Social demographics determinants for resistome and microbiome variation of a multiethnic community in Southern Malaysia.

The prevalence of antibiotic-resistant bacteria in Southeast Asia is a significant concern, yet there is limited research on the gut resistome and its correlation with lifestyle and environmental factors in the region. This study aimed to profile the gut resistome of 200 individuals in Malaysia using shotgun metagenomic sequencing and investigate its association with questionnaire data comprising demographic and lifestyle variables. A total of 1038 antibiotic resistance genes from 26 classes were detected with a mean carriage rate of 1.74 ± 1.18 gene copies per cell per person. Correlation analysis identified 14 environmental factors, including hygiene habits, health parameters, and intestinal colonization, that were significantly associated with the resistome (adjusted multivariate PERMANOVA, p < 0.05). Notably, individuals with positive yeast cultures exhibited a reduced copy number of 15 antibiotic resistance genes. Network analysis highlighted Escherichia coli as a major resistome network hub, with a positive correlation to 36 antibiotic-resistance genes. Our findings suggest that E. coli may play a pivotal role in shaping the resistome dynamics in Segamat, Malaysia, and its abundance is strongly associated with the community's health and lifestyle habits. Furthermore, the presence of yeast appears to be associated with the suppression of antibiotic-resistance genes.

Allele frequency spectrum of known ankylosing spondylitis associated variants in a Swedish population.

Objective: The genetic predisposition to ankylosing spondylitis (AS) has been most widely studied in cohorts with European ancestry. However, within Europe, disease prevalence is higher in Sweden. Given this, we aimed to characterize known AS susceptibility variants in a homogeneous Swedish data set, assessing reproducibility and direction of effect.Method: The power to detect association within an existing Swedish targeted sequencing study (381 controls; 310 AS cases) was examined, and a set of published associations (n = 151) was intersected with available genotypes. Association to disease was calculated using logistic regression accounting for population structure, and HLA-B27 status was determined with direct polymerase chain reaction genotyping.Results: The cases were found to be 92.3% HLA-B27 positive, with the data set showing ≥ 80% predictive power to replicate associations, with odds ratios ≥ 1.6 over a range of allele frequencies (0.1-0.7). Thirty-four markers, representing 23 gene loci, were available for investigation. The replicated variants tagged MICA and IL23R loci (p < 1.47 × 10-3), with variable direction of effect noted for gene loci IL1R1 and MST1.Conclusion: The Swedish data set successfully replicated both major histocompatibility complex (MHC) and non-MHC loci, and revealed a different replication pattern compared to discovery data sets. This was possibly due to population demographics, including HLA-B27 frequency and measured comorbidities.

Roan, ticked and clear coat patterns in the canine are associated with three haplotypes near usherin on CFA38.

White coat patterning is a feature of many dog breeds and is known to be coded primarily by the gene micropthalmia-associated transcription factor (MITF). This patterning in the coat can be modified by other factors to produce the attractive phenotypes termed 'ticked' and 'roan' that describe the presence of flecks of color that vary in distribution and intensity within otherwise 'clear' white markings. The appearance of the pigment in the white patterning caused by ticking and roaning intensifies in the weeks after birth. We applied genome-wide association to compare English Cocker Spaniels of roan phenotype (N = 34) with parti-color (non-roan) English Cocker Spaniels (N = 9) and identified an associated locus on CFA 38, CFA38:11 057 040 (Praw  = 8.9 × 10-10 , Pgenome  = 2.7 × 10-5 ). A local case-control association in English Springer Spaniels comparing 11 ticked and six clear dogs identified indicative association with a different haplotype, CFA38:11 122 467G>T (Praw  = 1.7 × 10-5 ) and CFA38:11 124 294A>C (Praw  = 1.7 × 10-5 ). We characterize three haplotypes in Spaniels according to their putative functional variant profiles at CFA38:11 111 286C>T (missense), CFA38:11 131 841-11 143 239DUP.insTTAA (using strongly linked marker CFA38:11 143 243C>T) and CFA38:11 156 425T>C (splice site). In Spaniels, the haplotypes work as an allelic series including alleles (t, recessive clear; T, dominant ticked/parti-color; and TR , incomplete dominant roan) to control the appearance of pigmented spots or flecks in otherwise white areas of the canine coat. In Spaniels the associated haplotypes are t (CCT), T (TCC) and TR (TTT) for SNP markers on CFA38 at 11 111 286C>T, 11 143 243C>T and 11 156 425T>C respectively. It is likely that other alleles exist in this series and together the haplotypes result in a complex range of patterning that is only visible when dogs have white patterning resulting from the epistatic gene Micropthalmia-associated transcription factor (the S-locus).

