Potential Implications of Angiotensin-converting Enzyme 2 Blockades on Neuroinflammation in SARS-CoV-2 Infection.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) has been reported as a portal for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Consequently, scientific strategies to combat coronavirus disease of 2019 (COVID-19) were targeted to arrest SARS-CoV-2 invasion by blocking ACE2. While blocking ACE2 appears a beneficial approach to treat COVID-19, clinical concerns have been raised primarily due to the various intrinsic roles of ACE2 in neurological functions. Selective reports indicate that angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) upregulate ACE2 levels. ACE2 metabolizes angiotensin II and several peptides, including apelin-13, neurotensin, kinetensin, dynorphin, (des-Arg9) bradykinin, and (Lys-des-Arg9)-bradykinin, which may elicit neuroprotective effects. Since ARBs and ACEIs upregulate ACE2, it may be hypothesized that patients with hypertension receiving ARBs and ACEIs may have higher expression of ACE2 and thus be at a greater risk of severe disease from the SARS-CoV-2 infections. However, recent clinical reports indicate the beneficial role of ARBs/ACEIs in reducing COVID-19 severity. Together, this warrants a further study of the effects of ACE2 blockades in hypertensive patients medicated with ARBs/ACEIs, and their consequential impact on neuronal health. However, the associations between their blockade and any neuroinflammation also warrant further research. OBJECTIVE: This review collates mechanistic insights into the dichotomous roles of ACE2 in SARSCoV- 2 invasion and neurometabolic functions and the possible impact of ACE2 blockade on neuroinflammation. CONCLUSION: It has been concluded that ACE2 blockade imposes neuroinflammation.

[PD-1 is associated with CD8(+)T lymphocyte dysfunction in patients with acute and chronic liver failure].

Objective: To explore whether peripheral blood CD8(+)T lymphocyte dysfunction is correlated with the programmed death receptor-1 (PD-1) expression in patients with acute-on-chronic liver failure (HBV-ACLF). Methods: Peripheral blood mononuclear cells (PBMC) were collected from patients with HBV-ACLF and healthy controls. CD8(+)T lymphocytes number and PD-1 expression condition in CD8(+)T lymphocytes were detected by flow cytometry. CD8(+)T lymphocytes isolated from peripheral blood of HBV-ACLF patients were further cultured in vitro. One group was added with PD-L1-IgG fusion protein (ACLF+PD-1 group), and the other group was added with IgG fusion protein (ACLF group). Proliferation ability (ki67), cell viability (CD69), and secretion ability of effector cytokines (IL-2, IFN-γ, TNF-α) were analyzed. Results: 30 cases with HBV-ACLF and healthy controls were enrolled. CD8(+)T lymphocytes absolute number was significantly lower in the peripheral blood of patients with ACLF group (333.88 ± 147.74)/µl than healthy controls (872.50 ± 206.64)/µl (P < 0.001). PD-1 expression in peripheral blood CD8(+)T lymphocytes were significantly increased in ACLF group (13.33% ± 2.52%), (P = 0.027) than healthy controls (7.02% ± 2.12%). In in vitro culture, compared with healthy controls, the peripheral blood CD8(+)T lymphocytes cell viability (CD69), proliferation ability (ki67) (all P ​​< 0.001), and the level of cytokine production (IL-2, IFN-γ, TNF-α) (all P < 0.05) were equally weakened in patients with ACLF group. Compared with ACLF group, CD8(+)T cell viability (CD69), proliferation ability (KI67) (all P < 0.05), and the level of cytokine production were weakened in ACLF+PD-1 group (all P < 0.05). Conclusion: HBV-ACLF patients have CD8(+)T lymphocyte dysfunction. Therefore, PD-1 may have correlation in the regulation of CD8(+)T lymphocyte dysfunction in ACLF patients.

[Transcranial sonographic characteristics of Parkinson's disease with symptoms of restless legs syndrome].

