Biochemical assay for amyloid beta deposits to distinguish Alzheimer's disease from other dementias

Biochemical markers for Alzheimer's disease (AD) are of great value for precise diagnosis and in studies of the pathogenetic processes of this disease. A new biochemical assay allowing to differentiate AD from other forms of dementia is described. The assay is based on the extraction of amyloid β (Aβ) from milligram amounts of brain tissue by using 20% acetonitrile in 0.1% trifluoroacetic acid and its detection by Western blotting. The presence of the 4 kDa Aβ was demonstrated in all cases of AD (n=8) that were diagnosed by the independent histopathological examination of the postmortem tissues. No Aβ was found in tissue extracts from seven out of eight cases of other forms of dementia. In contrast to other biochemical techniques of Aβ detection in brain, the developed assay is simple; it does not require any special equipment and allows detection of Aβ using milligram amounts of brain tissue.     

Strategies for the generation of parametric images of [C]PIB with plasma input functions considering discriminations and reproducibility

Pittsburgh compound B or [¹¹C]PIB is an amyloid imaging agent which shows a clear differentiation between subjects with Alzheimer's disease (AD) and controls. However the observed signal difference in other forms of dementia such as dementia with Lewy bodies (DLB) is smaller, and mild cognitively impaired (MCI) subjects and some healthy elderly normals may show intermediate levels of [¹¹C]PIB binding. The cerebellum, a commonly used reference region for non-specific tracer uptake in [¹¹C]PIB studies in AD may not be valid in Prion disorders or monogenic forms of AD. The aim of this work was to: 1—compare methods for generating parametric maps of [¹¹C]PIB retention in tissue using a plasma input function in respect of their ability to discriminate between AD subjects and controls and 2—estimate the test–retest reproducibility in AD subjects. 12  AD subjects (5 of which underwent a repeat scan within 6 weeks) and 10 control subjects had 90 minute [¹¹C]PIB dynamic PET scans, and arterial plasma input functions were measured. Parametric maps were generated with graphical analysis of reversible binding (Logan plot), irreversible binding (Patlak plot), and spectral analysis. Between group differentiation was calculated using Student's t-test and comparisons between different methods were made using p values. Reproducibility was assessed by intraclass correlation coefficients (ICC). We found that the 75 min value of the impulse response function showed the best group differentiation and had a higher ICC than volume of distribution maps generated from Logan and spectral analysis. Patlak analysis of [¹¹C]PIB binding was the least reproducible.     

A simple gold nanoparticle probes assay for identification of Mycobacterium tuberculosis and Mycobacterium tuberculosis complex from clinical specimens

We had previously developed a nested polymerase chain reaction (PCR)–immunochromatography test (ICT) for identification of Mycobacterium tuberculosis (MTB) and differentiation of MTB from other members of M. tuberculosis complex (MTBC) from clinical sputum samples (Soo P.C. et al., Journal of Microbiological Methods. 2006, 66(3):440–8.). To further improve the detection flexibility, simplicity and efficiency, and reduce the cost, in this study, an alternative molecular diagnosis assay that utilizes gold nanoparticles derivatized with thiol modified oligonucleotides was developed. The gold nanoparticles probes, GP-1/GP-2 for IS6110 and GP-3/GP-4 for Rv3618, were designed to specifically hybridize with target DNAs of MTBC and MTB strains, respectively. Efficacy of the gold nanoparticle probes assay was evaluated by directly and simultaneously detecting not only MTBC but also MTB from 600 clinical sputum specimens. Results were compared with traditional culture and biochemical identification methods together with patients' clinical assessments. This assay showed a 96.6% sensitivity and 98.9% specificity towards detection of MTBC, and a 94.7% sensitivity and 99.6% specificity for detection of MTB. In conclusion, the gold nanoparticle probes assay is a simple, rapid, cost-effective and accurate detection system and shows great potential in clinical application of MTBC and MTB detection, especially in developing countries.     