Transcriptomes from German shepherd dogs reveal differences in immune activity between atopic dermatitis affected and control skin.

Canine atopic dermatitis (CAD) is an inflammatory and pruritic allergic skin disease with both genetic and environmental risk factors described. We performed mRNA sequencing of non-lesional axillary skin biopsies from nine German shepherd dogs. Obtained RNA sequences were mapped to the dog genome (CanFam3.1) and a high-quality skin transcriptome was generated with 23,510 expressed gene transcripts. Differentially expressed genes (DEGs) were defined by comparing three controls to five treated CAD cases. Using a leave-one-out analysis, we identified seven DEGs: five known to encode proteins with functions related to an activated immune system (CD209, CLEC4G, LOC102156842 (lipopolysaccharide-binding protein-like), LOC480601 (regakine-1-like), LOC479668 (haptoglobin-like)), one (OBP) encoding an odorant-binding protein potentially connected to rhinitis, and the last (LOC607095) encoding a novel long non-coding RNA. Furthermore, high mRNA expression of inflammatory genes was found in axillary skin from an untreated mild CAD case compared with healthy skin. In conclusion, we define genes with different expression patterns in CAD case skin helping us understand post-treatment atopic skin. Further studies in larger sample sets are warranted to confirm and to transfer these results into clinical practice.

Presence of thoracic and lumbar vertebral malformations in pugs with and without chronic neurological deficits.

Congenital vertebral malformations (CVMs) are common in brachycephalic dogs such as the pug, and are often considered incidental findings. However, specific CVMs have been suggested to be associated with neurological deficits in pugs. The objective of this study was to investigate the clinical importance of CVMs in the pug by comparing computed tomography studies of the thoracolumbar spine from pugs without neurological deficits with those from pugs with a confirmed T3-L3 spinal cord lesion and neurological deficits consistent with a chronic T3-L3 myelopathy. A total of 57 pugs were recruited into the study from Sweden (n=33), United Kingdom (n=21) and Norway (n=3); 30 with neurological deficits and 27 without. Focal T3-L3 pathology was confirmed in all pugs with neurological deficits by magnetic resonance imaging (n=29) and/or pathology (n=15). Computed tomography studies of the thoracolumbar spine from pugs with and without neurological deficits were compared to investigate possible associations between presentation of neurological deficits consistent with chronic T3-L3 pathology and signalment variables, presence of CVMs and type of CVMs. Congenital vertebral malformations were as common in pugs with, as in pugs without, neurological deficits. Regardless of neurological status, the majority of pugs (96%) presented with one or more CVM. An association between presence, or type of CVM in the T1-L3 vertebral column, and neurological deficits consistent with T3-L3 pathology could not be confirmed.

Glycaemic control boosts glucosylated nanocarrier crossing the BBB into the brain.

Recently, nanocarriers that transport bioactive substances to a target site in the body have attracted considerable attention and undergone rapid progression in terms of the state of the art. However, few nanocarriers can enter the brain via a systemic route through the blood-brain barrier (BBB) to efficiently reach neurons. Here we prepare a self-assembled supramolecular nanocarrier with a surface featuring properly configured glucose. The BBB crossing and brain accumulation of this nanocarrier are boosted by the rapid glycaemic increase after fasting and by the putative phenomenon of the highly expressed glucose transporter-1 (GLUT1) in brain capillary endothelial cells migrating from the luminal to the abluminal plasma membrane. The precisely controlled glucose density on the surface of the nanocarrier enables the regulation of its distribution within the brain, and thus is successfully optimized to increase the number of nanocarriers accumulating in neurons.There are only a few examples of nanocarriers that can transport bioactive substances across the blood-brain barrier. Here the authors show that by rapid glycaemic increase the accumulation of a glucosylated nanocarrier in the brain can be controlled.

Interactions between CXCR4 and CXCL12 promote cell migration and invasion of canine hemangiosarcoma.

The CXCR4/CXCL12 axis plays an important role in cell locomotion and metastasis in many cancers. In this study, we hypothesized that the CXCR4/CXCL12 axis promotes migration and invasion of canine hemangiosarcoma (HSA) cells. Transcriptomic analysis across 12 HSA cell lines and 58 HSA whole tumour tissues identified heterogeneous expression of CXCR4 and CXCL12, which was associated with cell movement. In vitro, CXCL12 promoted calcium mobilization, cell migration and invasion that were directly proportional to surface expression of CXCR4; furthermore, these responses proved sensitive to the CXCR4 antagonist, AMD3100, in HSA cell lines. These results indicate that CXCL12 potentiates migration and invasion of canine HSA cells through CXCR4 signalling. The direct relationship between these responses in HSA cells suggests that the CXCR4/CXCL12 axis contributes to HSA progression.

Photoadduct Formation from the FMN Singlet Excited State in the LOV2 Domain of Chlamydomonas reinhardtii Phototropin.

The two light, oxygen, and voltage domains of phototropin are blue-light photoreceptor domains that control various functions in plants and green algae. The key step of the light-driven reaction is the formation of a photoadduct between its FMN chromophore and a conserved cysteine, where the canonical reaction proceeds through the FMN triplet state. Here, complete photoreaction mapping of CrLOV2 from Chlamydomonas reinhardtii phototropin and AsLOV2 from Avena sativa phototropin-1 was realized by ultrafast broadband spectroscopy from femtoseconds to microseconds. We demonstrate that in CrLOV2, a direct photoadduct formation channel originates from the initially excited singlet state, in addition to the canonical reaction through the triplet state. This direct photoadduct reaction is coupled by a proton or hydrogen transfer process, as indicated by a significant kinetic isotope effect of 1.4 on the fluorescence lifetime. Kinetic model analyses showed that 38% of the photoadducts are generated from the singlet excited state.

Extended exome sequencing identifies BACH2 as a novel major risk locus for Addison's disease.

BACKGROUND: Autoimmune disease is one of the leading causes of morbidity and mortality worldwide. In Addison's disease, the adrenal glands are targeted by destructive autoimmunity. Despite being the most common cause of primary adrenal failure, little is known about its aetiology. METHODS: To understand the genetic background of Addison's disease, we utilized the extensively characterized patients of the Swedish Addison Registry. We developed an extended exome capture array comprising a selected set of 1853 genes and their potential regulatory elements, for the purpose of sequencing 479 patients with Addison's disease and 1394 controls. RESULTS: We identified BACH2 (rs62408233-A, OR = 2.01 (1.71-2.37), P = 1.66 × 10-15 , MAF 0.46/0.29 in cases/controls) as a novel gene associated with Addison's disease development. We also confirmed the previously known associations with the HLA complex. CONCLUSION: Whilst BACH2 has been previously reported to associate with organ-specific autoimmune diseases co-inherited with Addison's disease, we have identified BACH2 as a major risk locus in Addison's disease, independent of concomitant autoimmune diseases. Our results may enable future research towards preventive disease treatment.

Absolute quantification reveals the stable transmission of a high copy number variant linked to autoinflammatory disease.