Objective: To evaluate the transcranial sonographic characteristics in patients with Parkinson's disease (PD) with symptoms of restless legs syndrome (RLS). Methods: Patients with diagnosis of definite PD from the Second Affiliated Hospital of Soochow University and 3 other participating hospitals between September 2018 and December 2019 were consecutively enrolled. Concurrent RLS symptoms were determined using Non-motor Symptoms Questionnaire. Transcranial sonography (TCS) and clinical assessments were performed during the same time and the related variables were compared between the two groups using t-test, non-parametric test, Chi-square test and Spearman correlation analysis, respectively. Results: Among 349 patients with PD, the prevalence of RLS symptoms was 22.6%. Compared to patients without RLS symptoms, those with RLS had longer disease duration (43.0 (24.0, 91.0) months vs 37.0 (20.0, 60.0) months, P<0.05) and higher Hoehn-Yahr stage (2.5 (2.0, 3.0) vs 2.0 (1.5, 2.5), P<0.01).TCS revealed that patients with RLS symptoms were more likely to have abnormality in the raphe nucleus (21.50% vs 7.78%, χ²=15.9, P<0.001) and increased third ventricle width ((6.22±1.97) mm vs (5.16±1.90) mm, P<0.001). No significant differences were found regarding parameters of substantia nigra. Conclusions: Concurrent RLS symptoms are common in PD patients. Abnormal echogenicity of raphe nucleus and increased third ventricle width could be characteristics of TCS in PD patients with RLS symptoms.

Mortality under early access to antiretroviral therapy vs. Eswatini's national standard of care: the MaxART clustered randomized stepped-wedge trial.

OBJECTIVES: Current WHO guidelines recommend the treatment of all HIV-infected individuals with antiretroviral therapy (ART) to improve survival and quality of life, and decrease infection of others. MaxART is the first implementation trial of this strategy embedded within a government-managed health system, and assesses mortality as a secondary outcome. Because primary findings strongly supported scale-up of the 'treat all' strategy (hereafter Treat All), this analysis examines mortality as an additional indicator of its impact. METHODS: MaxART was conducted in 14 Eswatinian health clinics through a clinic-based stepped-wedge design, by transitioning clinics from then-national standard of care (SoC) to the Treat All intervention. All-cause, disease-related, and HIV-related mortality were analysed using the Cox proportional hazards model, censoring SoC participants at clinic transition. Median follow-up time among study participants was 292 days. There were 36/2034 deaths in SoC (1.77%) and 49/1371 deaths in Treat All (3.57%). RESULTS: Between September 2014 and August 2017, 3405 participants were enrolled. In SoC and Treat All interventions, respectively, the multivariable-adjusted 12-month all-cause mortality rates were 1.42% [95% confidence interval (CI): 0.66-2.17] and 1.60% (95% CI: 0.78-2.40), disease-related mortality rates were 1.02% (95% CI: 0.40-1.64) and 1.10% (95% CI: 0.46-1.73), and HIV-related mortality rates were 1.03% (95% CI: 0.40-1.65) and 0.99% (95% CI: 0.40-1.58). Treat All had no impact on all-cause [hazard ratio (HR) = 1.12, 95% CI: 0.58-2.18, P = 0.73], disease-related (HR = 1.04, 95% CI: 0.52-2.11, P = 0.90), or HIV-related mortality (HR = 0.93, 95% CI: 0.46-1.87, P = 0.83). CONCLUSION: There was no immediate benefit of the Treat All strategy on mortality, nor evidence of harm. Longer follow-up of participants is needed to establish long-term consequences.

MiR-129-5p inhibits proliferation of gastric cancer cells through targeted inhibition on HMGB1 expression.

