Unbiased analyses of ITS folding motifs in a taxonomically confusing lineage: Anagnostidinema visiae sp. nov. (cyanobacteria).

Cyanobacteria are diverse prokaryotic, photosynthetic organisms present in nearly every known ecosystem. Recent investigations around the world have recovered vast amounts of novel biodiversity in seldom sampled habitats. One phylogenetically significant character, the secondary folding structures of the 16S-23S ITS rDNA region, has allowed an unprecedented capacity to erect new species. However, two questions arise: Is this feature as informative as is proposed, and how do we best employ these features? Submerged sinkholes with oxygen-poor, sulfur-rich ground water in Lake Huron (USA) contain microbial mats dominated by both oxygenic and anoxygenic cyanobacteria. We sought to document some of this unique cyanobacterial diversity. Using culture-based investigations, we recovered 45 strains, of which 23 were analyzed employing 16S-23S rDNA sequences, ITS folding patterns, ecology, and morphology. With scant morphological discontinuities and nebulous 16S rDNA gene sequence divergence, ITS folding patterns were effective at articulating cryptic biodiversity. However, we would have missed these features had we not folded all the available motifs from the strains, including those with highly similar 16S rDNA gene sequences. If we had relied solely on morphological or 16S rDNA gene data, then we might well have missed the diversity of Anagnostidinema. Thus, in order to avoid conformation basis, which is potentially common when employing ITS structures, we advocate clustering strains based on ITS rDNA region patterns independently and comparing them back to 16S rDNA gene phylogenies. Using a total evidence approach, we erected a new taxon according to the International Code of Nomenclature for Algae, Fungi, and Plants: Anagnostidinema visiae.

Differences in Spatiotemporal Patterns of Vehicle Collisions with Wildlife and Livestock.

Road ecology research has tended to focus on wildlife-vehicle collisions (WVCs) while omitting or failing to differentiate domestic (i.e., livestock) animal-vehicle collisions (DAVCs). This has limited our understanding of where, when, and how frequently DAVCs occur, and whether these patterns differ from those for WVCs. We used a 10-year collision data set for the U.S. state of Montana to compare temporal and spatial patterns of DAVCs versus WVCs at multiple scales. WVCs exhibited two diel peaks (dawn and dusk) versus only one prominent peak (late evening/early night) for DAVCs. Seasonal patterns of WVCs and DAVCs were broadly similar, but DAVCs exhibited a more pronounced late-fall peak. At the county scale, DAVCs were overrepresented relative to WVCs in most of eastern Montana and underrepresented in most of western Montana. WVC and DAVC hotpots did not show strong overlap at the 1-mile road segment scale. Our results suggest that DAVCs warrant greater attention, and they may represent a high priority for management and mitigation measures in some areas because (1) they can be locally common even when regionally rare, (2) they are more dangerous to motorists on a per-collision basis than WVCs, and (3) they can present a legal liability for livestock owners. Mitigation measures for DAVCs may differ from those for WVCs and require further development and testing. Future data collection efforts should include information not only on the location and timing of animal-vehicle collisions, but also on the species of animals killed.

Comparing methods for estimation of heterogeneous treatment effects using observational data from health care databases.

There is growing interest in using routinely collected data from health care databases to study the safety and effectiveness of therapies in "real-world" conditions, as it can provide complementary evidence to that of randomized controlled trials. Causal inference from health care databases is challenging because the data are typically noisy, high dimensional, and most importantly, observational. It requires methods that can estimate heterogeneous treatment effects while controlling for confounding in high dimensions. Bayesian additive regression trees, causal forests, causal boosting, and causal multivariate adaptive regression splines are off-the-shelf methods that have shown good performance for estimation of heterogeneous treatment effects in observational studies of continuous outcomes. However, it is not clear how these methods would perform in health care database studies where outcomes are often binary and rare and data structures are complex. In this study, we evaluate these methods in simulation studies that recapitulate key characteristics of comparative effectiveness studies. We focus on the conditional average effect of a binary treatment on a binary outcome using the conditional risk difference as an estimand. To emulate health care database studies, we propose a simulation design where real covariate and treatment assignment data are used and only outcomes are simulated based on nonparametric models of the real outcomes. We apply this design to 4 published observational studies that used records from 2 major health care databases in the United States. Our results suggest that Bayesian additive regression trees and causal boosting consistently provide low bias in conditional risk difference estimates in the context of health care database studies.

Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission.