Calcium-alginate beads for the oral delivery of transforming growth factor-β1 (TGF-β1): stabilization of TGF-β1 by the addition of polyacrylic acid within acid-treated beads

The susceptibility of the gastrointestinal tract to the toxic effects of chemotherapeutic drugs remains a complication in chemotherapy. Recent studies have suggested that transforming growth factor-β₁ (TGF-β₁) can be used as a cytoprotectant against cell cycle specific drugs. This work describes the use of alginate beads as a potential oral delivery system for TGF-β₁ designed to release the drug in the lumen of the small intestine. TGF-β₁ encapsulation and extent of release from alginate beads approached 100% as determined by ¹²⁵I-labelled TGF-β₁. However, when assayed by ELISA and a growth inhibition assay, nearly all immunoreactivity and bioactivity was lost, apparently due to a very high affinity of the alginate for TGF-β₁. This limitation was overcome by two novel methods: (1) incorporation of selected polyanions within the alginate beads to ‘shield’ TGF-β₁ from interaction with alginate and (2) exposure of the alginate beads containing TGF-β₁ to 0.1 N HCl (acid treatment) to simultaneously reduce the molecular weight of the alginate and its ability to interact with TGF- β₁. If the beads were only acid treated, just 8% of the immunoreactivity ofTGF-β₁ was retained. If polyacrylic acid (90 kDa) was added to the beads, 50% of the immunoreactivity of TGF-β₁ was retained. However, when TGF-β₁ was released from acid-treated beads also containing polyacrylic acid, more than 80% of the TGF-β₁ remained immunoreactive and bioactive. The retained TGF-β₁ activity after release from the beads was found to continue to increase with increasing concentrations of polyacrylic acid, until a concentration was reached where beads would not form. The dramatic increase in retained TGF-β₁ activity is attributed to the ability of polyacrylic acid to shield TGF-β₁ from interaction with lower molecular fragments of alginate.     

Association of a novel polymorphism in the bovine PPARGC1A gene with growth, slaughter and meat quality traits in Brangus steers

The PPARGC1A gene (peroxysome proliferator-activated receptor-γ coactivator 1α gene) controls muscle fiber type and brown adipocyte differentiation; therefore, it is a candidate gene for beef quality traits (tenderness and fat content). Two SNPs (Single Nucleotide Polymorphisms) were identified within exon 8 by multiple alignment of DNA sequences obtained from 24 bulls: a transition G/A (SNP 1181) and a transversion A/T (SNP 1299). The SNP 1181 is a novel SNP, corresponding to a non-conservative substitution (AGT/AAT) that could be the cause of amino acid substitution (³⁶⁴Serine/³⁶⁴Asparagine). A Mismatch PCR method was designed to determine genotypes of 73 bulls and 268 steers for SNP 1181. Growth, slaughter and meat quality information were available for the group of steers. Allele A of SNP 1181 was not found in Angus. In 243 steers, no significant differences (P > 0.05) were found for either final live body weight, gain in backfat thickness in Spring, kidney fat weight, kidney fat percentage, Warner–Bratzler shear force at 7 days postmortem, intramuscular fat percentage or meat colour between genotype GG and AG. This SNP could be included in breed composition and population admixture analyses because there are marked differences in allelic frequencies between Bos taurus and Bos indicus breeds.     

Gender- and Age-Dependent γ-Secretase Activity in Mouse Brain and Its Implication in Sporadic Alzheimer Disease

