An 11-year-old female presents to the sleep clinic for evaluation for possible sleep-disordered breathing (SDB). She has a history of frequently intractable seizures for which she was tried on multiple antiepileptic medications. She had vagus nerve stimulation (VNS) implantation two years ago to treat her focal seizures. Nine months later, her seizures were controlled, but the family raised concerns about louder snoring and more frequently witnessed apneas. She had polysomnography (PSG) that showed severe obstructive sleep apnea (OSA) related to her VNS. The patient was diagnosed with SDB secondary to electrical activations of the implanted VNS. We describe an epilepsy patient whose case illustrates the possible respiratory complications (primarily OSA) associated with VNS. We will discuss the possible mechanisms of VNS related SDB and the importance of screening for SDB and advocate for a PSG both before and after VNS implantation. Vagus nerve stimulation (VNS) can worsen sleep-disordered breathing (SDB) after implantation. Patients with VNS can have central apneas, obstructive hypopneas, and/or obstructive apneas. Patients with VNS often have an increase in apneic events after implantation . This caAn 11-year-old female presented to the sleep clinic for evaluation of snoring and witnessed apneas. She has had a history of focal epilepsy since age four years and was tried on multiple antiseizure medications, including levetiracetam, clobazam, zonisamide, and valproate. Her most recent antiepileptic medications include clonazepam, lacosamide, and topiramate for the last three years. She continued to have up to 10 seizures every week, and so two years ago, the VNS was implanted to manage her intractable epilepsy. Nine months following her VNS implantation her seizures were reduced dramatically, but her mother raised concerns about more frequent nocturnal respiratory pauses during her sleep as well as louder snoring. This was reported by the family for the first time-only after her VNS was implanted. Thus, the patient was referred to our Pediatric Sleep Medicine clinic for an initial evaluation. Epworth Sleepiness Scale (ESS) assessment was six of 24. She had a consistently regular sleep/wake cycle with 10 hours of sleep with a bedtime of 9:30 PM. She usually would fall asleep within 15 minutes and would wake up between 7:30 AM and 8:00 AM. She had no daily naps. The patient had no other significant medical history. Family history included paternal sleep apnea and maternal generalized seizures.2 . She had a Mallampati score of 1; tonsil size of 0. The remainder of the physical examination was normal.Examination revealed patient weight at 34.8 kg; height, 127.5 cm; Z-scores of -0.89 and -3.05 were based on the Centers for Disease Control (CDC) two to 20 years’ weight-for-age and stature-for-age data, respectively. Body mass index was 21.43 kg/m2 (EtCO2) was 33 mmHg. Periodic limb movement index was 1.0 events/hour. The patient’s polysomnography (PSG) demonstrates the respiratory events  was 30.7 events/hour; central apnea index, 0.1 events/hour. Sleep latency was five minutes, lowest oxygen saturation was 90%, and mean end-tidal CODifferential diagnosisThe patient has evidence of severe obstructive sleep apnea (OSA) on the PSG. Respiratory events were unusually rhythmic and occurred with the accuracy of a metronome . Each reThese findings on the polysomnographic signals would raise questions about their possible etiologies that can include possibilities like an underlying arrhythmia or an artifact not related to the VNS versus a VNS related phenomena. Recognizing the exact etiology and accurately answering these questions is pivotal to decide what will be the best next step for management.Questions and answersThe findings on the polysomnographic signals would raise questions about their possible etiologies that can include possibilities like an underlying arrhythmia or an artifact not related to the VNS versus a VNS related phenomena. Recognizing the exact etiology and accurate interpretation of these findings is pivotal to decide what will be the best next step for management. 1. What is the most possible etiology of the electromyogram (EMG) and the electrocardiogram (EKG) artifacts in the figures above?(A) Myocardial fiber stretch during episodes of apnea; (B) electrical activations of the implanted VNS; (C) undiagnosed supraventricular arrhythmia; (D) ungrounded recording equipment affected by a nearby air-conditioner .The answer is B. Electrical activations of the implanted VNS. 2. What will be the most appropriate next step to address this finding?These respiratory events can be reduced with changes in the VNS operational parameters or with the use of continuous positive airway pressure (CPAP). (This will be discussed further below in detail.)Treatment20 during which the (O-AHI) was 1.3. The patient was started on PAP therapy. The VNS could have also worsened a preexisting OSA and thus the patient was also referred to ear, nose, and throat (ENT) for evaluation of possible surgical options like Tonsillectomy and Adenoidectomy (T&A). The ENT evaluation did not identify any additional surgical options.The patient had a repeat PSG for positive airway pressure (PAP) therapy. Her repeat PSG one month later with the VNS “on” showed an optimal pressure of 7 cm HOutcome and follow-upOn the three-month follow-up after PAP therapy was started the patient tolerated the PAP therapy well with 99.3% of days usage-an average usage of seven hours and 51 minutes per night and 90% of days with usage >=4 hours. There were no reported side effects to PAP therapy. The patient remained seizure-free for the last year and is currently on clonazepam and lacosamide.There is a paucity of cases demonstrating this finding in children. A robust PubMed database search for the search terms VNS & sleep apnea yielded 69 articles on this topic, most of these articles were discussing either the VNS role in epilepsy and/ or the respiratory complications associated with vagus nerve stimulators, however, only one article matched citation for VNS and obstructive sleep apnea in adults. Also, a systematic review on the topic has come to light by Óscar Romero-Osorio et al. that describes only four case reports in adults between the ages of 20 years and 57 years at the time of the evaluation in the United States . A similFew case reports in the literature have reported a possible association between the VNS and the respiratory events. Shaman reported a 50-year-old male who presented with snoring, witnessed apneas, unrefreshing sleep, and excessive daytime sleepiness (ESS 17) despite adequate sleep duration . This pa2) of 41. Bilevel positive airway pressure (BiPAP) was initiated and titrated to 11/7 cm H20 with a backup rate of 12. BiPAP with the VNS “on” resulted in an O-AHI of 0.0 and oxygen nadir of 94%. This case, however, did not diagrammatically illustrate the VNS-related respiratory events similar to our case and did not emphasize the importance of screening before and after the VNS implantation. The AHI was also much higher in our case (13.5 events/hour versus 30.7 events/hour in this case presented). Our case adds to the existing literature and raises awareness about the importance of screening for OSA and SDB even before the VNS is implanted. Recently, an abstract by Ochoa et al. described a 12-year-old female with medically intractable epilepsy who had a VNS implantation as an additional therapy . She wasIntermittent VNS can reduce the frequency of seizures in patients with refractory epilepsy. Although the exact mechanism of seizure reduction remains unclear, Mithani, et al. reported that the vagus afferent network (VagAN) is thought to be the neural substrate of VNS efficacy. This recent study provides the first multi-institutional, multimodal, connectomic prediction algorithm to reliably identify VNS responders based on the reported group differences in their white matter microstructure that was demonstrated on the diffusion tensor imaging used in this study. This is important in order to mitigate surgical risks for those children predicted not to benefit from the VNS implantation and to ensure cost-effective allocation of health care resources . Stimula2O resulted in the effective treatment of his severe OSA. This study also suggests PSG before VNS implantation should be considered in order to identify patients with OSA [Manipulating VNS settings such as stimulus intensity, frequency, and cycle times may decrease the respiratory disturbance ,9. Howevwith OSA . A similwith OSA .There are complex relationships between epilepsy and OSA. Patients with refractory epilepsy need assessment for undiagnosed and untreated SDB before implantation of VNS devices. Patients with VNS often have an increase in their apneic events after implantation, and these patients need screening for sleep apnea before and after implantation .This case demonstrates the importance of screening for SDB, preferably before, but also after the VNS implantation. This case illustrates these concepts, especially in children, where scarce cases are describing these findings. We also elaborate on the possible mechanisms of VNS related SDB and discuss the variable management options that the providers can choose to pursue like CPAP utilization or possible changes in the VNS operational application to address the medical condition.Additional research including a larger group of patients is needed in order to conduct a subgroup analysis to determine the concrete clinical practice guidelines of which subgroup of patients for which routine screening for SDB and recommending the PSG either before or after the VNS implantation or both will be most beneficial. This case raises awareness about this phenomenon and serves as a valuable proponent in the literature for providers to consider screening for SDB as well as a PSG both before and after the VNS implantation.


