS: Downregulation of miR-340-5p was detected in the serum of ICH patients and the brain tissues of ICH rats. Overexpression of miR-340-5p reversed the influence of ICH on the neurological function score and cerebral water content and inhibited the production of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß), which were induced by ICH in vivo. In in vitro study, levels of TNF-α, IL-6, and IL-1ß were significantly enhanced in cells after LPS treatment, but these increases were eliminated by overexpression of miR-340-5p. PDCD4 was a direct target gene of miR-340-5p. CONCLUSION: miR-340-5p protects against brain injury after ICH. miR-340-5p might exert an anti-inflammatory effect during the occurrence of ICH via targeting PDCD4.

Molecular systematics and phylogenetic analysis of the Asian endemic freshwater sleepers (Gobiiformes: Odontobutidae).

The Odontobutidae is a group of freshwater sleepers endemic to East and Southeast Asia. The composition of the Odontobutidae is controversial and the systematics position of some species (e.g. Philypnus chalmersi) remains unknown. Phylogenetic relationship among the odontobutids has never been really tested due to the lack of informative morphological characters, and that molecular data have not been collected in many species. Here, we sampled 41 specimens, representing all known genera of the Odontobutidae except the Laotian genus Terateleotris, in addition to a disputable odontobutid species, Philypnus chalmersi and 14 outgroups (six families). We collected sequence data of 4434 single-copy nuclear coding loci using gene capture and Illumina sequencing. A robust phylogeny of the odontobutids and outgroups was built, confirming that the Odontobutidae is monophyletic and sister to the Rhyacichthyidae. We verified that Neodontobutis, Sineleotris and Philypnus chalmersi are members of the Odontobutidae based on the resulting phylogeny as well as patterns of pectoral girdle examined by X-ray microtomography. We proposed a new genus Microdous for Philypnus chalmersi based on the new morphological and molecular evidences. The family of the Odontobutidae can be divided into two clades: Microdous (=Philypnus) sister to a group consisting of Micropercops and Sineleotris, and Odontobutis sister to a group unifying Perccottus and Neodontobutis. Divergence time among the odontobutids was estimated based on 100 most clock-like loci and three fossil calibration points using BEAST. Ancestral range of the family was reconstructed using Reconstruct Ancestral States in Phylogenies (RASP) and BioGeoBEARS. The results suggest that the common ancestor of the odontobutids originated around 30.8 Ma (20.7-42.0 Ma, 95% HPDs) in South China. Orogeny, climatic change and river capture might account for diversification and current distribution of the odontobutids.

Phylogenomic analysis on the exceptionally diverse fish clade Gobioidei (Actinopterygii: Gobiiformes) and data-filtering based on molecular clocklikeness.

The use of genome-scale data to infer phylogenetic relationships has gained in popularity in recent years due to the progress made in target-gene capture and sequencing techniques. Data filtering, the approach of excluding data inconsistent with the model from analyses, presumably could alleviate problems caused by systematic errors in phylogenetic inference. Different data filtering criteria, such as those based on evolutionary rate and molecular clocklikeness as well as others have been proposed for selecting useful phylogenetic markers, yet few studies have tested these criteria using phylogenomic data. We developed a novel set of single-copy nuclear coding markers to capture thousands of target genes in gobioid fishes, a species-rich lineages of vertebrates, and tested the effects of data-filtering methods based on substitution rate and molecular clocklikeness while attempting to control for the compounding effects of missing data and variation in locus length. We found that molecular clocklikeness was a better predictor than overall substitution rate for phylogenetic usefulness of molecular markers in our study. In addition, when the 100 best ranked loci for our predictors were concatenated and analyzed using maximum likelihood, or combined in a coalescent-based species-tree analysis, the resulting trees showed a well-resolved topology of Gobioidei that mostly agrees with previous studies. However, trees generated from the 100 least clocklike frequently recovered conflicting, and in some cases clearly erroneous topologies with strong support, thus indicating strong systematic biases in those datasets. Collectively these results suggest that data filtering has the potential improve the performance of phylogenetic inference when using both a concatenation approach as well as methods that rely on input from individual gene trees (i.e. coalescent species-tree approaches), which may be preferred in scenarios where incomplete lineage sorting is likely to be an issue.

Morphology and Molecular Phylogeny of Two Poorly Known Species of Protocruzia (Ciliophora: Protocruziida).

The ciliate genus Protocruzia is a highly confused group, which was formerly placed in the class Heterotrichea or Karyorelictea, and is according to the most recent system tentatively assigned to the class Spirotrichea. In the present study, the morphology, ciliary pattern, and molecular phylogeny of two poorly known species, Protocruzia tuzeti Villeneuve-Brachon, 1940, and Protocruzia granulosa Kahl, 1933, isolated from coastal waters of China, were investigated. Protocruzia tuzeti differs from its congeners mainly in possessing 6 adoral membranelles, 8-11 somatic kineties, and postoral dikinetids. Protocruzia granulosa is characterized by its extremely slender body, three postoral kineties, and 13 or 14 somatic kineties. The morphogenesis of P. granulosa is similar to that of P. tuzeti, especially in the parakinetal mode of stomatogenesis and the reorganization of the parental paroral membrane; however, more than one somatic kinety joins in the formation of the oral primordium in P. granulosa. Phylogenetic analyses based on small subunit ribosomal RNA gene revealed that six Protocruzia species form a fully supported clade that does not belong to any ciliate class; therefore, our data support the establishment of the class Protocruziea Gao et al. (Sci. Rep., 6, 2016, 24874).

Arecoline Induces Neurotoxicity to PC12 Cells: Involvement in ER Stress and Disturbance of Endogenous H2S Generation.

Arecoline is a major alkaloid of areca nut and has been effect on central nervous system. Although arecoline-induced neurotoxicity has been reported, the possible underlying neurotoxic mechanisms have not yet been elucidated. Increasing evidences have shown that both excessive endoplasmic reticulum (ER) stress and disturbance of hydrogen sulfide (H2S) production are involved in the pathophysiology of numerous neurodegenerative diseases. Here, the purpose of present study was to verify whether ER stress and the disturbance of endogenous H2S generation are also involved in arecoline-caused neurotoxicity. We found that treatment of PC12 cells with arecoline induced the down-regulation of cells viability and up-regulation of apoptosis and the activity of caspase-3, indicating the neurotoxic role of arecoline to PC12 cells. In addition, arecoline also increased the expression of Bax (pro-apoptotic protein) and attenuated the expression of Bcl-2 (anti-apoptotic protein) in PC12 cells. Simultaneously, arecoline caused excessive ER stress in PC12 cells, as evidenced by the up-regulations of Glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP), and Cleaved caspase-12 expressions. Notably, the level of H2S in the culture supernatant and the expressions of cystathionine ß-synthase and 3-mercaptopyruvate sulfurtransferase (two major enzymes for endogenous H2S generation in PC12 cells) were also reduced by arecoline treatment. These results indicate that arecoline-caused neurotoxicity to PC12 cells is involved in ER stress and disturbance of endogenous H2S generation and suggest that the modulation of ER stress and endogenous H2S generation may be potential therapeutic approach in treatment of arecoline-caused neurotoxicity.

Morphology and Phylogeny of Three Pleuronema Species (Ciliophora, Scuticociliatia) from Hangzhou Bay, China, with Description of Two New Species, P. binucleatum n. sp. and P. parawiackowskii n. sp.

The morphology and phylogeny of Pleuronema binucleatum n. sp., P. parawiackowskii n. sp., and P. marinum Dujardin 1841, collected from Hangzhou Bay estuary, China, were investigated using standard methods. Pleuronema binucleatum n. sp. can be identified by possessing about 90-120 × 35-50 µm cell size in vivo, reniform body outline, two macronuclei, six to eight preoral kineties, 32-41 somatic kineties, and posterior end of the anterior fragment of membranelle 2 (M2a) hook-like. Pleuronema parawiackowskii n. sp. is characterized by the combination of the following characters: body size about 60-90 × 20-40 µm in vivo, elliptical in outline, four to eight preoral kineties, 20-29 somatic kineties, posterior portion of the M2a slightly curved but nonhooked, and single macronucleus sausage-like. After comparison with other populations of P. marinum, it is suggested that many misidentifications exist in previous studies. And an improved diagnosis of P. marinum was supplied: cell about 95-180 µm long, elliptical in outline; 2-4 preoral kineties and 53-70 somatic kineties; both membranelle 1 and membranelle 3 three-rowed; posterior end of the M2a straight; single contractile vacuole characteristically positioned near mid-body. The small subunit rRNA genes of three forms were sequenced. Phylogenetic analyses indicate that the monophyly of the genus Pleuronema is still not supported.

Description of the Halophile Euplotes qatarensis nov. spec. (Ciliophora, Spirotrichea, Euplotida) Isolated from the Hypersaline Khor Al-Adaid Lagoon in Qatar.

The morphology, ontogenesis, and phylogenetic relationships of a halophile euplotid ciliates, Euplotes qatarensis nov. spec., isolated from the Khor Al-Adaid Lagoon in Qatar were investigated based on live observation as well as protargol- and silver nitrate-impregnated methods. The new species is characterised by a combination of features: the halophile habitat, a cell size of 50-65 × 33-40 µm, seven dorsal ridges, 10 commonly sized frontoventral cirri, two widely spaced marginal cirri, 10 dorsolateral kineties, and a double silverline pattern. The morphogenesis is similar to that of its congeners: (i) the oral primordium develops hypoapokinetally and the parental oral apparatus is retained; (ii) the frontoventral-transverse field of five streaks gives rise to the frontal, ventral, and transverse cirri, but not to the cirri I/1 and the marginal cirri; (iii) the dorsal somatic ciliature develops by intrakinetal proliferation of basal bodies in two anlagen per kinety that are just anterior and posterior to the future division furrow; (iv) the caudal cirri are formed by the two rightmost dorsolateral kineties. The SSU rDNA sequence of E. qatarensis branches with full support in the Euplotopsis elegans-Euplotes nobilii-Euplotopsis raikovi clade. The closest related publicly available SSU rDNA sequence is the one of E. nobilii, with which E. qatarensis has 93.4% sequence similarity. Euplotes parawoodruffi Song & Bradbury, 1997 is transferred to the genus Euplotoides based on the absence of frontoventral cirrus VI/3.

Morphology and molecular phylogeny of Pleuronema orientale spec. nov. and Pleuronema paucisaetosum spec. nov. (Ciliophora, Scuticociliata) from Hangzhou Bay, China.

Two novel species, Pleuronema orientale spec. nov. and Pleuronema paucisaetosum spec. nov., isolated from coastal waters of Hangzhou Bay, China, were investigated with standard methods. Pleuronema orientale is characterized as follows: size in vivo 95-135 × 50-85 µm; usually one spherical macronucleus; 12-15 prolonged caudal cilia; two or three preoral kineties and 42-50 somatic kineties; membranelle 1 (M1) about 20 % of the anterior fragment of membranelle 2 (M2a) in length, consisting of three longitudinal rows of kinetosomes; posterior end of M2a hook-like; membranelle 3 (M3) three-rowed. Pleuronema paucisaetosum is characterized as follows: size in vivo about 55-85 × 25-55 µm; four or five preoral kineties and 21-23 somatic kineties; posterior end of M2a hook-like; M3 three-rowed. The small-subunit rRNA gene was sequenced for both species. Phylogenetic analyses revealed that P. orientale is most closely related to Pleuronema puytoraci and that P. paucisaetosum is sister to Pleuronema grolierei and Pleuronema setigerum (GenBank accession no. JX310015). With the inclusion of the two new sequences, the monophyly of the genus Pleuronema is not supported.

Disturbance of hippocampal H2S generation contributes to CUMS-induced depression-like behavior: involvement in endoplasmic reticulum stress of hippocampus.

The chronic unpredictable mild stress (CUMS) model is a widely used experimental model of depression. Exogenous stress-induced neuronal cell death in the hippocampus is closely associated with the pathogenesis of depression. Excessive and prolonged endoplasmic reticulum (ER) stress triggers cell death. Hydrogen sulfide (H2S), the third endogenous signaling gasotransmitter, plays an important role in brain functions as a neuromodulator and a neuroprotectant. We hypothesized that the disturbance of endogenous H2S generation and ER stress in the hippocampus might be involved in CUMS-induced depression-like behaviors. Thus, the present study focused on whether CUMS disturbs the generation of endogenous H2S and up-regulates ER stress in the hippocampus and whether exogenous H2S prevents CUMS-induced depressive-like behaviors. Results showed that CUMS-treated rats exhibit depression-like behavior and hippocampal ER stress responses including up-regulated levels of glucose-regulated protein 78, CCAAT/enhancer binding protein homologous protein, and cleaved caspase-12 expression, while the endogenous generation of H2S in the hippocampus is suppressed in CUMS-treated rats. Furthermore, exogenous H2S prevents CUMS-induced depression-like behavior. These data indicated that CUMS-induced depression-like behaviors are related to the disturbance of endogenous H2S generation and ER stress in the hippocampus and suggested that endogenous H2S and ER stress are novel treatment targets of depression.