Neuroimaging for Early Diagnosis of Alzheimer's Disease: a Review.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease that primarily affects people above the age of 60 all around the world. As of now, the cause is unknown and there is no effective cure. The pathological changes of AD have occurred many years before the onset of the disease, and current treatment techniques can only delay the progression of the disease. Because disease-modifying therapies may be most beneficial in the early stages of AD, the clinical significance of an early diagnosis is emphasized. So far, a variety of imaging technologies and related biomarkers have been used to identify and monitor AD, but there are many imaging technologies; finding the most effective imaging technology can assist medical personnel in interpreting the early stages of AD and can also improve patient treatment opportunities. This is, therefore, the main purpose and back-ground of this study. METHODS: PubMed and other repositories were used in this study to conduct a literature search with various keywords, and relevant articles were reviewed. In this review, different neuroimaging techniques are reviewed which are considered advanced tools to help establish the diagnosis, and in addition, the diagnostic utility, advantages, and limitations of contemporary AD imaging techniques are discussed. RESULTS: The results of the literature review and synthesis show that the prevalence of several in vivo biomarkers helps distinguish affected individuals from healthy controls in the early stages of the disease. Additionally, each current imaging method has its advantages and disadvantages, so no single imaging method is the best diagnostic modality. CONCLUSIONS: This article also reviews and draws conclusions on better ways to use the imaging techniques to improve the likelihood of an early diagnosis of AD. It is suggested that future research could focus on expanding the use of imaging technologies and on identifying novel biomarkers manifesting the earliest stages of AD pathology.

Expression of tyrosine phosphatases in relation to PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori.

Protein tyrosine phosphorylation is a reversible, dynamic process regulated by the activities of tyrosine kinases and tyrosine phosphatases. Although the involvement of tyrosine kinases in the prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs) has been documented, few studies have been conducted on the involvement of protein tyrosine phosphatases (PTPs) in PTTH-stimulated ecdysteroidogenesis. In the present study, we investigated the correlation between PTPs and PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs. Our results showed that the basal PTP enzymatic activities exhibited development-specific changes during the last larval instar and pupation stage, with high activities being detected during the later stages of the last larval instar. PTP enzymatic activity was stimulated by PTTH treatment both in vitro and in vivo. Pretreatment with phenylarsine oxide (PAO) and benzylphosphonic acid (BPA), two chemical inhibitors of tyrosine phosphatase, reduced PTTH-stimulated enzymatic activity. Determination of ecdysteroid secretion showed that treatment with PAO and BPA did not affect basal ecdysteroid secretion, but greatly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated PTP activity is indeed involved in ecdysteroid secretion. PTTH-stimulated phosphorylation of the extracellular signal-regulated kinase (ERK) and 4E-binding protein (4E-BP) was partially inhibited by pretreatment with either PAO or BPA, indicating the potential link between PTPs and phosphorylation of ERK and 4E-BP. In addition, we also found that in vitro treatment with 20-hydroxyecdysone did not affect PTP enzymatic activity. We further investigated the expressions of two important PTPs (PTP 1B (PTP1B) and the phosphatase and tension homologue (PTEN)) in Bombyx PGs. Our immunoblotting analysis showed that B. mori PGs contained the proteins of PTP1B and PTEN, with PTP1B protein undergoing development-specific changes. Protein levels of PTP1B and PTEN were not affected by PTTH treatment. The gene expression levels of PTP1B and PTEN showed development-specific changes. From these results, we suggest that PTTH-regulated PTP signaling may crosstalk with ERK and target of rapamycin (TOR) signaling pathways and is a necessary component for stimulation of ecdysteroid secretion.

Effects of computer-aided rowing exercise systems on improving muscle strength and function in older adults with mild knee osteoarthritis: a randomized controlled clinical trial.