Alzheimer disease (AD) is an age-related disorder. Aging and female gender are two important risk factors associated with sporadic AD. However, the mechanism by which aging and gender contribute to the pathogenesis of sporadic AD is unclear. It is well known that genetic mutations in γ-secretase result in rare forms of early onset AD due to the aberrant production of Aβ42 peptides, which are the major constituents of senile plaques. However, the effect of age and gender on γ-secretase has not been fully investigated. Here, using normal wild-type mice, we show mouse brain γ-secretase exhibits gender- and age-dependent activity. Both male and female mice exhibit increased Aβ42Aβ40 ratios in aged brain, which mimics the effect of familial mutations of Presenilin-1, Presenlin-2, and the amyloid precursor protein on Aβ production. Additionally, female mice exhibit much higher γ-secretase activity in aged brain compared to male mice. Furthermore, both male and female mice exhibit a steady decline in Notch1 γ-secretase activity with aging. Using a small molecule affinity probe we demonstrate that male mice have less active γ-secretase complexes than female mice, which may account for the gender-associated differences in activity in aged brain. These findings demonstrate that aging can affect γ-secretase activity and specificity, suggesting a role for γ-secretase in sporadic AD. Furthermore, the increased APP γ-secretase activity seen in aged females may contribute to the increased incidence of sporadic AD in women and the aggressive Aβ plaque pathology seen in female mouse models of AD. In addition, deceased Notch γ-secretase activity may also contribute to neurodegeneration. Therefore, this study implicates altered γ-secretase activity and specificity as a possible mechanism of sporadic AD during aging.     

Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes

A multiplex PCR assay was developed which allowed the simultaneous detection of five trait genes or virulence markers in enterohemorrhagic Escherichia coli (EHEC) serotypes. A primer pair, designed to detect a single base-pair mutation in the uidA gene, is specific only for the prototypic EHEC of O157:H7 serotype and its toxigenic, non-motile variants. In a similar way, primers to the eaeA gene of the γ-intimin derivative specifically detects strains in the EHEC 1 clonal group, which consists mostly of O157:H7 and some O55:H7 serotypes. The other three primer pairs, specific for stx1, stx2 and both variants of ehxA genes, will detect the presence of these virulence genes in all EHEC serotypes. Analysis of 34 strains, including various serotypes of EHEC, Shiga toxin-producing E. coli and enteropathogenic E. coli, confirmed that the multiplex PCR assay detected the presence of these genes in a manner consistent with the known genotype of each respective strains.     

The small acid soluble proteins (SASP α and SASP β) of Bacillus weihenstephanensis and Bacillus mycoides group 2 are the most distinct among the Bacillus cereus group

The Bacillus cereus group includes Bacillus anthracis, B. cereus, Bacillus thuringiensis, Bacillus mycoides and Bacillus weihenstephanensis. The small acid soluble spore protein (SASP) β has been previously demonstrated to be among the biomarkers differentiating B. anthracis and B. cereus; SASP β of B. cereus most commonly exhibits one or two amino acid substitutions when compared to B. anthracis. SASP α is conserved in sequence among these two species. Neither SASP α nor β for B. thuringiensis, B. mycoides and B. weihenstephanensis have been previously characterized as taxonomic discriminators. In the current work molecular weight (MW) variation of these SASPs were determined by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for representative strains of the 5 species within the B. cereus group. The measured MWs also correlate with calculated MWs of translated amino acid sequences generated from whole genome sequencing projects. SASP α and β demonstrated consistent MW among B. cereus, B. thuringiensis, and B. mycoides strains (group 1). However B. mycoides (group 2) and B. weihenstephanensis SASP α and β were quite distinct making them unique among the B. cereus group. Limited sequence changes were observed in SASP α (at most 3 substitutions and 2 deletions) indicating it is a more conserved protein than SASP β (up to 6 substitutions and a deletion). Another even more conserved SASP, SASP α–β type, was described here for the first time.     

Concatenation of molecular docking and molecular simulation of BACE-1, γ-secretase targeted ligands: in pursuit of Alzheimer’s treatment