Genetic manipulations of insect populations for pest control have been advocated for some time, but there are few cases where manipulated individuals have been released in the field and no cases where they have successfully invaded target populations. Population transformation using the intracellular bacterium Wolbachia is particularly attractive because this maternally-inherited agent provides a powerful mechanism to invade natural populations through cytoplasmic incompatibility. When Wolbachia are introduced into mosquitoes, they interfere with pathogen transmission and influence key life history traits such as lifespan. Here we describe how the wMel Wolbachia infection, introduced into the dengue vector Aedes aegypti from Drosophila melanogaster, successfully invaded two natural A. aegypti populations in Australia, reaching near-fixation in a few months following releases of wMel-infected A. aegypti adults. Models with plausible parameter values indicate that Wolbachia-infected mosquitoes suffered relatively small fitness costs, leading to an unstable equilibrium frequency <30% that must be exceeded for invasion. These findings demonstrate that Wolbachia-based strategies can be deployed as a practical approach to dengue suppression with potential for area-wide implementation.

Metabarcoding reveals unique microbial mat communities and evidence of biogeographic influence in low-oxygen, high-sulfur sinkholes and springs.

High-sulfur, low-oxygen environments formed by underwater sinkholes and springs create unique habitats populated by microbial mat communities. To explore the diversity and biogeography of these mats, samples were collected from three sites in Alpena, Michigan, one site in Monroe, Michigan, and one site in Palm Coast, Florida. Our study investigated previously undescribed eukaryotic diversity in these habitats and further explored their bacterial communities. Mat samples and water parameters were collected from sulfur spring sites during the spring, summer, and fall of 2022. Cyanobacteria and diatoms were cultured from mat subsamples to create a culture-based DNA reference library. Remaining mat samples were used for metabarcoding of the 16S and rbcL regions to explore bacterial and diatom diversity, respectively. Analyses of water chemistry, alpha diversity, and beta diversity articulated a range of high-sulfur, low-oxygen habitats, each with distinct microbial communities. Conductivity, pH, dissolved oxygen, temperature, sulfate, and chloride had significant influences on community composition but did not describe the differences between communities well. Chloride concentration had the strongest correlation with microbial community structure. Mantel tests revealed that biogeography contributed to differences between communities as well. Our results provide novel information on microbial mat composition and present evidence that both local conditions and biogeography influence these unique communities.

Dare to Care for the Elderly: Five Insights.

Care is often limited by patient age due to societal forces concerning expense and successful outcome. A mother's critical illness yields insights into care that worked despite advanced age. The five subsequent insights are viewed against data from randomized clinical trials illustrating how we know what we think we know. From this a healthcare model is proposed that insures a healthier elderly population.

Synthesis of proteins by automated flow chemistry.

Ribosomes can produce proteins in minutes and are largely constrained to proteinogenic amino acids. Here, we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids long over 327 consecutive reactions. The machine is rapid: Peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.

Direct radiolabeling of monoclonal antibodies with generator-produced rhenium-188 for radioimmunotherapy: labeling and animal biodistribution studies.

The use of 188Re from an alumina-based 188W/188Re generator has been investigated for antibody radiolabeling. It was found that, with simple labeling techniques, 188Re can be used immediately after elution. The direct radiolabeling of intact antibodies with 188Re is described. Lyophilized antibody preparations have been reconstituted with 188Re taken directly from the generator at specific activities of up to 15 mCi of 188Re per mg of antibody. Radiolabeling yields of 90 to 98% have been obtained, with the incorporation rate being dependent upon time and the relative concentrations of the reagents. It was determined that the conjugates were immunoreactive and stable when challenged by serum in vitro, with 188Re-immunoglobulin G showing adequate resistance to reoxidation with no transfer of 188Re to serum protein. 188Re-antibody conjugates were shown to clear from the blood faster than the corresponding 131I-labeled antibody, giving rise to good tumor/nontumor ratios at 24 to 72 h postinjection, while serum samples taken from the animals have shown that the circulating 188Re remained bound to immunoglobulin G. The combination of the technologies of the 188W/188Re generator, the direct labeling methodology, and the use of single-vial lyophilized antibody makes the use of 188Re-radiolabeled monoclonal antibodies a simple and convenient method of cancer radioimmunotherapy with a beta-emitting radionuclide.

A Preliminary Genetic Investigation of VOLVOX CARTERI.

A preliminary genetic analysis of a number of genetic variants of Volvox carteri f. nagariensis is presented. Techniques are outlined for mutagenesis of Volvox, isolation of mutants and routine genetic analysis. All of the mutants show simple Mendelian segregation patterns and have been tentatively placed in 14 linkage groups.

Erotomania or de Clérambault syndrome.

