Butterfly eyespots evolved via cooption of an ancestral gene-regulatory network that also patterns antennae, legs, and wings.

Butterfly eyespots are beautiful novel traits with an unknown developmental origin. Here we show that eyespots likely originated via cooption of parts of an ancestral appendage gene-regulatory network (GRN) to novel locations on the wing. Using comparative transcriptome analysis, we show that eyespots cluster most closely with antennae, relative to multiple other tissues. Furthermore, three genes essential for eyespot development, Distal-less (Dll), spalt (sal), and Antennapedia (Antp), share similar regulatory connections as those observed in the antennal GRN. CRISPR knockout of cis-regulatory elements (CREs) for Dll and sal led to the loss of eyespots, antennae, legs, and also wings, demonstrating that these CREs are highly pleiotropic. We conclude that eyespots likely reused an ancient GRN for their development, a network also previously implicated in the development of antennae, legs, and wings.

Evolution of the tan locus contributed to pigment loss in Drosophila santomea: a response to Matute et al.

We have shown previously that the loss of abdominal pigmentation in D. santomea relative to its sister species D. yakuba resulted, in part, from cis-regulatory mutations at the tan locus. Matute et al. claim, based solely upon extrapolation from genetic crosses of D. santomea and D. melanogaster, a much more divergent species, that at least four X chromosome regions but not tan are responsible for pigmentation differences. Here, we provide additional evidence from introgressions of D. yakuba genes into D. santomea that support a causative role for tan in the loss of pigmentation and present analyses that contradict Matute et al.'s claims. We discuss how the choice of parental species and other factors affect the ability to identify loci responsible for species divergence, and we affirm that all of our previously reported results and conclusions stand.

Near-infrared Absorption and Emission Probes with Optimal Connection Bridges for Live Monitoring of NAD(P)H Dynamics in Living Systems.

Two NAD(P)H-biosensing probes consisting of 1,3,3-trimethyl-3H-indolium and 3-quinolinium acceptors, linked by thiophene, A, and 3,4-ethylenedioxythiophene, B, bridges are detailed. We synthesized probes C and D, replacing the thiophene connection in probe A with phenyl and 2,1,3-benzothiadiazole units, respectively. Probe E was prepared by substituting probe A's 3-quinolinium unit with a 1-methylquinoxalin-1-ium unit. Solutions are non-fluorescent but in the presence of NADH, exhibit near-infrared fluorescence at 742.1 nm and 727.2 nm for probes A and B, respectively, and generate absorbance signals at 690.6 nm and 685.9 nm. In contrast, probes C and D displayed pronounced interference from NADH fluorescence at 450 nm, whereas probe E exhibited minimal fluorescence alterations in response to NAD(P)H. Pre-treatment of A549 cells with glucose in the presence of probe A led to a significant increase in fluorescence intensity. Additionally, subjecting probe A to lactate and pyruvate molecules resulted in opposite changes in NAD(P)H levels, with lactate causing a substantial increase in fluorescence intensity, conversely, pyruvate resulted in a sharp decrease. Treatment of A549 cells with varying concentrations of the drugs cisplatin, gemcitabine, and camptothecin (5, 10, and 20 µM) led to a concentration-dependent increase in intracellular fluorescence intensity, signifying a rise in NAD(P)H levels. Finally, fruit fly larvae were treated with different concentrations of NADH and cisplatin illustrating applicability to live organisms. The results demonstrated a direct correlation between fluorescence intensity and the concentration of NADH and cisplatin, respectively, further confirming the efficacy of probe A in sensing changes in NAD(P)H levels within a whole organism.

The evolution of gene regulation underlies a morphological difference between two Drosophila sister species.

