Antitumor Effect of Anti-c-Myc Aptamer-Based PROTAC for Degradation of the c-Myc Protein.

Targeting "undruggable" targets with intrinsically disordered structures is of great significance for the treatment of disease. The transcription factor c-Myc controls global gene expression and is an attractive therapeutic target for multiple types of cancers. However, due to the lack of defined ligand binding pockets, targeted c-Myc have thus far been unsuccessful. Herein, to address the dilemma of lacking ligands, an efficient and high throughput aptamer screening strategy is established, named polystyrene microwell plate-based systematic evolution of ligands by exponential enrichment (microwell-SELEX), and identify the specific aptamer (MA9C1) against c-Myc. The multifunctional aptamer-based Proteolysis Targeting Chimeras (PROTAC) for proteolysis of the c-Myc (ProMyc) is developed using the aptamer MA9C1 as the ligand. ProMyc not only significantly degrades c-Myc by the ubiquitin-proteasome system, but also reduces the Max protein, synergistically inhibiting c-Myc transcriptional activity. Combination of the artificial cyclization and anti-PD-L1 aptamer (PA1)-based delivery system, circular PA1-ProMyc chimeras achieve tumor regression in the xenograft tumor model, laying a solid foundation for the development of efficacious c-Myc degrader for the clinic. Therefore, this aptamer-based degrader provides an invaluable potential degrader in drug discovery and anti-tumor therapy, offering a promising degrader to overcome the challenge of targeting intractable targets.

Juvenile hormone inhibits adult cuticle formation in Drosophila melanogaster through Kr-h1/Dnmt2-mediated DNA methylation of Acp65A promoter.

Differentiation of imaginal epidermal cells of Drosophila melanogaster to form adult cuticles occurs at approximately 40-93 h after puparium formation. Juvenile hormone (JH) given at pupariation results in formation of a second pupal cuticle in the abdomen instead of the adult cuticle. Although the adult cuticle gene Acp65A has been reported to be down-regulated following JH treatment, the regulatory mechanism remains unclear. Here, we found that the JH primary response gene Krüppel homologue 1 (Kr-h1) plays a vital role in the repression of adult cuticle formation through the mediation of JH action. Overexpression of Kr-h1 mimicked-while knocking down of Kr-h1 attenuated-the inhibitory action of JH on the formation of the adult abdominal cuticle. Further, we found that Kr-h1 inhibited the transcription of Acp65A by directly binding to the consensus Kr-h1 binding site (KBS) within the Acp65A promoter region. Moreover, the DNA methyltransferase Dnmt2 was shown to interact with Kr-h1, combined with the KBS to promote the DNA methylation of sequences around the KBS, in turn inhibiting the transcription of Acp65A. This study advances our understanding of the molecular basis of the "status quo" action of JH on the Drosophila adult metamorphosis.

Growing old in China in socioeconomic and epidemiological context: systematic review of social care policy for older people.

BACKGROUND: From 2020 to 2050, China's population aged ≥65 years old is estimated to more than double from 172 million (12·0%) to 366 million (26·0%). Some 10 million have Alzheimer's disease and related dementias, to approach 40 million by 2050. Critically, the population is ageing fast while China is still a middle-income country. METHODS: Using official and population-level statistics, we summarise China's demographic and epidemiological trends relevant to ageing and health from 1970 to present, before examining key determinants of China's improving population health in a socioecological framework. We then explore how China is responding to the care needs of its older population by carrying out a systematic review to answer the question: 'what are the key policy challenges to China achieving an equitable nationwide long-term care system for older people?'. Databases were screened for records published between 1st June 2020 and 1st June 2022 in Mandarin Chinese or English, reflecting our focus on evidence published since introduction of China's second long-term care insurance pilot phase in 2020. RESULTS: Rapid economic development and improved access to education has led to widescale internal migration. Changing fertility policies and household structures also pose considerable challenges to the traditional family care model. To deal with increasing need, China has piloted 49 alternative long-term care insurance systems. Our findings from 42 studies (n = 16 in Mandarin) highlight significant challenges in the provision of quality and quantity of care which suits the preference of users, varying eligibility for long-term care insurance and an inequitable distribution of cost burden. Key recommendations include increasing salaries to attract and retain staff, introduction of mandatory financial contributions from employees and a unified standard of disability with regular assessment. Strengthening support for family caregivers and improving smart old age care capacity can also support preferences to age at home. CONCLUSIONS: China has yet to establish a sustainable funding mechanism, standardised eligibility criteria and a high-quality service delivery system. Its long-term care insurance pilot studies provide useful lessons for other middle-income countries facing similar challenges in terms of meeting the long-term care needs of their rapidly growing older populations.