De Clérambault focused attention on a syndrome in which a woman has the delusional belief that a man, usually of higher social status and considerably older, is much in love with her. If the patient's romantic ideas shaped private fantasies instead of determined public behavior, there would be little cause for concern. The situation becomes critical when the fantasies are dramatized in real life with an unsuspecting and usually unwilling man cast in the role of the lover. The woman dwells on the feelings she ascribes to her "suitor." Such delusional thinking, resulting from an ego defect and producting bizarre actions, may be shaped largely by feelings of being unloved or even unloveable; a narcissistic blow is overcome by a grandiose fantasy. Cases in which erotomania is prominent are usually diagnosed as paranoid state or paranoid schizophrenia.

Effects of intrathecal thyrotropin-releasing hormone (protirelin) in refractory depressed patients.

BACKGROUND: Therapeutic effects of the tripeptide protirelin (thyrotropin-releasing hormone) have been postulated in the affective disorders, but direct assessment in humans has been hindered by poor blood-brain barrier permeability. METHODS: Eight medication-free inpatients with refractory depression received 500 micrograms of protirelin via a lumbar intrathecal injection and an identical sham lumbar puncture procedure, separated by 1 week, in a double-blind crossover design. RESULTS: Five of eight patients responded to intrathecal protirelin, defined as a 50% or greater reduction in an abbreviated Hamilton Rating Scale for Depression score. Suicidality also was reduced significantly (P < .05). Responses were rapid and clinically robust, but short-lived. CONCLUSION: Administration of protirelin by an intrathecal route induced a rapid improvement in mood and suicidality in these refractory depressed patients, supporting the hypothesis that thyrotropin-releasing hormone could be a positive modulator of mood.

Pes planus and paediatric obesity: a systematic review of the literature.

Children with obesity report musculoskeletal pain more than normal-weight children; this may be linked with literature suggesting children with obesity have higher prevalence of pes planus (flatfoot). To further elucidate whether this relation occurs, we conducted a systematic literature review on the co-occurrence of pes planus and paediatric obesity. Empirical articles published until September 2013 were obtained through an electronic search of MEDLINE and SPORTDiscus; included articles examined the association between body weight and pes planus in children. Thirteen cross-sectional studies of varied designs were identified. Methods used to diagnose pes planus varied between studies: imaging modalities, anthropometric measurements and clinical examination. Across all studies, pes planus prevalence among children with obesity ranged widely from 14 to 67%. Nearly all studies indicated increasing pes planus in children with increasing weight. No studies evaluated pain/complications related to pes planus. Our review suggests increased prevalence of pes planus among children with obesity or increasing weight status. Because of differing methodologies, lack of consensus regarding the pes planus definition, the dearth of investigation into pain/complications and the few existing studies, more research is needed to determine a relation between children's body weight, pes planus and associated effects on pain and function.

Comparative antidepressant effects of intravenous and intrathecal thyrotropin-releasing hormone: confounding effects of tolerance and implications for therapeutics.

A significant amount of preclinical and human data indicate that thyrotropin-releasing hormone (TRH) has antidepressant effects. Although early studies showing these effects using intravenous TRH were not consistently replicated, it has been suggested that this could be explained by its poor blood-brain barrier penetration. For this reason we compared the antidepressant effect of intrathecal and intravenous TRH administered in a double-blind design to 2 treatment-refractory patients with bipolar II disorder. Each experienced a robust antidepressant response by both routes; subsequent open trials of intravenous TRH also were effective until apparent tolerance developed. Intrathecal TRH was readministered and both subjects again experienced robust antidepressant responses. These preliminary data suggest a differential mechanism of tolerance to the two routes of administration and raise the possibility that a subgroup of patients may be responsive to the antidepressant effects of TRH independent of its route of administration.

Relationships between thyroid hormone and antidepressant responses to total sleep deprivation in mood disorder patients.

BACKGROUND: Acute transient antidepressant effects of sleep deprivation are consistently observed in 50% of depressed patients, but the mechanisms of these, at times, dramatic improvements in mood have not been adequately elucidated. Some, but not all, studies suggest a relationship to increased thyroid-stimulating hormone (TSH) secretion. METHODS: TSH and other thyroid indices were measured at 8:00 AM after a baseline night's sleep and at 8:00 AM following a night of total sleep deprivation (S.D.) in 34 medication-free, affective disorder patients assessed with Hamilton, Beck, and Bunney-Hamburg depression ratings as well as two hourly self-ratings on a visual analog scale. RESULTS: Compared with baseline, S.D. induced highly significant increases in TSH, levothyroxine, free levothyroxine, and triiodothyronine. The 12 S.D. responders tended to have greater TSH increases than the 15 nonresponders (p < .10). The change in Beck depression ratings significantly correlated with the change in TSH (r = -.40, p = .0496, n = 24). CONCLUSIONS: These data are consistent with several other reports of a significant relationship between degree of antidepressant response to S.D. and increases in TSH measured at 8:00 AM near their usual nadir. Acute removal of the sleep-related break on the hypothalamic-pituitary-thyroid axis remains a promising candidate for the mechanism of sleep deprivation-induced improvement in mood in depressed patients.