Understanding the mechanisms underlying the morphological divergence of species is one of the central goals of evolutionary biology. Here, we analyze the genetic and molecular bases of the divergence of body pigmentation patterns between Drosophila yakuba and its sister species Drosophila santomea. We found that loss of pigmentation in D. santomea involved the selective loss of expression of the tan and yellow pigmentation genes. We demonstrate that tan gene expression was eliminated through the mutational inactivation of one specific tan cis-regulatory element (CRE) whereas the Tan protein sequence remained unchanged. Surprisingly, we identify three independent loss-of-function alleles of the tan CRE in the young D. santomea lineage. We submit that there is sufficient empirical evidence to support the general prediction that functional evolutionary changes at pleiotropic loci will most often involve mutations in their discrete, modular cis-regulatory elements.

The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila.

Sexually dimorphic traits play key roles in animal evolution and behavior. Little is known, however, about the mechanisms governing their development and evolution. One recently evolved dimorphic trait is the male-specific abdominal pigmentation of Drosophila melanogaster, which is repressed in females by the Bric-à-brac (Bab) proteins. To understand the regulation and origin of this trait, we have identified and traced the evolution of the genetic switch controlling dimorphic bab expression. We show that the HOX protein Abdominal-B (ABD-B) and the sex-specific isoforms of Doublesex (DSX) directly regulate a bab cis-regulatory element (CRE). In females, ABD-B and DSX(F) activate bab expression whereas in males DSX(M) directly represses bab, which allows for pigmentation. A new domain of dimorphic bab expression evolved through multiple fine-scale changes within this CRE, whose ancestral role was to regulate other dimorphic features. These findings reveal how new dimorphic characters can emerge from genetic networks regulating pre-existing dimorphic traits.

Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.

Mycotoxin tolerance affects larval competitive ability in Drosophila recens (Diptera: Drosophilidae).

Certain mycophagous Drosophila species are the only known eukaryotes that can tolerate some highly potent mycotoxins. This association between mycophagy and mycotoxin tolerance is well established because Drosophila species that switch hosts from mushrooms to other food sources lose their mycotoxin tolerance trait without any evolutionary lag. These findings suggest that mycotoxin tolerance may be a costly trait to maintain. In this study, we attempted to identify whether mycotoxin tolerance has a fitness cost. Larval competitive ability is a vital fitness trait, especially in holometabolous insects, where the larvae cannot move to a new host. Furthermore, larval competitive ability is known to be associated with many critical life-history traits. Here we studied whether mycotoxin tolerance adversely affects larval competitive ability on isofemale lines from 2 distinct locations. We observed that the extent of mycotoxin tolerance affected larval competitive ability, but only in isofemale lines from one location. Additionally, we observed that the high mycotoxin-tolerant isofemale lines from the same location showed poor survival to eclosion. This study shows that mycotoxin tolerance is associated with fitness costs and provides preliminary evidence of an association between local adaptation and mycotoxin tolerance.

Atherosclerosis Burdens in Diabetes Mellitus: Assessment by PET Imaging.

Arteriosclerosis and its sequelae are the most common cause of death in diabetic patients and one of the reasons why diabetes has entered the top 10 causes of death worldwide, fatalities having doubled since 2000. The literature in the field claims almost unanimously that arteriosclerosis is more frequent or develops more rapidly in diabetic than non-diabetic subjects, and that the disease is caused by arterial inflammation, the control of which should therefore be the goal of therapeutic efforts. These views are mostly based on indirect methodologies, including studies of artery wall thickness or stiffness, or on conventional CT-based imaging used to demonstrate tissue changes occurring late in the disease process. In contrast, imaging with positron emission tomography and computed tomography (PET/CT) applying the tracers 18F-fluorodeoxyglucose (FDG) or 18F-sodium fluoride (NaF) mirrors arterial wall inflammation and microcalcification, respectively, early in the course of the disease, potentially enabling in vivo insight into molecular processes. The present review provides an overview of the literature from the more than 20 and 10 years, respectively, that these two tracers have been used for the study of atherosclerosis, with emphasis on what new information they have provided in relation to diabetes and which questions remain insufficiently elucidated.

Ratiometric Detection of Glutathione Based on Disulfide Linkage Rupture between a FRET Coumarin Donor and a Rhodamine Acceptor.

