A lysimeter-based field study was done to monitor the transfer of culturable Escherichia coli, general (ALLBAC), human (Hf183) and swine (PIG-BAC-1) specific 16S rRNA Bacteroides spp. markers, nutrients and metals through soils and leachate over time following land application of a CP1/Class A as well as two CP2/Class B municipal biosolids (MBs). Hf183 markers were detected up to six days following application in soils receiving dewatered and liquid MBs, but not in leachate, suggesting their use in source tracking is better suited for recent pollution events. The CP2/Class B biosolids and swine manure contributed the highest microbial load with E. coli loads (between 2.5 and 3.7 log CFU (100 mL)(-1)) being greater than North American concentration recommendations for safe recreational water. ALLBAC persisted in soils and leachate receiving all treatments and was detected prior to amendment application demonstrating its unsuitability for identifying the presence of fecal pollution. A significant increase in NO₃-N (for Lystek and dewatered MBs) and total-P (for dewatered and liquid MBs) in leachate was observed in plots receiving the CP1/Class A and CP2/Class B type MBs which exceeded North American guidelines, suggesting impact to surface water. Metal (As, Cd, Cr, Co, Cu, Pb, Mo, Ni, Se, Zn and Hg) transfer was negligible in soil and leachate samples receiving all treatments. This study is one of the first to examine the fate of E. coli and Bacteroides spp. markers in situ following the land application of MBs where surface runoff does not apply.

Among swine reproductive traits, sow lifetime productivity (SLP) is considered a profitable trait in commercial pig farming. Notably, longevity and efficiency in SLP can be adopted as the key phenotype representing SLP. In this study, we conducted a co-association network analysis using results from a genome-wide association study for SLP-related traits. A total of 656 purebred Landrace female pigs were genotyped using a 60K SNP array. Significantly associated SNPs identified from the GWAS were annotated for the specific genes. Then, we constructed an association weight matrix to build a network based on the co-associations between the genes and 10 SLP traits. The entire network consisted of 495 nodes and 37 755 significant edges. We identified three key regulatory transcription factors: STAT2 (signal transducer and activator of transcription 2), MYF6 (myogenic factor 6) and TFCP2L1 (transcription factor CP2 like 1). The network revealed that the STAT2 and MYF6 regulatory modules cooperate with each other and specifically influence the longevity and efficiency of sows, whereas the TFCP2L1 family specifically affects the improvement of litter size.

OBJECTIVE

To determine the effect of multiple autoclave sterilization cycles on the integrity of titanium plates and screws used in craniofacial reconstruction.

METHODS

Torque to fracture was evaluated for 36 titanium 6AL-4V (Ti 6/4) screws divided evenly into 3 groups and tested as machined (control), after 10 cycles of autoclaving or after 50 cycles of autoclaving. Sterilization was carried out by autoclaving for 15 minutes followed by 8 minutes of drying at 270 degrees to 272 degrees F. The maximum torque attained before fracture was recorded. Rotating beam specimens were crafted from single lots of Ti 6/4, commercially pure titanium grade 4 (CP4) and commercially pure titanium grade 2 (CP2), and then subjected to testing in a standard rotating beam device as machined (control), after 10 cycles of autoclaving or after 50 cycles of autoclaving. The cycles required to fracture the specimen at a given applied stress were recorded for each material and for the number of autoclavings carried out before testing.

RESULTS

Although there was a trend toward decreased strength and increased ability to fracture with increased number of autoclave cycles, this did not reach statistical significance. Torque to fracture testing for 7 mm Ti 6/4 screws showed no significant difference in the maximum torque reached before fracture between controls, those screws that had been autoclaved 10 times (P < .500 +/- 5.70) and those that had been autoclaved 50 times (P < .398 +/- 4.08). Rotating beam specimens of Ti 6/4, CP4, and CP2 showed no significant difference in cycles to fracture regardless of the number of sterilization cycles to which the material was subjected.