The microstructure and phase composition of the composite material were analyzed, and the influence of laser energy density on the microstructure and mechanical properties of composite materials was discussed. The results showed that the needle-like ZrB2 ceramic reinforcement was successfully synthesized via an in-situ synthesis reaction. The composites were mainly composed of needle-like ZrB2, Ni dendrites and a Cu matrix. The morphological changes of Ni dendrites could be observed at the interface inside the composite material: cellular crystals → large-sized columnar dendrites → small-sized dendrites . The continuous Ni dendritic network connected the ZrB2 reinforcements together, which significantly improved the mechanical properties of the composite material. At a laser energy density of 0.20 kJ/mm2, the average microhardness of the composite material reached 294 HV0.2 and the highest tensile strength was 535 MPa. With the laser energy density increased to 0.27 kJ/mm2, the hardness and tensile strength decreased and the elongation of the Cu composites increased due to an increase in the size of the ZrB2 and a decrease in the continuity of the Ni dendritic.Laser additive manufacturing is an advanced material preparation technology, which has been widely used to prepare various materials, such as polymers, metals, ceramics and composites. Zirconium diboride (ZrB Laser additive manufacturing (LAM) is an advanced material preparation technology, which adopts laser as an energy source and powders or wires as raw material to construct a 3D specimen under the control of a computer ,2. LAM h2/Cu5Zr reinforced Cu composites have exhibited a good combination of electrical conductivity and mechanical properties [2 ceramic whisker is a single crystal with a very small diameter [Copper is a common and important metal material due to its excellent thermal conductivity, corrosion resistance and ductility ,17, and operties . ZrB2 cediameter . Its ato4C, and they acted as the heterogeneous nucleation sites for the Ti matrix, which promoted the formation of fine equiaxed α-Ti grains [3Fe, CrB, M23C6 and Cr2.8Fe1.2B4) were synthesized in the molten pool, which significantly improved the high temperature wear resistance of the coating [2 and CuZr were synthesized in the Cu matrix, which improved the high temperature hardness of Cu composites [With regard to ceramic reinforced metal matrix composites (CMC), the manufacture of defect-free CMCs with excellent mechanical properties has always presented a challenge and has been the focus of research due to large differences in the physical and chemical properties of ceramic reinforcements and metal matrices ,25. Accoi grains . In the  coating . During mposites .2 ceramic whisker reinforced copper matrix composites. The ZrB2 reinforcement was in-situ synthesized via the designed chemical reaction in the molten pool, and the crystal structure of its growth tip was analyzed. The microstructure, phase composition and interface characteristics of the composites were determined. The influence of energy input density on the microstructure of composites was discussed. In addition, the microhardness distribution and tensile properties of the samples prepared under different energy input densities were studied.In this study, the exploration goal was to use LDED technology to prepare ZrB2, Al, Ni-B4C , Ni and Cu were used as the raw material for laser direct energy deposition. The sizes and weight percentages of the composite powders were listed in 2, Al and B4C were the reactants of the in-situ synthesis reaction, and the molar ratio between them was set from the designed reaction:The composite powders ZrO2 ceramic reinforcements using this ratio of reactants [The Gibbs free energy  of 180 mm/min, a spot diameter (D) of 3 mm and a laser power (P) of 1.8 or 2.4 kW. The laser energy density (E = P/VD) represented the laser energy applied per unit area during the deposition process, which was used to comprehensively analyze the influence of process parameters for each sample. Two samples were prepared under different laser energy densities; the E of samples 1 and 2 were 0.20 and 0.27 kJ/mm2, respectively.Pure copper with a size of 50 × 50 × 15 mm3 (10 g), HCl  and H2O (100 mL). The phase composition of the composite material was analyzed using an X-ray diffractometer  at a speed of 0.5°/min. A tungsten wire scanning microscope  equipped with an energy spectrometer (EDS) was used to observe the microstructure of the composites and the morphology of the reinforcements. The growth direction and interface characteristics of the reinforcement were characterized using transmission electron microscopy . A disc with a diameter of 3 mm was cut on the SD-LD surface of the composites, and then a TEM sample was obtained via mechanical thinning and ion beam thinning. The microhardness distribution of the samples on the BD-LD was tested using a Vicker’s hardness tester (HVS-1000) under a load of 0.2 kg and the dwell time of 10 s; the distance between the test points was 100 μm. The tensile samples of the composites were cut on the SD-LD plane . According to the build direction, the left side of the interface was N + 1 track deposition, in which small-sized dendrites and large-sized columnar dendrites could be observed. Compared with the area in sample 1  was [112 crystals grew into regular geometric shapes with multiple symmetry planes. The interface structure and surface energy played key roles in the growth of the crystal plane. The interface structure could be divided into a smooth interface and a rough interface according to the crystal structure and bonding bonds between atoms. The Jackson factor (α) was used to determine the interface type  [k is Boltzmann’s constant; mT is the melting point; η is the coordination number between atoms in the layer and the atoms in the same layer (only the surface layer); v is the coordination number between the surface layer atoms and the next layer of solid atoms; R is the gas constant and 2 with a C32 hexagonal close-packed structure was P6/mmm (191). η/v could be calculated for each crystal plane from the positions of atoms in the hexagonal close-packed structure. The η/v of {0001}, {10R = 8.31 J/mol·K, and so the α of the ZrB2 low-index were α{0001} = 5.84, α{10-10} = 1.95 and α{11-20} = 1.95. The α of {10uvtw) of a crystal plane (uvtw) was inversely proportional to the atomic layer distance (duvtw) of the crystal plane. According to the interplanar spacing of the common low-index crystal plane families of ZrB2  :(2)α=ΔHf J/mol·K , R = 8.3H) model ,39, the  of ZrB2 , the ord2. The regular crystal planes at the tip of ZrB2, which are a typical morphology of the single crystal, could be observed. Furthermore, the angles between the crystal planes were approximately 120°, which is similar to the ideal hexagonal morphology. 2 as marked by the dashed box. Two symmetrical facets could be clearly observed, and the normal angle θ between the two facets was about 62°. The space group of ZrB2 with a C32 close-packed hexagonal structure was P6/mmm (191), and the 4-axis coordinate system of its crystal structure is shown in 2 crystal was mainly composed of {0001} and {102 crystal nucleus grew into a whisker reinforcement.0.2. The average microhardness, 294.26 HV0.2, was about five times higher than that of pure copper. The fine ZrB2 needle-like phase and Ni dendritic network structure improved the microhardness of sample 1. However, there were fewer needle-like phases and the large-size columnar Ni dendrites at the remelting interface , the tensile strength reduced, but the elongation increased.2 reinforced Cu composites were successfully fabricated using laser direct energy deposition. The microstructure of the composites was mainly a composite structure composed of needle-like ZrB2, Ni dendrites, and a Cu matrix.The needle-like ZrB2 was shown to be a high-strength whisker reinforcement. In sample 1, the diameter of ZrB2 was less than 2 μm, and the aspect ratio was about 30:1. The long axis direction with the fastest growth rate of ZrB2 ceramic whiskers was {11-20}.In-situ synthesized ZrB2. The morphological changes of Ni dendrites could be observed at the interface inside the composite material: cellular crystals → large-sized columnar dendrites → small-sized dendrites .In the Cu matrix, a network of Ni dendrites connected the needle-like ZrB2 ceramic reinforcement and Ni dendrites significantly improved the mechanical properties of the composite material. The microhardness and ultimate tensile strength of the composite material reached 322 HV0.2 and 535 MPa, respectively, and the tensile fracture of sample 1 was a mixed mechanism of ductile and brittle fracturing. As the laser energy density increased, the size of the in-situ synthesized ZrB2 increased, and the number and continuity of Ni dendrites decreased, which decreased the hardness and strength of the composite material, and the fracture mechanism of sample 2 was changed to ductile fracturing.ZrB