IntroductionAlzheimer''s disease (AD), the most predominant cause of dementia, has evolved tremendously with an escalating frequency, mainly affecting the elderly population. An effective means of delaying, preventing, or treating AD is yet to be achieved. The failure rate of dementia drug trials has been relatively higher than in other disease-related clinical trials. Hence, multi-targeted therapeutic approaches are gaining attention in pharmacological developments.AimsAs an extension of our earlier reports, we have performed docking and molecular dynamic (MD) simulation studies for the same 13 potential ligands against beta-site APP cleaving enzyme 1 (BACE-1) and γ-secretase as a therapeutic target for AD. The In-silico screening of these ligands as potential inhibitors of BACE-1 and γ-secretase was performed using AutoDock enabled PyRx v-0.8. The protein-ligand interactions were analyzed in Discovery Studio 2020 (BIOVIA). The stability of the most promising ligand against BACE-1 and γ-secretase was evaluated by MD simulation using Desmond-2018 (Schrodinger, LLC, NY, USA).ResultsThe computational screening revealed that the docking energy values for each of the ligands against both the target enzymes were in the range of −7.0 to −10.1 kcal/mol. Among the 13 ligands, 8 (55E, 6Z2, 6Z5, BRW, F1B, GVP, IQ6, and X37) showed binding energies of ≤−8 kcal/mol against BACE-1 and γ-secretase. For the selected enzyme targets, BACE-1 and γ-secretase, 6Z5 displayed the lowest binding energy of −10.1 and −9.8 kcal/mol, respectively. The MD simulation study confirmed the stability of BACE-6Z5 and γ-secretase-6Z5 complexes and highlighted the formation of a stable complex between 6Z5 and target enzymes.ConclusionThe virtual screening, molecular docking, and molecular dynamics simulation studies revealed the potential of these multi-enzyme targeted ligands. Among the studied ligands, 6Z5 seems to have the best binding potential and forms a stable complex with BACE-1 and γ-secretase. We recommend the synthesis of 6Z5 for future in-vitro and in-vivo studies.     

Development of γG, γA, γM, β1C/β1A, C′1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, α1-antitrypsin, orosomucoid, β-lipoprotein, α2-macroglobulin, and prealbumin in the human conceptus

The synthesis of γG, γA, γM, β_1C/β_1A, C′1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, α₁-antitrypsin, orosomucoid, β-lipoprotein, α₂-macroglobulin, and prealbumin was studied in 15 normal human embryos and fetuses of 29 days to 18 wk gestation and in the yolk sacs of four embryos from 5.5 to 11.5 wk gestation using tissue culture in ¹⁴C-labeled amino acids followed by radioimmunoelectrophoresis. The human embryo as early as 29 day gestation synthesized β_1C/β_1A, C′1 esterase inhibitor, transferrin, hemopexin, α₁-antitrypsin, β-lipoprotein, α₂-macroglobulin, and prealbumin in culture. At 32 days gestation ceruloplasmin and orosomucoid were also synthesized, but synthesis of fibrinogen was not observed before 5.5 wk. Synthesis of γM occurred as early as 10.5 wk gestation, and γG synthesis was found in cultures as early as 12 wk gestation; γA synthesis was not detected in any of the tissue cultures. With the exception of the γ-globulins, each of the proteins studied was synthesized by the liver, but additional sites of synthesis for some of these proteins were also found. Synthesis of γG and γM occurred primarily in the spleen, but other sites of synthesis were noted as well.     

Stimulus-induced Rotary Saturation (SIRS): a potential method for the detection of neuronal currents with MRI

Neuronal currents produce local transient and oscillatory magnetic fields that can be readily detected by MEG. Previous work attempting to detect these magnetic fields with MR focused on detecting local phase shifts and dephasing in T₂ or T₂-weighted images. For temporally biphasic and multi-phasic local currents the sensitivity of these methods can be reduced through the cancellation of the accrued phase induced by positive and negative episodes of the neuronal current. The magnitude of the phase shift is also dependent on the distribution of the current within the voxel. Since spins on one side of a current source develop an opposite phase shift relative to those on the other side, there is likely to be significant cancellation within the voxel.We introduce a potential method for detecting neuronal currents though their resonant T_1ρ saturation during a spin-lock preparation period. The method is insensitive to the temporal and spatial cancellation effects since it utilizes the multi-phasic nature of the neuronal currents and thus is not sensitive to the sign of the local field. To produce a T_1ρ reduction, the Larmor frequency in the rotating frame, which is set by γB_1lock (typically 20 Hz–5 kHz), must match the major frequency components of the stimulus-induced neuronal currents. We validate the method in MRI phantom studies. The rotary saturation spectra showed a sharp resonance when a current dipole within the phantom was driven at the Larmor frequency in the rotating frame. A 7 min block-design experiment was found to be sensitive to a current dipole strength of 56 nAm, an approximate magnetic field of 1 nT at 1.5 mm from the dipole. This dipole moment is similar to that seen using the phase shift method in a similar experimental setup by Konn et al. [Konn, D., Gowland, P., Bowtell, R., 2003. MRI detection of weak magnetic fields due to an extended current dipole in a conducting sphere: a model for direct detection of neuronal currents in the brain. Magn. Reson. Med. 50, 40–49], but is potentially less encumbered by temporal and spatial cancellation effects.     

Conformational state and receptor recognition of the C-terminal domain of human α2-macroglobulin after dissociation into half-molecules

Background: Dissociation of native human α₂-macroglobulin (α₂M) by sodium thiocyanate generates stable half-molecules with intact thiol esters. Significant conformational changes occur by the dissociation, which are similar to those occurring by transformation from native to methylamine-treated α₂-macroglobulin. Methods: The conformational state of the receptor-binding domain of the half-molecules was investigated by receptor binding and clearance studies, and by use of a panel of 11 monoclonal antibodies (mAbs) specific for the 18-kDa C-terminal receptor-binding fragment of α₂-macroglobulin. Results: The half-molecules simultaneously express epitopes specific for native, as well as epitopes specific for transformed α₂-macroglobulin. While it is possible to immunochemically discriminate between the different forms of tetrameric protein, the half-molecules retain a conformational state with no observed conformational changes in the C-terminal domain following cleavage of thiol esters or bait regions. The in vivo clearance rate in mice was consequently significantly slower for the half-molecules than for the tetrameric receptor-recognized forms of α₂-macroglobulin. Furthermore, half-molecules demonstrate lower affinity for binding to mouse macrophages than methylamine-treated tetrameric α₂-macroglobulin in competition studies. Conclusions: It is suggested that contact zones are functionally important for mediating conformational switches, which result in trapping and exposure of the receptor-binding sites.     

Specific detection and quantification of the phytopathogenic agent ‘Candidatus Phytoplasma prunorum’

‘Candidatus Phytoplasma prunorum’ is a wall-less bacterium associated with European stone fruit yellows (ESFY), a severe disease of Prunus spp. (mainly apricot and Japanese plum trees). It can be spread by one insect vector, Cacopsylla pruni, and by the trade of infected material. The availability of PCR-based methods allowing a sensitive and specific detection of ‘Ca. P. prunorum’ is crucial for this phytoplasma because, at present, it is uncultured and cannot be detected serologically. We developed a PCR test which, in contrast to the existing detection tools, provides a fast, specific and sensitive detection of ‘Ca. P. prunorum’ in plants and insects. For studies requiring an absolute quantification of the phytoplasma titer, the same primers were used to develop a real-time PCR assay, including a standard for C. pruni. The sensitivity of these molecular tools was compared by serial dilutions and their specificity was assessed both in silico and experimentally for reference strains and field samples of the closely related phytoplasma ‘Ca. P. prunorum’, ‘Ca. P. pyri’ (pear decline agent) and ‘Ca. P. mali’ (apple proliferation agent), as well as for representative strains of the ‘Ca. Phytoplasma’ genus.     

Fourth Canadian Consensus Conference on the Diagnosis and Treatment of Dementia: Recommendations for family physicians

Objective

To revise diagnostic strategies for Alzheimer disease (AD), update recommendations on symptomatic treatment of dementia, and provide an approach to rapidly progressive and early-onset dementias.

Composition of the committee

Experts and delegates representing relevant disciplines from diverse regions across Canada discussed and agreed upon revisions to the 2006 guidelines.

Methods

The GRADE (grading of recommendations, assessment, development, and evaluation) system was used to evaluate consensus on recommendations, which was defined as when 80% or more of participants voted for the recommendation. Evidence grades are reported where possible.

Report

Important for FPs, despite advances in liquid biomarkers and neuroimaging, the diagnosis of dementia in Canada remains fundamentally clinical. New core clinical criteria for the diagnosis of AD now recognize less common, nonamnestic forms. Early-onset dementia, a rare but important condition, should prompt referral to specialists with access to genetic counselors. Rapidly progressive dementia, poorly defined in the literature, is described to facilitate detection of this rare but important condition. There are new expanded indications for cholinesterase inhibitors beyond AD, as well as guidelines for their discontinuation, which had not been previously described. New evidence regarding use of memantine, antidepressants, and other psychotropic medications in dementia care is presented.

Conclusion

Several recommendations from the Fourth Canadian Consensus Conference on the Diagnosis and Treatment of Dementia are relevant to FPs. For guidelines to remain useful, family physicians should participate in all stages of the ongoing development process, including topic selection.The rising prevalence of dementia imposes substantial challenges for patients, families, and societies. In Canada, Alzheimer disease (AD), the most common cause of dementia, will increase from the current 500 000 cases to 1.1 million cases by 2038.¹ The World Health Organization urges countries to view dementia as a critical public health priority and notes that dementia “poses one of the greatest societal challenges for the 21st century.”²The Canadian Consensus Conference on the Diagnosis and Treatment of Dementia (CCCDTD) has proposed many recommendations to guide Canadian FPs.^3,4 Indeed, 3 previous CCCDTDs recommended that diagnosis and management of patients with dementia should mainly be the responsibility of primary health care and this remains implicitly valid.⁵ Optimal use of these guidelines has been partly limited by lack of citizen, caregiver, and FP participation in the guideline development process; scepticism regarding pharmaceutical sponsorship; and overly lengthy and complex guidelines.^6,7 The Fourth CCCDTD (CCCDTD4) has responded to many of these challenges, and the objective of this report is to summarize relevant recommendations for FPs from this conference. The intention is to provide a useful update informing Canadian FPs of recent advances in dementia care that are based on the best available evidence.Several factors prompted the CCCDTD4. Dramatic advances in neuroimaging and use of biomarkers have stimulated new ways of conceptualizing AD even in the absence of clinical symptoms. In 2011, this compelled the US National Institute on Aging and the Alzheimer’s Association (NIA-AA) to jointly propose new diagnostic criteria for research purposes.^8–10 A catalyst for the CCCDTD4 was concern about migration of such investigations into clinical care before the diagnostic and prognostic values were known, and consideration of the substantial ethical and financial implications of premature clinical use. Therefore, an objective of the committee was to update the previous diagnostic approach to AD based on these considerations. Additional topics included updated evidence on memantine, antidepressants, and other psychotropic medications in dementia, and guidelines for managing early-onset dementias and rapidly progressive dementias (RPDs). The conference assembled in Montreal, Que, on May 4 and 5, 2012.     

Prenatal diagnosis of thalassemia in 695 pedigrees from southeastern China: a 10‐year follow‐up study

Thalassaemia is highly prevalent in southeastern China. This 10‐year follow‐up study aimed to characterize the genotype and karyotype of thalassaemia in fetal samples derived from thalassemia carriers in Fujian province, southeastern China. A total of 476 prenatal samples from 472 couples carrying α‐thalassaemia traits and 224 samples from 223 couples carrying β‐thalassaemia traits were collected for STR analysis, detection of thalassemia genotypes and karyotyping. The common deletional α‐thalassemias and rare thalassemia genotypes were detected using Gap‐PCR assay, and the common β‐globin gene mutations were detected using PCR‐RDB assay. We detected 43.49% prevalence of α‐thalassaemia minor, 26.05% prevalence of α‐thalassaemia intermediate and major and 1.89% prevalence of rare form among the 476 prenatal samples from couples with α‐thalassaemia, and 85 fetuses with β‐thalassemia heterozygote, 16 with homozygote and 21 with double heterozygote, and a rare β^{IVS−2−654(C→T)}/Chinese G_γ (^Aγδβ)⁰ genotype among the 224 prenatal samples from couples with β‐thalassemia. Karyotyping showed 7 fetuses with abnormal karyotypes. Totally 153 pregnancies were terminated, and genetic diagnosis of thalassemia using fetal umbilical cord blood following induction of labor showed consistent results with prenatal diagnosis. No thalassemia phenotypes were identified in normal infants half a year after birth, and the infants with α‐thalassemia and β‐thalassemia minor had no or mild anemia symptoms, but normal development, while 15 babies with hemoglobin H disease presented moderate anemia symptoms. Our data suggest the pregestational screening of thalassemia, notably compound and rare forms of thalassemia, for couples carrying thalassemia traits.     

