Emerging Genomic Trends on Rabies Virus in Davao Region, Philippines, 2018-2021.

Rabies, caused by the rabies virus (RABV), remains a significant public health issue in the Philippines despite efforts to control it. To eliminate rabies by 2030, effective surveillance strategies are crucial. In this study, we examined RABV evolution and phylodynamics in the Davao Region using genome sequences from Davao City and nearby provinces. We adapted the RABV ARTIC Protocol for Oxford Nanopore High-Throughput Sequencing to optimize workflow efficiency under limited resources. Comparing new virus samples collected from June 2019 to June 2021 (n = 38) with baseline samples from June 2018 to May 2019 (n = 49), new sub-clades were observed in the phylogenetic tree, suggesting divergence from older variants that were previously undetected. Most of the new viruses belonged to the Asian SEA4_A1.1.1 lineage, but new (SEA4_B1 and SEA4_B1.1) and emerging (SEA4_B1.1_E1) lineages that have never been reported in the Philippines were also identified. The baseline study reported phylogeographic clustering of RABV isolates from the same areas. However, this pattern was disrupted in the current biosurveillance, with variants detected in areas outside the original cluster. Furthermore, our findings revealed significant transmission routes between Davao City and neighboring provinces, contrasting with the predominantly intra-city transmission observed in the baseline study. These results underscore the need for ongoing and timely genomic surveillance to monitor genetic diversity changes and the emergence of novel strains, as well as to track alterations in transmission pathways. Implementing cost-effective next-generation sequencing workflows will facilitate the integration of genomic surveillance into rabies control programs, particularly in resource-limited settings. Collaborations between different sectors can empower local laboratories and experts in genomic technologies and analysis.

Niche-specificity and the variable fraction of the Pectobacterium pan-genome.

We compare genome sequences of three closely related soft-rot pathogens that vary in host range and geographical distribution to identify genetic differences that could account for lifestyle differences. The isolates compared, Pectobacterium atrosepticum SCRI1043, P. carotovorum WPP14, and P. brasiliensis 1692, represent diverse lineages of the genus. P. carotovorum and P. brasiliensis genome contigs, generated by 454 pyrosequencing ordered by reference to the previously published complete circular chromosome of P. atrosepticum genome and each other, account for 96% of the predicted genome size. Orthologous proteins encoded by P. carotovorum and P. brasiliensis are approximately 95% identical to each other and 92% identical to P. atrosepticum. Multiple alignment using Mauve identified a core genome of 3.9 Mb conserved among these Pectobacterium spp. Each core genome is interrupted at many points by species-specific insertions or deletions (indels) that account for approximately 0.9 to 1.1 Mb. We demonstrate that the presence of a hrpK-like type III secretion system-dependent effector protein in P. carotovorum and P. brasiliensis and its absence from P. atrosepticum is insufficient to explain variability in their response to infection in a plant. Additional genes that vary among these species include those encoding peptide toxin production, enzyme production, secretion proteins, and antibiotic production, as well as differences in more general aspects of gene regulation and metabolism that may be relevant to pathogenicity.

Developmentally controlled genomic rearrangements in ciliated protozoa.

The ciliated protozoa undergo an extensive genome reorganization during the course of forming a transcriptionally active macronucleus. The process includes numerous chromosome fragmentation and DNA breakage and rejoining events. Recent work indicates that transposable elements play a role in the process.

Periodontal bacteria DNA findings in human cardiac tissue - Is there a link of periodontitis to heart valve disease?

BACKGROUND: The aim of the study was to detect periodontal pathogens DNA in atrial and myocardial tissue, and to investigate periodontal status and their connection to cardiac tissue inflammation. METHODS: In 30 patients, biopsy samples were taken from the atrium (A) and the ventricle myocardium (M) during aortic valve surgery. The dental examination included the dental and periodontal status (PS) and a collection of a microbiological sample. The detection of 11 periodontal pathogens DNA in oral and heart samples was carried out using PCR. The heart samples were prepared for detecting the LPS-binding protein (LBP), and for inflammation scoring on immunohistochemistry (IHC), comprising macrophages (CD68), LPS-binding protein receptor (CD14), and LBP (big42). RESULTS: 28 (93%) patients showed moderate to severe periodontitis. The periodontal pathogens in the oral samples of all patients revealed a similar distribution (3-93%). To a lesser extent and with a different distribution, these bacteria DNA were also detected in atrium and myocardium (3-27%). The LBP was detected in higher amount in atrium (0.22±0.16) versus myocardium (0.13±0.13, p=0.001). IHC showed a higher inflammation score in atrial than myocardial tissue as well as for CD14, CD68 and for LBP. Additional, periodontal findings showed a significant correlation to CD14 and CD68. CONCLUSION: The results provide evidence of the occurrence of oral bacteria DNA at the cardiac tissue, with a different impact on atrial and myocardial tissue inflammation. Influence of periodontal findings was identified, but their relevance is not yet distinct. Therefore further clinical investigations with long term implication are warranted.

