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The SARS-CoV-2 Delta variant has evolved as the dominant strain of the current pandemic. Studies have shown that this variant has increased infectivity/viral load, and reduced neutralization by the host antibodies from convalescent patients/vaccinees. Clinically, Delta variant infection has been observed/documented in convalescent patients/vaccinees, although with less incidence of severe diseases, but can serve as reservoir to spread the infection to the unvaccinated. The current understanding (as of 18 August 2021) on the virologic aspect (including the amino acid substitutions), clinical implications, and public health implications will be discussed in this mini review, and recommendations to health authorities will be provided.

Background: APOE ε4 is the best-known risk factor for late-onset alzheimer’s disease (AD). Population studies have demonstrated a relatively low prevalence of APOE ε4 among Chinese population, implying additional risk factors that are Chinese-specific may exist. Apart from - alleles, genetic variation profile along the full-length APOE has rarely been investigated. Objective: In this study, we filled this gap by comprehensively determining all genetic variations in APOE and investigated their potential associations with late-onset AD and mild cognitive impairment (MCI) in southern Chinese. Methods: Two hundred and fifty-seven southern Chinese participants were recruited, of whom 69 were AD patients, 83 had MCI, and 105 were normal controls. Full-length APOE from promoter to 3′UTR regions were sequenced. Genetic variants were identified and compared among the three groups. Results: While APOE ε4 was more significantly found in AD patients, the prevalence of APOE ε4 in southern Chinese AD patients was the lowest when compared to other areas of China and nearby regions, as well as other countries worldwide. We further identified 13 rare non-singleton variants in APOE. Significantly more AD patients carried any of the rare non-singleton variants than MCI and normal subjects. Such difference was observed in the non-carriers of ε4-allele only. Among the identified rare variants, the potential functional impact was predicted for rs532314089, rs553874843, rs533904656 and rs370594287. Conclusion: Our study suggests an ethnic difference in genetic risk composition of AD in southern Chinese. Rare variants on APOE are a potential candidate for AD risk stratification biomarker in addition to APOE-ε4.

The genetic bases of many common diseases have been identified through genome-wide association studies in the past decade. However, the application of this approach on public healthcare planning has not been well established. Using Macau with population of around 650,000 as a basis, we conducted a pilot study to evaluate the feasibility of population genomic research and its potential on public health decisions. By performing genome-wide SNP genotyping of over a thousand Macau individuals, we evaluated the population genetic risk profiles of 47 non-communicable diseases and traits, as well as two traits associated with influenza infection. We found that for most of the diseases, the genetic risks of Macau population were different from those of Caucasian, but with similar profile with mainland Chinese. We also identified a panel of diseases that Macau population may have a high or elevated genetic risks. This pilot study showed that (1) population genomic study is feasible in Asian regions like Macau; (2) Macau may have different profile of population-based genetic risks than Caucasians, (3) the different prevalence of genetic risk profile indicates the importance of Asian-specific studies for Asian populations; and (4) the results generated may have an impact for going forward healthcare planning.

Infectious diseases, mostly caused by bacteria and viruses but also a result of fungal and parasitic infection, have been one of the most important public health concerns throughout human history. The first step in combating these pathogens is to get a timely and accurate diagnosis at an affordable cost. Many kinds of diagnostics have been developed, such as pathogen culture, biochemical tests and serological tests, to help detect and fight against the causative agents of diseases. However, these diagnostic tests are generally unsatisfactory because they are not particularly sensitive and specific and are unable to deliver speedy results. Nucleic acid-based diagnostics, detecting pathogens through the identification of their genomic sequences, have shown promise to overcome the above limitations and become more widely adopted in clinical tests. Here we review some of the most popular nucleic acid-based diagnostics and focus on their adaptability and applicability to routine clinical usage. We also compare and contrast the characteristics of different types of nucleic acid-based diagnostics.

Alzheimers disease (AD) is a neurodegenerative disease with major clinical hallmarks of memory loss, dementia, and cognitive impairment. Neuroinflammation is involved in the onset of several neurodegenerative disorders. Astrocyte is the most abundant type of glial cells in the central nervous system (CNS) and appears to be involved in the induction of neuroinflammation. Under stress and injury, astrocytes become astrogliotic leading to an upregulation of the expression of proinflammatory cytokines and chemokines, which are associated with the pathogenesis of AD. Cytokines and related molecules play roles in both neuroprotection and neurodegeneration in the CNS. During early AD pathogenesis, amyloid beta (Aβ), S100B and IL-1β could bring about a vicious cycle of Aβ generation between astrocytes and neurons leading to chronic, sustained and progressive neuroinflammation. In advanced stages of AD, TRAIL secreted from astrocytes have been shown to bind to death receptor 5 (DR5) on neurons to trigger apoptosis in a caspase-8-dependent manner. Furthermore, astrocytes could be reactivated by TGFβ1 to generate more Aβ and to undergo the aggravating astrogliosis. TGFβ2 was also observed to cooperate with Aβ to cause neuronal demise by destroying the stability of lysosomes in neurons. Inflammatory molecules can be either potential biomarkers for diagnosis or target molecules for therapeutic intervention. Understanding their roles and their relationship with activated astrocytes is particularly important for attenuating neuroinflammation in the early stage of AD. The main purpose of this review is to provide a comprehensive insight into the role of astrocytes in the neuroinflammatory pathogenesis of AD.

Ischemia occurs in the brain as the result of stroke and other related injuries and few therapies are effective. If more is understood then potential treatments could be investigated. It was previously reported that 14-3-3gamma could be up-regulated by ischemia in astrocyte to protect cells from ischemia-induced apoptosis. In this study, we attempted to uncover the mechanism responsible for this 14-3-3gamma up-regulation in primary culture of astrocytes under ischemic-like conditions. It was found that in vitro ischemia may activate PI3K/Akt and MAPK signaling pathways. Astrocyte cultures were treated with LY294002 (PI3K inhibitor), U0126 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor). Only SP600125 could inhibit the ischemia-induced 14-3-3gamma up-regulation in astrocytes. At the same time, we observed an ischemia-induced nuclear translocation of p-c-Jun, a major downstream component of JNK. Inhibition of AP-1 with curcumin also inhibited 14-3-3gamma up-regulation indicating that ischemia-induced up-regulation of 14-3-3gamma in astrocyte involves activation of the JNK/p-c-Jun/AP-1 pathway.

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival and functions of neurons. It has been shown to be a promising candidate in the treatment of ischemia and other neurodegenerative diseases. We transfected mouse astrocytes in primary cultures with a human GDNF gene and found that their conditioned medium could not only support the growth and survival of cultured dopaminergic neurons but also protect astrocytes from staurosporine- and ischemia-induced apoptosis. This indicated that these transfected astrocytes could release GDNF. A similar protective effect on astrocytes against apoptosis was evident when recombinant human GDNF was used. Moreover, GDNF reduced caspase-3 activity but not that of caspase-1 in cultured astrocytes after ischemia treatment. Thus, GDNF protects astrocytes from apoptosis by inhibiting the activation of caspase-3.

Summary Background Human infection with avian influenza H5N1 is an emerging infectious disease characterised by respiratory symptoms and a high fatality rate. Previous studies have shown that the human infection with avian influenza H5N1 could also target organs apart from the lungs. Methods We studied post-mortem tissues of two adults (one man and one pregnant woman) infected with H5N1 influenza virus, and a fetus carried by the woman. In-situ hybridisation (with sense and antisense probes to haemagglutinin and nucleoprotein) and immunohistochemistry (with monoclonal antibodies to haemagglutinin and nucleoprotein) were done on selected tissues. Reverse-transcriptase (RT) PCR, real-time RT-PCR, strand-specific RT-PCR, and nucleic acid sequence-based amplification (NASBA) detection assays were also undertaken to detect viral RNA in organ tissue samples. Findings We detected viral genomic sequences and antigens in type II epithelial cells of the lungs, ciliated and non-ciliated epithelial cells of the trachea, T cells of the lymph node, neurons of the brain, and Hofbauer cells and cytotrophoblasts of the placenta. Viral genomic sequences (but no viral antigens) were detected in the intestinal mucosa. In the fetus, we found viral sequences and antigens in the lungs, circulating mononuclear cells, and macrophages of the liver. The presence of viral sequences in the organs and the fetus was also confirmed by RT-PCR, strand-specific RT-PCR, real-time RT-PCR, and NASBA. Interpretation In addition to the lungs, H5N1 influenza virus infects the trachea and disseminates to other organs including the brain. The virus could also be transmitted from mother to fetus across the placenta.

Astrogliosis is an inevitable and rapid response of astrocytes to physical, chemical and pathological injuries. To study astrogliosis, we developed a reproducible in vitro model in which low temperature injury to cultured astrocytes could be induced by placing the culture dish onto a copper pipe pre-cooled by liquid nitrogen. Using this model, the relationship between the temperature decline and the severity of cellular damage was analyzed. An increase in the expression of some known injury-related proteins, such as glial fibrillary acidic protein (GFAP), immediate early response genes (IEGs), and heat shock proteins 70 (HSP70), was demonstrated in astrocytes after low temperature trauma. With the use of this low temperature trauma model, the flexibility in the temperature control and injury area may allow researchers to evaluate cryotherapy and cryosurgery, which could be applicable to future development of quality health care.

The genome of a novel foot-and-mouth disease virus, HKN/2002, was 8104 nucleotides (nt) in length (excluding the poly(C) tract and poly(A) tail) and was composed of a 1042-nt 5′-untranslated region (UTR), a 6966-nt open reading frame, and a 93-nt 3′-UTR. Genome sequences of HKN/2002 and other known FMDV strains were compared. The VP1, VP2, and VP3-based neighbor-joining (NJ) trees were divided into distinct clusters according to different serotypes, while other region-based NJ trees exhibited some degree of intercross among serotypes. Mutations in HKN/2002 were revealed, including frequent deletions and insertions in the G–H loop of VP1, and deletion involving 10 amino acid residues in the 3A protein. An evolutionary relationship of HKN/2002 with an Asian FMDV lineage isolated from a Hong Kong swine host in 1970 was postulated. A 43-nt deletion identified in the 5′-UTR of HKN/2002 possibly contributed to the loss of one pseudo-knot domain.

The genetically modified (GM) Roundup Readysoybean event GTS40-3-2 contains the bacterial gene 5-enol-pyruvylshiki- mate-3-phosphate synthase. A 534 bp rearrangement of the DNA in the 3 0 region flanking the functional insert likely occurred as a consequence of the insertion event. The structure of the DNA surrounding the insertion has not been fully characterized. A semi- nested PCR method identified the rearranged soybean DNA in samples of raw, partially processed and highly processed food at a level of 0.01%. The 534 bp rearranged segment contained a contiguous portion at least 238 bp long that was also identified in non- GM soybeans. Specific combinations of semi-nested PCR primers differentiated GM and non-GM soybean DNA. These studies confirm that the rearranged DNA originates from the soybean genome and does not involve the introduction of non-soybean genetic material. � 2003 Elsevier Ltd. All rights reserved.

Infection of poultry with highly pathogenic avian influenza virus (AIV) can be devastating in terms of flock morbidity and mortality, economic loss, and social disruption. The causative agent is confined to certain isolates of influenza A virus subtypes H5 and H7. Due to the potential of direct transfer of avian influenza to humans, continued research into rapid diagnostic tests for influenza is therefore necessary. A nucleic acid sequence-based amplification (NASBA) method was developed to detect a portion of the haemagglutinin gene of avian influenza A virus subtypes H5 and H7 irrespective of lineage. A further NASBA assay, based on the matrix gene, was able to detect examples of all known subtypes (H1–H15) of avian influenza virus. The entire nucleic acid isolation, amplification, and detection procedure was completed within 6 h. The dynamic range of the three AIV assays was five to seven orders of magnitude. The assays were sensitive and highly specific, with no cross-reactivity to phylogenetically or clinically relevant viruses. The results of the three AIV NASBA assays correlated with those obtained by viral culture in embryonated fowl’s eggs.

An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 10(2)-fold higher than the standard real-time PCR assay and 10(7)-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) of avian influenza virus was compared with viral culture in embryonated chicken eggs. Virus was isolated from blood or anal swabs of chickens artificially infected with highly pathogenic avian influenza A/Chicken/Hong Kong/1000/97 (H5N1). Viral nucleic acid was detected in blood samples by NASBA/ECL immediately prior to death, whilst nucleic acid extracted from anal swabs was detected from the day following artificial infection until death. Thus, blood and/or anal swabs are a suitable source of material for the detection of avian influenza in dead birds, but anal swabs are more suitable for detection of viral genetic material in live birds. Dilution of a known viral standard was used to determine the limit of sensitivity for both NASBA/ECL and egg culture detection methods. The NASBA/ECL method was equivalent in sensitivity to egg culture. The NASBA/ECL results agreed with egg culture data in 71/94 (75.5%) tissue samples obtained from artificially infected birds.

Nucleic acid sequence-based amplification (NASBA) is a technique that allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A NASBA technique has been developed that allows the detection of avian influenza A subtype H5 from allantoic fluid harvested from inoculated chick embryos. The amplified viral RNA is detected by electrochemiluminescence. The NASBA technique described below is rapid and specific for the identification of influenza A subtype H5 viruses of the Eurasian lineage. More importantly, it can be used to distinguish highly pathogenic and low pathogenic strains of the H5 subtype.

The brain is no longer considered immune-privileged due to its capability of producing cytokines in response to neurotrauma; however, the cellular sources of cytokines have not been defined. This study focused on the production of four inflammatory cytokines, interleukin-1 (IL-1alpha), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), and interferon gamma (IFN-gamma) in primary culture of astrocytes under two different injury models which simulated in vivo mechanical trauma (scratch injury) and ischemia. Results demonstrated that astrocytes after scratch injury were positively immunostained with IL-1alpha, IL-6, and TNFalpha. A slot-blot study of culture media showed that the release of IL-1alpha, IL-6, TNFalpha, and IFN-gamma by astrocytes subsequent to scratch and ischemic injury reached approximately twice the control values. The temporal expression of these cytokines was different for the two models. All four cytokines began to increase 1 h postscratch and remained at high levels throughout the experiment. In the ischemic model, however, the increase of cytokine expression was delayed until 4-8 h of ischemia, when sharp increases were seen in all four cytokines. In this culture system, the exogenous influence of blood-borne factors and leukocytes, which occur with in vivo trauma and ischemia, was eliminated. Accordingly, the cytokines detected in the culture media were derived from astrocytes. This study provides the first evidence that astrocytes, without the influence from other cell types, can produce and release cytokines following mechanical and ischemic injury.

Astrocytes form an integral part of the blood brain barrier and are the first cell type in the central nervous system to encounter insult if there is an ischemic attack. The immunologic reaction of astrocytes to an ischemic insult would be affective to the subsequent responses of other nerve cells. We previously showed that ischemia caused an increase in the levels of interleukin 1alpha (IL-1alpha), tumor necrosis factor alpha (TNF alpha), and interleukin 6 (IL-6) in the culture medium of mouse cerebral cortical astrocyte. We did not have evidence on the source of these cytokines. This study aimed to investigate the expressions of these cytokine mRNAs in the astrocytes under ischemia. Results demonstrated that ischemia could induce necrosis and apoptosis in astrocytes. By using the RT-PCR method, we demonstrated for the first time that the mRNA levels of IL-1alpha, TNF alpha and IL-6 in normal astrocyte was very low, but their expressions could be induced quickly under ischemia. These cytokines might be interactive as indicated by the difference in time course of their expressions, with IL-1alpha being the earliest and IL-6 being the latest. The result provided some understanding of the induction and progression of these immunologic responses in astrocytes under ischemia. It also supported our previous findings that astrocytes contributed to the cytokines released under ischemia.

To the Editor: In view of recent concern about the recurrence of severe acute respiratory syndrome (SARS) in Guangdong, China, we would like to highlight the tremendous importance of sensitivity in...

Abe et al. reported the development of a real-time PCR assay for the absolute quantification of the hepatitis B virus (HBV) genome by TaqMan probe-based chemistry in 1999 ([1][1]). Their studies included two set of primers and probes, and the primer named HBSR1 (5′-GGTGAGTGATTGGAGGGTTG-3′) was

We observed nuclear swelling in glutamate (Glu)-treated astrocytes that was concomitant with but independent of astrocytic cell swelling. We confirmed Glu-induced nuclear swelling with nuclei isolated from astrocytes. Ammonia is metabolically related to Glu and could induce a nuclear swelling in intact astrocytes but shrinkage in isolated nuclei. Other compounds such as glutamine, aspartate, taurine, glycine, and ATP did not cause any nuclear swelling in isolated nuclei of astrocytes. Surprisingly, Glu and ammonia did not induce nuclear swelling in microglia, C6, HEK 293, or Hep G2 cell lines in cultures and their isolated nuclei. The Glu- and ammonia-induced nuclear size changes appear to be a specific response of astrocytes to these two closely related metabolic compounds. © 2011 Wiley-Liss, Inc.

A group of common lower respiratory tract infections, influenza A, influenza B, human parainfluenza virus 1-4 (HPIV1-4), respiratory syncytial virus (RSV), rubella virus (RV) and Coxsackie virus (CSV), were selected for the development of a multiplex nucleic acid sequence-based amplification (NASBA) assay. Quantifiable measurement utilizing an enzyme-linked oligonucleotide capture (EOC) optical detection method, which was described previously, alleviated the requirement of specialized instrumentation that is commonly used in other molecular techniques. Multiplex NASBA-EOC provided rapid and specific detection of a single virus from a multiplexed group, reducing laboratory testing time and enabling high throughput screening. The uniquely designed primers and probes proved to be highly sensitive and specific, exemplifying the robustness of the multiplex NASBA-EOC technique.

Polyphosphate (poly P) has been widely identified in both inorganic environment and living organisms. Research shows that poly P in bacteria enhances their resistance to severe environment, triggers their protective responses, increases biofilm formation and involves in predation and bacterial virulence. In eukaryotes, poly P has been found to enhance the proliferation of fibroblast and many tumor cell lines, induce the calcification of osteoblast and be involved in calcium ion release. Based on the existing information, we attempt to discuss the possible functions of poly P in the nervous system.

After cellular injury many endogenous toxins are released from injured cells and result in secondary injury. To elucidate mechanisms of such injury many of these toxins have been studied individually. However, the data obtained is only useful for reference and does not accurately represent the multifactorial situation under pathophysiological conditions. Primary astrocytic cultures were treated individually and simultaneously with two well-studied toxins, glutamate (Glu) and arachidonic acid (AA). Both are simultaneously released from neural cells during injury. Measurements of cellular protein content, intracellular water space, lactate dehydrogenase release, and malondialdehyde formation indicated that Glu and AA act through different mechanisms. Glu + AA applied together had a synergistic effect on the levels of Caspase-3 gene expression, and Bcl-2 and Hsp70 protein. Atomic force microscopy observed that Glu caused cell membrane roughness and nuclear swelling, while AA induced pores in the cell membrane and nuclear shrinkage. Glu + AA accelerated nuclear shrinkage and resulted in more serious cell damage. This study not only distinguishes the different responses of astrocytes to Glu and AA, but also provides a new view into the synergistic effect of these biochemicals; highlighting the need to be cautious in applying single factor experimental data to interpret complex physiological and pathological conditions in animals. Two or more factors may act not only on different targets but also on the same target synergistically.

To study the etiology of influenza-like illness (ILI) in Beijing, and to investigate the impact of antibiotic treatment on outcomes.This was a prospective cohort study. Patients with diagnosis of influenza-like illness were prospectively enrolled for study of bacterial and viral pathogens. Demographic characteristics, underlying diseases, respiratory and extrapulmonary symptoms, laboratory tests were also collected for analysis of relationship between drug therapy and outcomes.A total of 476 cases were enrolled between Dec. 2006 and Apr. 2007, of whom 454 cases were used for analysis. Influenza virus was the most common pathogen( n = 197, 43.4%), with other pathogens rarely seen. The mean age of the patients was (33 +/- 13) years, and the ratio of male to female was 1.1:1. Twenty four patients (5.3% ) received influenza vaccine. The rate of antibiotic prescription after onset of illness was 63.4%, but none received antiviral drugs such as Oseltamivir and amantadine. Compared with influenza-negative patients, patients with influenza were older, had more underlying diseases and had greater severity of symptoms such as cough, sore throat, headache and myalgia (but with no statistical differences). The influenza syndrome (Tor = 39 degrees C plus cough, sore throat and headache or myalgia) was more common in the influenza group compared to the influenza-negative patients (P0.05). The ratio of antibiotic prescription was 67% in the influenza group, and the total white blood cell and platelet count, percentage of neutrophils were higher in antibiotic treatment patients compared with non-antibiotic treatment patients (P0.01). The cost in patients who received antibiotics was twice as much as non-antibiotic treatment patients (P0.05), but the defervescence time and respiratory symptom alleviation time did not differ. Cox regression analysis showed that the total white blood count and the differentials (OR value 1.049 and 1.014, respectively), but not antibiotic use were the independent risk factors for longer defervescence time.Influenza virus was the most common pathogen for adult patients with ILI in Beijing city during the winter and the spring seasons. Antibiotic treatment of adult patients with ILI did not improve illness resolution, while the cost was increased significantly.

The mechanism of systemic spread of H5N1 virus in patients with avian influenza is unknown. Here, H5N1 nucleoprotein and hemagglutinin were identified by immunohistochemistry in the nucleus and cytoplasm of neutrophils in the placental blood of a pregnant woman. Viral RNA was detected in neutrophils by in situ hybridization and enhanced real-time polymerase chain reaction. Therefore, neutrophils may serve as a vehicle for viral replication and transportation in avian influenza.

A study was conducted to evaluate the performance of a nucleic acid sequence-based amplification (NASBA) assay for the detection of foot-and-mouth disease virus (FMDV). Two detection methods: NASBA-electrochemiluminescence (NASBA-ECL) and a newly developed NASBA-enzyme-linked oligonucleotide capture (NASBA-EOC) were evaluated. The diagnostic sensitivity of these assays was compared with other laboratory-based methods using 200 clinical samples collected from different regions of the world. Assay specificity was also assessed using samples (n = 43) of other viruses that cause vesicular disease in livestock and genetic relatives of FMDV. Concordant results were generated for 174/200 (87.0%) of suspect FMD samples between NASBA-ECL and real-time RT-PCR. In comparison with the virus isolation (VI) data, the sensitivity of the NASBA-ECL assay was 92.9%, which was almost identical to that of the real-time RT-PCR (92.4%) for the same set of samples. There was broad agreement between the results of the NASBA-ECL and the simpler NASBA-EOC detection method for 97.1% of samples. In conclusion, this study provides further data to support the use of NASBA as a rapid and sensitive diagnostic method for the detection and surveillance of FMDV.

Newcastle disease (ND) is a contagious and widespread avian disease affecting most species of birds. ND virus (NDV) is the only member of the avian paramyxovirus serotype 1 (APMV1) causing ND outbreak in bird flocks. The technique of nucleic acid sequence-based amplification (NASBA) is a potential method to rapidly and reliably detect NDV isolates. Here, we describe an effective and unprecedented method for detecting NDV strains of all pathotypes. A conserved region of the fusion protein gene was used for designing oligonucleotides specific to all NDV pathotypes. The dynamic range of this NDV NASBA detection method is comparable to virus culture and therefore the NDV NASBA method is a potential alternative for NDV screening and surveillance.

Retinal neurodegenerative disease involves an inflammatory response in the retina characterized by an increase in inflammatory cytokines and activation of microglia. The degree of microglia activation may influence the extent of retinal injury following an inflammatory stimulus. Cytokines released by activated microglia regulate the influx of inflammatory cells to the damaged area. Thus, a therapeutic strategy to reduce cytokine expression in microglia would be neuroprotective. Minocycline, a semisynthetic tetracycline derivative, is known to protect rodent brain from ischemia and to inhibit microglial activation. In this study, we activated retinal microglia in culture with lipopolysaccharide (LPS) and attempted to determine whether minocycline could reduce the production of cytokines from activated microglia at both gene and protein levels. Changes in inflammatory cytokines, TNF-alpha and IL-1beta, were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) in the presence or absence of LPS. We also measured the levels of nitric oxide (NO) by the nitrate reductase method under similar conditions. LPS treatment induced a significant upregulation of the mRNA and release of TNF-alpha, IL-1beta, and NO from retinal microglia. Minocycline inhibited these releases. Thus, minocycline might exert its antiinflammatory effect on microglia by inhibiting the expression and release of TNF-alpha, IL-1beta, and NO.

Neuronal differentiation and aging are known to involve many genes, which may also be differentially expressed during these developmental processes. From primary cultured cerebral cortical neurons, we have previously identified various differentially expressed gene transcripts from cultured cortical neurons using the technique of arbitrarily primed PCR (RAP-PCR). Among these transcripts, clone 0–2 was found to have high homology to rat and human synaptic glycoprotein. By in silico analysis using an EST database and the FACTURA software, the full-length sequence of 0–2 was assembled and the clone was named as mouse synaptic glycoprotein homolog 2 (mSC2). DNA sequencing revealed transcript size of mSC2 being smaller than the human and rat homologs. RT-PCR indicated that mSC2 was expressed differentially at various culture days. The mSC2 gene was located in various tissues with higher expression in brain, lung, and liver. Functions of mSC2 in neurons and other tissues remain elusive and will require more investigation.