Chronic bronchitis in patients with chronic obstructive pulmonary disease (COPD) is associated with poor respiratory health outcomes. However, controversy exists around whether non-obstructive chronic bronchitis (NOCB) is associated with airflow obstruction, lung function decline, and all-cause mortality in ever smoker or never smoker.This systematic review and meta-analysis aimed to clarify the relationship between NOCB and incident COPD, lung function decline, and all-cause mortality, and to quantify the magnitude of these associations.We searched PubMed, Embase, and Web of Science for studies published up to October 1, 2021. Eligibility screening, data extraction, and quality assessment of the retrieved articles were conducted independently by two reviewers. Studies were included if they were original articles comparing incident COPD, lung function decline, and all-cause mortality in normal spirometry with and without chronic bronchitis. The primary outcomes were incident COPD and all-cause mortality. The secondary outcomes were respiratory disease-related mortality and lung function decline. Pooled effect sizes and 95% confidence intervals (CIs) were calculated using the random-effects model.I2 = 76.3% and relative risk: 1.44, 95%CI: 1.13–1.85, I2 = 56.1%), all-cause mortality , and respiratory disease-related mortality . Data on the decline in lung function could not be quantitatively synthesized, but the five articles that assessed the rate of decline in lung function showed that lung function declines faster in individuals with NOCB. The mean difference in the additional decline in forced expiratory volume in 1 s ranged from 3.6 to 23.2 mL/year.We identified 17,323 related references and included 14 articles. Compared with individuals without NOCB, individuals with NOCB had an increased risk of incident COPD (odds ratio: 1.98, 95% CI: 1.21–3.22, Individuals with NOCB are at a higher risk of incident COPD and all-cause mortality than individuals without NOCB, highlighting the crucial need for strategies to screen for and reduce NOCB risk.https://www.crd.york.ac.uk/PROSPERO/ PROSPERO, identifier CRD42020202837 Chronic obstructive pulmonary disease (COPD) is a common, preventable, and treatable disease that is characterized by persistent respiratory symptoms and airflow obstruction. It is currently a leading cause of death and disability worldwide , 2. IdenChronic bronchitis is defined epidemiologically as cough and sputum production for ≥3 months each year for ≥2 consecutive years . The maiUnderstanding the association between NOCB and incident COPD and respiratory health outcomes has important implications for disease management, such as in terms of targeted treatments and cautions about drug usage. Independent studies have shown a significantly increased risk of airflow obstruction and all-cause mortality in individuals with NOCB, and this has been summarized in a recent narrative review , 15–17. The study protocol was registered with the International Prospective Register of Systematic Reviews (registration number: CRD42020202837). This systematic review and meta-analysis was performed in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .In this systematic review and meta-analysis, two reviewers (FH and JL) independently searched Embase, Web of Science, and PubMed to identify studies published from database inception to October 1, 2021. Keywords and subject terms were customized for each database. The search terms combined the respiratory symptoms related to chronic bronchitis  and prognostic terms . The references of related studies were also consulted to identify potentially relevant articles.The eligibility of identified studies was independently verified, and disagreements were resolved by discussion or, where necessary, by consulting a third researcher (FW). For primary inspection, the titles and abstracts were reviewed, and articles were excluded mainly for COPD patients with chronic bronchitis and non-extractable data . For secondary inspection, full-text review was performed, and articles were excluded based on the inclusion and exclusion criteria. Studies that satisfied the following criteria were included: (1) prospective cohort study or retrospective cohort study; (2) inclusion of odds ratios (ORs), relative risks (RRs), or hazard ratios (HRs) with corresponding 95% confidence intervals (CIs) to estimate COPD risk; the risk of mortality  in individuals with NOCB; or enough data to calculate these risks; (3) comparison of individuals without NOCB; (4) independent studies. Studies that were the same as the published dataset were not considered independent. We deemed studies as eligible if they were longitudinal cohort studies that enrolled adults and reported the association between NOCB and the study outcomes.1]/forced vital capacity [FVC] ratio of <0.7) was the preferred definition of COPD . The secondary outcomes were respiratory disease-related mortality and lung function decline.NOCB was defined as chronic cough and sputum production for ≥3 months for ≥2 consecutive years in subjects without airflow obstruction. Without NOCB was defined as normal spirometry and no chronic bronchitis. Without NOCB serves as a healthy control of NOCB, which does not limit smoking status. The primary study outcomes were incident COPD and all-cause mortality. The GOLD criterion (a post-bronchodilator forced expiratory volume in 1 s [FEVTwo reviewers (FH and JL) independently extracted the study information and independently verified the quality of each study. The extracted information included first author, year of publication, location, study design, year of enrolment, study outcome, follow-up time, and the characteristics of the study participants . Two reviewers (HF and JL) independently conducted quality assessments of the included studies. The quality of each study was based on the Newcastle–Ottawa Scale (NOS) for cohort studies . StudiesI2 statistic was used to evaluate study heterogeneity. An I2 value of 0–24% was considered as no heterogeneity. Greater values represented greater heterogeneity, with values of 25–49% representing low heterogeneity, 50–74% representing moderate heterogeneity, and >75% indicating high heterogeneity  software was used for the meta-analysis.The outcomes of the synthesis included the OR and the RR of incident COPD, the HR of all-cause mortality and respiratory disease-related mortality, and the mean difference in the rate of lung function decline. For eligible studies that reported the HR and 95% CI of incident COPD, the HR and 95% CI were pooled with the RR. We could use the calculation formula to convert the OR into the RR, but some studies did not report the proportion of the control group that developed COPD, and the OR of most studies was adjusted for confounding factors . Therefoogeneity . Publicaogeneity . All sta1/FVC ratio of ≥0.7 as the main definition of normal spirometry, and one study used a pre-bronchodilator FEV1/FVC ratio ≥ the LLN as the main definition of normal spirometry. Coronary Artery Risk Development in Young Adults Lung Study data were published in two articles  with significant inter-study heterogeneity   . The numP < 0.001) with low inter-study heterogeneity  (P < 0.001) with no inter-study heterogeneity   . Two stu= 0.722) .1 declined significantly faster in individuals with NOCB than normal spirometry in men without chronic bronchitis, but there was no significant difference in women   and FVC  among ever smokers  . In the oduction . In the  in FEV1 . The mos smokers .P < 0.001) and respiratory disease-related mortality  than in individuals with normal lung spirometry without NOCB (P = 0.206) or respiratory disease-related mortality  compared with individuals with normal spirometry and without NOCB . Finally, almost all studies included in our meta-analysis used pre-bronchodilator lung function data to diagnose COPD. A previous study has demonstrated that pre-bronchodilator lung function overestimates the prevalence of COPD compared with post-bronchodilator lung function . This maTo conclude, this systematic review and meta-analysis demonstrated that NOCB is associated with an increased risk of incident COPD, all-cause mortality, and respiratory disease-related mortality, as well as faster lung function decline. Individuals with NOCB should be identified early through screening, and strategies aimed at controlling NOCB should be implemented.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.PR, YZ, FW, HF, and JL contributed to study conception, protocol of the review, and interpretation of data. FW, HF, JL, SZ, and WZ performed the systematic review. FW and HF performed the statistical analysis. YZ, PR, FW, HF, and JL drafted the paper and all authors provided critical revisions and contributed to the editing of the paper. All authors are guarantors of this work.This study was supported by the National Key Research and Development Program (2016YFC1304101), the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01S155), the National Natural Science Foundation of China (81970045), and Zhongnanshan Medical Foundation of Guangdong Province .