Hole-Transporting Materials based on Oligo(hetero)aryls with a Naphthodithiophene Core - Succinct Synthesis by Twofold Direct C-H Olefination.

This work demonstrated the first synthetic application of direct C-H olefinations in the step-saving preparation of various hole-transporting materials (HTM) for efficient perovskite solar cells (PSC). Cross-dehydrogenative couplings of naphthodithiophene (NDT) with vinyl arenes under palladium-catalysis facilely generated various new oligo(hetero)aryls with internal alkenes. Reaction conditions were optimized, which gave the product isolated yields of up to 71 % with high (E)-stereoselectivity. These readily accessible NDT core-based small molecules involving olefin as π-spacers displayed immediate power conversion efficiencies of up to 17.2 % without a device oxidation process that is required for the commercially available spiro-OMeTAD and most other existing HTMs while fabricated in corresponding PSC devices.

The characterization and comorbidities of heterozygous Bardet-Biedl syndrome carriers.

Introduction: Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder with clinical features of retinal dystrophy, obesity, postaxial polydactyly, renal anomalies, learning disabilities, hypogonadism, and genitourinary abnormalities. Nevertheless, previous studies on the phenotypic traits of BBS heterozygous carriers have generated inconclusive results. The aim of our study was to investigate the impact of BBS heterozygosity on carriers when compared to non-carriers within the Taiwanese population. Materials and Methods: This study follows a hospital-based case-control design. We employed the Taiwan Biobank version 2 (TWBv2) array to identify three specific loci associated with BBS (rs773862084, rs567573386, and rs199910690). In total, 716 patients were included in the case group, and they were compared to a control group of 2,864 patients who lacked BBS alleles. The control group was selected through gender and age matching at a ratio of 1:4. The association between BBS-related loci and comorbidity was assessed using logistic regression models. Results: We found that BBS heterozygous carriers exhibited a significant association with elevated BMI levels, especially the variant rs199910690 in MKS1 (p=0.0037). The prevalence of comorbidities in the carriers' group was not higher than that in the non-carriers' group. Besides, the average values of the biochemistry data showed no significant differences, except for creatinine level. Furthermore, we conducted a BMI-based analysis to identify specific risk factors for chronic kidney disease (CKD). Our findings revealed that individuals carrying the CA/AA genotype of the BBS2 rs773862084 variant or the CT/TT genotype of the MKS1 rs199910690 variant showed a reduced risk of developing CKD, irrespective of their BMI levels. When stratified by BMI level, obese males with the MKS1 rs199910690 variant and obese females with the BBS2 rs773862084 variant exhibited a negative association with CKD development. Conclusion: We found that aside from the association with overweight and obesity, heterozygous BBS mutations did not appear to increase the predisposition of individuals to comorbidities and metabolic diseases. To gain a more comprehensive understanding of the genetic susceptibility associated with Bardet-Biedl Syndrome (BBS), further research is warranted.

Sex-specific genetic variants associated with adult-onset inguinal hernia in a Taiwanese population.

Introduction: Inguinal hernia repair is one of the most common surgeries worldwide. However, there is limited information on its underlying genetic mechanism. Studies on the genetic factors related to inguinal hernia in Han Chinese are lacking. Therefore, we aimed to conduct a hospital-based study to assess the genetic factors and comorbidities underlying inguinal hernia in Taiwan. Materials and Methods: This was a retrospective case-control study. Utilizing data from the Taiwan Precision Medicine Initiative, we identified 1000 patients with inguinal hernia and 10,021 matched controls without inguinal hernia between June 2019 and June 2020. Four susceptibility loci (rs2009262, rs13091322, rs6991952, and rs3809060) associated with inguinal hernia were genotyped by the Taiwan Biobank version 2 (TWBv2) array. Inguinal hernia, surgery types, and comorbidities were obtained from the electronic health records of Taichung Veterans General Hospital. Results: Adult-onset inguinal hernia was associated with WT1 rs3809060 GT/TT genotype in males and EFEMP1 rs2009262 TC/CC genotype in females. In addition, we identified sex-specific risk factors associated with inguinal hernia; benign prostatic hyperplasia in males (OR: 3.19, 95% CI: 2.73 - 3.73, p< 0.001), chronic obstructive pulmonary disease in females (OR: 2.34, 95% CI: 1.33 - 4.11, p = 0.003) and overweight, defined by body mass index ≧24 kg/m2 (OR: 0.75, 95% CI: 0.65 - 0.86, p<0.001 in males, and OR: 0.60, 95% CI:0.37 - 0.98, p = 0.042 in females), were inversely associated with inguinal hernia. After stratifying BMI, overweight males with EFEMP1 rs2009262 TC/CC genotype exhibited a higher risk of inguinal hernia (OR: 1.31, 95% CI: 1.07 - 1.61, p = 0.01). Additionally, rs3809060 was specifically associated with male patients with direct-type inguinal hernia (OR: 1.62, 95% CI: 1.19 - 2.22, p = 0.002). Conclusion: Genetic susceptibility appears to participate in the pathogenesis of inguinal hernia in the Taiwanese population in a sex-specific manner. Future studies are needed to illuminate the complex interplay between heredity and comorbidities.