OBJECTIVE: The aim of this study was to investigate the effects of micro ribonucleic acid (miR)-129-5p on the proliferation and apoptosis of gastric cancer cells via targeted repression on the expression of high mobility group protein B1 (HMGB1). PATIENTS AND METHODS: Expression levels of miR-129-5p and HMGB1 in gastric cancer tissues (n=25) and adjacent normal tissues were measured via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The regulatory effect of miR-129-5p on the proliferation of gastric cancer MGC-803 and SGC7901 cells was determined through Cell Counting Kit-8 (CCK-8) assay. Flow cytometry was employed to analyze the apoptosis rate of gastric cancer cells. To further discover the mechanism of miR-129-5p in regulating malignant behaviors of gastric cancer cells, the miRDB database was employed to predict the binding targets of miR-129-5p. Finally, binding sites of HMGB1 3'-untranslated region (3'-UTR) to miR-129-5p were discovered. Subsequently, HMGB1 wild-type or mutant 3'-UTR Luciferase reporter vectors were constructed, and transfected to MGC-803 and SGC7901 cells together with miR-129-5p or negative control miRNA. Next, Western blotting was adopted to measure the protein expression level of HMGB1 in MGC-803 and SGC7901 cells transfected with miR-129-5p or negative control miRNA, so as to investigate whether miR-129-5p affected HMGB1 protein expression. Additionally, to determine whether HMGB1 mediated the regulatory effect of miR-129-5p on the proliferation of gastric cancer cells, MGC-803 and SGC7901 cells were transfected with pcDNA-HMGB1 or pcDNA-vector, respectively. The expression level of HMGB1 was measured via RT-qPCR, and cell proliferation was determined by CCK-8 assay. RESULTS: The expression level of miR-129-5p in gastric cancer tissues was significantly lower than that in adjacent normal tissues (p<0.001). Meanwhile, the level of miR-129-5p was overtly lower in gastric cancer MGC-803 and SGC7901 cell lines than that in normal gastric mucosal epithelial GES-1 cells (p<0.001). These results indicated that miR-129-5p was lowly expressed in gastric cancer tissues and cell lines. Subsequent results demonstrated that the expression of HMGB1 increased remarkably in gastric cancer tissues compared with normal adjacent tissues (p<0.05). The proliferation ability of MGC-803 (p<0.001) and SGC7901 (p<0.01) cells with over-expressed miR-129-5p was remarkably weakened. Overexpression of miR-129-5p distinctly promoted the apoptosis rate of gastric cancer MGC-803 (p<0.01) and SGC7901 (p<0.001) cells. Moreover, miR-129-5p up-regulation significantly reduced the Luciferase activity of wild-type HMGB1 (p<0.001). However, no significant effect was observed on that of mutant HMGB1. The results suggested that overexpression of miR-129-5p significantly down-regulated the level of HMGB1 in gastric cancer cells. In addition, the messenger RNA (mRNA) level of HMGB1 in cells transfected with miR-129-5p also decreased significantly (p<0.001). HMGB1 overexpression overtly reversed the inhibitory effect of miR-129-5p on the proliferation of gastric cancer cells (p<0.05). All these results demonstrated that the miR-129-5p/HMGB1 axis played a key role in regulating the growth of gastric cancer cells. CONCLUSIONS: MiR-129-5p suppresses the progression of gastric cancer through targeted inhibition on the expression of HMGB1.

High throughput quantification of the functional genes associated with RDX biodegradation using the SmartChip real-time PCR system.

The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a contaminant at many military sites. RDX bioremediation as a clean-up approach has been gaining popularity because of cost benefits compared to other methods. RDX biodegradation has primarily been linked to six functional genes (diaA, nfsI, pnrB, xenA, xenB, xplA). However, current methods for gene quantification have the risk of false negative results because of low theoretical primer coverage. To address this, the current study designed new primer sets using the EcoFunPrimer tool based on sequences collected by the Functional Gene Pipeline and Repository and these were verified based on residues and motifs. The primers were also designed to be compatible with the SmartChip Real-Time PCR system, a massively parallel singleplex PCR platform (high throughput qPCR), that enables quantitative gene analysis using 5,184 simultaneous reactions on a single chip with low volumes of reagents. This allows multiple genes and/or multiple primer sets for a single gene to be used with multiple samples. Following primer design, the six genes were quantified in RDX-contaminated groundwater (before and after biostimulation), RDX-contaminated sediment, and uncontaminated samples. The final 49 newly designed primer sets improved upon the theoretical coverage of published primer sets, and this corresponded to more detections in the environmental samples. All genes, except diaA, were detected in the environmental samples, with xenA and xenB being the most predominant. In the sediment samples, nfsI was the only gene detected. The new approach provides a more comprehensive tool for understanding RDX biodegradation potential at contaminated sites.

Co-administration of conjugated linoleic acid and rosiglitazone increases atherogenic co-efficient and alters isoprenaline-induced vasodilatation in rats fed high fat diet.