Molecular detection and quantification of the protozoan Bonamia ostreae in the flat oyster, Ostrea edulis

Bonamia ostreae is an intracellular protozoan which is recognized as a cause of mortality in European populations of flat oysters (Ostrea edulis). Based on the recent characterization of actin genes of B. ostreae, specific primers were designed for real-time PCR using SYBR^® Green chemistry. Specificity was demonstrated by the unique melting temperature peak observed in positive samples and by the lack of amplification in samples of oysters infected by closely related parasites, including Bonamia exitiosa. A calibration curve using a cloned template was defined to estimate copy number. The assay had a 6 log- dynamic range, mean inter- and intra-assay variation coefficients of <1% and a minimum detection limit of 50 gene copies per reaction. Using infected oyster samples as templates, the assay was at least 10-fold more sensitive than conventional PCR. The quantitative assay was applied to test 132 oysters, and results were compared with the heart imprint method. There was a strong correlation between both techniques, and the results showed that the real-time PCR assay should be useful for studies of the ecology of B. ostreae and its host–parasite relationship.     

Emergence of multiresistant variants of the community-acquired methicillin-resistant Staphylococcus aureus lineage ST1-SCCmecIV in 2 hospitals in Rio de Janeiro, Brazil

Usually, community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is susceptible to a variety of non–β-lactam drugs. These isolates commonly display SCCmecIV and are associated with community-acquired infections. More recently, CA-MRSA has been isolated from health-care–associated diseases. We characterized MRSA isolates from 2 hospitals in Rio de Janeiro area to assess the entry of new lineages. The isolates were primary genotyped using a combination of molecular typing methods including SCCmec, restriction modification test, and Panton–Valentine leukocidin (PVL) detection. Pulsed-field gel electrophoresis was carried out for representatives of each lineages found. Disk diffusion test was performed as recommended by the Clinical and Laboratory Standards Institute. SCCmecIV was the predominant cassette mec detected. The most frequent MRSA lineage, a PVL nonproducer, was allocated in the CC1-SCCmecIV. It was found that 56% of these isolates were resistant to 3 or more non–β-lactam drugs. Multilocus sequence typing of a representative of the CC1 isolates supported our finds that multiresistant variants of a CA-MRSA lineage (ST1-SCCmecIV) emerged in this city.     

Angiotensin-converting enzyme overexpression in myelomonocytes prevents Alzheimer’s-like cognitive decline

Cognitive decline in patients with Alzheimer’s disease (AD) is associated with elevated brain levels of amyloid β protein (Aβ), particularly neurotoxic Aβ_1–42. Angiotensin-converting enzyme (ACE) can degrade Aβ_1–42, and ACE overexpression in myelomonocytic cells enhances their immune function. To examine the effect of targeted ACE overexpression on AD, we crossed ACE^10/10 mice, which overexpress ACE in myelomonocytes using the c-fms promoter, with the transgenic APP_SWE/PS1_ΔE9 mouse model of AD (AD⁺). Evaluation of brain tissue from these AD⁺ACE^10/10 mice at 7 and 13 months revealed that levels of both soluble and insoluble brain Aβ_1–42 were reduced compared with those in AD⁺ mice. Furthermore, both plaque burden and astrogliosis were drastically reduced. Administration of the ACE inhibitor ramipril increased Aβ levels in AD⁺ACE^10/10 mice compared with the levels induced by the ACE-independent vasodilator hydralazine. Overall, AD⁺ACE^10/10 mice had less brain-infiltrating cells, consistent with reduced AD-associated pathology, though ACE-overexpressing macrophages were abundant around and engulfing Aβ plaques. At 11 and 12 months of age, the AD⁺ACE^10/WT and AD⁺ACE^10/10 mice were virtually equivalent to non-AD mice in cognitive ability, as assessed by maze-based behavioral tests. Our data demonstrate that an enhanced immune response, coupled with increased myelomonocytic expression of catalytically active ACE, prevents cognitive decline in a murine model of AD.     