Juvenile hormone suppresses sensory organ precursor determination to block Drosophila adult abdomen morphogenesis.

Juvenile hormone (JH) has a classic "status quo" action at both the pupal and adult molts when administrated exogenously. In Drosophila, treatment with JH at pupariation inhibits the formation of abdominal bristles, which are derived from the histoblasts. However, the mechanism via which JH exerts this effect remains poorly understood. In this study, we analyzed the effect of JH on histoblast proliferation, migration, and differentiation. Our results indicated that whereas the proliferation and migration of histoblasts remained unaffected following treatment with a JH mimic (JHM), their differentiation, particularly the specification of sensor organ precursor (SOP) cells, was inhibited. This effect was attributable to downregulated proneural genes achaete (ac) and Scute (sc) expression levels, which prevented the specification of SOP cells in proneural clusters. Moreover, Kr-h1 was found to mediate this effect of JHM. Histoblast-specific overexpression or knockdown of Kr-h1, respectively mimicked or attenuated the effects exerted by JHM on abdominal bristle formation, SOP determination, and transcriptional regulation of ac and sc. These results indicated that the defective SOP determination was responsible for the inhibition of abdominal bristle formation by JHM, which, in turn, was mainly mediated via the transducing action of Kr-h1.

Exploring barriers to dementia screening and management services by general practitioners in China: a qualitative study using the COM-B model.

BACKGROUND: Dementia has become a global public health problem, and general practitioners (GPs) play a key role in diagnosing and managing dementia. However, in Chinese primary care settings, dementia is underdiagnosed and inefficiently managed, and dementia screening and management services provided by GPs are suboptimal. The reasons underlying this gap are poorly understood. This study aimed to determine the barriers that hinder GPs from actively promoting dementia screening and management, and thereby provide insights for the successful promotion of dementia screening and management services in primary care. METHODS: Purposive sampling was used. And focus groups and in-depth interviews were conducted face-to-face among GPs from community health service centers (CHSCs) in South China. Thematic analysis was used to identify barriers to screening and managing dementia and map them to the Capability/Opportunity/Motivation-Behavior model (COM-B model). RESULTS: Fifty-two GPs were included. The COM-B model found nine barriers to implementing dementia screening and management services in primary healthcare: (1) poor capability: lack of systematic knowledge of dementia and inadequate dementia screening skills; (2) little opportunity: unclear pathways for referral, insufficient time for dementia screening and management, lack of dementia-specific leaders, and no guarantee of services continuity; (3) low motivation: outside of GP scope, worries associated with dementia stigma rooted in culture beliefs, and insufficient financial incentives. CONCLUSIONS: Our study concluded that GPs were not yet ready to provide dementia screening and management services due to poor capability related to knowledge and skills of dementia, little opportunity associated with an unsupportive working environment, and low motivation due to unclear duty and social pressure. Accordingly, systematic implementation strategies should be taken, including standardized dementia training programs, standardized community-based dementia guidelines, expansion of primary care workforces, development of dedicated leaders, and the eradication of stigma attached to dementia to promote dementia screening and management services in primary care.

The Agreement Between Virtual Patient and Unannounced Standardized Patient Assessments in Evaluating Primary Health Care Quality: Multicenter, Cross-sectional Pilot Study in 7 Provinces of China.