Genotype-Phenotype Dissociation in Two Taiwanese Children with Molybdenum Cofactor Deficiency Caused by MOCS2 Mutation.

BACKGROUND: To describe the genotype-phenotype dissociation in two Taiwanese patients with molybdenum cofactor deficiency (MoCoD) caused by MOCS2 gene mutations. PATIENT DESCRIPTION: Patient 1 exhibited early-onset neurological symptoms soon after birth, followed by subsequent myoclonic seizures and movement disorder. The brain magnetic resonance imaging (MRI) showed diffuse brain injury with cystic encephalomalacia along with bilateral globus pallidi involvement, hypoplasia of corpus callosum, and cerebellar atrophy. Patient 2 had a mild phenotype with prominent movement disorder after intercurrent illness, and the brain MRI showed selective injury of the bilateral globus pallidi and the cerebellum. Both patients had markedly low levels of plasma uric acid and harbored the same MOCS2 homozygous c.16C > T mutation. Patient 1 showed chronic regression of developmental milestones and died of respiratory failure at the age of 8 years, whereas patient 2 demonstrated improvement in motor function. CONCLUSION: Genotype-phenotype dissociation could be noted in patients with MoCoD due to MOCS2 mutation. Patients with neonatal seizures, developmental delay, movement disorder, and motor regression after an illness, as well as focal or bilateral involvement of the globus pallidi on the neuroimages, should undergo biochemical testing of plasma uric acid. A pronounced plasma uric acid level is a good indicator of MoCoD. Early diagnosis can allow early provision of adequate genetic counseling.

Enhancement of Anticancer Potential of Pterostilbene Derivative by Chalcone Hybridization.

Pterostilbene, a natural metabolite of resveratrol, has been indicated as a potent anticancer molecule. Recently, several pterostilbene derivatives have been reported to exhibit better anticancer activities than that of the parent pterostilbene molecule. In the present study, a series of pterostilbene derivatives were designed and synthesized by the hybridization of pterostilbene, chalcone, and cinnamic acid. The cytotoxic effect of these hybrid molecules was determined using two oral cancer cell lines, HSC-3 and OECM-1. (E)-3-(2-((E)-4-Hydroxystyryl)-4,6-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (4d), with IC50 of 16.38 and 18.06 µM against OECM-1 and HSC-3, respectively, was selected for further anticancer mechanism studies. Results indicated that compound 4d effectively inhibited cell proliferation and induced G2/M cell cycle arrest via modulating p21, cyclin B1, and cyclin A2. Compound 4d ultimately induced cell apoptosis by reducing the expression of Bcl-2 and surviving. In addition, cleavage of PARP and caspase-3 were enhanced following the treatment of compound 4d with increased dose. To conclude, a number of pterostilbene derivatives were discovered to possess potent anticancer potentials. Among them, compound 4d was the most active, more active than the parent pterostilbene.

Identification of high-copy number long terminal repeat retrotransposons and their expansion in Phalaenopsis orchids.