CONCLUSIONS

Repeated cycles of autoclaving had no significant effect on the integrity of titanium plates and screws routinely used in craniofacial surgery.

mStaf is a zinc-finger protein that activates the transcription of the mouse selenocysteine tRNA gene. The mStaf gene is approx. 35 kb long and split into 16 exons. All exon-intron junction sequences conform to the GT/AG rule. The transcription start site is located 83 bp upstream of the initiation codon. Chromosomal mapping localized the gene to mouse chromosome 7, region E3-F1. Sequence analysis of the proximal promoter region revealed several potential regulatory elements; these include the recognition elements of Sp1, Nkx, CP2, E2A, SIF (SIS-inducible factor), TFII-I and cAMP-responsive element (CRE), but no TATA sequences. Transfection experiments demonstrated that the 5'-flanking region (-1894 to +37) of the mStaf gene drives transcription in mouse NMuMG cells and that a construct containing a fragment from -387 to +37 showed the highest transcriptional activity. Deletion and mutation experiments suggested that four Sp1 sites played an important role for the basal promoter activity. Furthermore, electrophoretic mobility-shift assays demonstrated that Sp3 but not other Sp (specificity protein) family members binds to three of the Sp1 sites. Our present study suggests that Sp3 is involved in the basal transcriptional activation of the mStaf gene.

OBJECTIVE

To examine contact point (CP) tightness of the mandibular dental arch in cases with a unilateral first molar (M1) missing, and also investigate the influence of time since extraction on CPs tightness.

METHODS

CPs tightness was measured with a tightness of dental contact point device in 29 patients (9 men, 20 women), mean age 32.6 +/- 6.5 years, with a unilateral missing M1 and no periodontal disease. ANOVA with repeated measures test was used to statistically compare CP tightness between extraction (E) and non-extraction (NE) sides, at significant level of P < or = 0.05, with CPNE serving as internal control.

RESULTS

The first CP adjacent to the extraction site between the first and second premolar (CP4-5,E) was significantly (P < or = 0.001) lower (29%) than CP4-5,NE. The difference (NE-E) was reduced to 20% at the more anterior CP (CP3-4,E) which was close to but non-significant (P < or = 0.09). Further anterior CPs (CP2-3, CP1-2) showed a non-significant difference between homologous CPs. Thus, reduction in CP tightness was confined to two adjacent teeth anterior to the extraction site. The tightness reduction of the posterior dentition (CP4-5 + CP3-4) was obtained 3 years post-extraction and subsequently maintained a constant asymmetry ratio (E/NE = 0.68-0.80) up to 14 years post-extraction.

Maize rayado fino virus (MRFV; genus Marafivirus; family Tymoviridae) is an isometric plant virus of 30 nm containing two components: empty shells and complete virus particles (encapsidating the 6.3 kb genomic RNA). Both particles are composed of two serologically related, carboxy co-terminal, coat proteins (CP) of apparent molecular mass 21-22 kDa (CP2) and 24-28 kDa (CP1) in a molar ratio of 3:1, respectively; CP1 contains a 37 amino acid amino terminal extension of CP2. In our study, expression of CP1 or CP2 in Escherichia coli resulted in assembly of each capsid protein into virus-like particles (VLPs), appearing in electron microscopy as stain-permeable (CP2) or stain-impermeable particles (CP1). CP1 VLPs encapsidated bacterial 16S ribosomal RNA, but not CP mRNA, while CP2 VLPs encapsidated neither CP mRNA nor 16S ribosomal RNA. Expression of CP1 and CP2 in E. coli using a co-expression vector resulted in the assembly of VLPs which were stain-impermeable and encapsidated CP mRNA. These results suggest that the N-terminal 37 amino acid residues of CP1, although not required for particle formation, may be involved in the assembly of complete virions and that the presence of both CP1 and CP2 in the particle is required for specific encapsidation of MRFV CP mRNA.