BACKGROUND: The unannounced standardized patient (USP) is the gold standard for primary health care (PHC) quality assessment but has many restrictions associated with high human and resource costs. Virtual patient (VP) is a valid, low-cost software option for simulating clinical scenarios and is widely used in medical education. It is unclear whether VP can be used to assess the quality of PHC. OBJECTIVE: This study aimed to examine the agreement between VP and USP assessments of PHC quality and to identify factors influencing the VP-USP agreement. METHODS: Eleven matched VP and USP case designs were developed based on clinical guidelines and were implemented in a convenience sample of urban PHC facilities in the capital cities of the 7 study provinces. A total of 720 USP visits were conducted, during which on-duty PHC providers who met the inclusion criteria were randomly selected by the USPs. The same providers underwent a VP assessment using the same case condition at least a week later. The VP-USP agreement was measured by the concordance correlation coefficient (CCC) for continuity scores and the weighted κ for diagnoses. Multiple linear regression was used to identify factors influencing the VP-USP agreement. RESULTS: Only 146 VP scores were matched with the corresponding USP scores. The CCC for medical history was 0.37 (95% CI 0.24-0.49); for physical examination, 0.27 (95% CI 0.12-0.42); for laboratory and imaging tests, -0.03 (95% CI -0.20 to 0.14); and for treatment, 0.22 (95% CI 0.07-0.37). The weighted κ for diagnosis was 0.32 (95% CI 0.13-0.52). The multiple linear regression model indicated that the VP tests were significantly influenced by the different case conditions and the city where the test took place. CONCLUSIONS: There was low agreement between VPs and USPs in PHC quality assessment. This may reflect the "know-do" gap. VP test results were also influenced by different case conditions, interactive design, and usability. Modifications to VPs and the reasons for the low VP-USP agreement require further study.

Targeting KRAS in PDAC: A New Way to Cure It?

Pancreatic cancer is one of the most intractable malignant tumors worldwide, and is known for its refractory nature and poor prognosis. The fatality rate of pancreatic cancer can reach over 90%. In pancreatic ductal carcinoma (PDAC), the most common subtype of pancreatic cancer, KRAS is the most predominant mutated gene (more than 80%). In recent decades, KRAS proteins have maintained the reputation of being "undruggable" due to their special molecular structures and biological characteristics, making therapy targeting downstream genes challenging. Fortunately, the heavy rampart formed by KRAS has been broken down in recent years by the advent of KRASG12C inhibitors; the covalent inhibitors bond to the switch-II pocket of the KRASG12C protein. The KRASG12C inhibitor sotorasib has been received by the FDA for the treatment of patients suffering from KRASG12C-driven cancers. Meanwhile, researchers have paid close attention to the development of inhibitors for other KRAS mutations. Due to the high incidence of PDAC, developing KRASG12D/V inhibitors has become the focus of attention. Here, we review the clinical status of PDAC and recent research progress in targeting KRASG12D/V and discuss the potential applications.

Kr-h1, a Cornerstone Gene in Insect Life History.

Insect life cycle is coordinated by hormones and their downstream effectors. Krüppel homolog1 (Kr-h1) is one of the crucial effectors which mediates the actions of the two critical hormones of insects, the juvenile hormone (JH) and 20-hydroxyecdysone (20E). It is a transcription factor with a DNA-binding motif of eight C2H2 zinc fingers which is found to be conserved among insect orders. The expression of Kr-h1 is fluctuant during insect development with high abundance in juvenile instars and lower levels in the final instar and pupal stage, and reappearance in adults, which is governed by the coordination of JH, 20E, and miRNAs. The dynamic expression pattern of Kr-h1 is closely linked to its function in the entire life of insects. Over the past several years, accumulating studies have advanced our understanding of the role of Kr-h1 during insect development. It acts as a universal antimetamorphic factor in both hemimetabolous and holometabolous species by directly inhibiting the transcription of 20E signaling genes Broad-Complex (Br-C) and Ecdysone induced protein 93F (E93), and steroidogenic enzyme genes involved in ecdysone biosynthesis. Meanwhile, it promotes vitellogenesis and ovarian development in the majority of studied insects. In addition, Kr-h1 regulates insect behavioral plasticity and caste identity, neuronal morphogenesis, maturation of sexual behavior, as well as embryogenesis and metabolic homeostasis. Hence, Kr-h1 acts as a cornerstone regulator in insect life.

Effectiveness of smartphone application-based self-management interventions in patients with type 2 diabetes: A systematic review and meta-analysis of randomized controlled trials.