Abnormal levels of glutathione, a cellular antioxidant, can lead to a variety of diseases. We have constructed a near-infrared ratiometric fluorescent probe to detect glutathione concentrations in biological samples. The probe consists of a coumarin donor, which is connected through a disulfide-tethered linker to a rhodamine acceptor. Under the excitation of the coumarin donor at 405 nm, the probe shows weak visible fluorescence of the coumarin donor at 470 nm and strong near-infrared fluorescence of the rhodamine acceptor at 652 nm due to efficient Forster resonance energy transfer (FRET) from the donor to the acceptor. Glutathione breaks the disulfide bond through reduction, which results in a dramatic increase in coumarin fluorescence and a corresponding decrease in rhodamine fluorescence. The probe possesses excellent cell permeability, biocompatibility, and good ratiometric fluorescence responses to glutathione and cysteine with a self-calibration capability. The probe was utilized to ratiometrically visualize glutathione concentration alterations in HeLa cells and Drosophila melanogaster larvae.

A critical review of radiotracers in the positron emission tomography imaging of traumatic brain injury: FDG, tau, and amyloid imaging in mild traumatic brain injury and chronic traumatic encephalopathy.

PURPOSE: Positron emission tomography (PET) has been widely utilized in the study of traumatic brain injury (TBI) for decades. While most applications of PET have attempted to assess neuronal function after TBI, more recently, novel radiotracers have sought to image biomarkers in the context of TBI and chronic traumatic encephalopathy (CTE). METHODS: This review will begin with an overview of TBI and CTE along with the acute and chronic pathophysiological consequences of TBI. Next, glycolysis, beta-amyloid, and tau protein radiotracers will be critically assessed in light of the most recent imaging studies available. CONCLUSIONS: Based on the scientific relevance of such radiotracers to the molecular processes of TBI and CTE along with the broader evidence of radiotracer specificity and selectivity, this review will weigh the strengths and weaknesses of each radiotracer. Nonetheless, the evidence indicates that PET will continue to be a powerful modality in the diagnosis of TBI-related conditions.

Quantitative thoracic aorta calcification assessment by 18F-NaF PET/CT and its correlation with atherosclerotic cardiovascular disorders and increasing age.

OBJECTIVES: We aimed to assess the correlation between age and cardiovascular risk factors with NaF-PET/CT imaging in the thoracic aorta (TA). METHODS: In this prospective study, 80 healthy controls and 44 patients with chest pain underwent NaF-PET/CT imaging, and three segments of the aorta (ascending, arch, and descending) were examined. Average SUVmax, SUVmean, and Alavi-Carlsen Score (ACS) were calculated in each segment and the entire vessel. The degree of NaF uptake in controls and patients and its correlation with age were determined. Multivariate linear regression and logistic regression models were employed to determine the predictabilities of Framingham Risk Score (FRS) and unfavorable cardiovascular disease (CVD) risk profile by these measurements. RESULTS: Average SUVmax, average SUVmean, and ACS were significantly higher in patients than in controls, and all correlated well with age. The correlation of average SUVmean with age was significant in both controls (r = 0.32, p = 0.04) and patients (r = 0.64, p < 0.001). ACS of the entire TA was a stronger predictor of FRS compared with average SUVmax and average SUVmean (adjusted R2 = 0.38, standardized ß = 0.58, p < 0.001). ACS was a significant predictor of unfavorable CVD risk profile as compared with other values (odds ratio = 1.006, 95% CI = 1.000-1.013, p = 0.05). CONCLUSIONS: Active calcification in TA correlates with age, and its correlation is higher among subjects with CVD risk factors. Global assessment (ACS) can predict unfavorable CVD risk profile. These data provide evidence for the potential role of NaF in assessing micro-calcification in arteries and its relations to cardiovascular events. KEY POINTS: • Global micro-calcification in the thoracic aorta as measured by NaF-PET/CT imaging correlates with increasing age. • The extent of the correlation was higher among patients with cardiovascular disease (CVD) risk factors. • These data provide evidence for the potential role of NaF in assessing active calcification in arteries and its relations to cardiovascular events.

Carotid artery molecular calcification assessed by [18F]fluoride PET/CT: correlation with cardiovascular and thromboembolic risk factors.

