Dietary anthocyanins are important health-promoting antioxidants that make a major contribution to the quality of fruits. It is intriguing that most tomato cultivars do not produce anthocyanins in fruit. However, the purple tomato variety Indigo Rose, which has the dominant Aft locus combined with the recessive atv locus from wild tomato species, exhibits light-dependent anthocyanin accumulation in the fruit skin. Here, we report that Aft encodes a functional anthocyanin activator named SlAN2-like, while atv encodes a nonfunctional version of the anthocyanin repressor SlMYBATV. The expression of SlAN2-like is responsive to light, and the functional SlAN2-like can activate the expression of both anthocyanin biosynthetic genes and their regulatory genes, suggesting that SlAN2-like acts as a master regulator in the activation of anthocyanin biosynthesis. We further showed that cultivated tomatoes contain nonfunctional alleles of SlAN2-like and therefore fail to produce anthocyanins. Consistently, expression of a functional SlAN2-like gene driven by the fruit-specific promoter in a tomato cultivar led to the activation of the entire anthocyanin biosynthesis pathway and high-level accumulation of anthocyanins in both the peel and flesh. Taken together, our study exemplifies that efficient engineering of complex metabolic pathways could be achieved through tissue-specific expression of master transcriptional regulators.

Messenger RNA (mRNA) has recently emerged as a promising class of nucleic acid therapy, with the potential to induce protein production to treat and prevent a range of diseases. However, the widespread use of mRNA as a therapeutic requires safe and effective in vivo delivery technologies. Libraries of ionizable lipid nanoparticles (LNPs) have been designed to encapsulate mRNA, prevent its degradation, and mediate intracellular delivery. However, these LNPs are typically characterized and screened in an in vitro setting, which may not fully replicate the biological barriers that they encounter in vivo. Here, we designed and evaluated a library of engineered LNPs containing barcoded mRNA (b-mRNA) to accelerate the screening of mRNA delivery platforms in vivo. These b-mRNA are similar in structure and function to regular mRNA, and contain barcodes that enable their delivery to be quantified via deep sequencing. Using a mini-library of b-mRNA LNPs formulated via microfluidic mixing, we show that these different formulations can be pooled together, administered intravenously into mice as a single pool, and their delivery to multiple organs (liver, spleen, brain, lung, heart, kidney, pancreas, and muscle) can be quantified simultaneously using deep sequencing. In the context of liver and spleen delivery, LNPs that exhibited high b-mRNA delivery also yielded high luciferase expression, indicating that this platform can identify lead LNP candidates as well as optimal formulation parameters for in vivo mRNA delivery. Interestingly, LNPs with identical formulation parameters that encapsulated different types of nucleic acid barcodes (b-mRNA versus a DNA barcode) altered in vivo delivery, suggesting that the structure of the barcoded nucleic acid affects LNP in vivo delivery. This platform, which enables direct barcoding and subsequent quantification of a functional mRNA, can accelerate the in vivo screening and design of LNPs for mRNA therapeutic applications such as CRISPR-Cas9 gene editing, mRNA vaccination, and other mRNA-based regenerative medicine and protein replacement therapies.

The NADPH oxidase reaction produces protons. In the case of the NADPH oxidase, NOX2, activity depends on secretion of these protons and is inhibited by blockade of the voltage-gated proton channel (Hv1). Duox1 and Duox2 activities similarly produce intracellular protons but synthesize hydrogen peroxide directly instead of superoxide. Hv1 contributes to acid secretion in some epithelia that express Duox. To test the hypothesis that Duox activity is also sensitive to Hv1 channel blockers, Duox was assayed in the presence of either Zn2+ or 5-chloro-2-guanidinobenzimidazole (ClGBI). Both compounds inhibited Duox activity in normal human bronchial epithelial cells but with an IC50 over 10-fold higher than that reported for Hv1 (IC50 Zn2+ = 0.68 mM; IC50 ClGBI = 0.07-0.14 mM). Homogenized HEK293T cells expressing either Duox1 or Duox2 showed similar IC50 values for ClGBI suggesting these compounds inhibit the enzymes through alternate mechanisms independent of Hv1 proton secretion. Inclusion of superoxide dismutase did not restore Duox hydrogen peroxide synthesis. Addition of nigericin to eliminate any possible transmembrane pH gradients in intracellular membrane-localized Duox did not alter activity in HEK293T homogenates. Extracellular Zn2+ blocked intracellular Ca2+ increases needed for Duox activity. Together the data suggest that Duox enzyme activities in epithelia are inhibited by compounds that block Hv1 but inhibition occurs through Hv1-independent mechanisms and support the idea that Hv1 is not required for Duox activity.