AIMS: To synthesize evidences on smartphone application-based intervention and determine its effectiveness on glycaemic control, self-management behaviours, psychological well-being, quality of life and cardiometabolic risk factors. DESIGN: A systematic review and meta-analysis of randomized controlled trials (RCTs). DATA SOURCES: Major English and Chinese electronic databases were searched from January 2008 to January 2021, including PubMed, Web of Science, Embase, Cochrane Central Register of Controlled Trials, Google Scholar, China National Knowledge Infrastructure (CNKI), Wanfang and Sinomed. REVIEW METHODS: RCTs were screened and selected if they used smartphone applications to support patients in the self-management of diabetes. Data extraction and methodological assessment were performed by two reviewers independently. Meta-analysis was performed to pool the intervention effect on outcomes of interest using RevMan 5.3. RESULTS: Across 19 included trials involving 2585 participants, smartphone application-based interventions were associated with a clinically and statistically significant reduction of glycated haemoglobin (HbA1c). Beneficial effects were also observed in participants' behavioural performance, especially in medication adherence. Intervention effects on psychological status, quality of life and cardiometabolic risk factors were nonsignificant. Subgroup analysis showed interactive approach with medium frequency or flexible facilitator-patient interaction induced a larger effect on HbA1c reduction. Besides, patients with baseline HbA1c ≥9% benefited more than those with HbA1c <9% from the use of smartphone applications. CONCLUSIONS: Smartphone application-based diabetes self-management intervention could optimize patients' glycaemic control and enhance participants' self-management performance. Further endeavour is required to examine the long-term effects and cost-effectiveness of smartphone application-based intervention before promoting the adoption and dissemination of such intervention. IMPACT: This review supports the potential of smartphone application-based intervention as effective approach to optimize glycaemic control and promote self-management engagement among patients with type 2 diabetes. Suggestions for future research and practice are provided and discussed.

Effectiveness of combined exercise in people with type 2 diabetes and concurrent overweight/obesity: a systematic review and meta-analysis.

OBJECTIVE: To synthesise the available scientific evidence on the effects of combined exercise on glycaemic control, weight loss, insulin sensitivity, blood pressure and serum lipids among patients with type 2 diabetes (T2D) and concurrent overweight/obesity. DESIGN AND SAMPLE: PubMed, EMBASE, Web of Science, the Cochrane library, WANFANG, CNKI, SinoMed, OpenGrey and ClinicalTrials.gov were searched from inception through April 2020 to identify randomised controlled trials (RCTs) that reported the effects of combined exercise in individuals with T2D and concurrent overweight/obesity. METHODS: Quality assessment was performed using the Cochrane Collaboration's risk of bias tool. The mean difference (MD) with its corresponding 95% CI was used to estimate the effect size. Meta-analysis was performed using Review Manager V.5.3. RESULTS: A total of 10 RCTs with 978 participants were included in the meta-analysis. Pooled results demonstrated that combined exercise significantly reduced haemoglobin A1c (MD=-0.16%, 95% CI: -0.28 to -0.05, p=0.006); body mass index (MD=-0.98 kg/m2, 95% CI: -1.41 to -0.56, p<0.001); homeostasis model assessment of insulin resistance (MD=-1.19, 95% CI: -1.93 to -0.46, p=0.001); serum insulin (MD=-2.18 µIU/mL, 95% CI: -2.99 to -1.37, p<0.001) and diastolic blood pressure (MD=-3.24 mm Hg, 95% CI: -5.32 to -1.16, p=0.002). CONCLUSIONS: Combined exercise exerted significant effects in improving glycaemic control, influencing weight loss and enhancing insulin sensitivity among patients with T2D and concurrent overweight/obesity.

A Case of Cushing Disease Masked by Symptoms of Bipolar Disorder.