BACKGROUND: Transposable elements (TEs) are fragments of DNA that can insert into new chromosomal locations. They represent a great proportion of eukaryotic genomes. The identification and characterization of TEs facilitates understanding the transpositional activity of TEs with their effects on the orchid genome structure. RESULTS: We combined the draft whole-genome sequences of Phalaenopsis equestris with BAC end sequences, Roche 454, and Illumina/Solexa, and identified long terminal repeat (LTR) retrotransposons in these genome sequences by using LTRfinder and classified by using Gepard software. Among the 10 families Gypsy-like retrotransposons, three families Gypsy1, Gypsy2, and Gypsy3, contained the most copies among these predicted elements. In addition, six high-copy retrotransposons were identified according to their reads in the sequenced raw data. The 12-kb Orchid-rt1 contains 18,000 copies representing 220 Mbp of the P. equestris genome. Southern blot and slot blot assays showed that these four retrotransposons Gypsy1, Gypsy2, Gypsy3, and Orchid-rt1 contained high copies in the large-genome-size/large-chromosome species P. violacea and P. bellina. Both Orchid-rt1 and Gypsy1 displayed various ratios of copy number for the LTR sequences versus coding sequences among four Phalaenopsis species, including P. violacea and P. bellina and small-genome-size/small-chromosome P. equestris and P. ahprodite subsp. formosana, which suggests that Orchid-rt1 and Gypsy1 have been through various mutations and homologous recombination events. FISH results showed amplification of Orchid-rt1 in the euchromatin regions among the four Phalaenopsis species. The expression levels of Peq018599 encoding copper transporter 1 is highly upregulated with the insertion of Orchid-rt1, while it is down regulated for Peq009948 and Peq014239 encoding for a 26S proteasome non-ATP regulatory subunit 4 homolog and auxin-responsive factor AUX/IAA-related. In addition, insertion of Orchid-rt1 in these three genes are all in their intron regions. CONCLUSION: Orchid-rt1 and Gypsy1-3 have amplified within Phalaenopsis orchids concomitant with the expanded genome sizes, and Orchid-rt1 and Gypsy1 may have gone through various mutations and homologous recombination events. Insertion of Orchid-rt1 is in the introns and affects gene expression levels.

Association of Programmed Death-Ligand 1 Expression with Fusion Variants and Clinical Outcomes in Patients with Anaplastic Lymphoma Kinase-Positive Lung Adenocarcinoma Receiving Crizotinib.

BACKGROUND: Programmed death-ligand 1 (PD-L1) expression is associated with clinical outcomes of epidermal growth factor receptor (EGFR) mutant lung adenocarcinoma (ADC) treated with tyrosine kinase inhibitors (TKIs). However, whether PD-L1 expression plays a role in anaplastic lymphoma kinase (ALK)-positive lung ADC is unknown. We aimed to evaluate the impact of PD-L1 in patients with ALK-positive lung ADC receiving crizotinib. MATERIALS AND METHODS: PD-L1 expression was identified by immunohistochemistry (IHC). Reverse transcriptase-polymerase chain reaction was used for ALK variant detection, and immunofluorescence-based multiplex staining was applied for exploring immune cells in tumor microenvironments. RESULTS: A total of 78 patients with ALK-positive advanced ADC were enrolled in our study, of whom 52 received crizotinib. Compared with EGFR/ALK wild-type tumors, PD-L1 expression was lower in ALK-positive ADC. ALK fusion variants were identified in 32 patients, and those with variant 3 and 5 (short variants) had higher PD-L1 expression than those with other variants. The crizotinib objective response rate (ORR) and progression-free survival (PFS) was better in tumors with negative PD-L1 expression (ORR/PFS in PD-L1 0% vs. 1%-49% vs. 50%-100%: 60.7%/11.8 months vs. 38.5%/6.5 months vs. 36.4%/4.0 months, p = .007/.022). The multivariate Cox proportional hazards model revealed that PD-L1 0% (vs. ≥1%) was an independent factor for longer PFS (adjusted hazard ratio 0.322, 95% confidence interval 0.160-0.650, p = .002). Multiplex IHC in three cases showed a varied extent of immune cell infiltrations in tumors with different PD-L1 expression. CONCLUSION: Positive PD-L1 expression was associated with unfavorable clinical outcomes in patients with ALK-positive lung ADC receiving crizotinib. IMPLICATIONS FOR PRACTICE: Not all lung adenocarcinoma with sensitizing driver mutations experienced durable responses to small-molecule tyrosine kinase inhibitors (TKIs). Similar to the negative impact of programmed death-ligand 1 (PD-L1) in epidermal growth factor receptor mutant tumors treated with TKIs, this study demonstrated that positive PD-L1 expression was also associated with worse response rate and shorter progression-free survival of anaplastic lymphoma kinase (ALK)-positive adenocarcinoma treated with crizotinib. Among different ALK fusion partners, tumors with short variants (V3 and V5) had higher PD-L1 compared with long variants (V1, V2, and V6). Testing PD-L1 before initiating crizotinib for ALK-positive lung cancer could be a simple method to provide important prognostic information.