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Insects tend to feed on related hosts. Coevolution tends to be dominated by interactions resulting from plant chemistry in defense strategies, and evolution of secondary metabolisms being in response to insect herbivory remains a classic explanation of coevolution. The present study examines whether evolutionary constraints existing in host associations of economically important fruit flies in the species‐rich tribe Dacini (Diptera: Tephritidae) and to what extent these species have evolved specialized dietary patterns. We found a strong effect of host phylogeny on associations on the 37 fruit flies tested, although the fruit fly species feeding on ripe commercially grown fruits that lost the toxic compounds after long‐term domestication are mostly polyphagous. We assessed the phylogenetic signal of host breadth across the fruit fly species, showing that the results were substantially different depending on partition levels. Further, we mapped main host family associations onto the fruit fly phylogeny and Cucurbitaceae has been inferred as the most likely ancestral host family for Dacini based on ancestral state reconstruction. The present study examines whether evolutionary constraints existing in host associations of economically important fruit flies in the species‐rich tribe Dacini (Diptera: Tephritidae) and to what extent these species have evolved specialized dietary patterns. We found a strong effect of host phylogeny on associations on the 37 fruit flies tested, although the fruit fly species feeding on ripe commercially grown fruits that lost the toxic compounds after long domestication are mostly polyphagous. Further, we discussed the role of secondary metabolisms in feeding preference, and other suites of traits in the specialization of fruit fly dietary patterns. To obtain the interacted hosts of the tephritid species, we extracted records from the pest‐oriented database of the Centre for Agricultural and Biosciences  which reports fruit flies occurring and feeding on plants. Host associations were cross‐checked for completeness with the COFFHI database . Synonyms on the host list were eliminated and only accepted names adopted for each plant species. In general, our database of interactions between fruit flies and host plants contains 1,841 associations, including 37 Dacini fruit flies and 706 host species belonging to 87 families.The tribe Dacini, primarily comprises species of three genera  model of evolution, values of K > 1 imply strong phylogenetic signals, and K = 0 implies the absence of phylogenetic conservatism. While there is no clearly defined K value cutoff in which to apply phylogenetic comparative methods, nonsignificant value of <1, or more conservative <0.5, are typical for characters that are phylogenetic independent. Pagel's λ examines the effect of ancestral relatedness on character evolution, with a value close to 1 indicates phylogenetic signal and a value approaching 0 indicates phylogenetic independence.To test whether host breadth and namely states of polyphagy would be phylogenetically distributed across the tephritid species tree, we used Blomberg's λ Pagel,  to asses10 (phylodistance + 1) according to empirical experiments from Gilbert et al. (β0) and slope coefficient (β1) and the 95% confidence interval were obtained from the mean coefficients across all 1,000 sets from these regressions for all fruit flies.We took a logistic regression approach to calculate the probability of a host plant being infected by, or found in association with, a particular tephritid species , and its relationship with phylogenetic distances. We compiled associations at the genus level, and the associations were modeled as a binary trait . In the regression analysis, we only considered fruit fly species with three or more plant associations to make it more reasonable, and host phylogenetic distances were calculated in million years. We integrated the associations between fruit flies and host plants with the matrix of pairwise phylogenetic distances among host genera to investigate the role of host phylogeny. To calculate the possibility of fruit fly host affiliation, we applied the phylogenetic distance as predictor variable , 2.4We applied the DEC  model  “two families” and “four families” were not apparent using the K estimate, but signals were detected under the λ estimate . The character (feeding on) “three families” showed strong phylogenetic signals using both the te Table . SignifiThe probability that two host genera sharing one tephritid species declined significantly with phylogenetic distance, supporting that Dacini fruit flies prefer to feed on closely related plants Figure . The logZeugodacus and Dacus.There were 87 families being used as hosts; however, only a handful of families are “important” host families which might play a decisive role in the evolutionary history of Dacini fruit flies. It contained nine main host families exclusively in the ancestral host state reconstruction analysis for studying purpose. Families of Cucurbitaceae and Myrtaceae were the most frequently used two families in terms of number of associated host plants belong to these families; families of Cucurbitaceae and Rutaceae were the most commonly used two families in terms of number of fruit fly species feeding upon these plant species. In addition, host use family Cucurbitaceae was distributed widely in the diet on genera of Bactrocera, and two Zeugodacus species Zeugodacus cucurbitae and Zeugodacus tau were not included in the reconstruction, because that these fruit flies feeding on more than 20 host families seemed to be out of limitations due to shifts in geographic distribution and could do little to estimate ancestral hosts. There were 25 tephritid species that had been expected to be contributed to estimate the ancestral host state in the DEC model. We mapped main host families onto simplified phylogeny of tephritid species, most likely ancestral host states were shown on each node  by host shifts, that fruit odors as key traits help distinguish among respective host plants  that can evolve as adaptive syndromes may be relevant to plant–insect interactions. Although ripe, fleshy fruits function primarily to attract seed dispersers, they must also defend against diverse communities of frugivores. In particular, fruits can employ a variety of strategies such as ripening in seasons in which herbivores population densities are low or, reducing nutrient content and evolving a thick protective exocarp . Alternatively, inducible volatile organic compounds involved in host recognition may function as indirect defenses  is an evolutionarily dynamic phenomenon and host breadth should be treated as a continuum (expand or shrink over evolutionary time), its evolution is thus particularly difficult to test due to the inherent uncertainties. It requires systems approaches that could be capable of handling the practical situation of preference and performance on a case‐by‐case basis especially for highly polyphagous species which exhibit a great degree of variability in their host use patterns. However, combining host utilization with information of bioinformatics and phylogenetic analyses is still a proven approach to investigate insect species dietary pattern and feeding strategy. Furthermore, although the association data included were cross‐checked, the identification of these fruit flies on hosts might actually be incorrect  or based upon accidentally incidence on putative hosts . The existence of complexes could also confuse fruit fly host affiliations. We assigned the host records at the level of host genus or family that could be more reliable and reasonable for the analyses, and dubious host records are omitted. Nonetheless, an extremely polyphagous fruit fly might consist of a complex of cryptic species, leading to overestimation of host breadth in the fruit flies. However, it means that the conservatism should be more evolutionarily constrained than we investigated.5We found that Dacini fruit flies feeding on economically important host plants which normally lost secondary metabolisms after long‐term domestication still show specialized host associations, implying a decrease in the possibility of host sharing with increasing phylogenetic distance of the host plant taxa. Earlier study proposed that polyphagous fruit flies should be able to infest a broad range of phylogenetically distant host plants without absolute limitations and overcome plant defenses with little effort. Our study disaccords with this view and suggests that host specialization cannot be explained solely by secondary metabolites. Cucurbitaceae was recognized as the most probably ancestral host family in ancestral host reconstruction analysis. Diverse associations outside ancestral hosts for extremely polyphagous fruit flies are potentially attributable to capabilities of long‐distance dispersal and being generalist opportunists.The authors declared no conflicts of interest.Jiayao He: Conceptualization ; Data curation (supporting); Funding acquisition ; Methodology (supporting); Visualization (supporting); Writing‐original draft (supporting); Writing‐review & editing (lead). Ke Chen: Conceptualization ; Funding acquisition (supporting); Project administration ; Supervision ; Validation ; Visualization . Fan Jiang: Conceptualization ; Funding acquisition (supporting); Investigation ; Validation (lead); Writing‐review & editing . Xubin Pan: Conceptualization ; Investigation ; Project administration ; Supervision (lead); Writing‐review & editing .Data S1Click here for additional data file.


Vitamin D deficiency is common, but no data have been reported on vitamin D levels in light chain (AL) amyloidosis.2D] and vitamin D binding protein (DBP). Measurements were made by liquid chromatography-tandem mass spectrometry. Kidney survival and overall survival (OS) were assessed in association to vitamin D status.In this exploratory study, stored serum samples from 173 patients with newly diagnosed AL amyloidosis were analyzed for vitamin studies which included 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)2D deficiency was noted in 37.6% of patients and was independently associated with low eGFR and hypoalbuminemia. Progression to ESRD occurred in 23.7% of evaluable patients. Patients who progressed to ESRD had lower serum 25(OH)D and 1,25(OH)2D levels compared to those who did not progress to ESRD. On a multivariate analysis, severe 25(OH)D deficiency was an independent predictor of progression to ESRD as was renal stage, while 1,25(OH)2D deficiency was not.Cardiac and kidney involvement occurred in 69% and 63% of patients, respectively. 25(OH)D deficiency (<20 ng/mL) was seen in 56.6% of the patients and was notably found among patients with heavy proteinuria (96%), hypoalbuminemia (84.3%) and morbidly obese patients (68.3%). Heavy proteinuria (>5 gr/24-h) and vitamin D supplementation were independent predictors of 25(OH)D level on nominal multivariate regression analysis. 1,25(0H)Hypovitaminosis D is common in AL amyloidosis, particularly among patients with heavy proteinuria. Severe 25(OH)D deficiency at time of diagnosis predicts progression to ESRD. Light chain (AL) amyloidosis is a rare B-cell secreting clonal disorder characterized by circulating immunoglobulin light chains with amyloidogenic properties . The clo2D]. 1,25(OH)2D reflects 1-alpha hydroxylation of 25(OH)D, a process that occurs primarily within the kidney. Circulating 1,25(OH)2D levels are approximately 1000-fold lower than those of 25(OH)D.Vitamin D deficiency is assessed by measurement of the storage form of vitamin D, namely 25-hydroxyvitamin D [25(OH)D]. The prevalence of vitamin D deficiency, defined as a 25(OH)D level below the optimal range of <20 ng/ml, has been reported to range from 15-50% within the general United States (US) population –8. IdentAside from its roles in maintaining serum calcium levels and skeletal homeostasis, vitamin D has been shown to play an important role in regulation of differentiation, proliferation, apoptosis, metastatic potential and angiogenesis in a variety of malignancies –11. Low No studies have explored vitamin D levels in light chain (AL) amyloidosis. We therefore performed a study to examine vitamin D levels among patients with AL and to explore the impact vitamin D has in this rare disease.2D] and vitamin D binding protein (DBP). All vitamin D measurements were made by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Total 25(OH)D and 1,25(OH)2D was assessed by the additive sums of the 25(OH)D2 + 25(OH)D3 and 1,25(OH)2D2 + 1,25(OH)2D3 components, respectively. Samples obtained for vitamin D studies were collected within 90 days of diagnosis (median 20 days) and were stored at -20°C.In this study patients with biopsy-proven AL amyloidosis diagnosed between January 1, 2000 and September 30, 2013 were included if they had stored serum samples. The stored serum samples were obtained for vitamin D studies, which included 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D  was ascertained for 162 patients (94% of the study population). Ten patients presented or progressed to ESRD within 3 months of AL diagnosis and were excluded from this analysis. Of the remaining patients, 36 patients developed ESRD, representing 23.7% of patients assessed for progression to ESRD. Patients who developed ESRD had lower baseline 25(OH)D levels compared to patients who did not progress to ESRD  are provided in vs 49.2 months; P=0.03; 2D ≥18 pg/mL and those with lower levels D levels >20 ng/mL had shorter overall survival (OS) compared to patients with lower 25(OH)D values D level <20 ng/mL showed a trend towards longer OS , whereas a 1,25(OH) did not Table 5.In this study we explored, for the first time, the prevalence and risk factors for vitamin D deficiency in patients with AL amyloidosis. We found that more than half of our study population had vitamin D deficiency (defined as 25(OH)D <20 ng/mL), while over a fifth of the study cohort had severe vitamin D deficiency [25(OH)D <10 ng/mL]. Vitamin D deficiency was particularly common among patients with significant proteinuria, an established risk factor for vitamin D deficiency. We have also found that severe vitamin D deficiency was associated with increased risk for progression to end-stage renal disease. Overall, the findings in this study support measurement of baseline vitamin 25(OH)D in patients with AL amyloidosis, particularly among those with significant proteinuria.The proportion of patients with vitamin D deficiency in this study (56.6%) is higher compared to the general US population. In the National Health and Nutrition Examination Survey (NHANES), conducted in a similar time period to our study (2001 to 2010), the prevalence of vitamin D deficiency among US adults age 18 or above ranged between 27.1% and 30.8% (depending on age group)  and popuWe identified several risk factors for hypovitaminosis D in AL amyloidosis. The most significant risk factor for low 25(OH)D levels was significant proteinuria. Patients with nephrotic syndrome are known to be at risk for vitamin D deficiency . Kidney via modulation of several pathways including the renin-angiotensin, NF-kB and Wnt/β-catenin signaling pathways D deficiency is an independent predictor of progression to ESRD. This finding has been reported in previous studies with patients with chronic kidney disease –34. The pathways . In addipathways . HoweverIrrespective of the mechanisms by which vitamin D deficiency increases the risk of ESRD, correction of vitamin D deficiency in patients with CKD , should be assessed in prospective studies given the potential benefit of such treatment. A large, randomized trial among type II diabetes mellitus participants did not demonstrate a renoprotective effect for vitamin D supplementation versus placebo . However2D deficiency, was associated with improved survival compared to patients with normal vitamin D levels. This observation stands in contrast to observations among cancer patients (25(OH)D deficiency, but not 1,25(OH)patients , 18–22. patients . As survpatients , 40 and patients , vitamin2D levels to be normal in subjects with t(11:14) as compared to those with non-t. t is the most common genetic abnormality in patients with AL amyloidosis  (In this study there was a trend for 25(OH)D and 1,25(OH)poptosis –43. As a (BCL-2) , an antivia the gold standard method of liquid chromatography-tandem mass spectrometry. We were also powered for several observations including the positive effect of concurrent vitamin D supplementation on serum 25(OH)D and the expected seasonal variation on serum 25(OH)D levels. The study follow-up period was long (median >16 years), allowing sufficient time of observation for events . The primary limitation to our study was its retrospective design. We did not preselect number of samples based on predefined hypothesis, which may limit the study power. These limitations negatively affect the generalizability of the above findings and require further studies.Our study has several strengths. These include that this is the first report on the incidence of vitamin D deficiency in patients with AL amyloidosis, and the ability to measure vitamin D metabolites and vitamin D binding protein In conclusion, we have shown that patients with renal AL amyloidosis have a high incidence of vitamin D deficiency. We anticipate our findings will serve as the basis for future prospective clinical efforts to improve vitamin D levels in these patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Mayo Clinic Institutional review Board. The patients/participants provided their written informed consent to participate in this study.EM, MTD, and MG designed the study, analyzed the data, wrote the first draft and approved the final version of the manuscript; NL, AD, ML, FB, DD, SH, PK, YH, AF, MH, WG, TK, RW, SR, RG, MB, SR, and SK provided care for patients, revised the manuscript critically, and approved the final version of the manuscript. RK performed patients’ follow-up, revised the manuscript critically, and approved final version of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported in part by CA90628-21 Pal Calabresi K12 Career Development Award.EM: honorarium from Janssen and consultation fee from Protego (fee paid to institution). NL served on advisory board for Takeda Pharmaceuticals. AD: research funding from Celgene, Millennium Pharmaceuticals, Pfizer, and Janssen and received a travel grant from Pfizer. ML: Research Funding from Celgene. PK is a principal investigator of research studies for which Mayo Clinic has received funding from AbbVie, Takeda, Sanofi, Janssen, Karyopharm, Glaxo SmithKline, Regeneron Pharmaceuticals, Ichnos Sciences and Amgen. He has served on the Medical advisory board meetings of Sanofi, Pharmacyclics, BeiGene, Cellectar, GSK, X4 and Karyopharm. SK received research funding for clinical trials to the institution from Abbvie, Amgen, BMS, Carsgen, Janssen, Astra-Zeneca, Novartis, Roche-Genentech, Takeda, Tenebio, Molecular Templates, received consulting/Advisory Board participation  from Abbvie, Amgen, BMS, Janssen, Roche-Genentech, Takeda, Astra-Zeneca, Bluebird Bio, Epizyme, Secure Biotherapeutics and  Oncopeptides, Beigene, Antengene. MG served as a consultant for Millennium Pharmaceuticals and received honoraria from Celgene, Millennium Pharmaceuticals, Onyx Pharmaceuticals, Novartis, GlaxoSmithKline, Prothena, Ionis Pharmaceuticals, and Amgen.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We analyzed the usefulness of vitamin D biomarkers for the evaluation of MBD in patients with CKD. We analyzed blood and urine samples from 208 outpatients with CKD stage G2–G5. 25(OH)D showed a poor correlation with the estimated glomerular filtration rate (eGFR). Conversely, the 24,25(OH)2D level and VMR were significantly correlated with eGFR and the intact parathyroid hormone level. In conclusion, 24,25(OH)2D and VMR have the potential to be vitamin D biomarkers for the detection of MBD in CKD patients.The appropriate management of vitamin D deficiency and hyperparathyroidism is essential to prevent metabolic bone disorder (MBD) and cardiovascular diseases in chronic kidney disease (CKD). Recently, the 24,25-dihydroxyvitamin D [24,25(OH) In case of vitamin D toxicity, negative feedback turns on the catabolism mechanism to reduce blood vitamin D levels. The activated 24(OH)D hydroxylase enzyme (CYP24A1) converts 25(OH)D to 24,25(OH)2D by the stimulation of 1,25(OH)2D [2D or VMR could be a physiologically better biomarker than the currently used 25(OH)D, in that they represent the result of a feedback mechanism. In addition, previous studies have suggested that VMR may have a stronger association with bone health than that of 25(OH)D [2D level or VMR might be biomarkers of vitamin D status, particularly in CKD patients [Recently, serum 24,25(OH) D level ,19. 24,225(OH)2D ,21. Ther 25(OH)D . The kid2D, and VMR in patients with CKD. In this present study, we compared various vitamin D biomarkers according to renal function in CKD patients, as well as the association of the biomarkers with intact PTH (iPTH). Since there are several limitations to the current method for evaluating vitamin D status with 25(OH)D, we aim to evaluate whether 24,25(OH)2D or VMR can be a biomarker alternative to 25(OH)D in CKD.There are rare studies comparing vitamin D biomarkers, including 25(OH)D, 24,25(OH)This prospective study enrolled outpatients with CKD aged >18 years who visited the Department of Nephrology, Gyeongsang National University Hospital, between June and December 2021. We analyzed a subset of blood samples from a total of 208 patients who provided informed consent. Patients taking calcimimetics, phosphate binders, any medications and supplements containing calcium, or phosphorus, as well as vitamin D, were excluded. This study was approved by the Institutional Review Board of Gyeongsang National University Hospital .2D was measured by the method mentioned in a previous study with slight modifications [2D can currently be measured only by the LC-MS/MS method. While 25(OH)D can also be measured by a electrochemiluminescence binding assay, it was measured by the LC-MS/MS method for consistency in the measurement method. Following the addition of the internal standard, stable isotope-labeled d6-24,25(OH)2D and d6-25(OH)D were added to 200 μL of serum sample. After this, methanol was mixed with the solution. To precipitate protein, the solution was mixed by vortexing and kept at 4 °C for 10 min. After centrifugation at 4 °C and 12,000 g for 10 min, phosphate-buffered saline was added into the supernatant and applied onto an SPE cartridge. After performing SPE, the vacuum system was applied to the eluted fraction for evaporation. The dried remnant was reconstituted in 75% methanol. In total, 5 μL of the reconstituted solution was analyzed by the LC-MS/MS system. The LC-MS/MS system in our study was an Agilent 1260 HPLC system  with an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies) and an electrospray ionization source. For the separation of 24,25(OH)2D and 25(OH)D by HPLC, a Kinetex® Biphenyl column  and Poroshell® 120 EC-C 18 column  were utilized, respectively. The mobile phase in HPLC used 0.1% aqueous formic acid and methanol, and was processed by a gradient program at a flow rate of 0.4 mL/min. The monitored transitions were 417 → 381 m/z for 24,25(OH)2D, 423 → 387 m/z for d6-24,25(OH)2D, 401 → 383 m/z for 25(OH)D, and 407 → 389 m/z for d6-25(OH)D. In the LC-MS/MS system for the present study, the limits of quantitation of 24,25(OH)2D and 25(OH)D were 0.2 and 2 ng/mL, respectively.The serum concentration of 25(OH)D and 24,25(OH)2D concentration by 25(OH)D concentration and then multiplying it by 100.The VMR was calculated according to a previous study . It was 2/dL2), and iPTH were recorded. Medical records were screened to identify patients with a history of diabetes mellitus and hypertension. The estimated glomerular filtration rate (eGFR) was calculated using the 2012 CKD-EPI creatinine equation. The CKD stage was classified as G2-G5, according to the Kidney Disease: Improving Global Outcomes (KDIGO) Clinical Practice Guidelines [The following data were collected through the Electrical Medical Record (EMR) to analyze the correlation between the CKD stage and vitamin D biomarkers in the patients with CKD. Age, sex, height, serum creatinine (SCr), serum albumin, urine protein to creatinine ratio , serum calcium (mg/dL), serum phosphate (mg/dL), calcium–phosphate product , the Akaike information criterion, and the Bayesian information criterion. All the tests were two tailed, and p < 0.05 was considered statistically significant. We used R software  and SPSS software  for statistical analyses and the construction of graphics.To assess the correlation between two variables, Pearson’s correlation test was used. For variables with a normal distribution, differences between the groups were evaluated using an analysis of variance. For multivariate analysis, multivariate linear regression and logistic regression analyses were used. We confirmed the goodness-of-fit of the regression models using an adjusted R-squared , 5 (10.2%), 18 (29.0%), 19 (35.2%), and 8 (57.1%), respectively.2D, and VMR levels were 22.81 ± 11.24 ng/mL, 0.83 ± 0.63 ng/mL, and 3.55 ± 1.91, respectively. In CKD stage G3a, the mean iPTH level was 42.10 ± 20.43 pg/mL, which was lower than that of G2. However, in general, the mean iPTH level increased as the CKD stage increased from G2 to G5 (p < 0.001). The 25(OH)D level did not differ according to the CKD stage (p = 0.353), whereas the 24,25(OH)2D level and VMR showed a significant difference according to the CKD stage .2 = 0.0874, p < 0.001)  . The eGFR and VMR were also positively correlated  (A negative correlation was found between iPTH level and eGFR (R< 0.001) a. The 25= 0.632) b. There < 0.001) c,d.2 = 0.0277, p = 0.016)   (The iPTH level was negatively correlated with the 25(OH)D (R= 0.016) a, 24,25(< 0.001) b, and VM< 0.001) c levels.< 0.001) . 2D, and VMR levels, according to the CKD stage. The mean iPTH level was the lowest in CKD stage G3a and gradually increased with the increasing stage to CKD stage G5, except in CKD stage G2. However, the 24,25(OH)2D level and the VMR decreased with the increasing CKD stage, from CKD stage G2; this trend was more prominent for the VMR. The 25(OH)D level did not change with the 24,25(OH)2D level or the VMR between different CKD stages.2, the Akaike information criterion, and the Bayesian information criterion. In models using the eGFR as a dependent variable (models 4–6), model 6, which explained the eGFR using the VMR, had the best fit. Of the three vitamin D biomarkers, the VMR was the most strongly associated with the eGFR. In the models using iPTH as a dependent variable (models 7–9), model 8, which explained iPTH using 24,25(OH)2D, had the best fit. Of the three vitamin D biomarkers, 24,25(OH)2D was most strongly associated with the iPTH level D, 24,25(OH)2D and VMR as the alternative biomarkers of 25(OH)D. In the present study, we compared biomarkers of the vitamin D level in patients with CKD, particularly to determine the usefulness of 24,25(OH)2D level and VMR tend to decrease consistently from stage G2 to G5. Although various biochemical indicators related to CKD-MBD are altered in CKD stage 3, clinical features, such as degree of severity and rate of change, vary among patients. The KDIGO guideline recommends that serum calcium, phosphorus, and PTH levels should be monitored from CKD stage 3 in adults and from CKD stage 2 in children [As shown in children .2; additionally, the proportion of patients with SHPT increased rapidly with a decreasing eGFR [2D level and VMR were more strongly correlated with eGFR than iPTH or the 25(OH)D level, particularly in the early stages of CKD.In children with slowly progressive kidney disease, the PTH level may be elevated from CKD stage 2 ,30. In aing eGFR . Therefo2D level and the VMR, but not the 25(OH)D level, were significantly correlated with the KDIGO CKD stage and the eGFR; this suggests that the 24,25(OH)2D level and the VMR are better indicators of vitamin D level than 25(OH)D, the currently used vitamin D biomarker in CKD patients. In several previous studies, the 24,25(OH)2D, VMR and 25(OH)D have been controversial as vitamin D biomarkers. A study found that VMR may be used as a complementary indicator of vitamin D level for the evaluation of endogenous metabolism in premenopausal women [Levin et al.  found loal women . In a stal women . On the al women . Therefo2D, and VMR, were significantly correlated with the iPTH level, the 24,25(OH)2D level showed the strongest correlation with the iPTH level in the model fitness analysis. A previous study showed that the 24,25(OH)2D level had a stronger association with iPTH than the 25(OH)D level [2D and the VMR are better predictors of the effects of vitamin D supplementation on MBD than 25(OH)D, particularly in CKD patients. Our study suggests that 24,25(OH)2D and the VMR may be more sensitive vitamin D biomarkers than 25(OH)D in CKD patients, because they started to decrease at an earlier stage than 25(OH)D in Although all the evaluated vitamin D biomarkers, including the 25(OH)D, 24,25(OH))D level . However2D or the VMR is a better biomarker than 25(OH)D, especially for bone health. Ginsberg et al. showed that the VMR had stronger correlation with both BMD and fracture risk compared to 25(OH)D [2D or the VMR differs from 25(OH)D in bone health are unclear. In Some studies suggest that 24,25(OH) 25(OH)D . Another 25(OH)D . This su2D is decreased in CKD due to a reduced 1α-hydroxylase function. The vitamin D level is regulated by a precise feedback mechanism that maintains homeostasis between its production and catabolism [2D and FGF-23, and is inhibited by