BACKGROUND: Cushing disease is a rare but serious endocrine disorder. It involves increased cortisol levels, which can damage the function of various systems throughout the body, including the central nervous system, affecting cognition, memory, and emotion. However, it is rare that Cushing disease presents as a mental disorder. This paper reports the complete diagnosis, treatment process, and follow-up of a patient with Cushing disease whose main symptom was bipolar disorder. CASE PRESENTATION: A 31-year-old woman was diagnosed with bipolar disorder 9 years ago and was admitted to hospital several times due to repeated episodes of depression and mania. In October 2017, after careful examination and consideration by doctors, the patient was diagnosed with Cushing syndrome and underwent transsphenoidal pituitary microadenoma resection. Glucocorticoid replacement therapy and antipsychotic therapy were administered postoperatively. After careful treatment and care by the medical staff, the patient's cortisol levels returned to normal, and her mental symptoms were significantly improved. CONCLUSION: This is a rare case of Cushing disease marked by psychiatric symptoms. Neuropsychiatric symptoms can precede the onset of Cushing disease, making diagnosis challenging.

17ß-Estradiol and progesterone decrease MDP induced NOD2 expression in bovine mammary epithelial cells.

During the periparturient period, many neuroendocrine changes develop in cows. Periparturient hormone fluxes may adversely affect mammary gland immunity and mastitis susceptibility. 17ß-Estradiol (E2) and progesterone (P4) have been reported to function on immune regulation, and their concentration fluctuates dramatically during the perinatal period. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) mediate numerous aspects of innate immunity in humans and experimental animals. This study aimed to explore the effects of E2 and P4 on NOD2 expression in bovine mammary epithelial cells (BMECs). BMECs were isolated and purified from bovine mammary tissue and treated with E2/P4 and muramyl dipeptide (MDP). After these treatments, the mRNA levels of NOD2, receptor-interacting protein kinase (RIP) 2, interleukin (IL) 1ß, IL-6, IL-8 and tumor necrosis factor (TNF) α were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) respectively, and the protein levels of NOD2 were analyzed by western blotting. The results showed that E2 and P4 decreased MDP-induced transcriptional expression of NOD2 and the downstream molecules. Moreover, E2 reduced MDP-induced NOD2 protein expression levels. Our study suggests that down-regulation of NOD2 by E2 and P4 may be one of the reasons for mastitis susceptibility in periparturient dairy cows.

Correction: Methyl Farnesoate Plays a Dual Role in Regulating Drosophila Metamorphosis.

[This corrects the article DOI: 10.1371/journal.pgen.1005038.].

Nucleoporin Nup358 facilitates nuclear import of Methoprene-tolerant (Met) in an importin ß- and Hsp83-dependent manner.

The bHLH-PAS transcription factor, Methoprene-tolerant (Met)1, functions as a juvenile hormone (JH) receptor and transduces JH signals by directly binding to E-box like motifs in the regulatory regions of JH response genes. Nuclear localization of Met is crucial for its transcriptional activity. Our previous studies have shown that the chaperone protein Hsp83 facilitates JH-induced Met nuclear import in Drosophila melanogaster. However, the exact molecular mechanisms of Met nuclear transport are not fully elucidated. Using DNA affinity chromatography, we have previously detected binding of the nucleoporin Nup358, in the presence of JH, to the JH response region (JHRR) sequences isolated from the Krüppel-homolog 1 (Kr-h1) promoter. Here, we have demonstrated that Nup358 regulates JH-Hsp83-induced Met nuclear localization. RNAi-mediated knockdown of Nup358 expression in Drosophila fat body perturbs Met nuclear transport during the 3 h after initiation of wandering, when the JH titer is high. The accompanying reduced expression of the transport receptor importin ß in Nup358 RNAi flies could be one of the reasons accounting for Met mislocalization. Furthermore, a tetratricopeptide repeat (TPR) domain at the N-terminal end of Nup358 interacts with Hsp83 and is indispensable for Met nuclear localization. Overexpression of the TPR domain in Drosophila fat body prevents Met nuclear localization resulting in a decrease in JHRR-driven reporter activity and Kr-h1 expression. These data show that Nup358 facilitates JH-induced Met nuclear transport in a manner dependent on importin ß and Hsp83.

Application of a microfluidic sperm sorter to in vitro production of dairy cattle sex-sorted embryos.