The cis(c)-9, trans(t)-11 (c9,t11) and t10,c12 isomers of conjugated linoleic acid (CLA) have been reported as agonists of peroxisome proliferator-activated receptor (PPAR) and beneficial in lipidemia and glycemia. However, it is unclear whether CLA isomers enhance or antagonize effects of conventional drugs targeting PPAR. Male Sprague-Dawley rats were fed high fat diet (HFD) for 8 weeks and treated without or with CLA, rosiglitazone or both for 4 weeks. Oral glucose tolerance and surrogate markers of insulin resistance were not significantly different for all treatments compared to untreated normal diet (ND) or HFD group, except lipoprotein levels. The combination of CLA and rosiglitazone had suppressed levels of low and high density lipoproteins (46 % and 25 %, respectively), compared to HFD-alone. Conversely, the atherogenic co-efficient of the animals received HFD or HFD+rosiglitazone+CLA was 2-folds higher than ND, HFD+rosiglitazone or HFD+CLA. Isolated aortic rings from the combined CLA and rosiglitazone treated animals were less sensitive to isoprenaline-induced relaxation among endothelium-denuded aortas with a decreased efficacy and potency (R(max)=53+/-4.7 %; pEC50=6+/-0.2) compared to endothelium-intact aortas (R(max)=100+/-9.9 %; pEC50=7+/-0.2). Our findings illustrate that the combination of CLA and rosiglitazone precede the atherogenic state with impaired endothelium-independent vasodilatation before the onset of HFD-induced insulin resistance.

[Molecular mechanism of microRNA125 regulating human coagulation factor IX gene with nonsense mutation].

OBJECTIVE: To construct human coagulation factor Ⅸ mini-gene (Mini-hF9) and some nonsense mutants, detect the levels of the Mini-hF9 mRNA, and analyze the molecular mechanism of microRNA125 regulating F9 gene with nonsense mutation. METHODS: Three nonsense mutants were obtained by using PCR mutagenesis to analyze the mechanism of plasma thromboplastin component recognition. The Mini-hF9 gene mRNA levels were detected by Real-time PCR in mammalian cells co-transfected with nonsense mutant expression vectors and miR-125 mimics. RESULTS: Mini-hF9 gene was constructed successfully and cloned into the mammalian expression vector. The only normal transcript was detected in cells transfected with the Mini-hF9 gene expression vectors. Three nonsense mutants, M1 (nt 34 G>T in Exon 7), M2 (nt 52 G>T in Exon 7) and M3 (nt 85 G>T in Exon 7), were obtained by using PCR mutagenesis. The levels of the Mini-hF9 mRNA decreased to 14.1% (t=15.464, P=0.004) in M1 and 22.4% (t=15.755, P=0.004) in M2 mutants after transfection, respectively. It was proved to be caused by nonsense-mediated mRNA decay (NMD) in CHX experiment. The levels of Mini-hF9 mRNA increased to 1.70 times (t=-4.883, P=0.039) and 2.40 times (t=-17.537, P=0.003) in M1 mutant after miR-125a or miR-125b mimics treatment, respectively. The levels of Mini-hF9 mRNA increased to 2.02 times (t=-19.264, P=0.003) and 2.07 times (t=-9.158, P=0.012) in M2 mutant after miR-125a or miR-125b mimics treatment, respectively. CONCLUSION: Nonsense mutant location is a key determinant for triggering NMD. MicroRNA125 could improve the stability of Mini-hF9 mRNA in M1 and M2 mutants by repressing NMD. MicroRNA125, a short non-coding RNA molecule, could be a potential therapeutic target in conditions caused by the NMD pathway.

A novel transcript is up-regulated by fasting in the hypothalamus and enhances insulin signalling.

A transcript of unknown function, regulated by fasting and feeding, was identified by microarray analysis. The transcript is up-regulated in the fasting state. An 1168-bp cDNA was cloned from rat hypothalamus and sequenced. This sequence is consistent with adipogenesis down-regulating transcript 3 (AGD3) (also known as human OCC-1) mRNA. A protein sequence identical to AGD3 was determined by mass spectrometry. In the rat brain, AGD3 mRNA is distributed in the arcuate nucleus, ventromedial hypothalamus, amygdaloid nuclei, hippocampus, and somatic cortex. Double in situ hybridisation showed that AGD3 mRNA is co-localised with pro-opiomelanocortin and neuropeptide Y in arcuate nucleus neurones. AGD3 binds with insulin receptor substrate 4 and increases insulin-stimulated phospho-Akt and regulates AMP-activated protein kinase and mammalian target of rapamycin downstream target S6 kinase phosphorylation.