BACKGROUND: Dissecting the role copy number variants (CNVs) play in disease pathogenesis is directly reliant on accurate methods for quantification. The Shar-Pei dog breed is predisposed to a complex autoinflammatory disease with numerous clinical manifestations. One such sign, recurrent fever, was previously shown to be significantly associated with a novel, but unstable CNV (CNV_16.1). Droplet digital PCR (ddPCR) offers a new mechanism for CNV detection via absolute quantification with the promise of added precision and reliability. The aim of this study was to evaluate ddPCR in relation to quantitative PCR (qPCR) and to assess the suitability of the favoured method as a genetic test for Shar-Pei Autoinflammatory Disease (SPAID). RESULTS: One hundred and ninety-six individuals were assayed using both PCR methods at two CNV positions (CNV_14.3 and CNV_16.1). The digital method revealed a striking result. The CNVs did not follow a continuum of alleles as previously reported, rather the alleles were stable and pedigree analysis showed they adhered to Mendelian segregation. Subsequent analysis of ddPCR case/control data confirmed that both CNVs remained significantly associated with the subphenotype of fever, but also to the encompassing SPAID complex (p < 0.001). In addition, harbouring CNV_16.1 allele five (CNV_16.1|5) resulted in a four-fold increase in the odds for SPAID (p < 0.001). The inclusion of a genetic marker for CNV_16.1 in a genome-wide association test revealed that this variant explained 9.7 % of genetic variance and 25.8 % of the additive genetic heritability of this autoinflammatory disease. CONCLUSIONS: This data shows the utility of the ddPCR method to resolve cryptic copy number inheritance patterns and so open avenues of genetic testing. In its current form, the ddPCR test presented here could be used in canine breeding to reduce the number of homozygote CNV_16.1|5 individuals and thereby to reduce the prevalence of disease in this breed.

Effect of Breed on Plasma Endothelin-1 Concentration, Plasma Renin Activity, and Serum Cortisol Concentration in Healthy Dogs.

BACKGROUND: There are breed differences in several blood variables in healthy dogs. OBJECTIVE: Investigate breed variation in plasma endothelin-1 (ET-1) concentration, plasma renin activity, and serum cortisol concentration. ANIMALS: Five-hundred and thirty-one healthy dogs of 9 breeds examined at 5 centers (2-4 breeds/center). METHODS: Prospective observational study. Circulating concentrations of ET-1 and cortisol, and renin activity, were measured using commercially available assays. Absence of organ-related or systemic disease was ensured by thorough clinical investigations, including blood pressure measurement, echocardiography, ECG, blood and urine analysis. RESULTS: Median ET-1 concentration was 1.29 (interquartile range [IQR], 0.97-1.82) pg/mL, median cortisol concentration 46.0 (IQR, 29.0-80.8) nmol/L, and median renin activity 0.73 (IQR, 0.48-1.10) ng/mL/h in all dogs. Overall, breed differences were found in ET-1 and cortisol concentrations, and renin activity (P < .0001 for all). Pair-wise comparisons between breeds differed in 67% of comparisons for ET-1, 22% for cortisol, and 19% for renin activity, respectively. Within centers, breed differences were found at 5/5 centers for ET-1, 4/5 centers for cortisol, and 2/5 centers for renin activity. Newfoundlands had highest median ET-1 concentration, 3 times higher than Cavalier King Charles Spaniels, Doberman Pinschers, and Dachshunds. Median renin activity was highest in Dachshunds, twice the median value in Newfoundlands and Boxers. Median cortisol concentration was highest in Finnish Lapphunds, almost 3 times higher than in Boxers. CONCLUSIONS AND CLINICAL IMPORTANCE: Breed variation might be important to take into consideration when interpreting test results in clinical studies.

FTIR Spectroscopy Revealing Light-Dependent Refolding of the Conserved Tongue Region of Bacteriophytochrome.

Bacteriophytochromes (BphPs) constitute a class of photosensory proteins that toggle between Pr and Pfr functional states through absorption of red and far-red light. The photosensory core of BphPs is composed of PAS, GAF, and PHY domains. Here, we apply FTIR spectroscopy to investigate changes in the secondary structure of Rhodopseudomonas palustris BphP2 (RpBphP2) upon Pr to Pfr photoconversion. Our results indicate conversion from a ß-sheet to an α-helical element in the so-called tongue region of the PHY domain, consistent with recent X-ray structures of Deinococcus radiodurans DrBphP in dark and light states (Takala H.; et al. Nature2014, 5, 245-248). A conserved Asp in the GAF domain that noncovalently connects with the PHY domain and a conserved Pro in the tongue region of the PHY domain are essential for the ß-sheet-to-α-helix conversion.