OBJECTIVES: There is growing evidence that sodium fluoride ([18F]fluoride) PET/CT can detect active arterial calcifications at the molecular stage. We investigated the relationship between arterial mineralization in the left common carotid artery (LCC) assessed by [18F]fluoride PET/CT and cardiovascular/thromboembolic risk. METHODS: In total, 128 subjects (mean age 48 ± 14 years, 51% males) were included. [18F]fluoride uptake in the LCC was quantitatively assessed by measuring the blood-pool-corrected maximum standardized uptake value (SUVmax) on each axial slice. Average SUVmax (aSUVmax) was calculated over all slices and correlated with 10-year risk of cardiovascular events estimated by the Framingham model, CHA2DS2-VASc score, and level of physical activity (LPA). RESULTS: The aSUVmax was significantly higher in patients with increased risk of cardiovascular (one-way ANOVA, p < 0.01) and thromboembolic (one-way ANOVA, p < 0.01) events, and it was significantly lower in patients with greater LPA (one-way ANOVA, p = 0.02). On multivariable linear regression analysis, age ( = 0.07, 95% CI 0.05 - 0.10, p < 0.01), body mass index ( = 0.02, 95% CI 0.01 - 0.03, p < 0.01), arterial hypertension ( = 0.15, 95% CI 0.08 - 0.23, p < 0.01), and LPA ( = -0.10, 95% CI -0.19 to -0.02, p=0.02) were independent associations of aSUVmax. CONCLUSIONS: Carotid [18F]fluoride uptake is significantly increased in patients with unfavorable cardiovascular and thromboembolic risk profiles. [18F]fluoride PET/CT could become a valuable tool to estimate subjects' risk of future cardiovascular events although still major trials are needed to further evaluate the associations found in this study and their potential clinical usefulness. KEY POINTS: • Sodium fluoride ([18F]fluoride) PET/CT imaging identifies patients with early-stage atherosclerosis. • Carotid [18F]fluoride uptake is significantly higher in patients with increased risk of cardiovascular and thromboembolic events and inversely correlated with the level of physical activity. • Early detection of arterial mineralization at a molecular level could help guide clinical decisions in the context of cardiovascular risk assessment.

AMPA-15N - Synthesis and application as standard compound in traceable degradation studies of glyphosate.

Stable isotope labeling of pollutants is a valuable tool to investigate their environmental transport and degradation. For the globally most frequently used herbicide glyphosate, such studies have, so far, been hampered by the absence of an analytical standard for its labeled metabolite AMPA-15N, which is formed during the degradation of all commercially available glyphosate isotopologues. Without such a standard, detection and quantitation of AMPA-15N, e.g. with LC-MS/MS, is not possible. Therefore, a synthetic pathway to AMPA-15N from benzamide-15N via the hemiaminal was developed. AMPA-15N was obtained in sufficient yield and purity to be used as a standard compound for LC-MS/MS analysis. Suitable MS-detection settings as well as a calibration using the internal standard (IS) approach were established for Fmoc-derivatized AMPA-15N. The use of different AMPA isotopologues as IS was complicated by the parallel formation of [M+H]+ and [M]+• AMPA-Fmoc precursor ions in ESI-positive mode, causing signal interferences between analyte and IS. We recommend the use of either AMPA-13C-15N, AMPA-13C-15N-D2 or a glyphosate isotopologue as IS, as they do not affect the linearity of the calibration curve. As a proof of concept, the developed analysis procedure for AMPA-15N was used to refine the results from a field lysimeter experiment investigating leaching and degradation of glyphosate-2-13C-15N. The newly enabled quantitation of AMPA-15N in soil extracts showed that similar amounts (0.05 - 0.22 mg·kg-1) of the parent herbicide glyphosate and its primary metabolite AMPA persisted in the topsoil over the study period of one year, while vertical transport through the soil column did not occur for either of the compounds. The herein developed analysis concepts will facilitate future design and execution of experiments on the environmental fate of the herbicide glyphosate.

Automated Design and Integration of Asset Administration Shells in Components of Industry 4.0.