UNEXPECTED PRESENCE OF SOLUTE-FREE ZONES AT METAL-WATER INTERFACES.

Solute-free zones, termed "exclusion zones" are routinely seen next to hydrophilic surfaces in aqueous solution. Here we report similar zones next to various metals. The largest, approximately 200 µm in width, was found adjacent to zinc. Other reactive metals, including aluminum, tin, lead, and tungsten exhibited distinct but smaller exclusion zones, while precious metals such as platinum and gold did not produce any. Electrical potential measurements showed positive potentials within the exclusion zones, while pH measurements revealed an abundance of OH- groups in the aqueous regions beyond the exclusion zones. A correspondence was found between exclusion-zone size and the respective metal's position within the galvanic series. The presence of these interfacial exclusion zones is unexpected, and may shed new light on electrochemical processes taking place at metal interfaces.

Induction of apoptosis by thrombin in the cultured neurons of dorsal motor nucleus of the vagus.

BACKGROUND: A previous study demonstrated the presence of protease-activated receptor (PAR) 1 and 2 in the dorsal motor nucleus of vagus (DMV). The aim of this study is to characterize the effect of thrombin on the apoptosis of DMV neurons. METHODS: The dorsal motor nucleus of vagus neurons were isolated from neonatal rat brainstems using micro-dissection and enzymatic digestion and cultured. Apoptosis of DMV neurons were examined in cultured neurons. Apoptotic neuron was examined by TUNEL and ELISA. Data were analyzed using anova and Student's t-test. KEY RESULTS: Exposure of cultured DMV neurons to thrombin (0.1 to 10 U mL(-1)) for 24 h significantly increased apoptosis. Pretreatment of DMV neurons with hirudin attenuated the apoptotic effect of thrombin. Similar induction of apoptosis was observed for the PAR1 receptor agonist SFLLR, but not for the PAR3 agonist TFRGAP, nor for the PAR4 agonist YAPGKF. Protease-activated receptors 1 receptor antagonist Mpr(Cha) abolished the apoptotic effect of thrombin, while YPGKF, a specific antagonist for PAR4, demonstrated no effect. After administration of thrombin, phosphorylation of JNK and P38 occurred as early as 15 min, and remained elevated for up to 45 min. Pretreatment of DMV neurons with SP600125, a specific inhibitor for JNK, or SB203580, a specific inhibitor for P38, significantly inhibited apoptosis induced by thrombin. CONCLUSIONS & INFERENCES: Thrombin induces apoptosis in DMV neurons through a mechanism involving the JNK and P38 signaling pathways.

Functional protease-activated receptors in the dorsal motor nucleus of the vagus.

BACKGROUND: Protease-activated receptors (PARs), a family member of G-protein coupled receptors, are present and functionally active in a wide variety of cells. The object of this study was to demonstrate the presence and function of PAR-1 and PAR-2 in the dorsal motor nucleus of the vagus (DMV). METHODS: DMNV neurons were isolated from neonatal rat brainstems using micro-dissection and enzymatic digestion. Neurons were cultured in Neurobasal medium A containing 2% B27 supplement. Intracellular calcium concentration ([Ca(2+)](i)) was measured using fura-2 based microspectrometry. Expression of PARs was detected by RT-PCR and immunofluorescent staining. KEY RESULT: Thrombin and PAR-1 agonist peptide activate PAR-1 with a maximum change in [Ca(2+)](i) expressed as DeltaF/F0 of 229 +/- 14% and 137 +/- 7%, respectively. Trypsin and PAR-2 agonist peptide activate PAR-2 with a maximum DeltaF/F0 change of 258 +/- 12% and 242 +/- 10%, respectively. Inhibition of phospholipase C (PLC) by U73312 (1 microm) decreased the maximal change in DeltaF/F0 induced by PAR-1 activation from 140 +/- 17% to 21 +/- 3%, while the PAR-2-mediated maximal change in DeltaF/F0 decreased from 185 +/- 21% to 19 +/- 6%. Blockade of IP3 receptor with 2APB inhibited the maximal change in DeltaF/F0 due to PAR-1 and PAR-2 activation by 72 +/- 13% and 71 +/- 20% respectively. PAR-1 immnuoreactivity was present in DMV neurons. Increase in transcripts for PAR-1 and PAR-2 were detected in DMV tissues derived from IBD rats relative to control animals. CONCLUSIONS & INFERENCES: Our results indicate that PAR-1 and PAR-2 are present in the DMV neurons, and their activation leads to increases in intracellular calcium via signal transduction mechanism that involves activation of PLC and the production of IP3.

The Ribosomal Database Project: improved alignments and new tools for rRNA analysis.

The Ribosomal Database Project (RDP) provides researchers with quality-controlled bacterial and archaeal small subunit rRNA alignments and analysis tools. An improved alignment strategy uses the Infernal secondary structure aware aligner to provide a more consistent higher quality alignment and faster processing of user sequences. Substantial new analysis features include a new Pyrosequencing Pipeline that provides tools to support analysis of ultra high-throughput rRNA sequencing data. This pipeline offers a collection of tools that automate the data processing and simplify the computationally intensive analysis of large sequencing libraries. In addition, a new Taxomatic visualization tool allows rapid visualization of taxonomic inconsistencies and suggests corrections, and a new class Assignment Generator provides instructors with a lesson plan and individualized teaching materials. Details about RDP data and analytical functions can be found at http://rdp.cme.msu.edu/.

The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data.

Substantial new features have been implemented at the Ribosomal Database Project in response to the increased importance of high-throughput rRNA sequence analysis in microbial ecology and related disciplines. The most important changes include quality analysis, including chimera detection, for all available rRNA sequences and the introduction of myRDP Space, a new web component designed to help researchers place their own data in context with the RDP's data. In addition, new video tutorials describe how to use RDP features. Details about RDP data and analytical functions can be found at the RDP-II website (http://rdp.cme.msu.edu/).

Arcuate nucleus transcriptome profiling identifies ankyrin repeat and suppressor of cytokine signalling box-containing protein 4 as a gene regulated by fasting in central nervous system feeding circuits.

The arcuate nucleus of the hypothalamus is a primary site for sensing blood borne nutrients and hormonal messengers that reflect caloric status. To identify novel energy homeostatic genes, we examined RNA extracts from the microdissected arcuate nucleus of fed and 48-h fasted rats using oligonucleotide microarrays. The relative abundance of 118 mRNA transcripts was increased and 203 mRNA transcripts was decreased during fasting. One of the down-regulated mRNAs was ankyrin-repeat and suppressor of cytokine signalling box-containing protein 4 (Asb-4). The predicted structure of Asb-4 protein suggested that it might encode an intracellular regulatory protein, and therefore its mRNA expression was investigated further. Reverse transcription quantitative polymerase chain reaction was used to validate down-regulation of Asb-4 mRNA in the arcuate nucleus of the fasted Sprague-Dawley rat (relative expression of Asb-4 mRNA: fed = 4.66 +/- 0.26; fasted = 3.96 +/- 0.23; n = 4, P < 0.01). Down-regulation was also demonstrated in the obese fa/fa Zucker rat, another model of energy disequilibrium (relative expression of Asb-4 mRNA: lean Zucker = 3.91 +/- 0.32; fa/fa = 2.93 +/- 0.26; n = 5, P < 0.001). In situ hybridisation shows that Asb-4 mRNA is expressed in brain areas linked to energy homeostasis, including the arcuate nucleus, paraventricular nucleus, dorsomedial nucleus, lateral hypothalamus and posterodorsal medial amygdaloid area. Double in situ hybridisation revealed that Asb-4 mRNA colocalises with key energy homeostatic neurones. In the fed state, Asb-4 mRNA is expressed by 95.6% of pro-opiomelanocortin (POMC) neurones and 46.4% of neuropeptide Y (NPY) neurones. By contrast, in the fasted state, the percentage of POMC neurones expressing Asb-4 mRNA drops to 73.2% (P < 0.001). Moreover, the density of Asb-4 mRNA per fasted POMC neurone is markedly decreased. Conversely, expression of Asb-4 mRNA by NPY neurones in the fasted state is modestly increased to 52.7% (P < 0.05). Based on its differential expression, neuroanatomical distribution and colocalisation, we hypothesise that Asb-4 is a gene involved in energy homeostasis.

A mutation in SART3 gene in a Chinese pedigree with disseminated superficial actinic porokeratosis.