Assessment of violet-blue color formation in Phalaenopsis orchids.

BACKGROUND: Phalaenopsis represents an important cash crop worldwide. Abundant flower colors observed in Phalaenopsis orchids range from red-purple, purple, purple-violet, violet, and violet-blue. However, violet-blue orchids are less bred than are those of other colors. Anthocyanin, vacuolar pH and metal ions are three major factors influencing flower color. This study aimed to identify the factors causing the violet-blue color in Phalaenopsis flowers and to analyze whether delphinidin accumulation and blue pigmentation formation can be achieved by transient overexpression of heterologous F3'5'H in Phalaenopsis. RESULTS: Cyanidin-based anthocyanin was highly accumulated in Phalaenopsis flowers with red-purple, purple, purple-violet, and violet to violet-blue color, but no true-blue color and no delphinidin was detected. Concomitantly, the expression of PeF3'H (Phalaenopsis equestrsis) was high, but that of PhF3'5'H (Phalaenopsis hybrid) was low or absent in various-colored Phalaenopsis flowers. Transient overexpression of DgF3'5'H (Delphinium grandiflorum) and PeMYB2 in a white Phalaenopsis cultivar resulted a 53.6% delphinidin accumulation and a novel blue color formation. In contrast, transient overexpression of both PhF3'5'H and PeMYB2 did not lead to delphinidin accumulation. Sequence analysis showed that the substrate recognition site 6 (SRS6) of PhF3'5'H was consistently different from DgF3'5'Hs at positions 5, 8 and 10. Prediction of molecular docking of the substrates showed a contrary binding direction of aromatic rings (B-ring) with the SRS6 domain of DgF3'5'H and PhF3'5'H. In addition, the pH values of violet-blue and purple Phalaenopsis flowers ranged from 5.33 to 5.54 and 4.77 to 5.04, respectively. Furthermore, the molar ratio of metal ions (including Al3+, Ca2+ and Fe3+) to anthocyanin in violet-blue color Phalaenopsis was 190-, 49-, and 51-fold higher, respectively, than those in purple-color Phalaenopsis. CONCLUSION: Cyanidin-based anthocyanin was detected in violet-blue color Phalaenopsis and was concomitant with a high pH value and high molar ratio of Al3+, Ca2+ and Fe3+ to anthocyanin content. Enhanced expression of delphinidin is needed to produce true-blue Phalaenopsis.

A HORT1 Retrotransposon Insertion in the PeMYB11 Promoter Causes Harlequin/Black Flowers in Phalaenopsis Orchids.

The harlequin/black flowers in Phalaenopsis orchids contain dark purple spots and various pigmentation patterns, which appeared as a new color in 1996. We analyzed this phenotype by microscopy, HPLC, gene functional characterization, genome structure analysis, and transient overexpression system to obtain a better understanding of the black color formation in Phalaenopsis orchids. Most mesophyll cells of harlequin flowers showed extremely high accumulation of anthocyanins as well as a high expression of Phalaenopsis equestris MYB11 (PeMYB11) as the major regulatory R2R3-MYB transcription factor for regulating the production of the black color. In addition, we analyzed the expression of basic helix-loop-helix factors, WD40 repeat proteins, and MYB27- and MYBx-like repressors for their association with the spot pattern formation. To understand the high expression of PeMYB11 in harlequin flowers, we isolated the promoter sequences of PeMYB11 from red and harlequin flowers. A retrotransposon, named Harlequin Orchid RetroTransposon 1 (HORT1), was identified and inserted in the upstream regulatory region of PeMYB11 The insertion resulted in strong expression of PeMYB11 and thus extremely high accumulation of anthocyanins in the harlequin flowers of the Phalaenopsis Yushan Little Pearl variety. A dual luciferase assay showed that the insertion of HORT1 enhanced PeMYB11 expression by at least 2-fold compared with plants not carrying the insertion. Furthermore, the presence of HORT1 explains the high mutation rates resulting in many variations of pigmentation patterning in harlequin flowers of Phalaenopsis orchids.