Taura syndrome virus (TSV) is an important virus infecting penaeid shrimp in the western hemisphere. Genetic variation and immunohistochemical differences of 20 TSV isolates collected from the USA, Taiwan, Mexico and Nicaragua were compared. Capsid protein genes CP1 (546 bp) and CP2 (584 bp) were amplified by RT-PCR and the cDNAs were sequenced. Pairwise comparison of nucleotide sequences showed a 0-2.4% difference in CP1 and a 0-3.5% difference in CP2. Phylogenetic analyses clustered the TSV isolates into two groups: one contained USA, Taiwan and some Mexican isolates, the other contained Mexican isolates only. Immunohistochemical analysis using a TSV-specific monoclonal antibody produced positive results for the USA and Taiwan isolates but negative results for the Mexican and Nicaraguan isolates. Molecular and immunohistochemical data suggest the existence of at least two TSV strains, one of which might have evolved following contact with a new penaeid host, Penaeus stylirostris.

A new series of Mn(II) coordination polymers, namely, [{Mn(L)(H2 O)2 }⋅2 Nap]∞ (CP1), [{Mn(L)(Ibu)2 (H2 O)2 }]∞ (CP2), [{Mn(L)(Flr)2 (H2 O)2 }]∞ (CP3), [{Mn(L)(Ind)2 (H2 O)2 }⋅H2 O]∞ (CP4), [{Mn2 (L)2 (μ-Flu)4 (H2 O)}⋅L]∞ (CP5), [{Mn2 (L)2 (μ-Tol)4 (H2 O)2 }]∞ (CP6) and [{Mn2 (L)2 (μ-Mef)4 (H2 O)2 }]∞ (CP7) (Nap=naproxen, Ibu=ibuprofen, Flr=flurbiprofen, Ind=indometacin, Flu=flufenamic acid, Tol=tolfenamic acid and Mef=mefenamic acid) derived from various non-steroidal anti-inflammatory drugs (NSAIDs) and the organic linker 1,2-bis(4-pyridyl)ethylene (L) have been synthesized with the aim of being used for cell imaging and drug delivery. Single-crystal X-ray diffraction (SXRD) studies revealed that the NSAID molecules were part of the coordination polymeric network either through coordination to the metal center (in the majority of the cases) or through hydrogen bonding. Remarkably, all the Mn(II) coordination polymers were found to be soluble in DMSO, thereby making them particularly suitable for the desired biological applications. Two of the coordination polymers (namely, CP1 and CP3) reported herein, were found to be photoluminescent both in the solid as well as in the solution state. Subsequent experiments (namely, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and PGE2 (prostaglandin E2 ) assays) established their biocompatibility and anti-inflammatory response. In vitro studies by using a macrophage cell line (i.e., RAW 264.7) revealed that both CP1 and CP3 were excellent cell imaging agents. Finally, biodegradability studies under simulated physiological conditions in phosphate-buffered saline (PBS) at pH 7.6 showed that slow and sustained release of the corresponding NSAID was indeed possible from both CP1 and CP3.