BACKGROUND: Disseminated superficial actinic porokeratosis (DSAP) is an uncommon autosomal dominant chronic disorder of keratinization, characterized by multiple superficial keratotic lesions surrounded by a slightly raised keratotic border. Thus far, although two loci for DSAP have been identified, and the genetic basis and pathogenesis of this disorder have not been elucidated. OBJECTIVES: To determine the locus of DSAP and identify the candidate gene(s) of the disease. METHODS: Genome-wide scan and linkage analysis were performed in a six-generation Chinese family with DSAP. The coding exons of the candidate genes were sequenced to analyse and detect the nucleotide variations. RESULTS: Linkage analysis showed that the maximum two-point lod score of 5.56 was obtained with the marker D12S79 at a recombination fraction theta of 0.00. Haplotype analysis defined the critical region for DSAP between D12S330 and D12S1612 on 12q24.1-24.2. By sequence analysis, we found a Val591Met mutation in SART3 in all affected individuals of the family. CONCLUSION: SART3 is a candidate gene for DSAP, and is possibly involved in the pathogenesis of DSAP.

The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis.

The Ribosomal Database Project (RDP-II) provides the research community with aligned and annotated rRNA gene sequences, along with analysis services and a phylogenetically consistent taxonomic framework for these data. Updated monthly, these services are made available through the RDP-II website (http://rdp.cme.msu.edu/). RDP-II release 9.21 (August 2004) contains 101,632 bacterial small subunit rRNA gene sequences in aligned and annotated format. High-throughput tools for initial taxonomic placement, identification of related sequences, probe and primer testing, data navigation and subalignment download are provided. The RDP-II email address for questions or comments is rdpstaff@msu.edu.

The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy.

The Ribosomal Database Project-II (RDP-II) pro-vides data, tools and services related to ribosomal RNA sequences to the research community. Through its website (http://rdp.cme.msu.edu), RDP-II offers aligned and annotated rRNA sequence data, analysis services, and phylogenetic inferences (trees) derived from these data. RDP-II release 8.1 contains 16 277 prokaryotic, 5201 eukaryotic, and 1503 mitochondrial small subunit rRNA sequences in aligned and annotated format. The current public beta release of 9.0 debuts a new regularly updated alignment of over 50 000 annotated (eu)bacterial sequences. New analysis services include a sequence search and selection tool (Hierarchy Browser) and a phylogenetic tree building and visualization tool (Phylip Interface). A new interactive tutorial guides users through the basics of rRNA sequence analysis. Other services include probe checking, phylogenetic placement of user sequences, screening of users' sequences for chimeric rRNA sequences, automated alignment, production of similarity matrices, and services to plan and analyze terminal restriction fragment polymorphism (T-RFLP) experiments. The RDP-II email address for questions or comments is rdpstaff@msu.edu.

Competence of oat ( Avena sativa L.) shoot apical meristems for integrative transformation, inherited expression, and osmotic tolerance of transgenic lines containing hva1.

Three oat ( Avena sativa L.) cultivars have been successfully transformed using an efficient and reproducible in vitro culture system for differentiation of multiple shoots from shoot apical meristems. The transformation was performed using microprojectile bombardment with two plasmids (pBY520 and pAct1-D) containing linked ( hva1-bar) and non-linked ( gus) genes. The hva1 and bar genes cointegrated with a frequency of 100% as expected, and 61.6% of the transgenic plants carried all three genes. Molecular and biochemical analyses in R0, R1 and R2 progenies confirmed stable integration and expression of all transgenes. Localization of the GUS protein in R0 and R1 plants revealed that high-expression of gus occurred in vascular tissues and in the pollen grains of mature flowers. The constitutive expression of HVA1 protein was observed at all developmental stages of transgenic plants, and was particularly stronger during the early seedling stages. R2 progeny of five independent transgenic lines was tested in vitro for tolerance to osmotic (salt and mannitol) stresses. As compared to non-transgenic control plants, transgenic plants maintained a higher growth and showed significantly ( P < 0.05) increased tolerance to stress conditions. Less than 10% of transgenic plants showed symptoms of wilting or death of leaves and, when these symptoms present were delayed in transgenic plants as compared to 80% of non-transgenic plants, either wilted or died. These symptoms confirmed the increased in vitro tolerance in hva1-expressing transgenic plants to non-transgenic plants, providing strong evidence that the HVA1 protein may play an important role in the protection of oats against salinity and possible water-deficiency stress conditions.