Enhanced Response to Drug-Induced QT Interval Lengthening in Patients with Heart Failure with Preserved Ejection Fraction.

BACKGROUND: Patients with heart failure (HF) with reduced ejection fraction demonstrate enhanced response to drug-induced QT interval lengthening and are at increased risk for torsades de pointes. The influence of HF with preserved ejection fraction (HFpEF) on response to drug-induced QT lengthening is unknown. METHODS AND RESULTS: We administered intravenous ibutilide 0.003 mg/kg to 10 patients with HFpEF and 10 age- and sex-matched control subjects without HF. Serial 12-lead electrocardiograms were obtained for determination of QT intervals. Demographics, maximum serum ibutilide concentrations, area under the serum ibutilide concentration vs time curves, and baseline Fridericia-corrected QT (QTF) (417 ± 14 vs 413 ± 15 ms, P = .54) were similar in the HFpEF and control groups. Area under the effect (QTFvs time) curve (AUEC) from 0 to 1.17 hours during and following the ibutilide infusion was greater in the HFpEF group (519 ± 19 vs 497 ± 18 ms·h, P= .04), as was AUEC from 0 to 8.17 hours (3576 ± 125 vs 3428 ± 161 ms·h, P = .03) indicating greater QTF interval exposure. Maximum QTF (454 ± 15 vs 443 ± 22 ms, P = .18) and maximum percent increase in QTF from baseline (8.2 ± 2.1 vs 6.7 ± 1.9%, P = .10) in the 2 groups were not significantly different. CONCLUSIONS: HFpEF is associated with enhanced response to drug-induced QT interval lengthening.

Cardiac manifestations and gene mutations of patients with RASopathies in Taiwan.

RASopathies are developmental diseases caused by mutations in rat sarcoma-mitogen-activated protein kinase pathway genes. These disorders, such as Noonan syndrome (NS) and NS-related disorders (NSRD), including cardio-facio-cutaneous (CFC) syndrome, Costello syndrome (CS), and NS with multiple lentigines (NSML; also known as LEOPARD syndrome), have a similar systemic phenotype. A wide spectrum of congenital heart disease and hypertrophic cardiomyopathy (HCMP) can exhibit major associated characteristics. A retrospective study was conducted at the Mackay Memorial Hospital, National Taiwan University Hospital, Buddhist Tzu-Chi General Hospital, Chang-Gung Memorial Hospital, Taichung Veterans General Hospital, and Chung Shan Medical University Hospital from January 2007 to December 2018. We reviewed the clinical records of 76 patients with a confirmed molecular diagnosis of RASopathies, including NS, CS, CFC syndrome, and NSML. We evaluated the demographic data and medical records with clinical phenotypes of cardiac structural anomalies using cross-sectional and color Doppler echocardiography, electrocardiographic findings, and follow-up data. A total of 47 (61.8%) patients had cardiac abnormalities. The prevalence of cardiac lesions according to each syndrome was 62.7, 50.0, 60.0, and 66.7% in patients with NS, CFC syndrome, CS, and NSML, respectively. An atrial septal defect was usually combined with other cardiac abnormalities, such as pulmonary stenosis (PS), HCMP, ventricular septal defect, or patent ductus arteriosus. Patients with NS most commonly showed PS. In patients with NSRD and cardiac abnormalities, HCMP (29.4%) was the most commonly observed cardiac lesion. PTPN11 was also the most frequently detected mutation in patients with NS and NSRD. Cardiac abnormalities were the most common symptoms observed in patients with RASopathies at the time of their first hospital visit. Performing precise analyses of genotype-cardiac phenotype correlations in a larger cohort will help us accurately diagnose RASopathy as soon as possible.

PePIF1, a P-lineage of PIF-like transposable element identified in protocorm-like bodies of Phalaenopsis orchids.