Tec2, a second transposon-like element demonstrating developmentally programmed excision in Euplotes crassus.

The analysis of a repetitive DNA interruption of the micronuclear precursor to a 0.85-kb macronuclear gene in the hypotrich Euplotes crassus has led to the identification of a second transposon-like element named Tec2. Two copies of this element, one inserted into the other, compose the interruption. The Tec2 element resembles the previously characterized Tec1 element in overall size, copy number, length, and extreme terminal sequence of its inverted repeats and in the apparent use of a 5'-TA-3' target site. In addition, extrachromosomal circular forms of Tec2 appear in DNA isolated from cells undergoing macronuclear development at the same time and with the same conformation as extrachromosomal circular forms of Tec1. These similarities suggest that the Tec1 and Tec2 elements may be under the same type of regulation during macronuclear development.

DNA rearrangements in Euplotes crassus coincide with discrete periods of DNA replication during the polytene chromosome stage of macronuclear development.

Macronuclear development in Euplotes crassus begins with polytenization of micronuclear chromosomes and is accompanied by highly precise excision of DNA sequences known as internal eliminated sequences and transposon-like elements (Tecs). Quantitation of radiolabeled-precursor incorporation into DNA indicates that DNA synthesis during formation of polytene chromosomes is not continuous and occurs during two distinct periods. We demonstrate that the timing of Tec excision coincides with these replication periods and that excision can occur during both periods even at a single locus. We also show that Tec and internal eliminated sequence excisions are coincident in the second replication period, thus providing further evidence for similarity in their excision mechanism. Inhibition of DNA synthesis with hydroxyurea diminishes Tec element excision, indicating that replication is an important aspect of the excision process.

Aerosol optical depth, aerosol composition and air pollution during summer and winter conditions in Budapest.

The dependence of aerosol optical depth (AOD) on air particulate concentrations in the mixing layer height (MLH) was studied in Budapest in July 2003 and January 2004. During the campaigns gaseous (CO, SO(2), NO(x), O(3)), solid components (PM(2.5), PM(10)), as well as ionic species (ammonium, sulfate and nitrate) were measured at several urban and suburban sites. Additional data were collected from the Budapest air quality monitoring network. AOD was measured by a ground-based sun photometer. The mixing layer height and other common meteorological parameters were recorded. A linear relationship was found between the AOD and the columnar aerosol burden; the best linear fit (R(2)=0.96) was obtained for the secondary sulfate aerosol due to its mostly homogeneous spatial distribution and its optically active size range. The linear relationship is less pronounced for the PM(2.5) and PM(10) fractions since local emissions are very heterogeneous in time and space. The results indicate the importance of the mixing layer height in determining pollutant concentrations. During the winter campaign, when the boundary layer decreases to levels in between the altitudes of the sampling stations, measured concentrations showed significant differences due to different local sources and long-range transport. In the MLH time series unexpected nocturnal peaks were observed. The nocturnal increase of the MLH coincided with decreasing concentrations of all pollutants except for ozone; the ozone concentration increase indicates nocturnal vertical mixing between different air layers.

Differentiation of chromatin during DNA elimination in Euplotes crassus.

In Euplotes crassus, most of the micronuclear genome is eliminated during formation of a transcriptionally active macronucleus. To understand how this is mediated throughout the genome, we have examined the chromatin structure of the macronucleus-destined sequences and Tec transposons, which are dispersed in 15,000 copies in the micronuclear genome and completely eliminated during formation of the macronuclear genome. Whereas the macronucleus-destined sequences show a typical pattern of nucleosomal repeats in micrococcal nuclease digests, the Tec element chromatin structure digests to a nucleosome-like repeat pattern that is not typical: the minimum digestion products are approximately 300-600 base pairs, or "subnucleosomal," in size. In addition, the excised, circular forms of the Tec elements are exceedingly resistant to nucleases. Nevertheless, an underlying nucleosomal structure of the Tec elements can be demonstrated from the size differences between repeats in partial micrococcal nuclease digests and by trypsin treatment of nuclei, which results in mononucleosome-sized products. Characterization of the most micrococcal nuclease-resistant DNA indicates that micronuclear telomeres are organized into a chromatin structure with digestion properties identical to those of the Tec elements in the developing macronucleus. Thus, these major repetitive sequence components of the micronuclear genome differ in their chromatin structure from the macronuclear-destined sequences during DNA elimination. The potential role of developmental stage-specific histone variants in this chromatin differentiation is discussed.

Differential replication and DNA elimination in the polytene chromosomes of Euplotes crassus.

The transposon-like Tec elements of Euplotes crassus are precisely excised during formation of polytene chromosomes in the developing macronucleus. To determine whether all Tec elements exhibit identical developmental timing of excision, we used polymerase chain reaction to visualize amplification and diminution at numerous randomly selected Tec insertion sites. Two classes of sites are evident. Early replicating sites show one or more rounds of amplification and diminution (corresponding to excision) and frequently occur within macronuclear-destined sequences. Late replicating sites do not undergo diminution until chromosome fragmentation and are predominantly associated with eliminated sequences. We conclude that the previously described clustering of macro-nuclear-destined sequences in the micronuclear genome allows for their differential replication at the polytene stage and results in targeting of these sequences for transcriptional activation and highly specific deletion and chromosome fragmentation processes.

[Why screening for a renal artery stenosis?]. / Pourquoi dépister une sténose artérielle rénale?

Diagnosis of renal artery stenosis (RAS) should be discussed in numerous clinical situations including refractory high blood pressure (HBP), HBP in a polyvascular patient, degradation of renal function following renin angiotensin inhibitor or flash pulmonary edema. Ultrasound-doppler coupled with gadolinium-enhanced MR or CT angiography has proven adequate for most patients with RAS. Digital subtraction angiography should be limited to revascularisation procedures. Functional testing are not sensitive or specific enough because the degree of renin activation differs widely among patients with RAS. Renal percutaneous angioplasty induces a light to moderate decrease in blood pressure, has no effect on renal function but allows to reduce the number of anti-hypertensive drugs. Stenting completed angioplasty is worthwhile in most patients with atherosclerotic RAS. ACE inhibitors decrease mortality and increase renal function in patients with RAS.

[Seasonal fluctuations and influence of nutrition on macular pigment density]. / Jahreszeitliche Schwankungen und Einfluss der Ernährung auf die makuläre Pigmentdichte.

PURPOSE: We have previously reported on measuring macular pigment density (MPD) with a scanning laser ophthalmoscope (HRA, Heidelberg Engineering, Heidelberg, Germany). This study war undertaken to evaluate the variation of MPD over a period of 1 year in healthy subjects. METHOD: We used autofluorescence images recorded with a HRA to evaluate MPD with a 2 degrees circle centered on the fovea. Healthy subjects were included in the study and MPD measurements were repeated every 2 months over a period of 1 year. RESULTS: We included a total of 30 healthy subjects aged 19-34 years (mean: 23+/-2 years). Mean MPD at time point 1 was 0.215+/-0.056 density units (DU), at time point 2 0.235+/-0.051 DU, at time point 3 0.218+/-0.055 DU, at time point 4 0.228+/-0.057 DU, at time point 5 0.225+/-0.053 DU, and at time point 6 0.203+/-0.050 DU. The statistical analysis revealed no significant variation of MPD over the follow-up period of 1 year. CONCLUSION: This study demonstrates that MPD shows no variation over a period of 1 year in healthy subjects.

Organization of the micronuclear genome of oxytricha nova.

In the hypotrichous ciliated protozoan Oxytricha nova, approximately 95% of the micronuclear genome, including all of the repetitive DNA and most of the unique sequence DNA, is eliminated during the formation of the macronuclear genome. We have examined the interspersion patterns of repetitive and unique and eliminated and retained sequences in the micronuclear genome by characterizing randomly selected clones of micronuclear DNA. Three major classes of clones have been defined: (1) those containing primarily unique, retained sequences; (2) those containing only unique, eliminated sequences; and (3) those containing only repetitive, eliminated sequences. Clones of type one and three document two aspects of organization observed previously: clustering of macronuclear destined sequences and the presence of a prevalent repetitive element. Clones of the second type demonstrate for the first time that eliminated unique sequence DNA occurs in long stretches uninterrupted by repetitive sequences. To further examine repetitive sequence interspersion, we characterized the repetitive sequence family that is present in 50% of the clones (class three above). A consensus map of this element was obtained by mapping approximately 80 phage clones and by hybridization to digests of micronuclear DNA. The repeat element is extremely large (approximately 24 kb) and is interspersed with both macronuclear destined sequences and eliminated unique sequences.