Lack of correlation between cerebrospinal fluid thyrotropin-releasing hormone (TRH) and TRH-stimulated thyroid-stimulating hormone in patients with depression.

BACKGROUND: It has been proposed that elevated central thyrotropin-releasing hormone (TRH) is associated with the blunted thyroid-stimulating hormone (TSH) response to TRH in patients with depression. Few studies have directly evaluated this relationship between central nervous system and peripheral endocrine systems in the same patient population. METHODS: 15 depressed patients (4 male, 11 female, 12 bipolar, and 3 unipolar) during a double-blind, medication-free period of at least 2 weeks duration, underwent a baseline lumbar puncture followed by a TRH stimulation test. Cerebrospinal fluid (CSF) TRH and serial serum TSH, free thyroxine, triiodothyronine, prolactin, and cortisol were measured. A blunted response to TRH was defined as a delta TSH less than 7 microU/mL. RESULTS: There was no significant difference in mean CSF TRH between "blunters" (2.82 +/- 1.36 pg/mL) and "non-blunters" (3.97 +/- 0.62 pg/mL, p = .40). There was no evidence of an inverse relationship between CSF TRH and baseline or delta TSH. There was no correlation between CSF TRH and the severity of depression or any other endocrine measure. CONCLUSIONS: These data are not consistent with the prediction of hypothalamic TRH hypersecretion and subsequent pituitary down-regulation in depression; however, CSF TRH may be from a nonparaventricular nucleus-hypothalamic source (i.e., limbic area, suprachiasmatic nucleus, brain stem-dorsal raphe) and thus, not necessarily related to peripheral neuroendocrine indices.

Inverse relationship of peripheral thyrotropin-stimulating hormone levels to brain activity in mood disorders.

OBJECTIVE: The author's goal was to investigate relationships between peripheral thyroid hormone levels and cerebral blood flow (CBF) and cerebral glucose metabolism in affectively ill patients. METHOD: Medication-free inpatients with major depression or bipolar disorder were studied with oxygen-15 water and positron emission tomography (PET) to measure CBF (N = 19) or with [18F] fluorodeoxyglucose and PET to measure cerebral glucose metabolism (N = 29). Linear regression was used to correlate global CBF and cerebral glucose metabolism with serum thyrotropin-stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4), and free T4 concentrations. Statistical parametric mapping was used to correlate regional CBF and cerebral glucose metabolism with these thyroid indexes. Post hoc t tests were used to further explore the relationships between serum TSH and global CBF and cerebral glucose metabolism. RESULTS: Serum TSH was inversely related to both global and regional CBF and cerebral glucose metabolism. These relationships persisted in the cerebral glucose metabolism analysis and, to a lesser extent, in the CBF analysis after severity of depression had been controlled for. In contrast, no significant relationships were observed between T3, T4, or free T4 and global or regional CBF and cerebral glucose metabolism. CONCLUSIONS: These data suggest that peripheral TSH (putatively the best marker of thyroid status) is inversely related to global and regional CBF and cerebral glucose metabolism. These findings indicate relationships between thyroid and cerebral activity that could provide mechanistic hypotheses for thyroid contributions to primary and secondary mood disorders and the psychotropic effects of thyroid axis manipulations.

Classification of ischemic-induced damage to Na+, K+-ATPase in gerbil forebrain. Modification by therapeutic agents.

The activity of three forms of ATPase were examined in fractions of the brain of the gerbil treated with ethylene glycol-N-N-tetra-acetic acid (EGTA) under a variety of conditions of primary and secondary (reflow) ischemia. In animals which were unilateral ischemic (ligation of the right common carotid), damage to Na+, K+-ATPase alone was observed only after at least 6 hr of ischemia had elapsed. The phenomenon occurred in only symptomatic gerbils and was absent in animals which were either asymptomatic or only displayed partial neurological symptoms. Under conditions of bilateral cerebral ischemia, in which both carotid arteries were clamped, only irreversible ischemia (60 min) followed by reflow, was associated with highly significant damage to cerebral Na+, K+-ATPase. In regional studies of the forebrain involving ischemia for 60 min plus 30 min reflow, damage to Na+, K+-ATPase was evident in the cerebrum, hippocampus, striatum and thalamus, while the hypothalamus and olfactory bulb were spared. Pretreatment of gerbils with allopurinol, clonazepam or combinations of thiopental plus either indomethacin or methylprednisolone offered protection to cerebral Na+, K+-ATPase subsequent to secondary ischemia. With only minor exceptions (striatum) neither Ca2+, Mg2+- nor Mn2+-ATPase were altered by stroke or treatment with drugs.