BACKGROUND: Orchids produce a colorless protocorm by symbiosis with fungi upon seed germination. For mass production of orchids, the prevailing approaches are both generation of protocorm-like bodies (PLBs) from callus and multiplication of adventitious buds on inflorescence. However, somaclonal variations occur during micropropagation. RESULTS: We isolated the two most expressed transposable elements belonging to P Instability Factor (PIF)-like transposons. Among them, a potential autonomous element was identified by similarity analysis against the whole-genome sequence of Phalaenopsis equestris and named PePIF1. It contains a 19-bp terminal inverted repeat flanked by a 3-bp target site duplication and two coding regions encoding ORF1- and transposase-like proteins. Phylogenetic analysis revealed that PePIF1 belongs to a new P-lineage of PIF. Furthermore, two distinct families, PePIF1a and PePIF1b, with 29 and 37 putative autonomous elements, respectively, were isolated, along with more than 3000 non-autonomous and miniature inverted-repeat transposable element (MITE)-like elements. Among them, 828 PePIF1-related elements were inserted in 771 predicted genes. Intriguingly, PePIF1 was transposed in the somaclonal variants of Phalaenopsis cultivars, as revealed by transposon display, and the newly inserted genes were identified and sequenced. CONCLUSION: A PIF-like element, PePIF1, was identified in the Phalaenopsis genome and actively transposed during micropropagation. With the identification of PePIF1, we have more understanding of the Phalaenopsis genome structure and somaclonal variations during micropropagation for use in orchid breeding and production.

Antisense oligonucleotides modulate dopa decarboxylase function in aromatic l-amino acid decarboxylase deficiency.

Aromatic l-amino acid decarboxylase deficiency (AADCD), attributed to mutations in the dopa decarboxylase (DDC) gene, is a rare neurometabolic disease resulting from a defect in the biosynthesis of dopamine and serotonin. The DDC c.714+4A>T mutation is the most prevalent mutation among patients with AADCD, and is also a founder mutation among Taiwanese patients. In this study, the molecular consequences and function of this mutation were examined in AADCD patient-derived lymphoblastoid cells. We identified novel DDC mRNA isoforms spliced with a new exon (exon 6a) in normal and c.714+4A>T lymphoblastoid cells. In addition, we identified the SR proteins (SRSF9 and SRSF6), as well as cis-elements involved in modulating the splicing of this mutated transcript. Notably, we demonstrated that antisense oligonucleotides (ASOs) were able to restore the normal mRNA splicing and increase the level of DDC protein, as well as its downstream product serotonin, in lymphoblastoid cells derived from the patient with AADCD, suggesting that these ASOs might represent a feasible alternative strategy for gene therapy of AADCD in patients with the common c.714+4A>T mutation.

Three R2R3-MYB transcription factors regulate distinct floral pigmentation patterning in Phalaenopsis spp.

Orchidaceae are well known for their fascinating floral morphologic features, specialized pollination, and distinctive ecological strategies. With their long-lasting flowers of various colors and pigmentation patterning, Phalaenopsis spp. have become important ornamental plants worldwide. In this study, we identified three R2R3-MYB transcription factors PeMYB2, PeMYB11, and PeMYB12. Their expression profiles were concomitant with red color formation in Phalaenopsis spp. flowers. Transient assay of overexpression of three PeMYBs verified that PeMYB2 resulted in anthocyanin accumulation, and these PeMYBs could activate the expression of three downstream structural genes Phalaenopsis spp. Flavanone 3-hydroxylase5, Phalaenopsis spp. Dihydroflavonol 4-reductase1, and Phalaenopsis spp. Anthocyanidin synthase3. In addition, these three PeMYBs participated in the distinct pigmentation patterning in a single flower, which was revealed by virus-induced gene silencing. In the sepals/petals, silencing of PeMYB2, PeMYB11, and PeMYB12 resulted in the loss of the full-red pigmentation, red spots, and venation patterns, respectively. Moreover, different pigmentation patterning was regulated by PeMYBs in the sepals/petals and lip. PeMYB11 was responsive to the red spots in the callus of the lip, and PeMYB12 participated in the full pigmentation in the central lobe of the lip. The differential pigmentation patterning was validated by RNA in situ hybridization. Additional assessment was performed in six Phalaenopsis spp. cultivars with different color patterns. The combined expression of these three PeMYBs in different ratios leads to a wealth of complicated floral pigmentation patterning in Phalaenopsis spp.

Mitochondrial dysfunction represses HIF-1α protein synthesis through AMPK activation in human hepatoma HepG2 cells.