One of the central concepts in the principles of Industry 4.0 relates to the methodology for designing and implementing the digital shell of the manufacturing process components. This concept, the Asset Administration Shell (AAS), embodies a systematically formed, standardized data envelope of a concrete component within Industry 4.0. The paper discusses the AAS in terms of its structure, its components, the sub-models that form a substantial part of the shell's content, and its communication protocols (Open Platform Communication-Unified Architecture (OPC UA) and MQTT) or SW interfaces enabling vertical and horizontal communication to involve other components and levels of management systems. Using a case study of a virtual assembly line that integrates AASs into the technological process, the authors present a comprehensive analysis centered on forming AASs for individual components. In the given context, the manual AAS creation mode exploiting framework-based automated generation, which forms the AAS via a configuration wizard, is assessed. Another outcome consists of the activation of a virtual assembly line connected to real AASs, a step that allows us verify the properties of the distributed manufacturing management. Moreover, a discrete event system was modeled for the case study, enabling the effective application of the Industry 4.0 solution.

18F-Sodium Fluoride PET as a Diagnostic Modality for Metabolic, Autoimmune, and Osteogenic Bone Disorders: Cellular Mechanisms and Clinical Applications.

In a healthy body, homeostatic actions of osteoclasts and osteoblasts maintain the integrity of the skeletal system. When cellular activities of osteoclasts and osteoblasts become abnormal, pathological bone conditions, such as osteoporosis, can occur. Traditional imaging modalities, such as radiographs, are insensitive to the early cellular changes that precede gross pathological findings, often leading to delayed disease diagnoses and suboptimal therapeutic strategies. 18F-sodium fluoride (18F-NaF)-positron emission tomography (PET) is an emerging imaging modality with the potential for early diagnosis and monitoring of bone diseases through the detection of subtle metabolic changes. Specifically, the dissociated 18F- is incorporated into hydroxyapatite, and its uptake reflects osteoblastic activity and bone perfusion, allowing for the quantification of bone turnover. While 18F-NaF-PET has traditionally been used to detect metastatic bone disease, recent literature corroborates the use of 18F-NaF-PET in benign osseous conditions as well. In this review, we discuss the cellular mechanisms of 18F-NaF-PET and examine recent findings on its clinical application in diverse metabolic, autoimmune, and osteogenic bone disorders.

Comparison of 18F-sodium fluoride uptake in the whole bone, pelvis, and femoral neck of multiple myeloma patients before and after high-dose therapy and conventional-dose chemotherapy.

AIM: To compare the effects of high-dose therapy (HDT consisting of high-dose chemotherapy followed by autologous stem cell transplantation) and conventional-dose chemotherapy (non-HDT) on the uptake of 18F-sodium fluoride (NaF) in the whole bone, pelvis, and femoral neck of multiple myeloma (MM) patients. METHOD: The data of 19 MM patients who received HDT (61.5 (SD 5.6) years) and 11 MM patients who received conventional-dose chemotherapy (70.9 (SD 7.2) years) were collected in a prospective study. NaF PET/CT imaging was performed at baseline, and 8 weeks and 2 weeks after treatment for the HDT group and the non-HDT group, respectively. A CT-based algorithm was applied to segment the bones, and the global mean SUV (GSUVmean) of the whole bone and pelvis was calculated (OsiriX MD v.9.0, Pixmeo SARL; Bernex, Switzerland). In addition, regions of interest for the whole, medial, and lateral femoral neck were delineated bilaterally. Whole bone and pelvis measurements were replicated by two observers. RESULTS: The average GSUVmean in the whole bone and pelvis of the patients who underwent HDT significantly decreased from before to after treatment (- 16.27%, p = 0.02 and - 16.54%, p = 0.01, respectively). A significant decrease in the whole and lateral femoral neck was also observed bilaterally in the HDT group. No significant decrease in average GSUVmean was observed in the non-HDT group. A high level of inter-observer reliability was found in intra-class correlation (ICC for pre-treatment whole bone 0.983, post-treatment whole bone 0.989, pre-treatment whole pelvis 0.998, post-treatment whole pelvis 0.996). CONCLUSION: NaF uptake significantly decreased after treatment in patients who received high-dose therapy. A high level of agreement was observed between two operators for whole bone and pelvis measurements.