Selective intracellular delivery of proteasome inhibitors through pH-sensitive polymeric micelles directed to efficient antitumor therapy.

The ubiquitin-proteasome system is central in the regulation of cellular proteins controlling cell cycle progression and apoptosis, drawing much interest for developing effective targeted cancer therapies. Herein, we developed a novel pH-responsive polymeric-micelle-based carrier system to effectively deliver the proteasome inhibitor MG132 into cancer cells. MG132 is covalently bound to the block copolymer composed of polyethylene glycol (PEG) and polyaspartate through an acid-labile hydrazone bond. This bond is stable at physiological condition, but hydrolytically degradable in acidic compartments in the cell, such as late-endosomes and lysosomes, and thus, it was used for controlled release of MG132 after EPR-mediated preferential accumulation of the micelles into the tumor. MG132-loaded micelles have monodispersed size distribution with an average diameter of 45nm, and critical micelle concentration is well below 10(-7)M. In vitro studies against several cancer cell lines confirmed that MG132-loaded micelles retained the cytotoxic effect, and this activity was indeed due to the inhibition of proteasome by released MG132 from the micelles. Real-time in vitro confocal-microscopy experiments clearly indicated that MG132-conjugated micelles disintegrated only inside the target cells. By intravital confocal micro-videography, we also confirmed the prolonged circulation of MG132 loaded micelles in the bloodstream, which lead to tumor specific accumulation of micelles, as confirmed by in vivo imaging 24h after injection. These micelles showed significantly lower in vivo toxicity than free MG132, while achieving remarkable antitumor effect against a subcutaneous HeLa-luc tumor model. Our findings create a paradigm for future development of polymeric-micelle-based carrier system for other peptide aldehyde type proteasome inhibitors to make them effective cohort of the existing cancer therapeutic regiments.

Breed distribution of SOD1 alleles previously associated with canine degenerative myelopathy.

BACKGROUND: Previous reports associated 2 mutant SOD1 alleles (SOD1:c.118A and SOD1:c.52T) with degenerative myelopathy in 6 canine breeds. The distribution of these alleles in other breeds has not been reported. OBJECTIVE: To describe the distribution of SOD1:c.118A and SOD1:c.52T in 222 breeds. ANIMALS: DNA from 33,747 dogs was genotyped at SOD1:c.118, SOD1:c.52, or both. Spinal cord sections from 249 of these dogs were examined. METHODS: Retrospective analysis of 35,359 previously determined genotypes at SOD1:c.118G>A or SOD1:c.52A>T and prospective survey to update the clinical status of a subset of dogs from which samples were obtained with a relatively low ascertainment bias. RESULTS: The SOD1:c.118A allele was found in cross-bred dogs and in 124 different canine breeds whereas the SOD1:c.52T allele was only found in Bernese Mountain Dogs. Most of the dogs with histopathologically confirmed degenerative myelopathy were SOD1:c.118A homozygotes, but 8 dogs with histopathologically confirmed degenerative myelopathy were SOD1:c.118A/G heterozygotes and had no other sequence variants in their SOD1 amino acid coding regions. The updated clinical conditions of dogs from which samples were obtained with a relatively low ascertainment bias suggest that SOD1:c.118A homozygotes are at a much higher risk of developing degenerative myelopathy than are SOD1:c.118A/G heterozygotes. CONCLUSIONS AND CLINICAL IMPORTANCE: We conclude that the SOD1:c.118A allele is widespread and common among privately owned dogs whereas the SOD1:c.52T allele is rare and appears to be limited to Bernese Mountain Dogs. We also conclude that breeding to avoid the production of SOD1:c.118A homozygotes is a rational strategy.

Breed differences in natriuretic peptides in healthy dogs.