Amyloid-Beta: A Crucial Factor in Alzheimer's Disease

Alzheimer''s disease (AD) is the most prevalent form of dementia which affects people older than 60 years of age. In AD, the dysregulation of the amyloid-beta (Aβ) level leads to the appearance of senile plaques which contain Aβ depositions. Aβ is a complex biological molecule which interacts with many types of receptors and/or forms insoluble assemblies and, eventually, its nonphysiological depositions alternate with the normal neuronal conditions. In this situation, AD signs appear and the patients experience marked cognitional disabilities. In general, intellect, social skills, personality, and memory are influenced by this disease and, in the long run, it leads to a reduction in quality of life and life expectancy. Due to the pivotal role of Aβ in the pathobiology of AD, a great deal of effort has been made to reveal its exact role in neuronal dysfunctions and to finding efficacious therapeutic strategies against its adverse neuronal outcomes. Hence, the determination of its different molecular assemblies and the mechanisms underlying its pathological effects are of interest. In the present paper, some of the well-established structural forms of Aβ, its interactions with various receptors and possible molecular and cellular mechanisms underlying its neurotoxicity are discussed. In addition, several Aβ-based rodent models of AD are reviewed.Key Words: Alzheimerߣs disease, Amyloid-beta, Pathobiology     

Genetic research and clinical analysis of β‐globin gene cluster deletions in the Chinese population of Fujian province: A 14‐year single‐center experience

BackgroundHeterozygotes of HPFH and δβ thalassemia are clinically asymptomatic or have mild hemoglobin (Hb) values. However, when both HPFH and δβ‐thalassemia are coinherited with heterozygous β‐thalassemia, patients may progress to a clinical phenotype of thalassemia intermedia or thalassemia major. The purpose of this study was to characterize the genotypes and analyze the phenotypes of these disorders in Fujian Province, to offer advice for genetic counseling and accurate prenatal diagnosis in this region. A total of 55 001 subjects were participated in thalassemia screening. 142 subjects with HbF levels ≥10%, before the blood transfusion, were selected for further investigation.MethodsMultiplex ligation‐dependent probe amplification (MLPA) and Gap‐PCR were used to screen for three β‐globin gene cluster deletions: Chinese ^Gγ(^Aγδβ)⁰ thalassemia and Southeast Asia HPFH (SEA‐HPFH) deletion and 1357 bp deletion (NG‐000007.3:g.69997‐71353 del 1357).ResultsA total of 142 patients with HbF (≥10%) were enrolled to characterize the molecular basis of β‐globin gene cluster deletions in our study; 22 cases 0.04% (22/55 001) were definitively diagnosed with β‐globin gene cluster deletions. Ten cases were heterozygous for the Chinese ^Gγ(^Aγδβ)⁰‐thal mutations, 10 cases were heterozygous for SEA‐HPFH, and one case was compound heterozygous for SEA‐HPFH and the α‐thal mutation. The 1357 bp deletion (NG‐000007.3:g.69997‐71353 del 1357) was detected in one case. Moreover, the hemoglobin A₂ levels in patients who were heterozygous for Chinese ^Gγ(^Aγδβ)⁰‐thal were statistically lower than in cases with SEA‐HPFH deletion(p < 0.05).ConclusionIn Fujian Province, the prevalence of common β‐globin gene cluster deletions was 0.04%. What''s more, the most common β‐globin cluster deletions are the Chinese ^Gγ(^Aγδβ)⁰ and SEA‐HPFH.