The prime issue derived from prostate cancer (PCa) is its high prevalence to metastasize to bone. MicroRNA-204-5p (miR-204-5p) has been reported to be involved in the development and metastasis in a variety of cancers. However, the clinical significance and biological functions of miR-204-5p in bone metastasis of PCa are still not reported yet. In this study, we find that miR-204-5p expression is reduced in PCa tissues and serum sample with bone metastasis compared with that in PCa tissues and serum sample without bone metastasis, which is associated with advanced clinicopathological characteristics and poor bone metastasis-free survival in PCa patients. Moreover, upregulation of miR-204-5p inhibits the migration and invasion of PCa cells in vitro, and importantly, upregulating miR-204-5p represses bone metastasis of PCa cells in vivo. Our results further demonstrated that miR-204-5p suppresses invasion, migration, and bone metastasis of PCa cells via inactivating nuclear factor κB (NF-κB) signaling by simultaneously targeting TRAF1, TAB3, and MAP3K3. In clinical PCa samples, miR-204-5p expression negatively correlates with TRAF1, TAB3, and MAP3K3 expression and NF-κB signaling activity. Therefore, our findings reveal a new mechanism underpinning the bone metastasis of PCa, as well as provide evidence that miR-204-5p might serve as a novel serum biomarker in bone metastasis of PCa. This study identifies a novel functional role of miR-204-5p in bone metastasis of prostate cancer and supports the potential clinical value of miR-204-5p as a serum biomarker in bone metastasis of PCa.

Although our knowledge of human diseases has increased dramatically, the molecular basis, phenotypic traits, and therapeutic targets of most diseases still remain unclear. An increasing number of studies have observed that similar diseases often are caused by similar molecules, can be diagnosed by similar markers or phenotypes, or can be cured by similar drugs. Thus, the identification of diseases similar to known ones has attracted considerable attention worldwide. To this end, the associations between diseases at the molecular, phenotypic, and taxonomic levels were used to measure the pairwise similarity in diseases. The corresponding performance assessment strategies for these methods involving the terms "category-based," "simulated-patient-based," and "benchmark-data-based" were thus further emphasized. Then, frequently used methods were evaluated using a benchmark-data-based strategy. To facilitate the assessment of disease similarity scores, researchers have designed dozens of tools that implement these methods for calculating disease similarity. Currently, disease similarity has been advantageous in predicting noncoding RNA (ncRNA) function and therapeutic drugs for diseases. In this article, we review disease similarity methods, evaluation strategies, tools, and their applications in the biomedical community. We further evaluate the performance of these methods and discuss the current limitations and future trends for calculating disease similarity.

Gaucher Disease (GD) type 3 is a neurological form of a multisystemic autosomal recessive disorder belonging to the group of lysosomal storage diseases. Causal mutations in the glucocerebrosidase 1 (GBA1) commonly lead to abnormal protein and GD, heterozygosity is a genetic risk factor for Parkinson's disease. This work describes the use of a non-integrative approach using Sendai Virus delivery to establish induced Pluripotent Stem Cells (iPSCs) from fibroblasts from a GD type 3 patient. Differentiation of iPSCs can be employed to generate a variety of complex cell types with a high degree of genetic complexity that would otherwise be unattainable.

Mucopolysaccharidosis type I-Hurler (MPS1-H) is the most severe form of inherited metabolic diseases caused by mutations in the IDUA gene. The resulting deficiency of alpha L-iduronidase enzyme leads to a progressive accumulation of glycosaminoglycans in lysosomes which damages multiple organs and highly reduces life expectancy of affected children. Skin fibroblasts of a 2-year-old MPS1-H male, carrying two mutations in each IDUA alleles (H358_T364del; W402X), were reprogrammed into induced pluripotent stem cells (iPSCs) using the CytoTune-iPS Sendai Reprogramming method applying Yamanaka-factors (OCT4, SOX2, KLF4, c-MYC). iPSCs expressed pluripotency transcription factors while iPSC-derived embryoid bodies reveal markers of the three germ layers.