Background: Apolipoprotein E (APOE) ε4 is the only established risk gene for late-onset, sporadic Alzheimer's disease (AD). Previous studies have provided inconsistent evidence for the effect of APOE ε4 status on the visuospatial working memory (VSWM). Objective: The aim was to investigate the effect of APOE ε4 on VSWM with an event-related potential (ERP) study in healthy controls (HC) and amnestic mild cognitive impairment (aMCI) patients. Methods: The study recorded 39 aMCI patients (27 APOE ε4 non-carriers and 12 APOE ε4 carriers) and their 43 matched controls (25 APOE ε4 non-carriers and 18 APOE ε4 carriers) with an 64-channel electroencephalogram. Participants performed an N-back task, a VSWM paradigm that manipulated the number of items to be stored in memory. Results: The present study detected reduced accuracy and delayed mean correct response time (RT) in aMCI patients compared to HC. P300, a positive component that peaks between 300 and 500 ms, was elicited by the VSWM task. In addition, aMCI patients showed decreased P300 amplitude at the central-parietal (CP1, CPz, and CP2) and parietal (P1, Pz, and P2) electrodes in 0- and 1-back task compared to HC. In both HC and aMCI patients, APOE ε4 carriers showed reduced P300 amplitude with respect to non-carriers, whereas no significant differences in accuracy or RT were detected between APOE ε4 carriers and non-carriers. Additionally, standardized low-resolution brain electromagnetic tomography analysis (s-LORETA) showed enhanced brain activation in the right parahippocampal gyrus (PHG) during P300 time range in APOE ε4 carriers with respect to non-carriers in aMCI patients. Conclusion: It demonstrated that P300 amplitude could predict VSWM deficits in aMCI patients and contribute to early detection of VSWM deficits in APOE ε4 carriers.

Plants in the juvenile state are more tolerant to adverse conditions. Constitutive expression of MicroRNA156 (miR156) prolonged the juvenile phase and increased resistance to abiotic stress, but also affected the architecture of transgenic plants. In this study, we investigated the possibility of subtle manipulation of miR156 expression in flowering plants, with the goal to increase tolerance to abiotic stress without altering the normal growth and development of transgenic plants. Transgenic tobacco plants expressing ZmmiR156 from maize were generated, driven either by the cauliflower mosaic virus (CaMV) 35S promoter or the stress-inducible ZmRab17 promoter. Expression of ZmmiR156 led to improved drought and salt tolerance in both <i>35S::MIR156</i> and <i>Rab17::MIR156</i> transgenic plants, as shown by more vigorous growth, greater biomass production and higher antioxidant enzyme expression after a long period of drought or salt treatment, when compared to wild type and transgenic vector control plants. However, constitutive expression of ZmmiR156 also resulted in retarded growth, increased branching and delayed flowering of transgenic plants. These undesirable developmental changes could be mitigated by using the stress-inducible ZmRab17 promoter. Furthermore, under drought or salt stress conditions, expression of ZmmiR156 reduced the transcript level of <i>NtSPL2</i> and <i>NtSPL9</i>, the genes potentially targeted by ZmmiR156, as well as that of <i>CP1</i>, <i><em>CP2</em></i>, and <i>SAG12</i>, the senescence-associated genes in tobacco. Collectively, our results indicate that ZmmiR156 can be temporally manipulated for the genetic improvement of plants resistant to various abiotic stresses.

OBJECTIVE

To determine the prevalence of the different capsular polysaccharide (CP) and major surface-associated non-CP antigen 336 (SP-336) types among Staphylococcus aureus isolated from bovine mastitis cases in Australia and India.

METHODS

A total of 414 strains (154 from Australia, 260 from India) isolated from clinical bovine mastitis were included in the study. Mouse antisera raised against CP types (CP1, CP2, CP5, and CP8) or SP-336 were used in slide agglutination tests and compared with detection of cap1, cap5 and cap8 gene fragments by PCR.

RESULTS

Serological studies revealed the presence of CP2, CP5, CP8 and SP-336 in 9.1%, 23.4%, 31.8%, and 5.8% of the Australian versus 0.8%, 46.9%, 13.1% and 0% of the Indian isolates, respectively. By PCR, CP1, CP5 and CP8 accounted for 0%, 26.6% and 32.4% of the Australian versus 3.9%, 85% and 8.1% of the Indian isolates, respectively.

CONCLUSIONS

Both PCR and the serological method demonstrated that CP5 and CP8 are the predominant capsular types in Australia, whereas CP5 is the predominant capsular type in India. The study also demonstrated a strong correlation between both methods of typing for CP1, CP5, CP8 and non-typeable S. aureus strains. High-percentage prevalence of non-typeable isolates in both the countries highlights the importance of continued investigations of the identification of unique surface-associated polysaccharide antigens prevalent among S. aureus isolates for the formulation of CP- and SP-based vaccines for bovine mastitis.