PTH [2D [2D reflects the vitamin D level more accurately than 25(OH)D, particularly because it is an indicator of catabolism that maintains the concentration of active vitamin D in vivo through feedback. Additionally, 24,25(OH)2D is an appropriate vitamin D biomarker. First, the circulating half-life of 24,25(OH)2D in blood is relatively long . Second, it has a relatively high level, which is why it is measured in ng/mL [In general, the synthesis of 1,25(OH)tabolism ,35. The d by PTH ,37,38. D PTH [2D . Therefoin ng/mL . For comThis study has several strengths. First, 24,25(OH)2D and the VMR were compared with 25(OH)D as vitamin D biomarkers in the clinical setting with CKD patients. Second, our study included patients of various CKD stages from G2 to G5, suggesting the possibility of 24,25(OH)2D and the VMR as a potential vitamin D biomarker in a wide range of CKD situations. Third, the measurement of 25(OH)D in a clinical laboratory commonly proceeds with an electrochemiluminescence assay, a type of immunoassay. However, in our study, the measurement of 25(OH)D and 24,25(OH)2D was performed with the same test platform, LC-MS/MS, to minimize errors due to differences in the analyzers and to increase the accuracy. Our study had two main limitations. First, this was a cross-sectional study, and follow-ups of patients or blood investigations were not performed. Second, several factors that could affect the serum vitamin D level, including seasonal variation, food, outdoor activity duration, and use of sunscreen, were not analyzed. However, to minimize seasonal variation, we conducted the study over two seasons, from summer to autumn, in South Korea. According to the website, which provides year-round weather statistics for Seoul, the capital of South Korea, there is an average of 2428 h of sunlight per year (of a possible 4383) with an average of 6:38 of sunlight per day . Because2D and the VMR were significantly correlated with the eGFR and iPTH levels in patients with CKD stage G2–G5. Therefore, we suggest that 24,25(OH)2D and the VMR have the potential to be alternative vitamin D biomarkers to 25(OH)D for the diagnosis of MBD in CKD patients.Although 25(OH)D is the most commonly performed test to assess vitamin D levels in CKD patients, it was poorly correlated with the eGFR. On the other hand, 24,25(OH)


Saturating concentrations of 1,25(OH)2D3, 25(OH)D3 and 25(OH)D2 resulted after 24 h stimulation in a comparable number and identity of target genes, but below 250 nM 25(OH)D3 and 25(OH)D2 were largely insufficient to affect the transcriptome. The average EC50 values of 206 common target genes were 322 nM for 25(OH)D3 and 295 nM for 25(OH)D2 being some 600-fold higher than 0.48 nM for 1,25(OH)2D3. The type of target gene, such as primary/secondary, direct/indirect or up-/down-regulated, had no significant effect on vitamin D metabolite sensitivity, but individual genes could be classified into high, mid and lower responders. Since the 1α-hydroxylase CYP27B1 is very low expressed in PBMCs and early (4 and 8 h) transcriptome responses to 25(OH)D3 and 25(OH)D2 were as prominent as to 1,25(OH)2D3, both vitamin D metabolites may directly control gene expression. In conclusion, at supra-physiological concentrations 25(OH)D3 and 25(OH)D2 are equally potent in modulating the transcriptome of PBMCs possibly by directly activating the vitamin D receptor.Human peripheral blood mononuclear cells (PBMCs) represent a highly responsive primary tissue that is composed of innate and adaptive immune cells. In this study, we compared modulation of the transcriptome of PBMCs by the vitamin D metabolites 25-hydroxyvitamin D The vitia cells , as wellia cells , 31. Prihe donor .3 is 100- to 1,000-fold lower than for 1,25(OH)2D3 2D3 (0.05–0.15 nM) D5(OH)2D3 , 34, whi0.15 nM) . This re the VDR . The molg pocket , 38. In g pocket .3 and 25(OH)D2 in comparison to that of 1,25(OH)2D3 in freshly isolated human PBMCs. We will demonstrate that 25(OH)D3 and 25(OH)D2 are equally potent in modulating the transcriptome of PBMCs and cannot exclude the possibility that both vitamin D metabolites directly activate the VDR.In this study, we analyzed the transcriptome-wide effects of 25(OH)DPeripheral blood was collected from a single healthy individual  the vitamin D responsiveness of whom had already been characterized in the VitDbol trial (NCT02063334) , 40. The2 incubator and treated for 4, 8 or 24 h either with 1,25(OH)2D3  25(OH)D3 , 25(OH)D2  or solvent (0.1% EtOH). All experiments were performed in three repeats. Deconvolution of RNA-seq data using the algorithm CIBERSORTx (+ T cells (32%), CD4+ T cells (20%), natural killer cells (21%) and monocytes/macrophages (20%) within the PBMC pool.PBMCs were isolated immediately after collecting 100 ml peripheral blood using Vacutainer CPT Cell Preparation Tubes with sodium citrate (Becton Dickinson) according to manufacturer's instructions. After washing with phosphate-buffered saline, the cells were grown at a density of 0.5 million/ml in 5 ml RPMI1640 medium supplemented with 10% charcoal-depleted fetal calf serum, 2 mM L-glutamine, 0.1 mg/ml streptomycin and 100 U/ml penicillin at 37 °C in a humidified 95% air/5% COBERSORTx  and its www.ncbi.nlm.nih.gov/geo) with accession number GSE199273.Total RNA was isolated using the High Pure RNA Isolation Kit (Roche) according to manufacturer's instructions. RNA quality was assessed on an Agilent 2100 Bioanalyzer system (RNA integrity number ≥ 8). rRNA depletion and cDNA library preparation were performed using the New England Biolabs kits NEBNext rRNA Depletion, NEBNext Ultra II Directional RNA Library Prep for Illumina and NEBNext Multiplex Oligos for Illumina (Index Primers Sets 1 and 2) according to manufacturer's protocols. RNA-seq libraries went through quality control on an Agilent 2100 Bioanalyzer and were sequenced on a NextSeq 500 system (Illumina) at 75 bp read length using standard protocols at the Gene Core facility of the EMBL . The libraries were sequenced as four batches. Fastq files of the 66 libraries can be found at Gene Expression Omnibus  (via the R package BiomaRt (version 2.46.0)  with Ensembl annotation (version 103) by using default settings of the nf-core/rnaseq STAR-Salmon pipeline (version 3.0) 4323183) . The num23183 . TEdgeR (version 3.36.0)   between the samples. This distance was calculated as the root mean square deviation (Euclidean distance) of the largest 500 log2FCs between a given pair of samples, i.e., for each pair a different set of top genes was selected. The inspection of the MDS plot showed that (i) the time-dependent divergence from the native transcriptome state and (ii) the concentration-dependent modulation by treatment with 1,25(OH)2D3, 25(OH)D3 or 25(OH)D2 are the two principal factors driving differences in the gene expression profiles of PBMCs. The gene-wise statistical test for differential expression was computed using the generalized linear model quasi-likelihood pipeline  and FC > 2 at 24 h. Taking all treatments together, 553 genes with a Benjamini-Hochberg corrected p-value  < 0.05 and a total trimmed mean of M-value normalized CPM count > 10 2D3 and EtOH-treated samples at each time point) were considered as vitamin D targets .Differential gene expression analysis was computed in R (version 4.1.2) in the Rocky Linux 8.5 operating system using the tool  3.36.0) . The anang (MDS) . MDS waspipeline . The emppipeline . The glm targets . Mean-Digets 3A;  for the 2FC) was modeled with the three-parameter log-logistic function LL.3 from the R package drc  and family-wise error rate (FWER)-adjusted p-values retrieved. Comparisons with a FWER < 0.05 were considered as significant. The code of the analysis can be found at https://github.com/andreahanel/2022_Doseresponse.The effect of vitamin D metabolites on the change in mRNA levels D3, 1,000 nM 25(OH)D2 or solvent (0.1% EtOH). In three repeats RNA-seq was performed on the basis of total RNA. As in comparable studies D3  and 385 genes modulated by 25(OH)D2  , FN1 (fibronectin 1), LOXHD1 (lipoxygenase homology PLAT domains 1) and SLC24A4 (solute carrier family 24 member 4) were the top up-regulated and the genes CXCL10 (C-X-C motif chemokine ligand 10), STEAP4 , CXCL9 and OLFM1 (olfactomedin 1) the most down-regulated.In order to obtain maximal responses of the transcriptome, PBMCs from an healthy individual were treated immediately after isolation for 24 h with either 10 nM 1,25(OH) studies , 47, 48,gulated) . For all2D3, with 100, 250, 500, and 750 nM 25(OH)D3 as well as with 100, 250, 500, and 750 nM 25(OH)D2. This resulted in 7 and 179 target genes for 0.1 and 1 nM 1,25(OH)2D3, no targets for 100 and 250 nM 25(OH)D3, 322 and 392 responding genes for 500 and 750 nM 25(OH)D3, no targets for 100 nM 25(OH)D2 as well as 30, 342, and 397 genes that reacted on a stimulation with 250, 500, and 750 nM 25(OH)D2 D3 as well as 25, 278, 307, and 299 to 250, 500, 750, and 1,000 nM 25(OH)D2 D2 25(OH)D2 . These i)D2 (2D3 . This le25(OH)D2 . Venn di25(OH)D2 . From bo3 and 25(OH)D2 were insufficient for general gene regulation, while 500 nM of both vitamin D metabolites was clearly above this threshold.The 206 common targets represent a stable set of genes responding to variant concentrations of all three tested vitamin D metabolites . Combine2D3, 25(OH)D3 and 25(OH)D2 are very comparable. At concentrations of 250 nM and below, 25(OH)D3 and 25(OH)D2 are largely insufficient to significantly modulate the expression of vitamin D target genes.In summary, both by number as well as by most responsive target genes the transcriptome-wide responses to