Bcl-2-associated death protein (Bad), a member of the Bcl family, directs astrocytes in primary cultures to enter or resist apoptosis during ischemia in vitro. Under ischemia, Bad was the only Bcl family member whose expression was upregulated significantly during the early stages of an ischemic insult. Increased endogenous Bad was translocated from the cytoplasm to mitochondria to induce apoptosis in astrocytes. Concurrently, ischemia also induced Bad phosphorylation specifically on Ser112 to promote survival. This site-specific phosphorylation of Bad was mediated by an early activation of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) intracellular signaling pathway. This study demonstrates that ischemia-induced Bad plays a dual role in determining whether astrocytes enter or resist apoptosis after an ischemic insult. © 2005 Wiley-Liss, Inc.

Neuroglobin (Ngb), a recently discovered intracellular respiratory globin in neurons, may play a crucial role in oxygen homeostasis in the brain. We report preliminary findings indicating the presence of functional neuroglobin in primary cultures of cerebral cortical astrocytes. Reverse transcription real-time polymerase chain reaction (RRT-PCR) and immunostaining confirmed such presence in cultured astrocytes isolated from newborn mouse brain. Ngb antisense treatment increased apoptosis in ischemic astrocytes. The discovery of Ngb in astrocytes may provide some insight into how oxygen homeostasis is regulated in the brain.

Nucleic acid sequence-based amplification (NASBA) allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A NASBA technique was developed that allows the detection of avian influenza A subtype H5 from allantoic fluid harvested from inoculated chick embryos. The amplified viral RNA is detected by electrochemiluminescence. The described NASBA technique is a specific, rapid, and sensitive method of detection of influenza A subtype H5 viruses. More importantly, it can be used to distinguish high- and low-pathogenicity strains of the H5 subtype.

An in vitro ischemia model was established and the effect of the metabolic inhibitors cycloheximide (CHX) and actinomycin D (ActD) on apoptosis in astrocytes under ischemia studied. CHX decreased by 75% the number of cells dying after 6 hr of ischemia compared with control cultures. TdT-mediated dUTP nick end labelling (TUNEL) staining of comparable cultures was reduced by 40%. ActD decreased cell death by 60% compared with controls. The number of TUNEL-positive cells was reduced by 38%. The nuclear shrinkage in TUNEL-positive astrocytes in control cultures did not occur in ActD-treated astrocytes, indicating that nuclear shrinkage and DNA fragmentation during apoptosis are two unrelated processes. Expression of bcl-2 (α and β), bax, and Ice in astrocytes under similar ischemic conditions, as measured by quantitative reverse transcription-polymerase chain reaction, indicated that ischemia down-regulated bcl-2 (α and β) and bax. Ice was initially down-regulated from 0 to 4 hr, before returning to control levels after 8 hr of ischemia. ActD decreased the expression of these genes. CHX reduced the expression of bcl-2 (α and β) but increased bax and Ice expression. It is hypothesized that the balance of proapoptotic (Bad, Bax) and antiapoptotic (Bcl-2, Bcl-Xl) proteins determines apoptosis. The data suggest that the ratio of Bcl-2/Bad in astrocytes following ActD and CHX treatment does not decrease as much in untreated cells during ischemia. Our data indicate that it is the ratio of Bcl-2 family members that plays a critical role in determining ischemia-induced apoptosis. It is also important to note that ischemia-induced apoptosis involves the regulation of RNA and protein synthesis. © 2003 Wiley-Liss, Inc.

A foot-and-mouth disease virus (FMDV, HKN/2002) was isolated in Hong Kong in 2002. The nucleotide sequence of the 3D(pol) gene encoding the viral RNA-dependent RNA polymerase was determined and compared with that of the same gene from other FMDVs. The 3D(pol) gene was 1410 nucleotides in length encoding a protein of 470 amino acid residues. Sequence comparisons indicated that HKN/2002 belonged to serotype O. An evolutionary tree based on the 3D(pol) sequences of 20 FMDV isolates revealed that the nucleotide sequence of the HKN/2002 3D(pol) gene was most similar to those of isolates found in Taiwan in 1997, suggesting that they share a common ancestor. The amino acid sequence of the HKN/2002 3D(pol) gene was determined and aligned with those of representative isolates from seven other Picornaviridae genera. Eight highly conserved regions were detected, indicating a conserved functional relevance for these motifs. Alignment of 20 FMDV 3D(pol) amino acid sequences revealed a hypermutation region near the N-terminus that may help the virus evade host immune systems.