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α) is an important transcription factor that modulates cellular responses to hypoxia and also plays critical roles in cancer progression. Recently, somatic mutations and decreased copy number of mitochondrial DNA (mtDNA) were detected in hepatocellular carcinoma (HCC). These mutations were shown to have the potential to cause mitochondrial dysfunction. However, the effects and mechanisms of mitochondrial dysfunction on HIF-1α function are not fully understood. This study aims to explore the underlying mechanism by which mitochondrial dysfunction regulates HIF-1α expression. METHODS: Human hepatoma HepG2 cells were treated with various mitochondrial respiration inhibitors and an uncoupler, respectively, and the mRNA and protein expressions as well as transactivation activity of HIF-1α were determined. The role of AMP-activated protein kinase (AMPK) was further analyzed by compound C and AMPK knock-down. RESULTS: Treatments of mitochondrial inhibitors and an uncoupler respectively reduced both the protein level and transactivation activity of HIF-1α in HepG2 cells under normoxia or hypoxia. The mitochondrial dysfunction-repressed HIF-1α protein synthesis was associated with decreased phosphorylations of p70(S6K) and 4E-BP-1. Moreover, mitochondrial dysfunction decreased intracellular ATP content and elevated the phosphorylation of AMPK. Treatments with compound C, an AMPK inhibitor, and knock-down of AMPK partially rescued the mitochondrial dysfunction-repressed HIF-1α expression. CONCLUSIONS: Mitochondrial dysfunctions resulted in reduced HIF-1α protein synthesis through AMPK-dependent manner in HepG2 cells. GENERAL SIGNIFICANCE: Our results provided a mechanism for communication from mitochondria to the nucleus through AMPK-HIF-1α. Mitochondrial function is important for HIF-1α expression in cancer progression.

SIRT3 expression as a biomarker for better prognosis in gastric cancer.

BACKGROUND: SIRT3-mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase sirtuin-3-plays an important role in regulating cell metabolism and carcinogenesis. The role of SIRT3 in gastric cancer has not yet been investigated. METHODS: A total of 221 gastric cancer patients who underwent curative surgery were enrolled at the Department of Surgery, Taipei Veterans General Hospital. SIRT3 expression in gastric tissues and tumors were examined in these patients using immunohistochemical staining. Clinicopathologic characteristics and survival were analyzed and compared in gastric cancer patients with or without SIRT3 expression. RESULTS: The 5-year survival rates of patients with or without SIRT3 expression were 51.2 and 39.1 %, respectively (p = 0.005). The 5-year disease-free survival rates of patients with or without SIRT3 expression were 49.6 and 38.0 %, respectively (p = 0.010). Microscopic features showed that there are more poor cell differentiation (p = 0.001), more diffuse-type Lauren's histology (p = 0.018), and more scirrhous-type stromal reactions (p = 0.027) in gastric cancer without SIRT expression. Multivariate analysis with overall survival as an endpoint showed that age (p < 0.001), Lauren's histology (p = 0.007), stromal reaction (p = 0.035), TNM pathologic N category (p < 0.001), and SIRT3 expression (p < 0.001) were significantly correlated with gastric cancer. CONCLUSIONS: Gastric cancer patients with SIRT3 expression have a better prognosis than those without. SIRT3 expression is an independent prognostic marker for overall survival and may act as a tumor suppressor in gastric cancer.

Two frequent mutations associated with the classic form of propionic acidemia in Taiwan.

Propionyl-CoA carboxylase (PCC) is involved in the catabolism of branched chain amino acids, odd-numbered fatty acids, cholesterol, and other metabolites. PCC consists of two subunits, α and ß, encoded by the PCCA and PCCB genes, respectively. Mutations in the PCCA or PCCB subunit gene may lead to propionic acidemia. In this study, we performed mutation analysis on ten propionic acidemia patients from eight unrelated and nonconsanguineous families in Taiwan. Two PCCA mutations, c.229C→T (p.R77W) and c.1262A→C (p.Q421P), were identified in a PCCA-deficient patient. Six mutations in the PCCB gene, including c.-4156_183+3713del, c.580T→C (p.S194P), c.838dup (p.L280Pfs 11), c.1301C→T (p.A434V), c.1316A→G (P.Y439C), and c.1534C→T (p.R512C), were identified in seven PCCB-deficient families. The c.-4156_183+3713del mutation is the first known large deletion that affects the PCCB gene functions. Furthermore, the c.1301C→T and c.-4156_183+3713del mutations in the PCCB gene have not been reported previously. Clinical features demonstrated that these two frequent mutations are associated with low enzyme activity and a classic propionic acidemia phenotype.

Spectrum of PHEX Mutations and FGF23 Profiles in a Taiwanese Cohort With X-Linked Hypophosphatemia Including 102 Patients.

BACKGROUND/AIM: X-linked hypophosphatemia (XLH), the most common form of hereditary rickets, results from loss-of-function mutations in the phosphate-regulating PHEX gene. Elevated fibroblast growth factor 23 (FGF23) contributes to hypophosphatemia in XLH. This study aimed to characterize PHEX variants and serum FGF23 profiles in Taiwanese patients with XLH. PATIENTS AND METHODS: We retrospectively reviewed the records of 102 patients clinically suspected of having hypophosphatemic rickets from 2006 to 2022. Serum intact Fibroblast growth factor-23 (iFGF23) levels were measured on clinic visit days. PHEX mutations were identified using Sanger sequencing, and negative cases were analyzed using whole-exome sequencing. RESULTS: The majority (92.1%) of patients exhibited elevated FGF23 compared with normal individuals. Among 102 patients, 44 distinct PHEX mutations were identified. Several mutations recurred in multiple unrelated Taiwanese families. We discovered a high frequency of novel PHEX mutations and identified variants associated with extreme FGF23 elevation and tumorigenesis. CONCLUSION: Our findings revealed the PHEX genotypic variants and FGF23 levels in Taiwanese patients with XLH. These results are crucial given the recent approval of burosumab, a monoclonal FGF23 antibody, for XLH therapy. This study provides key insights into the clinical management of XLH in Taiwan.

Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids.

Orchidaceae, one of the largest angiosperm families, has significant commercial value. Isolation of genes involved in orchid floral development and morphogenesis, scent production, and colouration will advance knowledge of orchid flower formation and facilitate breeding new varieties to increase the commercial value. With high-throughput virus-induced gene silencing (VIGS), this study identified five transcription factors involved in various aspects of flower morphogenesis in the orchid Phalaenopsis equestris. These genes are PeMADS1, PeMADS7, PeHB, PebHLH, and PeZIP. Silencing PeMADS1 and PebHLH resulted in reduced flower size together with a pelaloid column containing petal-like epidermal cells and alterations of epidermal cell arrangement in lip lateral lobes, respectively. Silencing PeMADS7, PeHB, and PeZIP alone resulted in abortion of the first three fully developed flower buds of an inflorescence, which indicates the roles of the genes in late flower development. Furthermore, double silencing PeMADS1 and PeMADS6, C- and B-class MADS-box genes, respectively, produced a combinatorial phenotype with two genes cloned in separate vectors. Both PeMADS1 and PeMADS6 are required to ensure the normal development of the lip and column as well as the cuticle formation on the floral epidermal cell surface. Thus, VIGS allows for unravelling the interaction between two classes of MADS transcription factors for dictating orchid floral morphogenesis.

YAP co-localizes with the mitotic spindle and midbody to safeguard mitotic division in lung-cancer cells.

YES-associated protein (YAP) is a part of the Hippo pathway, with pivotal roles in several developmental processes and dual functionality as both a tumor suppressor and an oncogene. In the present study, we identified YAP activity as a microtubular scaffold protein that maintains the stability of the mitotic spindle and midbody by physically interacting with α-tubulin during mitotic progression. The interaction of YAP and α-tubulin was evident in co-immunoprecipitation assays, as well as observing their co-localization in the microtubular structure of the mitotic spindle and midbody in immunostainings. With YAP depletion, levels of ECT2, MKLP-1, and Aurora B are reduced, which is consistent with YAP functioning in midbody formation during cytokinesis. The concomitant decrease in α-tubulin and increase in acetyl-α-tubulin during YAP depletion occurred at the post-transcriptional level. This suggests that YAP maintains the stability of the mitotic spindle and midbody, which ensures appropriate chromosome segregation during mitotic division. The increase in acetyl-α-tubulin during YAP depletion may provide a lesion-halting mechanism in maintaining the microtubule structure. The depletion of YAP also results in multinuclearity and aneuploidy, which supports its role in stabilizing the mitotic spindle and midbody.