The reactions of the Group 4 metallocene dichlorides [Cp'2 MCl2 ] (1 a: M=Ti, Cp'=Cp*=η(5) -pentamethylcyclopentadienyl, 1 b: M=Zr, Cp'=Cp=η(5) -cyclopentadienyl) with lithiated MesCH2-C≡N gave [Cp*2 TiCl(N=C=C(HMes))] (3; Mes=mesityl) in the case of 1 a. For compound 1 b, a nitrile-nitrile coupling resulted in a five-membered bridge in 4. The reaction of the metallocene alkyne complex [Cp*2 Zr(η(2) -Me3 SiC2 SiMe3 )] (2) with PhCH2 C≡N led in the first step to the unstable product [Cp*2 Zr(η(2) -Me3 SiC2 SiMe3 )(NC=CH2 Ph)] (5). After the elimination of the alkyne, a mixture of products was formed. By variation of the solvent and the reaction temperature, three compounds were isolated: a diazadiene complex 6, a bis(keteniminate) complex 7, and 8 with a keteniminate ligand and a five-membered metallacycle. Subsequent variation of the Cp ligand and the metal center by using [Cp2 Zr] and [Cp*2 Ti] with Me3 SiC2 SiMe3 in the reactions with PhCH2-C≡N gave complex mixtures.

In recent years, there was an increasing interest on candidate genes may play an important role in the development of Alzheimer's disease (AD). Several genome wide screens have undertaken so far or expanded recently, and suggested a number of genomic areas that may contain novel susceptibility genes for AD, in particular most compelling have been the findings on chromosome 12. Polymorphisms in different susceptibility genes on chromosome 12 (A2M, LRP1, CP2 and OLR1) are now being suggested as possible genetic markers for increased risk of developing AD. However, many of these studies are controversial and have shown conflicting results. Thus far, the search for the chromosome 12 Alzheimer's gene must continue and there are several other genes in this region that we are looking at. In this article, we focused on the current knowledge of the genetics of familial late-onset and sporadic AD linked to the chromosome 12, and the future search for other candidate genes.

In the present work the synergistic relationship between different strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus was studied at optimal (44 degrees C) and suboptimal temperatures (30 degrees C). Acidification, viscosity, whey syneresis, and bacterial concentration of the final product were evaluated on single-strain and mixed cultures after 24 h at 30 degrees C and 6 h at 44 degrees C. Three pairs of strains (LBB + CP2, LBP + CP2, and LBR + CP2) showed synergistic effect, which was reflected by the viscosity and syneresis of the coagulum. These results were more significant when cultures were incubated at 30 degrees C, reaching apparent viscosity values of 19 to 28 mPa x s. On the other hand, lactobacilli cultures enhanced the growth of two streptococci strains (CP2 and CP4). These results were confirmed by cultures of streptococci supplemented with supernatants of culture of lactobacilli. Those supernatants stimulate the viscosity produced by CP2 and CP4 strains and reduce the syneresis of all cultures of streptococci. Neither the increase of viscosity nor reduction of syneresis could be attributed to a decrease of pH.

