Middle-Aged Worker Bees Express Higher Innate Immunity than Young Worker Bees in the Abdomen without the Digestive Tract of Worker Bees Reared in an Incubator.

Honey bees (Apis mellifera) can be reared in an incubator to study the mechanisms of aging and longevity; however, whether breeding in an incubator and using the abdomen without the digestive tract influences the expression of immune genes is unclear. In this study, we assayed the immune genes including abaecin, hymenoptaecin, defensin-2, glucose dehydrogenase, phenoloxidase, and lysozyme from the whole body of young and middle-aged worker bees reared in field hives, the whole body of young and middle-aged worker bees reared in a 34 °C incubator, and the abdomen without the digestive tract of young and middle-aged worker bees reared in a 34 °C incubator. The results showed that three groups of middle-aged worker bees have higher immunity than young worker bees. Furthermore, the similarity of immune genes expression in three groups indicated that the abdomen without the digestive tract of honey bees reared in an incubator can be used to study the relationship between immunity and aging and longevity to avoid the interference of pathogens and parasites from field hives.

Detection of hypermethylation at H19DMR at amniocentesis in a fetus with overgrowth, distended abdomen and Beckwith-Wiedemann syndrome.

OBJECTIVE: We present detection of hypermethylation at H19 differentially methylated region (DMR) at amniocentesis in a fetus with overgrowth, distended abdomen and Beckwith-Wiedemann syndrome (BWS). CASE REPORT: A 31-year-old, gravida 2, para 1, woman was referred for genetic counseling at 22 weeks of gestation because of fetal overgrowth with fetal biometry equivalent to 24 weeks of gestation and a distended abdomen with an abdominal circumference equivalent to 26 weeks of gestation. She did not undergo any assisted reproductive technology during this pregnancy. Amniocentesis was performed at 23 weeks of gestation. Conventional cytogenetic analysis revealed a karyotype of 46,XX. Array comparative genomic hybridization analysis on the DNA extracted from uncultured amniocytes revealed no genomic imbalance. Methylation analysis on the DNA extracted from amniocytes revealed hypermethylation at H19DMR [imprinting center 1 (IC1)] and normal methylation at KvDMR1 (IC2). The methylation test confirmed the diagnosis of BWS in the fetus. The parents decided to continue the pregnancy. At 36 weeks of gestation, a 4000-g female baby was delivered with macroglossia, ear tags and creases, and an enlarged liver, consistent with the phenotype of BWS. CONCLUSION: Prenatal diagnosis of fetal overgrowth should include a differential diagnosis of BWS, and methylation analysis of H19DMR (IC1) and KvDMR1 (IC2) is useful under such a circumstance.

The Association of Diabetic Retinopathy and Cardiovascular Disease: A 13-Year Nationwide Population-Based Cohort Study.

OBJECTIVES: Previous studies have demonstrated that patients with diabetic retinopathy (DR) have a higher prevalence of risk factors known to be associated with cardiovascular disease (CVD). We hypothesized that patients with more severe DR could have a higher relative risk of CVD. METHODS: To test this hypothesis, we used the National Health Insurance Research Database (NHIRD) to evaluate whether associations exist between DR and CVD. The data for this nationwide population-based retrospective cohort study were obtained from the NHIRD in Taiwan from 2001 to 2013. The assessed study outcome used was the incidence and other statistical analyses of CVD in patients with DR during a 13-year follow-up period. RESULTS: Our findings obtained from 2001 to 2013 suggest that the incidence rates of CVD are 2.026 times that of diabetes mellitus (DM) without DR (95% C.I. = 1.876-2.187) and 2.75 times that of DM with DR (95% C.I. = 2.487-3.04) compared with the Non-DM group. CONCLUSION: The relative risk of CVD in DR was greater than that in the Non-DM group for both men and women. Targeted monitoring of DM, especially the co-existence of diabetic retinopathy, is of utmost importance in the clinical care of the DM population.

Long-term inhibition of ferritin2 synthesis in trophocytes and oenocytes by ferritin2 double-stranded RNA ingestion to investigate the mechanisms of magnetoreception in honey bees (Apis mellifera).