saturating concentrations of 1,25(OH)50 values 0.48 nM for 1,25(OH)2D3, 322 nM for 25(OH)D3 and 295 nM for 25(OH)D2  between the potencies of 25(OH)D3 and 25(OH)D2, but their average EC50 is some 640-fold higher than that of 1,25(OH)2D3.Plotting the FC of the 206 common vitamin D target genes over vitamin D metabolite concentration and using the three-parameter log-logistic model, determined the EC25(OH)D2 . There i2D3 the EC50-values of the eight different categories varied from 0.29 to 0.73 nM but did not differ significantly  between each other (3 the range was 318 to 389 nM and for 25(OH)D2 192 to 313 nM, but the difference was not statistically significant .Based on the reference dataset , the 206ch other . Similar2D3 the EC50-values ranged from 0.10 nM [ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2)] to 2.39 nM [LMNA (lamin A/C)], for 25(OH)D3 from 121 nM [NXPH4 (neurexophilin 4)] to 461 nM [ENTPD7 (ectonucleoside triphosphate diphosphohydrolase 7)] and for 25(OH)D2 from 132 nM (SLC11A1) to 421 nM [STAB1 (stabilin 1)] , 59 mid responders (0.41 to 1.06 nM) and 12 low responders (1.20 to 2.39 nM). Representative genes for the three categories are HBEGF (heparin binding EGF like growth factor) as high responding gene (PPARGC1B (PPARG coactivator 1 beta) as mid responding gene  as low responding gene D3 and 25(OH)D2 did not allow a categorization of the vitamin D target genes by their response to these metabolites.Since neither gene categories nor up- or down-regulation allowed a distinction of the sensitivity of vitamin D target genes to vitamin D metabolites, the dose responses of the 206 genes were investigated on an individual gene level. Manual inspection of the dose response curves provided for 130 genes convincing fits for all three vitamin D metabolites. Interestingly, for 1,25(OH)ilin 1)] . The widing gene , PPARGC1ing gene  and MYCLing gene . Importa50 values of the response of vitamin D target genes to 25(OH)D3 and 25(OH)D2 are not significantly different and are in the order of 300 nM, i.e., some 600-fold higher as those for 1,25(OH)2D3. Categorization of the target genes into primary/secondary, direct/indirect or up-regulated/down-regulated does not allow any significant distinction in their response to the three vitamin D metabolites. However, individual target genes can be classified by their response to 1,25(OH)2D3 (but not to 25(OH)D3 and 25(OH)D2) as high, mid and low responding genes.Taken together, the average EC3 and 25(OH)D2 may activate vitamin D signaling without enzymatic conversion by CYP27B1 to 1,25(OH)2D3 and 1,25(OH)2D2, respectively, the transcriptome-wide response to saturating concentrations of all three vitamin D metabolites was assessed by RNA-seq at earlier time points. After 4 h stimulation with 10 nM 1,25(OH)2D3, 16 genes (13 up- and 3 down-regulated) passed the statistical threshold , with 1,000 nM 25(OH)D3 32 genes (27 up- and 5 down-regulated) and with 1,000 nM 25(OH)D2 20 genes (19 up- and 1 down-regulated) (HBEGF and G0S2 (G0/G1 switch 2). After filtering with the reference dataset of vitamin D target genes in PBMCs D3 or 1,000 nM 25(OH)D2 at 4, 8 and 24 h (In order to investigate whether 25(OH)Dgulated) . Common in PBMCs  31), Ve, Ve3 andin PBMCs . As obsein PBMCs , 31, at and 24 h .DHCR7, CYP2R1 and CYP27A1 show comparable mid-range expression, while the average expression of CYP27B1 is 10- to 32-fold lower. In this way, CYP27B1 belongs to the 5% lowest expressed genes in PBMCs. Since the CYP24A1 gene is a well-known vitamin D target gene 2D on the basis of 25(OH)D is far less prominently covered by enzyme expression as the degradation of 25(OH)D and 1,25(OH)2D to 24,25(OH)D and 1,24,25(OH)3D, respectively.The enzymes DHCR7 (7-dehydrocholesterol reductase), CYP2R1, CYP27A1, CYP27B1 and CYP24A1 are critical nodes in the vitamin D metabolism pathway , left. Tget gene , its expiD study  are dispiD study . The ind3 and 25(OH)D2 as prominently as to 1,25(OH)2D3. The expression of CYP27B1 protein in PBMCs is very likely too low for an efficient conversion of 25(OH)D3 and 25(OH)D2 into 1,25(OH)2D3 and 1,25(OH)2D2, respectively, in particular in the presence of highly expressed CYP24A1 protein. However, it cannot be excluded that metabolic formation of 1,25(OH)2D is partly contributing to the results obtained.In summary, already at early time points (4 and 8 h) the PBMC transcriptome responds to a stimulation with saturating concentrations of 25(OH)D3 and 25(OH)D2 to each other and in reference to 1,25(OH)2D3. At supra-physiological concentrations of 10 nM 1,25(OH)2D3  and 1,000 nM 25(OH)D (10-times the recommended serum level) the response of the PBMC transcriptome is very comparable, i.e., the expression of some 300 genes is significantly modulated showing after 24 h stimulation some 3-times more down-regulation than up-regulation. Moreover, the target gene lists of 25(OH)D3, 25(OH)D2 and 1,25(OH)2D3 are to 85% identical, i.e., our experimental series had relatively low transcriptional noise. These observations suggest that all three vitamin D metabolites use identical mechanisms in the modulation of vitamin D target gene expression. Form a structural point of view this is obvious, since 25(OH)D will bind in the same agonistic VDR conformation as 1,25(OH)2D D25(OH)2D .2D3 time course study in PBMCs D3 and 295 nM for 25(OH)D2 compared to 0.48 nM for 1,25(OH)2D3 is the first report on the sensitivity of the transcriptome to vitamin D metabolites. These results provide an additional argument that there is no difference in the response of the transcriptome to 25(OH)D3 and 25(OH)D2. Moreover, our findings indicate with the factor of ~600 a good estimation of the relative gene regulatory potential of 25(OH)D compared to 1,25(OH)2D. Since serum levels of 25(OH)D are even 1,000-times higher than that of 1,25(OH)2D, this supports the option that 25(OH)D can directly modulate the expression of vitamin D target genes. However, 25(OH)D concentrations in the order of 300 nM are far higher than the recommended 100 nM. Therefore, irrespective of the mechanism of action, for persons with a normal vitamin D status the transcriptome as a whole may not be regulated by 25(OH)D. However, there are a few very sensitive genes, such as NXPH4, SLC11A1, ADGR3 (adhesion G protein-coupled receptor G3), G0S2, HBEGF, and PMEPA1 , which showed EC50 values for 25(OH)D below 150 nM. Thus, in healthy persons with a very high vitamin D status, a few genes may be directly affected by elevated 25(OH)D serum levels.The 206 common vitamin D target genes, on which we focus in this study, represent the majority of the vitamin D sensitive part of the PBMC transcriptome, although  they had been filtered by a reference dataset from a recent 1,25(OH)in PBMCs . The est3 or D2 supplementation, there are clinical settings, where supplementation with higher doses of 25(OH)D3 or 25(OH)D2 are recommended D supplementation.In addition to the control of the vitamin D status of healthy individuals through careful sun exposure and vitamin Dommended . These p chicken . Also inHBEGF D2, it does not matter, if the observed effects on the PBMC transcriptome are explained either (i) by the enzymatic conversion of 25(OH)D into 1,25(OH)2D during the 4–24 h duration of stimulation phase, which then activates the VDR, (ii) by a direct binding of 25(OH)D to the VDR or (iii) a combination of both. The very low expression of the CYP27B1 gene, in particular in relation to CYP24A1 expression, in PBMCs of one person used in this study is representative for other individuals. This calls into question, whether there was enough 1α-hydroxylase activity to convert within 4 h a sufficient amount of 25(OH)D into 1,25(OH)2D, which then stimulated primary vitamin D target genes. For example, in order to reach a 1,25(OH)2D level of 10 nM, 1% of the 1,000 nM 25(OH)D pool need to be handled within 4 h. However, as it is typical for in vitro cell culture stimulation experiments, supra-physiological concentrations of 1,25(OH)2D3 and 25(OH)D are compared. In fact, the tight regulation of the 1,25(OH)2D3 level in vivo via the molecule's rapid degradation by the enzyme CYP24A1 D CYP24A1 , indicat3 and 25(OH)D2 are equally potent in modulating the transcriptome of PBMCs and regulate the same set of vitamin D target genes as the most potent VDR ligand, 1,25(OH)2D3. However, in order to observe consequences of the gene regulatory potential of 25(OH)D, concentrations of 300 nM or higher need to be available. This is three times the recommended serum level, i.e., it does not apply to healthy individuals with a regular vitamin D status.In conclusion, 25(OH)DFastq files of the raw data can be found at GEO with accession number GSE199273.The studies involving human participants were reviewed and approved by the Ethics Committee of the Northern Savo Hospital District had approved the study protocol (#9/2014). The patients/participants provided their written informed consent to participate in this study.AH performed RNA-seq data analysis. CC did RNA isolation and RNA-seq library preparation. CV synthesized the tested compounds. AH and CC wrote the manuscript, which was reviewed by CV. All authors contributed to the article and approved the submitted version.Parts of this study were sponsored by Carbogen Amics, Ltd. CC was supported by the WELCOME2—ERA Chair European Union's Horizon2020 research and innovation program under Grant Agreement No. 952601.This study received funding from Carbogen Amics, Ltd. The funder had the following involvement with the study: Providing vitamin D metabolites. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.