BACKGROUND: Osteoarthritis (OA) is common in aged adults and can result in muscle weakness and function limitations in lower limbs. Knee OA affects the quality of life in the elderly. Technology-supported feedback to achieve lower impact on knee joints and individualized exercise could benefit elderly patients with knee OA. Herein, a computer-aided feedback rowing exercise system is proposed, and its effects on improving muscle strength, health conditions, and knee functions of older adults with mild knee OA were investigated. METHODS: Thirty-eight older adults with mild knee OA and satisfying the American College of Rheumatology (ACR) clinical criteria participated in this randomized controlled clinical trial. Each subject was randomly assigned to a computer-aided rowing exercise (CRE) group (n = 20) or a control group (CON) (n = 18) that received regular resistance exercise programs two times per week for 12 weeks. Outcome measurements, including the Western Ontario and MacMaster Universities (WOMAC), muscle strength and functional fitness of the lower limbs, were evaluated before and after the intervention. RESULTS: Participants' functional fitness in the CRE group exhibited significantly higher adjusted mean post-tests scores, including the WOMAC (p = 0.006), hip abductors strength (kg) (MD = 2.36 [1.28, 3.44], p = 5.67 × 10-5), hip adductors strength (MD = 3.04 [1.38, 4.69], p = 0.001), hip flexors strength (MD = 4.01 [2.24, 5.78], p = 6.46 × 10-5), hip extensors strength (MD = 2.88 [1.64, 4.12], p = 4.43 × 10-5), knee flexors strength (MD = 2.03 [0.66, 3.41], p = 0.005), knee extensors strength (MD = 1.80 [0.65, 2.94], p = 0.003), and functional-reach (cm) (MD = 3.74 [0.68, 6.80], p = 0.018), with large effect sizes (η2 = 0.17-0.42), than those in the CON group after the intervention. CONCLUSIONS: Older adults with knee OA in the CRE group exhibited superior muscle strength, health conditions, and functional fitness improvements after the 12-week computer-aided rowing exercise program than those receiving the conventional exercise approach. TRIAL REGISTRATION: The Institutional Review Board of the Taipei Medical University approved the study protocol (no. N201908020, 27/05/2020) and retrospectively registered at ClinicalTrials.gov (trial registry no. NCT04919486, 09/06/2021).

Role of protein phosphatase 2A in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori.

In the present study, the roles of a major serine/threonine protein phosphatase 2A (PP2A) in prothoracicotropic hormone (PTTH)-stimulated prothoracic glands (PGs) of Bombyx mori were evaluated. Immunoblotting analysis showed that Bombyx PGs contained a structural A subunit (A), a regulatory B subunit (B), and a catalytic C subunit (C), with each subunit undergoing development-specific changes. The protein levels of each subunit were not affected by PTTH treatment. However, the highly conserved tyrosine dephosphorylation of PP2A C subunit (PP2Ac), which appears to be related to activity, was increased by PTTH treatment in a time-dependent manner. We further demonstrated that phospholipase C (PLC), Ca2+, and reactive oxygen species (ROS) are upstream signaling for the PTTH-stimulated dephosphorylation of PP2Ac. The determination of PP2A enzymatic activity showed that PP2A enzymatic activity was stimulated by PTTH treatment both in vitro and in vivo. Okadaic acid (OA), a specific PP2A inhibitor, prevented the PTTH-stimulated dephosphorylation of PP2Ac and reduced both basal and PTTH-stimulated PP2A enzymatic activity. The determination of ecdysteroid secretion showed that treatment with OA did not affect basal ecdysteroid secretion but did significantly inhibit PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated PP2A activity is involved in ecdysteroidogenesis. Treatment with OA stimulated the basal phosphorylation of the extracellular signal-regulated kinase (ERK) and 4E-binding protein (4E-BP) without affecting PTTH-stimulated ERK and 4E-BP phosphorylation. From these results, we hypothesize that PTTH-regulated PP2A signaling is a necessary component for the stimulation of ecdysteroidogenesis, potentially by mediating the link between ERK and TOR signaling pathways.

Frailty as a Predictor of Future Fracture in Older Adults: A Systematic Review and Meta-Analysis.