Benzoxepinones: A new isoform-selective class of tumor associated carbonic anhydrase inhibitors.

Benzoxepinones ("homocoumarins") are identified as a new class of selective inhibitors for tumor associated human carbonic anhydrases (hCA, EC 4.2.1.1) isoforms IX and XII. Similar to coumarins, they do not inhibit or poorly inhibit cytosolic human (h) isoforms hCA I and II, but act as nanomolar inhibitors of the trans-membrane, tumor associated isoforms hCA IX and XII.

Reduction of Activated Alkenes by PIII /PV Redox Cycling Catalysis.

The carbon-carbon double bond of unsaturated carbonyl compounds was readily reduced by using a phosphetane oxide catalyst in the presence of a simple organosilane as the terminal reductant and water as the hydrogen source. Quantitative hydrogenation was observed when 1.0 mol % of a methyl-substituted phosphetane oxide was employed as the catalyst. The procedure is highly selective towards activated double bonds, tolerating a variety of functional groups that are usually prone to reduction. In total, 25 alkenes and two alkynes were hydrogenated to the corresponding alkanes in excellent yields of up to 99 %. Notably, less active poly(methylhydrosiloxane) could also be utilized as the terminal reductant. Mechanistic investigations revealed the phosphane as the catalyst resting state and a protonation/deprotonation sequence as the crucial step in the catalytic cycle.

BRAFV600E mutation: A promising target in colorectal neuroendocrine carcinoma.

To determine the role of BRAFV600E mutation and MAPK signaling as well as the effects of BRAF and MEK directed therapy in gastroenteropancreatic neuroendocrine neoplasia (GEP-NEN), with a focus on highly aggressive gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC). Using Sanger sequencing of BRAF exon 15 we determined the frequency of BRAFV600E mutations in 71 primary GEP-NENs. MEK phosphorylation was examined by immunohistochemistry in corresponding tissue samples. To evaluate the biological relevance of BRAFV600E mutation and MAPK signaling in GEP-NECs, effects of a pharmacological BRAF and MEK inhibition were analyzed in NEC cell lines both in vitro and in vivo. BRAFV600E mutation was detected in 9.9% of all GEP-NENs. Interestingly, only NECs of the colon harbored BRAFV600E mutations, leading to a mutation frequency of 46.7% in this subgroup of patients. In addition, a BRAFV600E mutation was significantly associated with high levels of MEK phosphorylation (pMEK) and advanced tumor stages. Pharmacological inhibition of BRAF and MEK abrogated NEC cell growth, inducing G1 cell cycle arrest and apoptosis only in BRAFV600E mutated cells. BRAF inhibitor dabrafenib and MEK inhibitor trametinib prevented growth of BRAFV600E positive NEC xenografts. High frequencies of BRAFV600E mutation and elevated expression levels of pMEK were detected in biologically aggressive and highly proliferative colorectal NECs. We provide evidence that targeting BRAF oncogene may represent a therapeutic strategy for patients with BRAF mutant colorectal NECs.

A FRET-Based Near-Infrared Fluorescent Probe for Ratiometric Detection of Cysteine in Mitochondria.

We report a near-infrared fluorescent probe A for the ratiometric detection of cysteine based on FRET from a coumarin donor to a near-infrared rhodamine acceptor. Upon addition of cysteine, the coumarin fluorescence increased dramatically up to 18-fold and the fluorescence of the rhodamine acceptor decreased moderately by 45 % under excitation of the coumarin unit. Probe A has been used to detect cysteine concentration changes in live cells ratiometrically and to visualize fluctuations in cysteine concentrations induced by oxidation stress through treatment with hydrogen peroxide or lipopolysaccharide (LPS). Finally, probe A was successfully applied for the in vivo imaging of Drosophila melanogaster larvae to measure cysteine concentration changes.