Mitochondrial processing peptidase (MPP) consists of α and β subunits that catalyze the cleavage of N-terminal mitochondrial-targeting sequences (N-MTSs) and deliver preproteins to the mitochondria. In plants, both MPP subunits are associated with the respiratory complex bc1, which has been proposed to represent an ancestral form. Subsequent duplication of MPP subunits resulted in separate sets of genes encoding soluble MPP in the matrix and core proteins (cp1 and cp2) of the membrane-embedded bc1 complex. As only α-MPP was duplicated in Neurospora, its single β-MPP functions in both MPP and bc1 complexes. Herein, we investigated the MPP/core protein family and N-MTSs in the kinetoplastid Trypanosoma brucei, which is often considered one of the most ancient eukaryotes. Analysis of N-MTSs predicted in 336 mitochondrial proteins showed that trypanosomal N-MTSs were comparable with N-MTSs from other organisms. N-MTS cleavage is mediated by a standard heterodimeric MPP, which is present in the matrix of procyclic and bloodstream trypanosomes, and its expression is essential for the parasite. Distinct Genes encode cp1 and cp2, and in the bloodstream forms the expression of cp1 is downregulated along with the bc1 complex. Phylogenetic analysis revealed that all eukaryotic lineages include members with a Neurospora-type MPP/core protein family, whereas cp1 evolved independently in metazoans, some fungi and kinetoplastids. Evolution of cp1 allowed the independent regulation of respiration and protein import, which is essential for the procyclic and bloodstream forms of T. brucei. These results indicate that T. brucei possesses a highly derived MPP/core protein family that likely evolved in response to its complex life cycle and does not appear to have an ancient character proposed earlier for this eukaryote.

Prior to 2004, Colombian shrimp farming benefited from a selection program in which Penaeus vannamei stocks were developed with resistance to Taura syndrome disease (TS). However since 2004, TS reappeared as a significant disease. In 2010, an apparently new strain of TSV (designated as CO 10) was collected in Colombia. Its genome was sequenced and compared with six other fully sequenced isolates. This analysis revealed that the TSV CO 10 is closely related to the isolates from Hawaii and Venezuela. Phylogenetic analysis based on capsid protein 2 (CP2) region from 59 TSV isolates shows that the recent Colombian isolates (2006-2010) form a new cluster and differ from the previous Colombia isolates (1994-1998) by 4% in nucleotide sequence. The virulence of this CO 10 isolate was similar to a Belize TSV determined through experimental infection in P. vannamei showing 100% mortalities and similar survival curves. By RT-qPCR for TSV, the viral loads were also close in the infected shrimp from both CO 10 and Belize at the order of 1×10(10) copies per μl RNA. To develop TSV-resistant lines, the candidate shrimp should be challenged with virus strains that have been isolated most recently from the regions where they will be cultured. This study suggests that the TSV present in Colombian shrimp farms during the last 5 years is a new TSV strain with high virulence.

Ipsdienone (2-methyl-6-methylene-2,7-octadien-4-one) is an important intermediate in the biosynthesis of pheromonal ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) and ipsenol (2-methyl-6-methylene-7-octen-4-ol) in male pine engraver beetles, Ips pini (Say). A novel ipsdienol dehydrogenase (IDOLDH) with a pheromone-biosynthetic gene expression pattern was cloned, expressed, functionally characterized, and its cellular localization analyzed. The cDNA has a 762nt ORF encoding a 253 amino acid predicted translation product of 28kDa and pI 5.8. The protein has conserved motifs of the Cp2 subfamily of "classical" short-chain dehydrogenases. Transcript levels were highest in pheromone producing tissue: the anterior midgut of fed males. The protein was detected only in male midguts and localized in the cytosolic fraction of midgut cells. Recombinant IDOLDH was produced in Sf9 cells using a baculovirus expression system. Enzyme assays of protein preparations showed IDOLDH used both NAD⁺ and NADP⁺ as coenzymes with specific activities in the nanomole range. Enzyme assays and GC/MS analysis showed that IDOLDH catalyzed the oxidation of racemic ipsdienol and (4R)-(-)-ipsdienol to form ipsdienone, while (4S)-(+)-ipsdienol was not a substrate. These data strongly implicate IDOLDH as an enzyme involved in terminal pheromone-biosynthetic steps, likely functioning to "tune" ipsdienol enantiomeric ratios.