BACKGROUND: Measurement of plasma concentration of natriuretic peptides (NPs) is suggested to be of value in diagnosis of cardiac disease in dogs, but many factors other than cardiac status may influence their concentrations. Dog breed potentially is 1 such factor. OBJECTIVE: To investigate breed variation in plasma concentrations of pro-atrial natriuretic peptide 31-67 (proANP 31-67) and N-terminal B-type natriuretic peptide (NT-proBNP) in healthy dogs. ANIMALS: 535 healthy, privately owned dogs of 9 breeds were examined at 5 centers as part of the European Union (EU) LUPA project. METHODS: Absence of cardiovascular disease or other clinically relevant organ-related or systemic disease was ensured by thorough clinical investigation. Plasma concentrations of proANP 31-67 and NT-proBNP were measured by commercially available ELISA assays. RESULTS: Overall significant breed differences were found in proANP 31-67 (P < .0001) and NT-proBNP (P < .0001) concentrations. Pair-wise comparisons between breeds differed in approximately 50% of comparisons for proANP 31-67 as well as NT-proBNP concentrations, both when including all centers and within each center. Interquartile range was large for many breeds, especially for NT-proBNP. Among included breeds, Labrador Retrievers and Newfoundlands had highest median NT-proBNP concentrations with concentrations 3 times as high as those of Dachshunds. German Shepherds and Cavalier King Charles Spaniels had the highest median proANP 31-67 concentrations, twice the median concentration in Doberman Pinschers. CONCLUSIONS AND CLINICAL IMPORTANCE: Considerable interbreed variation in plasma NP concentrations was found in healthy dogs. Intrabreed variation was large in several breeds, especially for NT-proBNP. Additional studies are needed to establish breed-specific reference ranges.

Molecular profiling reveals prognostically significant subtypes of canine lymphoma.

We performed genomewide gene expression analysis of 35 samples representing 6 common histologic subtypes of canine lymphoma and bioinformatics analyses to define their molecular characteristics. Three major groups were defined on the basis of gene expression profiles: (1) low-grade T-cell lymphoma, composed entirely by T-zone lymphoma; (2) high-grade T-cell lymphoma, consisting of lymphoblastic T-cell lymphoma and peripheral T-cell lymphoma not otherwise specified; and (3) B-cell lymphoma, consisting of marginal B-cell lymphoma, diffuse large B-cell lymphoma, and Burkitt lymphoma. Interspecies comparative analyses of gene expression profiles also showed that marginal B-cell lymphoma and diffuse large B-cell lymphoma in dogs and humans might represent a continuum of disease with similar drivers. The classification of these diverse tumors into 3 subgroups was prognostically significant, as the groups were directly correlated with event-free survival. Finally, we developed a benchtop diagnostic test based on expression of 4 genes that can robustly classify canine lymphomas into one of these 3 subgroups, enabling a direct clinical application for our results.

Fluorescence quantum yield and photochemistry of bacteriophytochrome constructs.

Bacteriophytochromes (Bphs) are red-light photoreceptor proteins with a photosensory core that consists of three distinct domains, PAS, GAF and PHY, and covalently binds biliverdin (BV) to a conserved cysteine in the PAS domain. In a recent development, PAS-GAF variants were engineered for use as a near-infrared fluorescent marker in mammalian tissues (Tsien and co-workers, Science, 2009, 324, 804-807). Here, we report the fluorescence quantum yield and photochemistry of two highly-related Bphs from Rps. palustris, RpBphP2 (P2) and RpBphP3 (P3) with distinct photoconversion and fluorescence properties. We applied ultrafast spectroscopy to wild type P3 and P2 PAS-GAF proteins and their P3 D216A, Y272F and P2 D202A PAS-GAF-PHY mutant proteins. In these mutants hydrogen-bond interactions between a conserved aspartate (Asp) which connects the BV chromophore with the PHY domains are disrupted. The excited-state lifetime of the truncated P3 and P2 PAS-GAF proteins was significantly longer than in their PAS-GAF-PHY counterparts that constitute the full photosensory core. Mutation of the conserved Asp to Ala in the PAS-GAF-PHY protein had a similar but larger effect. The fluorescence quantum yields of the P3 D216A and Y272F mutants were 0.066, higher than that of wild type P3 (0.043) and similar to the engineered Bph of Tsien and co-workers. We conclude that elimination of a key hydrogen-bond interaction between Asp and a conserved Arg in the PHY domain is responsible for the excited-state lifetime increase in all Bph variants studied here. H/D exchange resulted in a 1.4-1.7 fold increase of excited-state lifetime. The results support a reaction model in which deactivation of the BV chromophore proceeds via excited-state proton transfer from the BV pyrrole nitrogens to the backbone of the conserved Asp or to a bound water. This work may aid in rational structure- and mechanism-based conversion of constructs based on P3 and other BPhs into efficient near-IR, deep tissue, fluorescent markers.