CUL4B gene mutation can cause intelligence deficiency 15, a syndromic form of X-linked mental retardation characterized by severe intellectual deficit associated with short stature, craniofacial dysmorphism, speech delay and impairment, tremor and gait ataxia. Here, we generated iPSCs from a Chinese patient with c.1007_1011del (p.(Ile336fs)) in CUL4B gene by reprogramming peripheral blood mononuclear cells with non-integrating vectors. The generated iPSC line (SDQLCHi015-A) expresses pluripotency markers, presents a normal karyotype and is able to differentiate into three germ layers.

"Cell-cycle hypothesis" is emerging in recent years to suggest that aberrant cell cycle re-entry of differentiated neurons leads to a remarkable genetic disequilibrium which is likely to be the primary cause of neuronal apoptosis. DNA polymerase-β is involved in neuronal DNA replication during cell cycle re-entry, thus constituting a promising target for Alzheimer's disease treatment. Recently, 5-methoxyflavone was identified as a candidate molecule endowed with good biological activity and selectivity on the DNA pol-β in multiple in vitro AD models. In vivo assays, especially the brain uptake of 5-methoxyflavone, is need to be evaluated for further development for AD treatment. We report herein the synthesis of 11C-labeled 5-methoxyflavone, and the evaluation of in vivo properties of 5-[11C]methoxyflavone in rodents.

To assess the impact of vagotomy on the IgA-response, male BALB/c mice underwent anterior vagotomy or a sham procedure were sacrificed on day 14 post-operation and the proximal and distal small-gut segments were dissected. In intestinal lavages IgA/IgM antibodies were analysed by ELISA; in Peyer's-patches and lamina-propria cell suspensions the intracellular IgA-associated interleukins (ILs) and pro-inflammatory cytokines in CD4+ T cells were analysed by cytofluorometry. Vagotomy reduced the IgA or increased the IgM antibody concentration in both segments and reduced or increased the lamina- propria CD4+ T cell pro-inflammatory cytokine responses in the distal or proximal segments, respectively. Data show the role of the vagus nerve on the IgA response.

To date, research examining the relationship between brain structure and hearing acuity is sparse, especially given the context of a broad age range. To investigate this relationship, we applied an automated surface-based morphometry (SBM) approach (FreeSurfer) in this study to re-examine a sample of normal-hearing (n = 17) and hearing-impaired (n = 17) age- and education-matched adults, aged between 20 and 63 years (Alfandari et al., 2018). The SBM approach allows the disentanglement of cortical surface area (CSA) from cortical thickness (CT), the 2 independent constituents of cortical volume (CV). We extend the findings of Alfandari and colleagues by showing several clusters in auditory-related areas as well as in the left and right angular gyrus that showed reduced CT, CSA and CV in hearing-impaired compared to normal-hearing listeners. Nevertheless, none of the clusters found correlated significantly with hearing acuity, measured by pure-tone thresholds, in the 2 groups. An additional vertex-wise correlation analysis between hearing acuity and morphometric parameters over all participants revealed a single significant cluster encompassing the left Heschl's gyrus. Higher hearing thresholds were associated with a thinner cortex within this cluster. Our results imply that hearing impairment is associated with reduced thickness in primary and secondary auditory cortex regions, those regions especially involved in perceiving and processing relevant speech cues. This decrease was observed not only in older but also in younger and middle-aged adults, independent of age-related decline in the cognitive domain and age-dependent whole-brain atrophy. Further, the results show the value added when considering CV, CT and CSA separately, relative to previous studies which have solely relied on voxel-based morphometry to investigate brain structure and hearing acuity across the lifespan.