Design, synthesis, and evaluation of omega-iodovinyl- and omega-iodoalkyl-substituted methyl-branched long-chain fatty acids.

The synthesis of a new methyl-branched fatty acid, (E)-19-iodo-3(RS)-methyl-18-nonadecenoic acid (19), is described. Methyl branching has been introduced at the 3-position to inhibit beta-oxidation and radioiodide has been attached as a trans-vinyl iodide. Preparation of 19 involved a 15-step sequence of reactions climaxing with formation of the methyl ester 18 by iododestannylation of methyl (E)-19-(tri-n-butylstannyl)-3(RS)-methyl-18-nonadecenoate (17) resulting from the reaction of n-Bu3SnH with methyl 3(RS)-methyl-18-nonadecynoate (16). Methyl branching was introduced at an early stage by Friedel-Crafts acylation of thiophene with 3(RS)-methyl-4-carbomethoxybutanoyl chloride (3) generated from 3-methylglutaric anhydride. The new agent, [125I]-19, showed high myocardial uptake (5 min, 4.89% dose/g; 30 min, 3.32% dose/g), good heart/blood (H/B) ratios (5 min, 5.4/1; 30 min, 4.3/1), and significantly greater myocardial retention in fasted rats than the corresponding straight-chain analogue 19-[125I]-iodo-18-nonadecenoic acid (5 min, 3.52% dose/g, H/B = 4.8/1; 30 min, 1.19% dose/g, H/B = 1.6/1). Excellent myocardial images were obtained in rats after administration of [123I]-19 and confirmed the slow myocardial washout over a 60-min period. These data suggest that 19 is a good candidate for evaluation of heart disease involving aberrations in fatty acid metabolism by use of imaging techniques such as single photon emission computerized tomography (SPECT) where redistribution or washout should be minimized.

Potential pancreatic imaging agents. Tellurium-123m labeled DL-alpha-amino-gamma-(phenyltelluro)butyric acid.

This report describes the first successful preparation of a 123mTe-labeled alpha-amino acid as a potential pancreatic imaging agent. Tellurium-123m labeled DL-alpha-amino-gamma-(phenyltelluro)butyric acid was prepared by basic hydrolysis of the radiolabeled 5-[beta-(phenyltelluro)ethyl]hydantoin. The hydantoin was prepared by the reacitron of 123mTe-labeled phenyltellurol, generated by sodium borohydride reduction of diphenyl ditelluride, with 5-(beta-bromoethyl)hydantoin. Tissue distribution studies in rats with the 123mTe-labeled amino acid for periods varying from 30 min to 24 h demonstrated only marginal pancreatic accumulation of radioactivity. The significant result of these studies is that a general synthetic method has been developed for the preparation of 123mTe-labeled amino acids.

Synthesis and biodistribution of iodine-125-labeled 1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate. A new ligand for the potential imaging of muscarinic receptors by single photon emission computed tomography.

1-Azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)- alpha-phenylacetate (IQNP, 3), an analogue of QNB in which a phenyl ring has been replaced with an iodopropenyl substituent, was prepared and evaluated in vitro and in vivo for m-AChR selectivity and specificity. High specific activity [125]IQNP ([125I]-3) was synthesized in greater than 60% yield utilizing an electrophilic iododestannylation reaction with hydrogen peroxide for the oxidation of iodide. In in vitro receptor binding studies, 3 demonstrated high affinity for M1 (Ki = 0.78 nM), M2 (Ki = 1.06 nM), and M3 (Ki = 0.27 nM) subtypes. In vivo biodistribution studies in female rats [125I]-3 demonstrated high uptake in areas rich in muscarinic receptors such as the brain (cortex and striatum) and the heart. Blocking studies were performed with a series of receptor specific agents and demonstrated that the uptake of [125I]-3 was selective and specific for cerebral muscarinic receptor rich areas and that the binding to m-AChR is reversible. The high-yield preparation and specificity and selectivity of high specific activity [125I]IQNP for muscarinic receptors suggest that this is an attractive new agent for potential imaging of cerebral receptors using single photon tomographic imaging (SPECT).