Primary reactions of bacteriophytochrome observed with ultrafast mid-infrared spectroscopy.

Phytochromes are red-light photoreceptor proteins that regulate a variety of responses and cellular processes in plants, bacteria, and fungi. The phytochrome light activation mechanism involves isomerization around the C(15)═C(16) double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important recent development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues. Bphs covalently bind a biliverdin (BV) chromophore, naturally abundant in mammalian cells. Here, we report an ultrafast time-resolved mid-infrared spectroscopic study on the Pr state of two highly related Bphs from Rps. palustris , RpBphP2 (P2) and RpBphP3 (P3) with distinct photoconversion and fluorescence properties. We observed that the BV excited state of P2 decays in 58 ps, while the BV excited state of P3 decays in 362 ps. By combining ultrafast mid-IR spectroscopy with FTIR spectroscopy on P2 and P3 wild type and mutant proteins, we demonstrate that the hydrogen bond strength at the ring D carbonyl of the BV chromophore is significantly stronger in P3 as compared to P2. This result is consistent with the X-ray structures of Bph, which indicate one hydrogen bond from a conserved histidine to the BV ring D carbonyl for classical bacteriophytochromes such as P2, and one or two additional hydrogen bonds from a serine and a lysine side chain to the BV ring D carbonyl for P3. We conclude that the hydrogen-bond strength at BV ring D is a key determinant of excited-state lifetime and fluorescence quantum yield. Excited-state decay is followed by the formation of a primary intermediate that does not decay on the nanosecond time scale of the experiment, which shows a narrow absorption band at ∼1540 cm(-1). Possible origins of this product band are discussed. This work may aid in rational structure- and mechanism-based conversion of BPh into an efficient near-IR fluorescent marker.

Diabetes mellitus in elkhounds is associated with diestrus and pregnancy.

BACKGROUND: Female Elkhounds are shown to be at increased risk for diabetes mellitus, and occurrence of diabetes during pregnancy has been described in several cases. HYPOTHESIS: Onset of diabetes mellitus in Elkhounds is associated with diestrus. ANIMALS: Sixty-three Elkhounds with diabetes mellitus and 26 healthy controls. METHODS: Medical records from 63 Elkhounds with diabetes were reviewed and owners were contacted for follow-up information. Blood samples from the day of diagnosis were available for 26 dogs. Glucose, fructosamine, C-peptide, growth hormone (GH), insulin-like growth factor-1, progesterone, and glutamate decarboxylase isoform 65-autoantibodies were analyzed and compared with 26 healthy dogs. Logistic models were used to evaluate the association of clinical variables with the probability of diabetes and with permanent diabetes mellitus after ovariohysterectomy (OHE). RESULTS: All dogs in the study were intact females and 7 dogs (11%) were pregnant at diagnosis. The 1st clinical signs of diabetes mellitus occurred at a median of 30 days (interquartile range [IQR], 3-45) after estrus, and diagnosis was made at a median of 46 days (IQR, 27-62) after estrus. Diabetes was associated with higher concentrations of GH and lower concentrations of progesterone compared with controls matched for time after estrus. Forty-six percent of dogs that underwent OHE recovered from diabetes with a lower probability of remission in dogs with higher glucose concentrations (odds ratio [OR], 1.2; P=.03) at diagnosis and longer time (weeks) from diagnosis to surgery (OR, 1.5; P=.05). CONCLUSIONS: Diabetes mellitus in Elkhounds develops mainly during diestrus and pregnancy. Immediate OHE improves the prognosis for remission of diabetes.