Behavioral studies indicate that honey bees (Apis mellifera) have a capacity for magnetoreception and superparamagnetic magnetite is suggested to be a magnetoreceptor. The long-term inhibition of magnetite formation can be employed to explore the bee's magnetoreception. A recent study shows that magnetite formation, ferritin2 messenger RNA (mRNA) expression, and the protein synthesis of ferritin2 in trophocytes and oenocytes were all inhibited by a single injection of ferritin2 double-stranded RNA (dsRNA) into the hemolymph of honey bees but how to maintain this knockdown of ferritin2 for the long-term is unknown. In this study, we injected ferritin2 dsRNA into the hemolymph of worker bees three times every six days to maintain long-term inhibition; however, multi-microinjections accelerated the death of the bees. To overcome this problem, we further reared newly emerged worker bees daily with ferritin2 dsRNA throughout their lives, demonstrating no impact on their lifespans. Follow-up assays showed that the mRNA expression and protein synthesis of ferritin2 were persistently inhibited. These findings verified that daily ferritin2 dsRNA ingestion not only displays the long-term inhibition of mRNA expression and protein synthesis of ferritin2, but also did not damage the bees. This method of long-term inhibition can be used in behavioral studies of magnetoreception in honey bees.

The trophocytes and oenocytes of worker and queen honey bees (Apis mellifera) exhibit distinct age-associated transcriptome profiles.

Despite the identical genomic context, trophocytes and oenocytes in worker bees exhibit aging-related phenotypes, in contrast to the longevity phenotypes in queen bees. To explore this phenomenon at the molecular level, we evaluated the age-associated transcriptomes of trophocytes and oenocytes in worker bees and queen bees using high-throughput RNA-sequencing technology (RNA-seq). The results showed that (i) while gene expression profiles were different between worker and queen bees, they remained similar between young and old counterparts; (ii) worker bees express a high proportion of low-abundance genes, whereas queen bee transcriptomes display a high proportion of moderate-expression genes; (iii) genes were upregulated to a greater extent in queen bees vs. worker bees; and (iv) distinct aging-related and longevity-related candidate genes were found in worker and queen bees. These results provide new insights into the cellular aging and longevity of trophocytes and oenocytes in honey bees. Identification of aging-associated biomarker genes also constitutes a basis for translational research of aging in higher organisms.

Prenatal diagnosis of a 1.651-Mb 19q13.42-q13.43 microdeletion in a fetus with micrognathia and bilateral pyelectasis on prenatal ultrasound.

OBJECTIVE: We present prenatal diagnosis of a de novo 1.651-Mb 19q13.42-q13.43 microdeletion in a fetus with micrognathia and bilateral pyelectasis on prenatal ultrasound. CASE REPORT: A 32-year-old woman underwent amniocentesis at 28 weeks of gestation because of fetal micrognathia and bilateral pyelectasis on prenatal ultrasound. Amniocentesis revealed a karyotype of 46,XX. Simultaneous array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniocytes revealed the result of arr 19q13.42q13.43 (55,028,722-56,680,564) × 1.0 [GRCh37 (hg19)] or a 1.651-Mb microdeletion encompassing 44 Online Mendelian Inheritance in Man (OMIM) genes including NLRP7, GP6, TNNT1, TNNI3 and DNAAF3. The parents did not have such a deletion and decided to continue the pregnancy. At 37 weeks of gestation, a 2560-g female baby was delivered by cesarean section because of oligohydramnios and decreased fetal movements. The baby manifested cleft palate, micrognathia and retrognathia at birth. She was doing well at age three months. Her body weight was 5.3 Kg (15th-25th centile), and body length was 59.2 cm (25th-50th centile). Renal sonogram showed bilateral mild pelvic dilation. She manifested no psychomotor retardation and no other internal organ abnormalities during pediatric follow-ups. CONCLUSION: A 19q13.42-q13.43 microdeletion can be associated with micrognathia, retrognathia, cleft palate and bilateral pyelectasis at birth.

Prenatal diagnosis of mosaic trisomy 8 by amniocentesis in a fetus with ventriculomegaly and dysgenesis of the corpus callosum.

OBJECTIVE: We present prenatal diagnosis of mosaic trisomy 8 by amniocentesis in a fetus with central nervous system abnormalities. CASE REPORT: A 39-year-old woman was found to have fetal bilateral ventriculomegaly and enlargement of the third ventricle on prenatal ultrasound at 32 weeks of gestation. Fetal magnetic resonance imaging examination confirmed bilateral ventriculomegaly and dysgenesis of the corpus callosum. Amniocentesis was performed subsequently. Array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniotic cells revealed trisomy 8 mosaicism with a result of arr [GRCh37] (8) × 3[0.19], (X,Y) × 1. Conventional cytogenetic analysis on cultured amniocytes showed that among 108 cells in 12 colonies of three cultures, only one cell was abnormal with trisomy 8, trisomy 9 and monosomy 13, while the rest 107 cells had a normal karyotype. Repeat amniocentesis and cord blood sampling revealed a result of arr 8p23.3q24.3 (191,530-146,280,020) × 2.3 with a log2 ratio of 0.2 compatible with 20-30% mosaicism for trisomy 8 on the uncultured amniocytes, and a result of arr 8p23.3q24.3 (191,530-146,280,020) × 2.1 with a log2 ratio of 0.08 compatible with <10% mosaicism for trisomy 8 on the cord blood lymphocytes. Polymorphic DNA marker analysis excluded uniparental disomy 8. A malformed 2440-g dead fetus was delivered at 34 weeks of gestation with facial dysmorphism. CONCLUSION: Cytogenetic discrepancy can occur between cultured and uncultured amniocytes in mosaic trisomy 8 at amniocentesis. aCGH analysis on uncultured amniocytes is useful for confirmation of mosaic trisomy 8 at amniocentesis. Fetuses with low-level mosaicism for trisomy 8 may prenatally present ventriculomegaly and dysgenesis of the corpus callosum.

Ferritin RNA interference inhibits the formation of iron granules in the trophocytes of worker honey bees (Apis mellifera).

Iron granules containing superparamagnetic magnetite act as magnetoreceptor for magnetoreception in honey bees. Biomineralization of iron granules occurs in the iron deposition vesicles of trophocytes and requires the participation of actin, myosin, ferritin2, and ATP synthase. The mechanism of magnetoreception in honey bees can be explored by suppressing the formation of iron granules. Toward this goal, we injected double-stranded RNA of ferritin2 and ferritin1 into newly emerged worker honey bees to knock down these genes via RNA interference. We confirmed that mRNA and protein production of the ferritins was inhibited, leading to immature iron granules. Downregulating ferritin2 and ferritin1, moreover, leads to different deposition morphology of 7.5-nm diameter iron particles, indicating that the two genes play different roles in the formation of iron granules in worker honey bees.

Cellular energy metabolism maintains young status in old queen honey bees (Apis mellifera).

Trophocytes and oenocytes of queen honey bees are used in studies of cellular longevity, but their cellular energy metabolism with age is poorly understood. In this study, the molecules involved in cellular energy metabolism were evaluated in the trophocytes and oenocytes of young and old queen bees. The findings indicated that there were no significant differences between young and old queen bees in ß-oxidation, glycolysis, and protein synthesis. These results indicate that the cellular energy metabolism of trophocytes and oenocytes in old queen bees is similar to young queen bees and suggests that maintaining cellular energy metabolism in a young status may be associated with the longevity of queen bees. Fat and glycogen accumulation increased with age indicating that old queen bees are older than young queen bees.

Late-onset fetal bilateral pleural effusions associated with Down syndrome.

OBJECTIVE: We present two cases of late-onset bilateral fetal pleural effusions associated with fetal Down syndrome. CASE REPORTS: Case 1. A 33-year-old Vietnamese woman had undergone regular sonographic examinations since 23 weeks of gestation and no abnormality had been noted. However, bilateral moderate pleural effusions were found at 33 weeks of gestation, and massive pleural effusion, ascites and polyhydramnios developed at 34 weeks of gestation. Aspiration of the pleural effusion was subsequently performed. Clinical laboratory surveys of the aspiration fluid excluded toxoplasmosis and cytomegalovirus infection. Cytogenetic analysis of cultured lymphocytes derived from pleural effusion revealed a karyotype of 47,XX,+21. The parents elected to continue the pregnancy. Intrauterine fetal demise occurred at 37 weeks of gestation, and a macerated female baby was delivered. Postnatal cytogenetic analysis of the umbilical cord confirmed the prenatal diagnosis. Case 2. A 41-year-old Pakistani woman had undergone regular sonographic examinations and no abnormality had been noted. However, isolated bilateral mild pleural effusions were noted at 27 weeks of gestation. Amniocentesis revealed a karyotype of 47,XY,+21 and simultaneous array comparative genomic hybridization analysis of uncultured amniocytes confirmed the diagnosis of Down syndrome. The pregnancy was subsequently terminated. CONCLUSION: Fetuses with Down syndrome may present late-onset bilateral pleural effusions. Prenatal diagnosis of late-onset bilateral pleural effusions should raise the possibility of fetal Down syndrome and cytogenetic investigation is warranted.

Cellular degradation activity is maintained during aging in long-living queen bees.

Queen honeybees (Apis mellifera) have a much longer lifespan than worker bees. Whether cellular degradation activity is involved in the longevity of queen bees is unknown. In the present study, cellular degradation activity was evaluated in the trophocytes and oenocytes of young and old queen bees. The results indicated that (i) 20S proteasome activity and the size of autophagic vacuoles decreased with aging, and (ii) there were no significant differences between young and old queen bees with regard to 20S proteasome expression or efficiency, polyubiquitin aggregate expression, microtubule-associated protein 1 light chain 3-II (LC3-II) expression, 70 kDa heat shock cognate protein (Hsc70) expression, the density of autophagic vacuoles, p62/SQSTM1 expression, the activity or density of lysosomes, or molecular target of rapamycin expression. These results indicate that cellular degradation activity maintains a youthful status in the trophocytes and oenocytes of queen bees during aging and that cellular degradation activity is involved in maintaining the longevity of queen bees.

Ambient temperature reduction extends lifespan via activating cellular degradation activity in an annual fish (Nothobranchius rachovii).

Ambient temperature reduction (ATR) can extend the lifespan of organisms, but the underlying mechanism is poorly understood. In this study, cellular degradation activity was evaluated in the muscle of an annual fish (Nothobranchius rachovii) reared under high (30 °C), moderate (25 °C), and low (20 °C) ambient temperatures. The results showed the following: (i) the activity of the 20S proteasome and the expression of polyubiquitin aggregates increased with ATR, whereas 20S proteasome expression did not change; (ii) the expression of microtubule-associated protein 1 light chain 3-II (LC3-II) increased with ATR; (iii) the expression of lysosome-associated membrane protein type 2a (Lamp 2a) increased with ATR, whereas the expression of the 70-kD heat shock cognate protein (Hsc 70) decreased with ATR; (iv) lysosome activity increased with ATR, whereas the expression of lysosome-associated membrane protein type 1 (Lamp 1) did not change with ATR; and (v) the expression of molecular target of rapamycin (mTOR) and phosphorylated mTOR (p-mTOR) as well as the p-mTOR/mTOR ratio did not change with ATR. These findings indicate that ATR activates cellular degradation activity, constituting part of the mechanism underlying the longevity-promoting effects of ATR in N. rachovii.

Energy-regulated molecules maintain young status in the trophocytes and fat cells of old queen honeybees.

Queen honeybees (Apis mellifera) have much longer lifespans than worker bees. Energy-regulated molecules in the trophocytes and fat cells of workers during aging have been determined, but are unknown in queen bees. In the present study, energy-regulated molecules were evaluated in the trophocytes and fat cells of young and old queen bees. Adenosine monophosphate-activated protein kinase α2 (AMPK-α2), phosphorylated AMPK-α2 (pAMPK-α2), and cAMP-specific phosphodiesterases activity increased with aging. The pAMPK-α2/AMPK-α2 ratio and AMPK activity; adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) concentrations; the ADP/ATP ratio and the AMP/ATP ratio; the cyclic adenosine monophosphate concentration; forkhead box protein O expression; Silent information regulator T1 (SirT1) expression and activity; and peroxisome proliferator-activated receptor-α (PPAR-α) expression were not significantly different between young and old queen bees. These results show that energy-regulated molecules maintain a youthful status in the trophocytes and fat cells of queen bees during aging. These cells seem to have longevity-promoting mechanisms and may clarify the secret of longevity in queen bees.

Changes in cellular degradation activity in young and old worker honeybees (Apis mellifera).

The trophocytes and fat cells of honeybees (Apis mellifera) have been used in cellular senescence studies, but the changes of cellular degradation activity with aging in workers are unknown. In this study, cellular degradation activity was evaluated in the trophocytes and fat cells of young and old workers reared in a field hive. The results showed the following: (1) 20S proteosome activity decreased with aging, whereas its expression increased with aging; (2) the expression of microtubule-associated protein 1 light chain 3-II (LC3-II) and the 70 kD heat shock cognate protein (Hsc70) decreased with aging; (3) the size and number of autophagic vacuoles decreased with aging; (4) p62/SQSTM1 and polyubiquitin aggregate expression decreased with aging; (5) lysosomal efficiency decreased with aging; and (6) molecular target of rapamycin (mTOR) expression increased with aging. These results indicate that young workers have higher levels of cellular degradation activity than old workers and that aging results in a decline in the cellular degradation activity in worker honeybees.

Changes in energy-regulated molecules in the trophocytes and fat cells of young and old worker honeybees (Apis mellifera).

Trophocytes and fat cells of honeybees (Apis mellifera) have been used for cellular senescence studies, but the changes in the expression, concentration, and activity of cellular energy-regulated molecules that occur with aging in worker bees is unknown. In this study, energy-regulated molecules were evaluated in the trophocytes and fat cells of young and old workers. The results showed that (i) adenosine monophosphate-activated protein kinase-α2 (AMPK-α2) expression increased with aging, whereas phosphorylated AMPK-α2 expression, the phosphorylated AMPK/AMPK ratio, and AMPK activity decreased with aging; (ii) adenosine diphosphate and adenosine triphosphate concentrations decreased with aging, the AMP concentration was unchanged, the adenosine diphosphate/adenosine triphosphate ratio did not change with aging, and the AMP/adenosine triphosphate ratio increased with aging; (iii) the cyclic AMP concentration decreased with aging, and cyclic AMP-specific phosphodiesterases activity increased with aging; (iv) silent information regulator 2 (Sir2) expression increased with aging, whereas its activity decreased with aging; and (v) peroxisome proliferator-activated receptor-α expression decreased with aging. These results show that the trophocytes and fat cells of young workers have higher cellular energy status and express higher levels of energy-regulated molecules than those of old workers and that aging results in a decline in the energy status of trophocytes and fat cells in worker honeybees.

Oxidative stress decreases in the trophocytes and fat cells of worker honeybees during aging.

Trophocytes and fat cells of honeybees have been used for cellular senescence studies, but their oxidative stress and antioxidant enzyme activities with aging in workers is unknown. Here, we assayed reactive oxygen species and the activities of antioxidant enzymes in the trophocytes and fat cells of young and old workers. Young workers had higher reactive oxygen species levels, higher superoxide dismutase and thioredoxin reductase activities as well as lower catalase and glutathione peroxidase activities compared to old workers. Adding these results up, we propose that oxidative stress decreases with aging in the trophocytes and fat cells of workers.

Oxidative stress and anti-oxidant enzyme activities in the trophocytes and fat cells of queen honeybees (Apis mellifera).

Trophocytes and fat cells of queen honeybees have been used for delayed cellular senescence studies, but their oxidative stress and anti-oxidant enzyme activities with advancing age are unknown. In this study, we assayed reactive oxygen species (ROS) and anti-oxidant enzymes in the trophocytes and fat cells of young and old queens. Young queens had lower ROS levels, lower superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities, and higher thioredoxin reductase (TR) activity compared to old queens. These results show that oxidative stress and anti-oxidant enzyme activities in trophocytes and fat cells increase with advancing age in queens and suggest that an increase in oxidative stress and a consequent increase in stress defense mechanisms are associated with the longevity of queen honeybees.

Changes in mitochondrial energy utilization in young and old worker honeybees (Apis mellifera).

Trophocytes and fat cells in honeybees (Apis mellifera) have served as targets for cellular senescence studies, but mitochondrial energy utilization with advancing age in workers is unknown. In this study, mitochondrial energy utilization was evaluated in the trophocytes and fat cells of young and old workers reared in a field hive. The results showed that (1) mitochondrial density increased with advancing age; (2) mitochondrial membrane potential (∆Ψm), nicotinamide adenine dinucleotide oxidized form (NAD(+)) concentration, adenosine triphosphate (ATP) concentration, and NAD(+)/nicotinamide adenine dinucleotide reduced form (NADH) ratio decreased with advancing age; and (3) the expression of NADH dehydrogenase 1 (ND1), ATP synthase, and voltage-dependent anion channel 1 (VDAC1) increased with advancing age, whereas ND1 and ATP synthase did not differ with advancing age after normalization to mitochondrial density and VDAC1. These results show that the trophocytes and fat cells of young workers have higher mitochondrial energy utilization efficiency than those of old workers and that aging results in a decline in mitochondrial energy utilization in the trophocytes and fat cells of worker honeybees.

The use of honeybees reared in a thermostatic chamber for aging studies.

Honeybees (Apis mellifera) are an attractive model system for studying aging. However, the aging level of worker honeybees from the field hive is in dispute. To eliminate the influence of task performance and confirm the relationship between chronological age and aging, we reared newly emerged workers in a thermostat at 34°C throughout their lives. A survivorship curve was obtained, indicating that workers can be reared away from the field hive, and the only difference between these workers is age. To confirm that these workers can be used for aging studies, we assayed age-related molecules in the trophocytes and fat cells of young and old workers. Old workers expressed more senescence-associated ß-galactosidase, lipofuscin granules, lipid peroxidation, and protein oxidation than young workers. Furthermore, cellular energy metabolism molecules were also assayed. Old workers exhibited less ATP concentration, ß-oxidation, and microtubule-associated protein light chain 3 (LC3) than young workers. These results demonstrate that honeybees reared in a thermostatic chamber can be used for aging studies and cellular energy metabolism in the trophocytes and fat cells of workers changes with advancing age.