In order to investigate the shape and size differences in feet caused by daily footwear, a comparative study was conducted on foot morphology in two populations. The data from six measurements in general physique and 18 measurements in the feet and their contours were obtained from 34 Filipino women in Isabela Province and 40 Japanese women in Tokyo. Despite the fact that the Tokyo women had larger physique than the Isabela women, there were no significant differences in foot size between two groups. Both relative size of foot for general physique and intragroup deviation of foot proportion were larger in the Isabela women than those in the Tokyo women. In comparing foot contour, many measurements relating particularly to foot proportion, represented by angles, showed significant differences between the two groups. In gross observation some of the Isabela women showed marked deformity of the grand toe to the lateral side, "like a hallux valgus' without any complaints. In principal-component analysis (PCA), CP1 was interpreted as size factor, CP2 was considered as position of foot axis, CP3 and CP4 were estimated as degree of angle between foot axis and ball axis. Means of individual score by PCA showed a completely inverse pattern between Isabela and Tokyo women. The differences in foot morphology recognized in these two groups were considered from the point of view of differences of daily footwear, which have not changed in the Philippines but have changed dramatically in Japan since World War II. We concluded that the deformity like a hallux valgus, frequently found in previous generations of Japanese who used to wear traditional footwear, geta and zori, must have been a healthy deformity, however, the pathological deformity hallux valgus is observed only in the Isabela women of today.

Various anticoagulant-preservative solutions were investigated with a view to determining which would give optimal shelf life of packed cells. Satisfactory 24-hour post-transfusion survival (greater than 70%) after 28-day storage was obtained with citrate phosphate 277 mM dextrose, 0.25 mM adenine (CP2 X D-adenine). After addition of a rejuvenating electrolyte solution through a multiple closed plastic pack system, there was further improvement in viability, oxygen delivery capacity and metabolic activity of stored erythrocytes, with reduction in both plasma potassium and microaggregate numbers. No untoward reactions were observed during or after transfusion of 440 units of rejuvenated blood.

Bovine trichomonosis caused by Tritrichomonas foetus is a significant reproductive disease of cattle. Preputial samples were collected using sheath washing technique in bulls in Namibia. Thirty-six trichomonad cultures were characterized using the TaqMan-probe commercial real-time polymerase chain reaction (PCR) diagnostic assay (VetMAX™-Gold Trich Detection Kit) and CYBR real-time PCR assay based on TFR3/4 primers. Diagnostic real-time PCRs and DNA sequencing of the internal transcribed region confirmed presence of T. foetus in 35 out of 36 samples. Multilocus genotyping using cysteine proteases (CP1, CP2, CP4, CP5, CP6, CP7, CP8, CP9) and malate dehydrogenase (MDH1) gene sequences demonstrate that the T. foetus in Namibia are genetically distinct from those characterized elsewhere. We report the discovery of a novel genotype of T. foetus in Namibian cattle, distinct from other T. foetus genotypes in Europe, South and North America and Australia. We suggest recognition of a 'Southern African' genotype of T. foetus. Identification of the new genotype of T. foetus demonstrates the need for wider global sampling to fully understand the diversity and origin of T. foetus causing disease in cattle or cats.

Taura syndrome virus (TSV) is highly pathogenic to Litopenaeus vannamei (Pacific white shrimp) and has caused significant economic loss in the shrimp culture industry. It was first reported from Ecuador in 1992 and has since become widely distributed throughout the Americas and southeast Asia (SE Asia). To determine the genetic relationship among various geographic isolates, we amplified and sequenced a 1.3 kb fragment of the TSV capsid protein gene 2 (CP2) from each of 34 isolates collected from cultured penaeid shrimp stocks in Ecuador, Colombia, Honduras, USA, Mexico, Belize, Thailand, China, and Indonesia. An additional six CP2 sequences obtained from GenBank were included in the analysis. The results indicated low genetic variation (0--5.6% for nucleotide sequence and 0--7.0% for deduced amino acid sequence) among these 40 isolates. A phylogenetic analysis based on the deduced CP2 amino acid sequence revealed three distinct groups: Americas, Belize, and SE Asia. The Belize and SE Asia groups were separated from each other by a 4.7% difference in amino acid sequence. The Belize and Americas groups differed by 4.4%. The Americas and SE Asia groups were the closest, separated by a difference of only 3.3%. Comparison between Belize and Hawaii TSV (reference strain for Americas group) indicated that Belize TSV was more virulent than Hawaii TSV. In bioassays, the Belize isolate caused 50% mortality by 3 days, while the Hawaii isolate caused 50% mortality over 4--6 days. Based on the phylogenetic analysis and virulence comparison, the Belize TSV isolate should be considered as a new variant.

The profiling of microRNAs (miRNAs) is greatly significant for cellular events or disease diagnosis. Electrochemical methods for miRNAs analysis mostly can only measure one kind of miRNA, which is unambiguous to indicate the disease type and state. Here a label-free and PCR-free electrochemical method is presented for multiplexed evaluation of miRNAs in a single-tube experiment. The method is based on the combination of the high base-mismatch selectivity of ligase chain reaction (LCR) and the remarkable voltammetric signature of electrochemical QDs barcodes. Two reporting probes of RP1 and RP2 were labeled with PbS and CdS quantum dots (QDs) to prepare PbS-RP1 and CdS-RP2 conjugates, and two capture probes of CP1 and CP2 were co-immobilized on magnetic beads (MBs) to fabricate MB-CP1CP2 conjugate. The miRNAs samples were simply incubated with MB-CP1CP2, PbS-RP1, and CdS-RP2 conjugates, and then added with T4 DNA ligase. After release of the disjoined QDs barcodes from the MB-conjugates, two target miRNAs of miR-155 and miR-27b were simultaneously detected by square wave voltammetry with linear ranges of 50 fM-30 pM and 50 fM-1050 pM, and limits of detection (LODs) of 12 fM and 31 fM (S/N=3). The method fulfilled the assay in less than 70 min, and showed acceptable testing recoveries for the determination of miRNAs in biological matrix. Currently there are rare reports about electrochemical multiplexed quantification of miRNAs. The method is likely to provide a new platform for identification of multiple miRNAs in a simple way.

The total electronic energy and nucleus-independent chemical shift (NICS) of 95 isomers of N-confused porphyrin (NCP: normal porphyrin (N(0)CP), singly N-confused porphyrin (N(1)CP), doubly N-confused porphyrin (N(2)CP), triply N-confused porphyrin (N(3)CP), and fully N-confused porphyrin (N(4)CP)) have been calculated by the density functional theory (DFT) method. The stability of NCP decreased by increasing the number of confused pyrrole rings. Namely, the relative energies of the most stable isomers in each confusion level increased in a stepwise manner approximately by +18 kcal/mol: 0 (N(0)CP1), +17.147 (N(1)CP2), +37.461 (N(2)CPb3), +54.031 (N(3)CPd6), and +65.636 kcal/mol (N(4)CPc8). In this order, the mean plane deviation of these isomers increased from 0.000 to 0.123, 0.170, 0.215, and 0.251 A, respectively. The unusual tautomeric forms of pyrrole ring with an sp(3)-carbon were found in the stable forms of N(3)CP and N(4)CP. The NICS values at the mean position of the 24 core atoms were nearly the same for the most aromatic isomers regardless of the confusion level: -15.1280 (N(0)CP1), -13.8493 (N(1)CP2), -13.7267 (N(2)CPd1), -11.7723 (N(3)CPb5), and -13.6224 ppm (N(4)CPa6). The positive correlation between aromaticity and stability was inferred from the plots of NICS and the relative energy of NCP for N(0)CP, N(1)CP, and trans-N(2)CP. On the other hand, the correlation was negative for cis-N(2)CP, N(3)CP, and N(4)CP isomers.