BACKGROUND: Studies have identified frailty as an effective predictor of fracture; however, the correlation between frailty and fracture differs between various stages of frailty. OBJECTIVES: The main aim is to determine the correlation between various stages of frailty and fracture risk; a secondary purpose is to determine the correlation between subgroups (e.g., females, females with a hip fracture, or aged 65 years or older) within the stages of frailty and fracture risk. Finally, effect of frailty criteria on the association between stages of frailty and fracture risk was tested. METHODS: We conducted a systematic review and meta-analysis. The inclusion criteria were as follows: (a) a prospective study design; (b) subjects aged 55 years or older; (c) a division into robust, prefrail, and frail groups; and (d) reported confidence intervals of hazard ratio. Two investigators independently assessed quality and discussed their findings to reach consensus. The quality of the literature was assessed and the level of evidence was also determined. RESULTS: In total, five studies included 103,783 older people and recorded 2,960 fractures. The results identified that the risk of fracture in the frail people was higher than that in both the robust people (summary HR: 1.67; 95% CI [1.46-1.91]) and prefrail people (summary HR: 1.28; 95% CI [1.16-1.40], and that the risk of fracture in the prefrail people was higher than that in the robust people (summary HR: 1.30; 95% CI [1.20-1.41]). A subgroup analysis revealed that among female adults, older females with hip fracture, or those aged 65 years or more, those who were categorized as frail showed the highest fracture risk, followed by those who were categorized as prefrail. LINKING EVIDENCE TO ACTION: Professional nurses caring for frail or prefrail people should actively develop fracture prevention measures to reduce the risk of death caused by fractures.

Prefrailty in community-dwelling older adults is associated with nutrition status.

AIMS AND OBJECTIVES: This study investigated the differences of demographic characteristics and nutrition status between prefrail and nonfrail older adults. Meanwhile, the factors related to the nutrition status of community-dwelling older adults were also studied. BACKGROUND: The World Health Organisation has actively developed prevention programmes to address geriatric frailty. However, few previous studies have focused on the relationship between prefrailty and nutrition status. DESIGN: This study adopted a cross-sectional and correlational research design. METHODS: Community-dwelling older adults were recruited for this study. The participants' nutrition status was assessed using the Mini-Nutritional Assessment tool, and their frailty status was evaluated based on the Study of Osteoporotic Fractures indicators. The Study of Osteoporotic Fractures criteria comprise the following three items: >5% loss in total body weight within one year; inability to rise five times from a chair without assistance and a self-reported lack of energy. Inclusion criteria were age ≥65 years, living independently at home and exhibiting neither mental impairment nor acute disease. RESULTS: Data were gathered from 152 participants; most were men, lived alone and had a level of education lower than elementary school. Multiple linear regressions were used to evaluate the association between variables and nutritional status, the results of which revealed that prefrailty and body mass index were independently related to the participants' nutrition status. CONCLUSIONS: The results showed that nutritional assessment can be employed with community-dwelling older adults. In particular, prefrailty and body mass index were key factors that affected nutritional status in older adults. RELEVANCE TO CLINICAL PRACTICE: Community nurses must understand the risk factors related to nutritional status in community-dwelling older people. If nurses use this approach, prefrail older adults are likely to experience improved confidence in maintaining their health and a decreased incidence of frailty and malnutrition.

Systematic Literature Review and Meta-Analysis of the Association of Sarcopenia With Mortality.

BACKGROUND: The World Health Organization (2002) indicates that the prevention of frailty is a key indicator of successful aging, and the assessment of the muscular system is the core for determining the frailty syndrome in older adults. Sarcopenia in older adults will affect more than 50 million people and will affect more than 200 million people after 40 years. Systematic literature reviews have yet to focus on the association between sarcopenia and mortality. AIMS: The aims of this study were to determine the correlation between sarcopenia and mortality by using various evaluation methods and to analyze any differences among the resulting correlations of the different assessment tools. METHODS: This study conducted a systematic review of relevant studies in four databases (Cochrane Library, MEDLINE, Cumulative Index to Nursing and Allied Health Literature, and Ovid). The inclusion criteria were prospective research, the results included mortality or death, and risk estimates included risk ratio or hazard ratio estimates and 95% confidence intervals. Exclusion criteria included studies that provided no sarcopenia data, the risk of mortality or death, or data indicating deaths from cancers and in hospitals. RESULTS: This study identified 10 studies that have investigated the association between sarcopenia and mortality with an average follow up of 4.17 years. In total, these studies have sampled 3,797 people and recorded 1,010 deaths. The result suggested that the risk of mortality in the sarcopenia group was higher than that in the nonsarcopenia group. LINKING EVIDENCE TO ACTION: Although the results of evidence-based studies have suggested that sarcopenia is a geriatric syndrome associated with increased mortality, few studies have focused on decreasing sarcopenia mortality. Therefore, nurse practitioners must be aware of the correlation between sarcopenia and the mortality rate and become actively involved in developing intervention methods for reducing the mortality risk of sarcopenia.

Expressions of sugar transporters/trehalases in relation to PTTH-stimulated ecdysteroidogenesis in the silkworm, Bombyx mori.

The prothoracic gland (PG) is the source of ecdysteoids in larval insects. Although numerous studies have been conducted on signaling networks involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in PGs, less is known about regulation of metabolism in PGs. In the present study, we investigated correlations between expressions of sugar transporter (St)/trehalase (Treh) genes and PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs. Our results showed that in vitro PTTH treatment stimulated expression of the St1 gene, but not other transporter genes. Expression of the Treh1 gene was also stimulated by PTTH treatment. An immunoblotting analysis showed that St1 protein levels in Bombyx PGs increased during the later stage of the last larval instar and were not affect by PTTH treatment. PTTH treatment enhanced Treh enzyme activity in a time-dependent manner. Blocking either extracellular signal-regulated kinase (ERK) signaling with U0126 or phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 decreased PTTH-stimulated Treh enzyme activity, indicating a link from the ERK and PI3K signaling pathways to Treh activity. Treatment with the Treh inhibitor, validamycin A, blocked PTTH-stimulated Treh enzyme activity and partially inhibited PTTH-stimulated ecdysteroidogenesis. Treatment with either a sugar transport inhibitor (cytochalasin B) or a specific glycolysis inhibitor (2-deoxy-D-glucose, 2-DG) partially inhibited PTTH-stimulated ecdysteroidogenesis. Taken together, these results indicate that increased expressions of St1/Treh1 and Treh activity, which lie downstream of PTTH signaling, are involved in PTTH stimulation in B. mori PGs.

Grey matter alterations in generalized anxiety disorder: A voxel-wise meta-analysis of voxel-based morphometry studies.

OBJECTIVE: Grey matter, a crucial component of the brain, has been found altered in generalized anxiety disorder (GAD) of several voxel-based morphometry studies. The conclusive and consistent grey matter alterations in GAD have not been confirmed. METHOD: Eleven voxel-based morphometry studies of GAD patients were included in the current systematic review and meta-analysis. The linear model of anxiety severity scores was applied to explore the relationship of grey matter alterations and anxiety severity. The subgroup analysis of adult GAD and adolescent GAD was also performed. RESULTS: Significantly modest grey matter alterations in the left superior temporal gyrus of patients with GAD were found. The anxiety severity score was significantly correlated with grey matter alterations in the right insula, lenticular nucleus, putamen and striatum. The subgroup analysis of adult GAD and adolescent GAD all failed to show significant grey matter alterations. However, in the adult GAD subgroup, anxiety severity score was significantly correlated with grey matter alterations in the right insula. CONCLUSION: GAD might have the modest grey matter alterations in the left superior temporal gyrus. Anxiety severity might be related to the grey matter alterations in the limbic regions, such as the right insula, lenticular nucleus, putamen and striatum. This kind of correlation might be related to the effects of adult GAD. Future studies with adequate sample sizes and sophisticated GAD categories will be needed.

Monorail snare technique for retrieval of stretched coil.

Coil stretching is a recognized complication during cerebral aneurysm embolization.1, 3- 5 For over a decade, the microsnare has proven effective in retrieving migrated coils.1- 5 Fiorella et al. unveiled the "Monorail Snare Technique" in 2005, offering a specialized approach to stretched coil recovery.1 However, to gain a complete understanding of this technique, more than just textual descriptions are necessary; a thorough, practical demonstration is essential. In our technical video (video 1), we illustrated an episode of coil stretching during aneurysm embolization, where the "Monorail Snare Technique" was successfully employed to retrieve a stretched coil. Our video emphasizes the meticulous preparation and modification of the microsnare, showcasing enhanced steps to mitigate the potential blood backflow triggered by the exclusive use of one-arm hemostasis valve during the "Monorail Snare Technique."1, 5 This pivotal adjustment substantially lowers the threat of thromboembolic events. We highlighted essential precautions to ensure the procedure's safety and efficacy.4, 5.

[HPLC characteristic chromatographic profile of Sarcandra glabra].

OBJECTIVE: To develop the characteristic chromatographic profile of Sarcandra glabra by HPLC for its quality control. METHOD: The HPLC analysis was performed on an Agilent Zorbax Eclipse XDB-C18 column (4. 6 mm x 250 mm, 5 microm) with column temperature at 40 degree C. The mobile phase was consisted of water containing 0. 5% formic acid and acetonitrile to methanol (1:9) in gradient mode, and the detection wavelength was set at 344 nm. RESULT: A common mode of the HPLC characteristic chromatographic profile has been establised. There were 20 common peaks , seven of which were identified, and 46 samples from different habitats were classified into five groups based on principal component cluster analysis. CONCLUSION: The method was time-saving and can represent the chemical information and provide a scientific basis for quality control of S. glabra.

Signaling in cAMP-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori.

In the present study, we investigated downstream pathways of cyclic adenosine monophosphate (cAMP) signaling (which is related to prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis) in Bombyx mori prothoracic glands (PGs). Results showed that treatment with either dibutyryl cAMP (dbcAMP) or 1-methyl-3-isobutylxanthine (MIX) inhibited phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and activated phosphorylation of the translational repressor, 4E-binding protein (4E-BP), a marker of target of rapamycin (TOR) signaling. A chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside, AICAR) increased dbcAMP-inhibited AMPK phosphorylation and blocked dbcAMP-stimulated phosphorylation of 4E-BP, indicating that inhibition of AMPK phosphorylation lies upstream of dbcAMP-stimulated TOR signaling. Treatment of PGs with dbcAMP and MIX also stimulated phosphorylation of a 37-kDa protein, as recognized by a protein kinase C (PKC) substrate antibody, indicating that cAMP activates PKC signaling. Treatment with either LY294002 or AICAR did not affect dbcAMP-stimulated phosphorylation of the PKC-dependent 37-kDa protein, indicating that cAMP-stimulated PKC signaling is not related to phosphoinositide 3-kinase (PI3K) or AMPK. In addition, dbcAMP-stimulated ecdysteroidogenesis in PGs was partially inhibited by pretreatment with either LY294002, AICAR, or calphostin C. From these results, we concluded that AMPK/TOR/4E-BP and PKC pathways are involved in ecdysteroidogenesis of PGs stimulated by cAMP signaling in B. mori.

Expressions of sugar transporter genes during Bombyx mori embryonic development.

Sugar transporters (Sts) play important roles in controlling carbohydrate transport and are responsible for mediating the movement of sugars into cells. Few studies have been conducted on expressions of Sts during insect embryonic development. In the present study, we investigated temporal expressions of St genes during the embryonic diapause process in Bombyx mori. We found that in HCl-treated developing eggs, high gene expressions of trehalose transporter 1 (Tret1) were detected during middle and later embryonic development. St4 and St3 gene expressions gradually increased during the early stages, reached a small peak on Day 3, and large peaks were again detected on Day 7. However, in diapause eggs, expression levels of the Tret1, St4, and St3 genes all remained at low levels. Differential temporal changes in expressions of the Tret1, St4, and St3 genes found between diapause and HCl-treated eggs were further confirmed using nondiapause eggs. Our results showed that nondiapause eggs exhibited similar changing patterns as those of HCl-treated eggs, thus clearly indicating potential correlations between expressions of these genes and embryonic development. In addition, high gene expressions of Tret1 were also detected when dechorionated eggs were incubated in the medium. The addition of LY294002 (a specific phosphatidylinositol 3-kinase [PI3K] inhibitor) and U0126 (a mitogen-activated protein kinase/extracellular signal-regulated kinase [ERK] kinase [MEK] inhibitor) partially inhibited Tret1 gene expression in dechorionated eggs, but did not affect either ecdysteroid-phosphate phosphatase gene expression or ecdysteroid biosynthesis, clearly indicating that both PI3K and ERK are involved in increased gene expression of Tret1 that was independent of ecdysteroid levels. To our knowledge, this is the first comprehensive report to demonstrate the transcriptional regulation of St genes during embryonic development, thus providing useful information for a clearer understanding of insect egg diapause mechanisms.

Coil embolization of distal vascular lesion using a 1.5 Fr Marathon microcatheter and 'free-running' technique.

In distal vascular lesions, such as the distal anterior inferior cerebellar artery or posterior inferior cerebellar artery (PICA) dissecting aneurysm, and dural arteriovenous fistula (dAVF) and arteriovenous malformation (AVM), super-selective catheterization and embolization using liquid agents, such as NBCA or Onyx liquid embolic system, is the preferred treatment.1 2 We used a flow-directed 1.5 Fr Marathon microcatheter (Medtronic, Minneapolis, MN, USA) for embolization because commonly used detachable coil-compatible microcatheters can be too short or rigid for superselection.3-6 We designed an in vitro coil compatibility test for the Marathon microcatheter and developed a 'free-running' technique (video 1). Using this technique, we trapped the distal PICA dissecting aneurysm and embolized the fistula points of dAVF precisely and safely without affecting adjacent normal structures, which can occur when applying liquid embolizing agents.1-3 After reviewing the case, we determined that this technique can also potentially be applied for implementing the pressure cooker technique7 and combining the management of AVM.4neurintsurg;jnis-2023-020893v1/V1F1V1Video 1Free-running technique via 1.5 Fr Marathon microcatheter.

Specificity of the femoral slump test for the assessment of experimentally induced anterior knee pain.

OBJECTIVE: To assess the specificity of the femoral slump test (FST) when assessing experimentally induced anterior knee pain. DESIGN: Cross-sectional, exploratory study. SETTING: Research laboratory. PARTICIPANTS: Asymptomatic subjects (N=12; 6 men; 6 women) for the study. An experimental pain model was used to simulate anterior knee pain by injecting .25 mL of hypertonic saline solution (5% NaCl) into the medial infrapatellar fat pad. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURE: The changes in pain intensity and diameter after applying the structure differential maneuver (neck flexion/extension) during the FST were recorded and analyzed. RESULTS: Results revealed that the structure differential maneuver of the FST did not alter the pain intensity or diameter in 9 (neck extension) and 10 (neck flexion) out of 12 subjects, which meant that the FST provided appropriate testing responses in 75% to 83% cases when the anterior knee pain did not originate in neural tissues. CONCLUSIONS: The FST had a specificity of more than .75 when detecting nerve mechanosensitivity problems of anterior knee pain.

Are you SLiM? Developing an instrument for civic scientific literacy measurement (SLiM) based on media coverage.

The purpose of this study is to develop an instrument to assess civic scientific literacy measurement (SLiM), based on media coverage. A total of 50 multiple-choice items were developed based on the most common scientific terms appearing in media within Taiwan. These questions covered the subjects of biology (45.26%, 22 items), earth science (37.90%, 19 items), physics (11.58%, 6 items) and chemistry (5.26%, 3 items). A total of 1034 students from three distinct groups (7th graders, 10th graders, and undergraduates) were invited to participate in this study. The reliability of this instrument was 0.86 (KR 20). The average difficulty of the SLiM ranged from 0.19 to 0.91, and the discrimination power was 0.1 to 0.59. According to participants' performances on SLiM, it was revealed that 10th graders (Mean = 37.34±0.23) performed better than both undergraduates (Mean = 33.00±0.33) and 7th graders (Mean = 26.73±0.45) with significant differences in their SLiM.

Expressions of the Protein Phosphatases PP1 and PP4 during the Embryonic Diapause Process of the Silkworm Bombyx mori.

Reversible protein phosphorylation is accomplished by the opposing activities of kinases and phosphatases. We previously demonstrated the regulation of serine/threonine protein phosphatase (PP) type 2A (PP2A) and 2B (PP2B or calcineurin) during the embryonic diapause process of Bombyx mori. In the present study, we further examine the expressions of other PPs (PP1 and PP4) during embryonic stages. An immunoblot analysis showed that Bombyx eggs contained a 38-kDa PP1 catalytic subunit (PP1-C), a 38-kDa PP4 catalytic subunit (PP4-C), and a 120-kDa PP1 nuclear targeting subunit (PNUTS), each of which underwent differential changes between diapause and developing eggs during the embryonic process. In non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling diapausing eggs at 5°C for 70 days and then were transferred to 25°C, protein levels of PP1-C and PP4-C remained relatively high during the early embryonic stages and then decreased during middle (for PP1-C) or later (for PP4-C) embryonic stages. However, protein levels of PP1-C and PP4-C in diapause eggs remained at high levels during the first 8 days after oviposition. PNUTS protein levels showed inverse temporal changes, with increased levels being detected during the later embryonic stages of developing eggs. The direct determination of PP1 enzymatic activity showed higher activity in developing eggs than in diapause eggs. Examination of temporal changes in mRNA expression levels of PP1-C and PP4-C showed no difference between HCl-treated and diapause eggs. These results indicated that differential protein levels of PP1-C/PNUTS and PP4-C, and increased enzymatic activity of PP1 were likely related to the embryonic development of B. mori.

Bombyxin-stimulated ecdysteroidogenesis in relation to sugar transporter/trehalase expressions in Bombyx prothoracic glands.

Our previous studies showed that bombyxin stimulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs) during a long-term incubation period in a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner. In the present study, we further investigated the downstream signaling cascade in bombyxin-stimulated PGs. Our results showed that upon treatment with bombyxin, expression levels of the sugar transport 1 (St1) and St4 genes and trehalase 1 (Treh1) gene, but not ecdysteroid biosynthesis genes were greatly enhanced compared to the controls. Treatment with LY294002 (an inhibitor of PI3K) reduced the enhanced St1 and Treh1 expression levels, clearly indicating the involvement of PI3K. Treatment with 1 mM of mpV(pic) (a potent inhibitor of protein phosphotyrosine phosphatase and activator of insulin receptor (InR) kinase) also stimulated expression levels of the St1 and Treh1 genes, thus further confirming the involvement of the InR. Determining Treh enzyme activity showed that bombyxin treatment stimulated Treh enzyme activity in time- and PI3K-dependent manners. Validamycin A (a Treh inhibitor) blocked bombyxin-stimulated Treh enzyme activity and partly decreased bombyxin-stimulated ecdysteroidogenesis. A specific sugar transport inhibitor (cytochalasin B) and a glycolysis inhibitor (2-deoxy-D-glucose (2-DG)) also reduced bombyxin-stimulated ecdysteroidogenesis. Taken together, these results indicated that increased expressions of Sts and Treh1 and enhanced Treh enzyme activity downstream of InR/PI3K are involved in bombyxin-stimulated ecdysteroidogenesis in B. mori PGs.

Expression of protein tyrosine phosphatases and Bombyx embryonic development.

Protein phosphorylation is an integral component of signal transduction pathways within eukaryotic cells, and it is regulated by coordinated interactions between protein kinases and protein phosphatases. Our previous study demonstrated differential expressions of serine/threonine protein phosphatases (PP2A and calcineurin) between diapause and developing eggs in Bombyx mori. In the present study, we further investigated expression of protein tyrosine phosphatases (PTPs) in relation to the Bombyx embryonic development. An immunoblot analysis showed that eggs contained the proteins of the 51-kDa PTP 1B (PTP1B), the 55-kDa phosphatase and tensin homologue (PTEN), and the 70-kDa Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2), which undergo differential changes between diapause and developing eggs. Protein level of PTP1B and PTEN in eggs whose diapause initiation was prevented by HCl gradually increased toward embryonic development. The protein level of SHP2 also showed a dramatic increase on days 7 and 8 after HCl treatment. However, protein levels of PTP1B, PTEN, and SHP2 in diapause eggs remained at low levels during the first 9 days after oviposition. These differential changing patterns in protein levels were further confirmed using both non-diapause eggs and eggs in which diapause had been terminated by chilling of diapausing eggs at 5 °C for 70 days and then were transferred to 25 °C. Direct determination of PTP enzymatic activities showed higher activities in developing eggs (HCl-treated eggs, non-diapause eggs, and chilled eggs) compared to those in diapause eggs. Examination of temporal changes in mRNA expression levels of PTP1B, PTEN, and SHP2 did not show significant differences between diapause eggs and HCl-treated eggs except high expression in SHP2 variant B during the later embryonic development in HCl-treated eggs. These results demonstrate that higher protein levels of PTP1B, PTEN, and SHP2 and increased tyrosine phosphatase enzymatic activities in developing eggs are likely related to embryonic development of B. mori.

Changes in expressions of ecdysteroidogenic enzyme and ecdysteroid signaling genes in relation to Bombyx embryonic development.

Although the role of ecdysteroids in regulating egg diapause process in Bombyx mori is well documented, temporal changes in expression levels of genes involved in ecdysteroid biosynthesis and its downstream signaling are less well understood. In the present study, we studied changes in expression levels of genes involved in ecdysteroid biosynthesis and its downstream signaling during embryonic development of B. mori. Results showed that in diapause eggs, the expression of ecdysteroid-phosphate phosphatase (EPPase) gene and Halloween genes (Spook [Spo] and Shade [Shd]) remained at very low levels. However, in eggs whose diapause initiation was prevented by HCl, significant increases in the messenger RNA (mRNA) levels of EPPase, Spo, and Shd were detected during embryonic development. Other Halloween genes (Neverland [Nvd] and Phantom [Phm]) also showed different changes between diapause and HCl-treated eggs. However, genes of Disembodied (Dib) and Shadow (Sad) showed similar changes in both diapause and HCl-treated eggs. We further investigated changes in expression levels of ecdysone receptor genes (EcRA, EcRB1, and USP) and downstream signaling genes (E75A, E75B, E74A, E74B, Br-C, HR3, HR4, KR-H1, and FTZ-F1). Results showed that genes of EcRA and the other nuclear receptors (E75A, E75B, E74A, HR3, HR4, KR-H1, and FTZ-F1) exhibited significant differential patterns between diapause and HCl-treated eggs, with increased levels being detected during later stages of embryonic development in HCl-treated eggs. Differential temporal changes in expressions of genes involved ecdysteroid biosynthesis and its downstream signaling found between diapause and HCl-treated eggs were further confirmed using nondiapause eggs. Our results showed that nondiapause eggs exhibited the same changing patterns as those in HCl-treated eggs, thus clearly indicating potential correlations between expressions of these genes and embryonic development in B. mori. To our knowledge, this is the first comprehensive report to study the transcriptional regulation of ecdysteroidogenic and ecdysteroid signaling genes, thus providing useful information for a clearer understanding of insect egg diapause mechanisms.