Genetically determined cerebellar ataxias (CA) are a heterogeneous group of disorders with progressive decline of cerebellar functions. The cerebellum influences internal forward models that play a role in cognitive control, but whether these processes are dysfunctional in CA is unclear. Here, we examined sensory predictive coding processes and response adaptation in CA and healthy controls (HC) using behavioral tests with concomitant EEG recordings. N = 23 patients and N = 29 age- and sex-matched HC were studied. Sensory prediction coding was tested with an auditory distraction paradigm and error-related behavioral adaptation with a visual flanker task. As neurophysiological markers we studied different event-related potentials: the P3a for orientation of attention; the N2 and the error-related negativity (ERN) for cognitive adaptation processes/consequences of response errors; error-related positivity (Pe) for error-awareness; the mismatch negativity (MMN) for sensory predictive coding; and reorientation negativity (RON) for reorientation after unexpected events. Overall reaction times were slower in patients compared to HC, but error rates did not differ. Both in patients and HC, P3a amplitudes were larger in distraction trials, but the P3a amplitude was smaller in patients compared to HC. The MMN as well as behavioral and EEG-correlates of response adaptation (ERN/N2) did not differ between groups, while the Pe was attenuated in patients. During sensory predictive coding, RON amplitudes were significantly larger in HC compared to patients. In HC, but not in patients, RON amplitudes were also larger in deviant compared to frequent trials. Processes generating internal forward models are largely intact in genetically determined CA, whereas updating of mental models and error awareness are disturbed in these patients.

Insomnia Disorder (ID) is a prevalent and persistent condition, yet its neural substrate is not well understood. The cognitive, emotional, and behavioral characteristics of ID suggest that vulnerability involves distributed brain networks rather than a single brain area or connection. The present study utilized probabilistic diffusion tractography to compare the whole-brain structural connectivity networks of people with ID and those of matched controls without sleep complaints. Diffusion-weighted images and T1-weighed images were acquired in 51 people diagnosed with ID (21-69 years of age, 37 female) and 48 matched controls without sleep complaints (22-70 years of age, 31 female). Probabilistic tractography was performed to construct the whole-brain structural connectivity network of each participant. Case-control differences in connectivity strength and network efficiency were evaluated with permutation tests. People with ID showed structural hyperconnectivity within a subnetwork that spread over frontal, parietal, temporal, and subcortical regions and was anchored at the right angular gyrus. The result was robust across different edge-weighting strategies. Moreover, converging support was given by the finding of heightened right angular gyrus nodal efficiency (harmonic centrality) across varying graph density in people with ID. Follow-up correlation analyses revealed that subnetwork connectivity was associated with self-reported reactive hyperarousal. The findings demonstrate that the right angular gyrus is a hub of enhanced structural connectivity in ID. Hyperconnectivity within the identified subnetwork may contribute to increased reactivity to stimuli and may signify vulnerability to ID.

To assess the association between flooding/repeat flooding and: (1) psychological morbidity (anxiety, depression, post-traumatic stress disorder (PTSD)) and (2) health-related quality of life (HRQoL) at 6 months post-flooding.

BACKGROUND The association between serum vitamin D level and vertebral fracture (VFx) remains controversial. The purpose of this study was to determine whether serum 25-hydroxy vitamin D (25(OH)D) level is associated with osteoporotic thoracolumbar junction VFx in elderly patients. MATERIAL AND METHODS From Jan 2013 to Dec 2017, this retrospective case-control study included 534 patients with primary osteoporotic thoracolumbar junction VFx (T10-L2) and 569 elderly orthopedic patients with back pain (without osteoporotic VFx) as controls. Serum 25(OH)D levels were measured and the association with osteoporotic VFx was analyzed. Other clinical data, including BMI, comorbidities, and bone mineral density (BMD), were also collected and compared between these 2 groups. RESULTS It was shown that 25(OH)D levels were significantly lower in patients with T10-L2 VFx than in control patients. Among 534 VFx patients, 417 (78.1%) patients showed grade 2-3 fracture. Serum 25(OH)D levels were significantly related to affected vertebral numbers and VFx severities. The VFx risk was 28% lower (OR=0.72, 95% CI 0.62-0.83) per increased SD in serum 25(OH)D. Compared with the 1st quartile (mean 25(OH)D: 29.67±6.18 nmol/L), the VFx risk was significantly lower in the 3rd (mean 25(OH)D: 60.91±5.12nmol/L) and 4th quartiles (mean 25(OH)D: 103.3±44.21nmol/L), but not in the 2nd quartile (mean 25(OH)D: 45.40±3.95 nmol/L). In contrast, the VFx risk was significantly increased in the 1st quartile (OR=1.87, 95% CI 1.42-2.45) compared with the 2nd-4th quartiles. CONCLUSIONS Vitamin D deficiency/insufficiency was associated with risk of osteoporotic thoracolumbar junction vertebral fractures in elderly patients.

It is uncertain how electroconvulsive therapy-induced generalized seizures exert their potent therapeutic effects on various neuropsychiatric disorders. Adenosine monophosphate-activated protein kinase (AMPK) plays a major role in maintaining metabolic homeostasis and activates autophagic processes via unc-51-like kinase (ULK1). Evidence supports the involvement of autophagy system in the action mechanisms of antidepressants and antipsychotics. The effect of electroconvulsive therapy on autophagy-related signaling requires further clarification.

Blinatumomab (BLINCYTO®) is a novel bispecific T cell engager (BiTE®) approved in the USA for the treatment of relapsed or refractory B cell precursor acute lymphoblastic leukemia (ALL) in children and adults, as well as minimal residual disease ALL in adults. This analysis characterized the population pharmacokinetics of intravenous blinatumomab in pediatric and adult patients.

With the growing use of immune checkpoint inhibitors (ICIs), case reports of rare yet life-threatening pituitary-adrenal dysfunctions, particularly for hypopituitarism, are increasingly being published. In this analysis, we focus on these events by including the most recent publications and reports from early phase I/II and phase III clinical trials and comparing the incidence and risks across different ICI regimens. PubMed, Embase, and the Cochrane Library were systematically searched from inception to April 2019 for clinical trials that reported on pituitary-adrenal dysfunction. The rates of events, odds ratios (ORs), and 95% confidence intervals (CIs) were obtained using random effects meta-analysis. The analyses included data from 160 trials involving 40 432 participants. The rate was 2.43% (95% CI, 1.73%-3.22%) for all-grade adrenal insufficiency and 3.25% (95% CI, 2.15%-4.51%) for hypophysitis. Compared with the placebo or other therapeutic regimens, ICI agents were associated with a higher incidence of serious-grade adrenal insufficiency (OR 3.19, 95% CI, 1.84 to 5.54) and hypophysitis (OR 4.77, 95% CI, 2.60 to 8.78). Among 71 serious-grade hypopituitarism instances in 12 336 patients, there was a significant association between ICIs and hypopituitarism (OR 3.62, 95% CI, 1.86 to 7.03). Substantial heterogeneity was noted across the studies for the rates of these events, which in part was attributable to the different types of ICIs and varied phases of the clinical trials. Although the rates of these events were low, the risk was increased following ICI-based treatment, particularly for CTLA-4 inhibitors, which were associated with a higher incidence of pituitary-adrenal dysfunction than PD-1/PD-L1 inhibitors.

The use of baseline tumor burden (TB) as a prognostic factor for non-small cell lung cancer (NSCLC) patients treated with immune checkpoint inhibitors (ICIs) and associations between TB and other prognostic biomarkers remain unclear. In this study, we investigated the association between TB and survival in NSCLC patients treated with ICIs in comparison with other biomarkers.

KCNE β-subunits play critical roles in modulating cardiac voltage-gated potassium channels. Among them, KCNE1 associates with KCNQ1 channel to confer a slow-activated IKs current, while KCNE2 functions as a dominant negative modulator to suppress the current amplitude of KCNQ1. Any anomaly in these channels will lead to serious myocardial diseases, such as the long QT syndrome (LQTS). Trafficking defects of KCNE1 have been reported to account for the pathogenesis of LQT5. However, the molecular mechanisms underlying KCNE forward trafficking remain elusive. Here, we describe an arginine/lysine-based motif ([R/K](S)[R/K][R/K]) in the proximal C-terminus regulating the endoplasmic reticulum (ER) export of KCNE1 and KCNE2 in HEK293 cells. Notably, this motif is highly conserved in the KCNE family. Our results indicate that the forward trafficking of KCNE2 controlled by the motif (KSKR) is essential for suppressing the cell surface expression and current amplitude of KCNQ1. Unlike KCNE2, the motif (RSKK) in KCNE1 plays important roles in modulating the gating of KCNQ1 in addition to mediating the ER export of KCNE1. Furthermore, truncations of the C-terminus did not reduce the apparent affinity of KCNE2 for KCNQ1, demonstrating that the rigid C-terminus of KCNE2 may not physically interact with KCNQ1. In contrast, the KCNE1 C-terminus is critical for its interaction with KCNQ1. These results contribute to the understanding of the mechanisms of KCNE1 and KCNE2 membrane targeting and how they coassemble with KCNQ1 to regulate the channels activity.