[Post-denervation renal artery stenosis - a matter of concern?]. / Les sténoses artérielles rénales post-dénervation - une problématique préoccupante ?

Renal denervation, an invasive technique indicated in resistant hypertension patients insufficiently controlled by antihypertensive drugs, has a good safety profile. However, an increasing number of post-denervation renal artery stenosis cases has recently been reported. We describe the case of a 49-year-old woman with resistant hypertension who was referred to our university hypertension center for renal sympathetic denervation. Her daily treatment included six antihypertensive drugs. CT angiography prior to denervation showed no renal artery stenosis or vessel wall lesions. A standard renal denervation procedure using the St Jude protocol was performed. After an initial improvement in blood pressure profile, she presented with a blood pressure impairment at 3 months after renal denervation leading to the diagnosis of a severe right renal artery stenosis.

Actin, tubulin and H4 histone genes in three species of hypotrichous ciliated protozoa.

In hypotrichous ciliated protozoa, genes are transcribed in the macronucleus where the genome consists of 'gene-sized' linear DNA molecules. We have isolated clones of actin, tubulin and H4 histone macronuclear genes from Oxytricha nova, Stylonychia lemnae and Euplotes crassus in an effort to determine if they possess molecules of similar size for a given coding function, and also to determine the size range of non-coding DNA present on these molecules. Our results indicate that while the length of their non-coding DNA can vary slightly, both between different hypotrichs and within the gene family of a single organism, actin and tubulin macronuclear molecules are similarly sized. The sizes observed for these molecules support the hypothesis that each macronuclear molecule encodes a single gene. However, the H4 histone macronuclear molecules show a much wider size range and generally are much longer than necessary to encode the H4 histone. We therefore sequenced a 1700-bp H4 histone macronuclear molecule from O. nova to determine if it might possibly encode additional gene products. Sequence data reveals the presence of nine open reading frames (ORFs) greater than 100 bp in length; however, Northern hybridization analysis of the products of this DNA molecule reveals only a single transcript.

Sequence and transcript analysis of macronuclear histone H2B genes from Euplotes crassus.

Two 1.5-kb macronuclear chromosomes bearing histone H2B genes from the ciliated protozoan Euplotes crassus were cloned and sequenced. Although the noncoding sequences on these macronuclear chromosomes are very different, the genes encode an identical 113-aa histone H2B protein that has a shortened N-terminus and a highly conserved C-terminus relative to histone H2B proteins in other organisms. Primer extension was used to determine the transcription start points. Northern analysis shows that the abundance of H2B mRNA changes relative to DNA replication periods during the sexual phase of the life cycle. Analysis of 3' RACE products indicates that the H2B genes are coexpressed.

Overamplification of macronuclear linear DNA molecules during prolonged vegetative growth of Oxytricha nova.

During prolonged vegetative growth of a clonal line of Oxytricha nova, several macronuclear linear DNA molecules increased greatly in copy number over the rest of the approx. 24,000 kinds of molecules comprising the macronuclear genome. One of the amplified sequences was the linear DNA molecule encoding rRNA (rDNA). We have cloned and sequenced the other, smaller, amplified molecules and found that they comprise a gene family, with different allelic versions of one of the family members being amplified. Thus, increased replication is a general property of the molecules comprising this gene family. To date, no function has been assigned to these genes; thus, whether the amplification of these sequences has functional significance is unknown. The rDNA molecule and the two small amplified sequences increased 11-, 24- and 107-fold, respectively, during clonal growth of this line, eventually comprising up to 15% of the macronuclear DNA molecules. Seven other macronuclear DNA molecules did not vary substantially in copy number at different times during the clonal growth of this strain. Analysis of cell-to-cell differences in copy numbers in this clonally aged strain indicated more extensive variation than is evident when large populations from different times are compared.

Sequence of the macronuclear DNA encoding large subunit ribosomal protein 29 (L29) in Euplotes crassus and cycloheximide sensitivity.