Viable sperm from sex-sorted semen without centrifugal treatment was separated by a microfluidic sperm sorter (MFSS) for IVF to improve in vitro embryo production of dairy cattle. The MFSS was originally developed to isolate motile human sperm by two laminar flows in the micro-channel (there are four chambers in an MFSS. Chamber A is the inlet for semen, chamber B is the inlet for the medium, chamber C is the exit chamber for motile sperm, and chamber D is the outlet for nonmotile sperm). Sex-sorted sperm were adjusted to 1 × 10(7) spermatozoa/mL (2 million cells/dose, sperm motility was 30% above after thawing). In a first experiment, diluted sex-sorted semen was mixed with modified Medium199(mM199) containing 5-mM caffeine for 5 minutes, resulting in variations in sperm concentration and quality parameters at chambers A, C, and D. In a second experiment, medium containing sperm from three MFSS chambers was collected and mitochondrial activity of the sperm was determined by flow cytometry, the relative activity of sperm mitochondria in chamber C (1.56 ± 0.03) was the highest in three observation areas (P < 0.05). Thus, sperm motility and mitochondrial activity of sperm was high in chamber C. In a third experiment, different concentrations of sperm were added to chamber A and dairy cattle IVM oocytes were placed in chamber C, where motile spermatozoa will accumulate, with mM199 containing 5-mM caffeine for 5 minutes, and then cultured in caffeine-free mM199 for 8 hours. The results showed that sperm penetration rate, the monospermic penetration rate, and blastocyst rate of the 10 × 10(6) group (10 × 10(6) sperm/mL) were higher than in the 1 × 10(6) and 5 × 10(6) groups (P < 0.05). In the last experiment, we compared sperm penetration in the MFSS-IVF system with a modified standard IVF method (cocultured in droplets for 8 hours). The normal fertilization index (the ratio of monospermic oocytes to the number of oocytes examined) 8 hours after insemination was higher in the MFSS-IVF system than the modified standard IVF system (P < 0.05). Developmental competence of fertilized oocytes to the blastocyst stage was also higher in the MFSS-IVF system (40.12% ± 2.61%) than the modified standard IVF technique (24.55% ± 4.54%). These results demonstrate that a short coculture of dairy cattle oocytes with isolated motile sex-sorted spermatozoa gradually accumulated in the MFSS device improves the efficiencies of normally produced fertilized embryos and blastocyst formation.

Methyl farnesoate plays a dual role in regulating Drosophila metamorphosis.

Corpus allatum (CA) ablation results in juvenile hormone (JH) deficiency and pupal lethality in Drosophila. The fly CA produces and releases three sesquiterpenoid hormones: JH III bisepoxide (JHB3), JH III, and methyl farnesoate (MF). In the whole body extracts, MF is the most abundant sesquiterpenoid, followed by JHB3 and JH III. Knockout of JH acid methyl transferase (jhamt) did not result in lethality; it decreased biosynthesis of JHB3, but MF biosynthesis was not affected. RNAi-mediated reduction of 3-hydroxy-3-methylglutaryl CoA reductase (hmgcr) expression in the CA decreased biosynthesis and titers of the three sesquiterpenoids, resulting in partial lethality. Reducing hmgcr expression in the CA of the jhamt mutant further decreased MF titer to a very low level, and caused complete lethality. JH III, JHB3, and MF function through Met and Gce, the two JH receptors, and induce expression of Kr-h1, a JH primary-response gene. As well, a portion of MF is converted to JHB3 in the hemolymph or peripheral tissues. Topical application of JHB3, JH III, or MF precluded lethality in JH-deficient animals, but not in the Met gce double mutant. Taken together, these experiments show that MF is produced by the larval CA and released into the hemolymph, from where it exerts its anti-metamorphic effects indirectly after conversion to JHB3, as well as acting as a hormone itself through the two JH receptors, Met and Gce.

Heat shock protein 83 (Hsp83) facilitates methoprene-tolerant (Met) nuclear import to modulate juvenile hormone signaling.