As a first step towards developing a DNA transformation method for the ciliated protozoan Euplotes crassus we determined the minimum inhibitory concentration (MIC) for cell division in the presence of cycloheximide (Chx) for several cell lines and the range of Chx sensitivity for 106 different progeny cell lines derived by mating two lines. All of the cell lines are highly sensitive to Chx. Progeny cell lines show a wider range of sensitivities than the parental lines. Because site-directed mutagenesis of the RPL29 gene encoding the large subunit ribosomal protein 29 (L29) has been used to generate a Chx-resistance marker (ChxR) for another ciliate, Tetrahymena thermophila [Yao and Yao, Proc. Natl. Acad. Sci. USA 88 (1991) 9493-9497], we isolated and sequenced the entire E. crassus macronuclear DNA carrying RPL29. The encoded peptide is 52-73% identical in sequence to L29 sequences from organisms ranging from T. thermophila and Saccharomyces cerevisiae to mouse. In E. crassus, the codon that has been mutated to confer Chx resistance in both S. cerevisiae and T. thermophila already encodes the amino-acid residue of one of the mutant forms identified in these other organisms. Thus, E. crassus RPL29 is not a convenient source of a selectable marker. Notable features of the macronuclear DNA carrying RPL29 are its extremely short non-coding regions and a TAG stop codon.

Structures of the Euplotes crassus Tec1 and Tec2 elements: identification of putative transposase coding regions.

The Tec1 and Tec2 transposon-like element families of Euplotes crassus are highly unusual in that all 30,000 copies of each family are excised from the genome during a discrete time period of macronuclear development. Complete nucleotide sequences were generated for the Tec1-1 and Tec2-1 elements, representing the Tec1 and Tec2 families. Open reading frames (ORFs) are conserved in position and sequence between the two elements, although sequences that comprise one ORF (ORF2) of Tec1-1 are split into two overlapping ORFs (ORFs 2A and 2B) in Tec2-1. ORF1 in Tec1-1, its homolog in Tec2-1 and one of the overlapping ORFs from Tec2-1 (ORF2B) contain TGA codons, which may be translated as Cys, as observed for two other Euplotid genes. Sequence analyses of ORFs from other members of each element family indicate that the families are distinct from each other and are highly conserved within each family. Computer searches of sequence databases have revealed sequence similarity between Tec ORF1s and the previously described Tc1-IS630 family of transposases which includes ORFs from bacterial, nematode and insect transposons.

Differential expression of linker histone variants in Euplotes crassus.

Two genes have been cloned from the ciliate Euplotes crassus that encode proteins with sequence similarity to the linker histones from a variety of organisms. One gene, H1-1, is present on a 1.3-kb macronuclear DNA molecule and encodes a 16.2- kDa protein. The second gene, H1-2, is present on a 0.7-kb DNA molecule and encodes an 18.8-kDa protein. Both H1-1 and H1-2 are expressed in vegetative cells, but the two genes exhibit very different patterns of expression during macronuclear development. H1-1 transcripts accumulate during conjugation and during the final rounds of DNA amplification. H1-2 transcripts accumulate after the onset of polytene chromosome formation and remain high throughout the remainder of macronuclear development. H1-1 is the major perchloric-acid-soluble protein from macronuclei. The pattern of gene expression and the macronuclear location of the H1-1 protein indicate that H1-1 is the predominant linker histone in vegetative macronuclei.

Increased catabolism of VLDL-apolipoprotein B and synthesis of bile acids in a case of hypobetalipoproteinemia.

A 29-year-old man is described who has reduced concentrations of low density lipoprotein (LDL)-cholesterol seemingly due to an unusual variant of hypobetalipoproteinemia. The patient developed retinitis pigmentosa at age 14. When studied at age 28, his total cholesterol was 104 mg/dL, triglycerides 58 mg/dL, LDL-cholesterol 44 mg/dL, and HDL-cholesterol 51 mg/dL. Lipid and lipoprotein levels of his parents and sister were normal. His excretion of bile acids (13.9 mg/kg/d) was markedly elevated at about three times normal, although absorption rates of cholesterol and bile acids appeared to be in the normal range. His high excretion of bile acids equates to a threefold increase in bile acid synthesis. Isotope kinetic studies of his lipoproteins produced unexpected findings. Total production of VLDL-apolipoprotein B (apo B) was estimated to be 20.8 mg/kg/d, which was in the normal range. Synthesis of VLDL-triglycerides was also normal at 12.0 mg/kg/h. However, 75% of VLDL-apo B was removed directly from the circulation, which was much higher than values for direct removal of VLDL-apo B in control subjects. His production rate of LDL-protein (5.2 mg/kg/d) consequently was below normal, although his fractional catabolic rate for LDL (0.40 pools/d) was not distinctly elevated. These data suggest that the patient's hypobetalipoproteinemia was due to increased direct removal of VLDL remnants and not to reduced synthesis of VLDL-apo B; this abnormality may have been the result of enhanced activity of LDL receptors, which in turn was secondary to increased synthesis of bile acids.