Juvenile hormone (JH) receptors, methoprene-tolerant (Met) and Germ-cell expressed (Gce), transduce JH signals to induce Kr-h1 expression in Drosophila. Dual luciferase assay identified a 120-bp JH response region (JHRR) in the Kr-h1α promoter. Both in vitro and in vivo experiments revealed that Met and Gce transduce JH signals to induce Kr-h1 expression through the JHRR. DNA affinity purification identified chaperone protein Hsp83 as one of the proteins bound to the JHRR in the presence of JH. Interestingly, Hsp83 physically interacts with PAS-B and basic helix-loop-helix domains of Met, and JH induces Met-Hsp83 interaction. As determined by immunohistochemistry, Met is mainly distributed in the cytoplasm of fat body cells of the larval when the JH titer is low and JH induces Met nuclear import. Hsp83 was accumulated in the cytoplasm area adjunct to the nucleus in the presence of JH and Met/Gce. Loss-of-function of Hsp83 attenuated JH binding and JH-induced nuclear import of Met, resulting in a decrease in the JHRR-driven reporter activity leading to reduction of Kr-h1 expression. These data show that Hsp83 facilitates the JH-induced nuclear import of Met that induces Kr-h1 expression through the JHRR.

MET is required for the maximal action of 20-hydroxyecdysone during Bombyx metamorphosis.

Little is known about how the putative juvenile hormone (JH) receptor, the bHLH-PAS transcription factor MET, is involved in 20-hydroxyecdysone (20E; the molting hormone) action. Here we report that two MET proteins found in the silkworm, Bombyx mori, participate in 20E signal transduction. Met is 20E responsive and its expression peaks during molting and pupation, when the 20E titer is high. As found with results from RNAi knockdown of EcR-USP (the ecdysone receptor genes), RNAi knockdown of Met at the early wandering stage disrupts the 20E-triggered transcriptional cascade, preventing tissue remodeling (including autophagy, apoptosis and destruction of larval tissues and generation of adult structures) and causing lethality during the larval-pupal transition. MET physically interacts with EcR-USP. Moreover, MET, EcR-USP and the 20E-response element (EcRE) form a protein-DNA complex, implying that MET might modulate 20E-induced gene transcription by interacting with EcR-USP. In conclusion, the 20E induction of MET is required for the maximal action of 20E during Bombyx metamorphosis.

Drosophila Met and Gce are partially redundant in transducing juvenile hormone action.

The Drosophila Methoprene-tolerant (Met) and Germ cell-expressed (Gce) bHLH-PAS transcription factors are products of two paralogous genes. Both proteins potentially mediate the effect of juvenile hormone (JH) as candidate JH receptors. Here we report that Met and Gce are partially redundant in transducing JH action. Both Met and gce null single mutants are fully viable, but the Met gce double mutant, Met(27) gce(2.5k), dies during the larval-pupal transition. Precocious and enhanced caspase-dependent programmed cell death (PCD) appears in fat body cells of Met(27) gce(2.5k) during the early larval stages. Expression of Kr-h1, a JH response gene that inhibits 20-hydroxyecdysone (20E)-induced broad (br) expression, is abolished in Met(27) gce(2.5k) during larval molts. Consequently, expression of br occurs precociously in Met(27) gce(2.5k), which may cause precocious caspase-dependent PCD during the early larval stages. Defective phenotypes and gene expression changes in Met(27) gce(2.5k) double mutants are similar to those found in JH-deficient animals. Importantly, exogenous application of JH agonists rescued the JH-deficient animals but not the Met(27) gce(2.5k) mutants. Our data suggest a model in which Drosophila Met and Gce redundantly transduce JH action to prevent 20E-induced caspase-dependent PCD during larval molts by induction of Kr-h1 expression and inhibition of br expression.

Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila.

Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Here we report that genetic ablation of the corpus allatum cells of the Drosophila ring gland (the JH source) resulted in JH deficiency, pupal lethality and precocious and enhanced programmed cell death (PCD) of the larval fat body. In the fat body of the JH-deficient animals, Dronc and Drice, two caspase genes that are crucial for PCD induced by the molting hormone 20-hydroxyecdysone (20E), were significantly upregulated. These results demonstrated that JH antagonizes 20E-induced PCD by restricting the mRNA levels of Dronc and Drice. The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of the 20E receptor EcR. Moreover, MET and GCE, the bHLH-PAS transcription factors involved in JH action, were shown to induce PCD by upregulating Dronc and Drice. In the Met- and gce-deficient animals, Dronc and Drice were downregulated, whereas in the Met-overexpression fat body, Dronc and Drice were significantly upregulated leading to precocious and enhanced PCD, and this upregulation could be suppressed by application of the JH agonist methoprene. For the first time, we demonstrate that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila.