Decrotonylation of cGAS K254 prompts homologous recombination repair by blocking its DNA binding and releasing PARP1.

Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, also exhibits nuclear genomic localization and is involved in DNA damage signaling. In this study, we investigated the impact of cGAS crotonylation on the regulation of the DNA damage response, particularly homologous recombination repair, following exposure to ionizing radiation (IR). Lysine 254 of cGAS is constitutively crotonylated by the CREB-binding protein; however, IR-induced DNA damage triggers SIRT3-mediated decrotonylation. Lysine 254 decrotonylation decreased the DNA-binding affinity of cGAS and inhibited its interaction with PARP1, promoting HR repair. Moreover, SIRT3 suppression led to HR repair inhibition and markedly sensitized cancer cells to IR and DNA-damaging chemicals, highlighting SIRT3 as a potential target for cancer therapy. Overall, this study revealed the crucial role of cGAS crotonylation in the DNA damage response. Furthermore, we propose that modulating cGAS and SIRT3 activities could be potential strategies for cancer therapy.

GCN5 mediates DNA-PKcs crotonylation for DNA double-strand break repair and determining cancer radiosensitivity.

BACKGROUND: DNA double-strand break (DSB) induction and repair are important events for determining cell survival and the outcome of cancer radiotherapy. The DNA-dependent protein kinase (DNA-PK) complex functions at the apex of DSBs repair, and its assembly and activity are strictly regulated by post-translation modifications (PTMs)-associated interactions. However, the PTMs of the catalytic subunit DNA-PKcs and how they affect DNA-PKcs's functions are not fully understood. METHODS: Mass spectrometry analyses were performed to identify the crotonylation sites of DNA-PKcs in response to γ-ray irradiation. Co-immunoprecipitation (Co-IP), western blotting, in vitro crotonylation assays, laser microirradiation assays, in vitro DNA binding assays, in vitro DNA-PK assembly assays and IF assays were employed to confirm the crotonylation, identify the crotonylase and decrotonylase, and elucidate how crotonylation regulates the activity and function of DNA-PKcs. Subcutaneous xenografts of human HeLa GCN5 WT or HeLa GCN5 siRNA cells in BALB/c nude mice were generated and utilized to assess tumor proliferation in vivo after radiotherapy. RESULTS: Here, we reveal that K525 is an important site of DNA-PKcs for crotonylation, and whose level is sharply increased by irradiation. The histone acetyltransferase GCN5 functions as the crotonylase for K525-Kcr, while HDAC3 serves as its dedicated decrotonylase. K525 crotonylation enhances DNA binding activity of DNA-PKcs, and facilitates assembly of the DNA-PK complex. Furthermore, GCN5-mediated K525 crotonylation is indispensable for DNA-PKcs autophosphorylation and the repair of double-strand breaks in the NHEJ pathway. GCN5 suppression significantly sensitizes xenograft tumors of mice to radiotherapy. CONCLUSIONS: Our study defines K525 crotonylation of DNA-PKcs is important for the DNA-PK complex assembly and DSBs repair activity via NHEJ pathway. Targeting GCN5-mediated K525 Kcr of DNA-PKcs may be a promising therapeutic strategy for improving the outcome of cancer radiotherapy.

Profile of Chinese Cluster Headache Register Individual Study (CHRIS): Clinical characteristics, diagnosis and treatment status data of 816 patients in China.

BACKGROUND: The clinical profile of cluster headache may differ among different regions of the world, warranting interest in the data obtained from the initial Chinese Cluster Headache Register Individual Study (CHRIS) for better understanding. METHODS: We conducted a multicenter, prospective, longitudinal cohort study on cluster headache across all 31 provinces of China, aiming to gather clinical characteristics, treatment approaches, imaging, electrophysiological and biological samples. RESULTS: In total 816 patients were enrolled with a male-to-female ratio of 4.33:1. The mean age at consultation was 34.98 ± 9.91 years, and 24.89 ± 9.77 years at onset. Only 2.33% were diagnosed with chronic cluster headache, and 6.99% had a family history of the condition. The most common bout was one to two times per year (45.96%), lasting two weeks to one month (44.00%), and occurring frequently in spring (76.23%) and winter (73.04%). Of these, 68.50% experienced one to two attacks per day, with the majority lasting one to two hours (45.59%). The most common time for attacks was between 9 am and 12 pm (75.86%), followed by 1 am and 3 am (43.48%). Lacrimation (78.80%) was the most predominant autonomic symptom reported. Furthermore, 39.22% of patients experienced a delay of 10 years or more in receiving a correct diagnosis. Only 35.67% and 24.26% of patients received common acute and preventive treatments, respectively. CONCLUSION: Due to differences in ethnicity, genetics and lifestyle conditions, CHRIS has provided valuable baseline data from China. By establishing a dynamic cohort with comprehensive multidimensional data, it aims to advance the management system for cluster headache in China.

The effects of the coronavirus disease pandemic on intravenous thrombolytic therapy among patients with acute ischemic stroke in Dalian, China.

BACKGROUND: We investigated the influence of the coronavirus disease 2019 (COVID-19) pandemic on the number of patients with acute ischemic stroke who received intravenous thrombolytic therapy (ITT) in Dalian, China, in 2020. METHODS: This retrospective descriptive study, conducted from February 1, 2020, to August 31, 2020, examined 13 hospitals in Dalian that participated in the "stroke emergency map". To use this "stroke emergency map" of China, patients followed the official "Stroke Map" WeChat account and dialed 120 for emergency medical services. We analyzed the number of patients with acute ischemic stroke who underwent ITT. In particular, we examined the onset-to-door time (ODT), door-to-needle time (DNT), onset-to-needle time (ONT), mode of transportation to the hospital, and National Institutes of Health Stroke Scale (NIHSS) scores before and after ITT. Data were collected for the aforementioned period and compared with the 2021 baseline data from the same time of year. The Mann‒Whitney U test was performed for data analysis. RESULTS: Compared with the data from 2020, the number of patients with acute ischemic stroke who underwent ITT increased (from 735 to 1719 cases) in 2021, but the DNT decreased (from 59 to 45 min; P = 0.002). Moreover, 83.9% of patients in 2020 presented to the hospital without ambulance transport, compared to 81.1% of patients in the 2021 non-COVID-19 pandemic period. Patients with NIHSS scores of 6-14 were more likely to call an ambulance for transport to the hospital than to transport themselves to the emergency department. CONCLUSIONS: During the 2020 COVID-19 pandemic, the DNT was prolonged as a result of strengthened fever surveillance. In 2021, the number of patients with acute ischemic stroke who underwent ITT increased compared to the previous year. Notably, the growth in the number of patients with acute ischemic stroke who underwent ITT benefited from both the "stroke emergency map" of China and the "green channel," a novel treatment approach that focuses on the rational design of the rescue process. TRIAL REGISTRATION: Our study was a retrospective descriptive study, not a clinical trial, thus we did not have to register for clinical trials.

The prevalence and clinical features of fibromyalgia in Chinese hospital patients with primary headache: The survey of fibromyalgia comorbid with headache.

OBJECTIVE: The aims were to explore the prevalence and clinical features of fibromyalgia in Chinese hospital patients with primary headache. BACKGROUND: Studies done in non-Chinese populations suggest that around one-third of patients with primary headache have fibromyalgia, but data from mainland China are limited. Investigations into the prevalence and clinical features of fibromyalgia in Chinese patients with primary headache would improve our understanding of these two complex disease areas and help guide future clinical practice. METHODS: This cross-sectional study included adults with primary headache treated at 23 Chinese hospitals from September 2020 to May 2021. Fibromyalgia was diagnosed using the modified 2010 American College of Rheumatology criteria. Mood and insomnia were evaluated employing the Hospital Anxiety and Depression Scale and the Insomnia Severity Index. RESULTS: A total of 2782 participants were analyzed. The fibromyalgia prevalence was 6.0% (166/2782; 95% confidence interval: 5.1%, 6.8%). Compared to primary headache patients without combined fibromyalgia, patients with primary headache combined with fibromyalgia were more likely to be older (47.8 vs. 41.7 years), women (83.7% [139/166] vs. 72.8% [1904/2616]), less educated (65.1% [108/166] vs. 45.2% [1183/2616]), and with longer-duration headache (10.0 vs. 8.0 years). Such patients were more likely to exhibit comorbid depression (34.3% [57/166] vs. 9.9% [260/2616]), anxiety (16.3% [27/166] vs. 2.7% [70/2612]), and insomnia (58.4% [97/166] vs. 17.1% [447/2616]). Fibromyalgia was more prevalent in those with chronic (rather than episodic) migraine (11.1% [46/414] vs. 4.4% [72/1653], p < 0.001) and chronic (rather than episodic) tension-type headache (11.5% [27/235] vs. 4.6% [19/409], p = 0.001). Most fibromyalgia pain was in the shoulders, neck, and upper back. CONCLUSIONS: The prevalence of fibromyalgia in mainland Chinese patients with primary headache was 6.0%. Fibromyalgia was more common in those with chronic rather than episodic headache. The most common sites of fibromyalgia pain were the neck, shoulders, and back.

The Analysis of the Glycosyltransferase Gene Function From a Novel Granaticin Producer, Streptomyces Vilmorinianum. YP1.

Glycosylation is common among the synthesis of natural product and imparts the bioactivity for natural product. As for granaticin, a natural product with great bioactivity, glycosylation is an unusual sugar attachment and remains enigmatic. Orf14 in the gra cluster is the predicted glycosyltransferase but without being identified. Recently, we isolated and identified a novel granaticin producer Streptomyces vilmorinianum YP1. Orf14 gene in gra cluster of YP1 is knocked out and complemented. The instrumental analysis of the blue product synthesized by orf14-deficient mutant exhibits the none-granaticin detection and deglycosylated intermediates accumulation. The bioactivity and stability test suggests the weaker or none antibacterial activity and cytotoxicity of this blue product with greater ultraviolet stability and thermostability than granaticin and derivatives produced by YP1. All the result indicates that orf14 encodes glycosyltransferase and glycosylation played an important role in the bioactivity of granaticin. Meanwhile, the blue pigment, deglycosylated intermediates, has favorable processing characteristics. Our finding supplies the function of orf14 and glycosylation, but also indicates a promising candidate of edible blue pigment applicated in food industry.

Cytosolic Release of Mitochondrial DNA and Associated cGAS Signaling Mediates Radiation-Induced Hematopoietic Injury of Mice.

Mitochondrion is an important organelle of eukaryotic cells and a critical target of ionizing radiation (IR) outside the nucleus. The biological significance and mechanism of the non-target effect originating from mitochondria have received much attention in the field of radiation biology and protection. In this study, we investigated the effect, role, and radioprotective significance of cytosolic mitochondrial DNA (mtDNA) and its associated cGAS signaling on hematopoietic injury induced by IR in vitro culture cells and in vivo total body irradiated mice in this study. The results demonstrated that γ-ray exposure increases the release of mtDNA into the cytosol to activate cGAS signaling pathway, and the voltage-dependent anion channel (VDAC) may contribute to IR-induced mtDNA release. VDAC1 inhibitor DIDS and cGAS synthetase inhibitor can alleviate bone marrow injury and ameliorate hematopoietic suppression induced by IR via protecting hematopoietic stem cells and adjusting subtype distribution of bone marrow cells, such as attenuating the increase of the F4/80+ macrophage proportion in bone marrow cells. The present study provides a new mechanistic explanation for the radiation non-target effect and an alternative technical strategy for the prevention and treatment of hematopoietic acute radiation syndrome.

Status of diagnosis and preventative treatment for primary headache disorders: real-world data of unmet needs in China.

BACKGROUND: Headache disorders are widely prevalent and pose a considerable economic burden on individuals and society. Globally, misdiagnosis and inadequate treatment of primary headache disorders remain significant challenges, impeding the effective management of such conditions. Despite advancements in headache management over the last decade, a need for comprehensive evaluations of the status of primary headache disorders in China regarding diagnosis and preventative treatments persists. METHODS: In the present study, we analyzed the established queries in the Survey of Fibromyalgia Comorbidity with Headache (SEARCH), focusing on previous diagnoses and preventative treatment regimens for primary headache disorders. This cross-sectional study encompassed adults diagnosed with primary headache disorders who sought treatment at 23 hospitals across China between September 2020 to May 2021. RESULTS: The study comprised 2,868 participants who were systematically examined. Migraine and tension-type headaches (TTH) constituted a majority of the primary headache disorders, accounting for 74.1% (2,124/2,868) and 23.3% (668/2,868) of the participants, respectively. Medication overuse headache (MOH) affected 8.1% (231/2,868) of individuals with primary headache disorders. Over half of the individuals with primary headache disorders (56.6%, 1,624/2,868) remained undiagnosed. The previously correct diagnosis rates for migraine, TTH, TACs, and MOH were 27.3% (580/2,124), 8.1% (54/668), 23.2% (13/56), and 3.5% (8/231), respectively. The misdiagnosis of "Nervous headache" was found to be the most prevalent among individuals with migraine (9.9%, 211/2,124), TTH (10.0%, 67/668), trigeminal autonomic cephalalgias (TACs) (17.9%, 10/56), and other primary headache disorders (10.0%, 2/20) respectively. Only a minor proportion of individuals with migraine (16.5%, 77/468) and TTH (4.7%, 2/43) had received preventive medication before participating in the study. CONCLUSIONS: While there has been progress made in the rate of correct diagnosis of primary headache disorders in China compared to a decade ago, the prevalence of misdiagnosis and inadequate treatment of primary headaches remains a veritable issue. As such, focused efforts are essential to augment the diagnosis and preventive treatment measures related to primary headache disorders in the future.

Relation between serum ß-endorpin, neuropeptide Y and thyroid hormone in children with anorexia.

The purpose of this study was to investigate whether serum ß-endorpin and neuropeptide Y were associated with changes in levels of thyroid hormones in children suffering from anorexia. One hundred and five anorexic children admitted to Xianning City Central Hospital, China, from August 2019 to July 2021, were selected as case group, while 105 normal children were selected as normal control group. Serum ß-endorpin and neuropeptide Y levels in the case group were lower than those in the normal control group (both p<0.001), and serum triiodothyronine and thyroxine levels were also lower (both p<0.001). Serum ß-endorpin and neuropeptide Y levels in the case group were positively correlated with triiodothyronine and thyroxine. There is a reduced level of serum ß-endorpin, neuropeptide Y, and thyroid hormones in anorexic children, and it is possible that they are connected and work together in regulating ingestion.

Targeting the untargetable: RB1-deficient tumours are vulnerable to Skp2 ubiquitin ligase inhibition.

Proteins that regulate the cell cycle are accumulated and degraded in a coordinated manner during the transition from one cell cycle phase to the next. The rapid loss of a critical protein, for example, to allow the cell to move from G1/G0 to S phase, is often regulated by its ubiquitination and subsequent proteasomal degradation. Protein ubiquitination is mediated by a series of three ligases, of which the E3 ligases provide the specificity for a particular protein substrate. One such E3 ligase is SCFSkp1/Cks1, which has a substrate recruiting subunit called S-phase kinase-associated protein 2 (Skp2). Skp2 regulates cell proliferation, apoptosis, and differentiation, can act as an oncogene, and is overexpressed in human cancer. A primary target of Skp2 is the cyclin-dependent kinase inhibitor p27 (CDKN1b) that regulates the cell cycle at several points. The RB1 tumour suppressor gene regulates Skp2 activity by two mechanisms: by controlling its mRNA expression, and by an effect on Skp2's enzymatic activity. For the latter, the RB1 protein (pRb) directly binds to the substrate-binding site on Skp2, preventing protein substrates from being ubiquitinated and degraded. Inactivating mutations in RB1 are common in human cancer, becoming more frequent in aggressive, metastatic, and drug-resistant tumours. Hence, RB1 mutation leads to the loss of pRb, an unrestrained increase in Skp2 activity, the unregulated decrease in p27, and the loss of cell cycle control. Because RB1 mutations lead to the loss of a functional protein, its direct targeting is not possible. This perspective will discuss evidence validating Skp2 as a therapeutic target in RB1-deficient cancer.

Randomized, Double-Blinded, Placebo-Controlled Phase 2 Trial of an Inactivated Severe Acute Respiratory Syndrome Coronavirus 2 Vaccine in Healthy Adults.

BACKGROUND: We evaluated an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine for immunogenicity and safety in adults aged 18-59 years. METHODS: In this randomized, double-blinded, controlled trial, healthy adults received a medium dose (MD) or a high dose (HD) of the vaccine at an interval of either 14 days or 28 days. Neutralizing antibody (NAb) and anti-S and anti-N antibodies were detected at different times, and adverse reactions were monitored for 28 days after full immunization. RESULTS: A total of 742 adults were enrolled in the immunogenicity and safety analysis. Among subjects in the 0, 14 procedure, the seroconversion rates of NAb in MD and HD groups were 89% and 96% with geometric mean titers (GMTs) of 23 and 30, respectively, at day 14 and 92% and 96% with GMTs of 19 and 21, respectively, at day 28 after immunization. Anti-S antibodies had GMTs of 1883 and 2370 in the MD group and 2295 and 2432 in the HD group. Anti-N antibodies had GMTs of 387 and 434 in the MD group and 342 and 380 in the HD group. Among subjects in the 0, 28 procedure, seroconversion rates for NAb at both doses were both 95% with GMTs of 19 at day 28 after immunization. Anti-S antibodies had GMTs of 937 and 929 for the MD and HD groups, and anti-N antibodies had GMTs of 570 and 494 for the MD and HD groups, respectively. No serious adverse events were observed during the study period. CONCLUSIONS: Adults vaccinated with inactivated SARS-CoV-2 vaccine had NAb as well as anti-S/N antibody and had a low rate of adverse reactions. CLINICAL TRIALS REGISTRATION: NCT04412538.

Cyclin-dependent kinase 4/6 inhibitors require an arcuate-to-paraventricular hypothalamus melanocortin circuit to treat diet-induced obesity.

The arcuate nucleus (ARC) of the hypothalamus comprises two antagonistic neuron populations critical for energy balance, namely, the anorexigenic pro-opiomelanocortin (POMC) and the orexigenic agouti-related peptide (AgRP) neurons that act as agonists and antagonists, respectively, for neurons expressing the type IV melanocortin receptor (MC4R) (Andermann ML and Lowell BB. Neuron 95: 757-778, 2017). MC4R activation increases energy expenditure and decreases food intake during positive energy balance states to prevent diet-induced obesity (DIO). Work from our group identified aberrant neuronal cell cycle events both as a novel biomarker and druggable target in the ARC for the treatment of DIO, demonstrating pharmacological restoration of retinoblastoma protein function in the ARC using cyclin-dependent kinase 4/6 (CDK4/6) inhibitors could treat DIO in mice by increasing lipid oxidation to selectively decrease fat mass. However, the role of CDK4/6 inhibitors on food intake was not examined. Four-week-old Mc4r-loxTB mice were continuously administered high-fat diet (60% kcal fat). At 8 wk of age, animals were administered 60 mg/kg abemaciclib orally or a saline control and monitored every 2 wk for fat mass changes by MRI. At 11 wk of age, all animals were injected bilaterally in the paraventricular hypothalamus with AAV8 serotype virus expressing a Cre-mCherry and monitored for another 5 wk. Restoration of Mc4r expression in the paraventricular hypothalamic nucleus (PVN/PVH) reduced food intake in hyperphagic obese mice when given CDK4/6 inhibitor therapy. The reduced food intake was responsible for reduced fat mass in mice treated with abemaciclib. These results indicate that targeting POMC neurons could be an effective strategy in treating diet-related obesity.NEW & NOTEWORTHY We have defined some of the necessary components to prevent high-fat diet-induced obesity at the molecular and cellular level. Within POMC neurons, the retinoblastoma protein must remain active and prevented from phosphoinactivation by cyclin-dependent kinases. The downstream neurons within the PVH must also properly express MC4R for the circuit to appropriately regulate feeding behavior.

[Genetic analysis of three patients with Kleefstra syndrome].

OBJECTIVE: To analyze the clinical and genetic features of three patient diagnosed with Kleefstra syndrome. METHODS: Whole exome sequencing (WES) was carried out for the probands and their parents. Suspected variants were validated by Sanger sequencing. Copy number variations (CNV) were detected by CNV-seq and validated by real-time PCR. RESULTS: Proband 1 was found to carry a de novo heterogeneous variant (c.823+1G>T) of the EHMT1 gene, which may affect its expression. Based on the guidelines of the American College of Medical Genetics and Genomics, the variant was predicted to be pathogenic (PVS1+PS2+PM2). Proband 2 was found to carry a de novo missense variant c.439C>G (p.L147V) of the EHMT1 gene, which was predicted to be likely pathogenic (PS2+PM1+PM2+PP3). Proband 3 was found to carry a heterozygous 520 kb deletion at 9q34.3 by CNV-seq. The deletion has encompassed the whole of the EHMT1 gene. Real-time PCR has detected no CNV of this region in her parents. CONCLUSION: Variants of the EHMT1 gene probably underlay the disease in these patients. Genetic testing has provided a basis for their clinical diagnosis.

[Clinical and genetic analysis of four patients with congenital neutropenia].

OBJECTIVE: To delineate the clinical feature and genetic basis of four patients with congenital neutropenia. METHODS: All patients were subjected to whole exome sequencing (WES). Suspected variants were verified by Sanger sequencing. RESULTS: The patients (two boys and two girls), aged 7 to 15 months, suffered from neutropenia and recurrent infections. Bone marrow smears showed a significant decrease in the proportion of rod-shaped and lobulated granulocytes, which suggested impaired development and maturation of bone marrow neutrophils. WES has discovered heterozygous variants (c.496G>A, c.58C>G, c.391G>A and IVS1+5T>A) of the ELANE gene in the patients. Among these, c.58C>G and IVS1+5T>A were unreported previously. Follow up revealed patients 1 and 3 had periodic neutropenia, while patients 2 and 4 had severe congenital neutropenia. After attaining the definite diagnosis, the patients were treated symptomatically. CONCLUSION: The main clinical feature of congenital neutropenia is refractory recurrent bacterial infections, for which mutations of the ELANE gene are a common cause. Two novel pathogenic ELANE variants have been discovered in this study.

Transferrin Receptor Targeted Cellular Delivery of Doxorubicin Via a Reduction-Responsive Peptide-Drug Conjugate.

PURPOSE: Transferrin receptors (TfRs) are overexpressed in tumor cells but are scarce in normal tissues, which makes TfR an attractive target for drug treatment of cancer. The objective of this study was to evaluate the potential of BP9a (CAHLHNRS) as a peptide vector for constructing TfR targeted peptide-drug conjugates and selective drug delivery. METHODS: Doxorubicin (DOX) was connected to BP9a via a disulfide-intercalating linker to afford a reduction-responsive BP9a-SS-DOX conjugate. By using HepG2 human liver cancer cells and L-O2 normal hepatic cells as TfR over-expressing and low-expressing in vitro models, respectively, TfR mediated cellular uptake of this conjugate was studied by using flow cytometry and confocal laser scanning microscopy. The in vitro cytotoxicities of the conjugate against HepG2 and L-O2 cells were examined by cell counting kit-8 (CCK-8) assay to evaluate its tumorous specificity. RESULTS: Cellular uptake and TfR blockage test results showed that the BP9a-SS-DOX conjugate gained entry into HepG2 cells via endocytosis mediated by TfR and mainly accumulated in cytoplasm. The in vitro antiproliferative activity of this conjugate against HepG2 cells (IC50 6.21 ± 1.12 µM) was approximately one-sixth of that of free DOX (IC50 1.03 ± 0.13 µM). However, its cytotoxic effect on L-O2 cells was obviously reduced compared with that of free DOX. CONCLUSIONS: The BP9a-SS-DOX conjugate showed specific antiproliferative activity against HepG2 liver cancer cells. Our study suggests that BP9a has the potential to target chemotherapeutic agents to tumor cells over-expressing TfR and facilitate selective drug delivery.

Synthesis, in vitro stability, and antiproliferative effect of d-cysteine modified GnRH-doxorubicin conjugates.

Overexpression of gonadotropin-releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d-Cys6 , desGly10 , Pro9 -NH2 ]-GnRH, [d-Cys6 , desGly10 , Pro9 -NHEt]-GnRH, and [d-Cys6 , α-aza-Gly10 -NH2 ]-GnRH were conjugated with doxorubicin (Dox), respectively, through N-succinimidyl-3-maleimidopropionate as a linker to afford three new GnRH-Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP-HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor-positive MCF-7 human breast cancer cell line by MTT assay. The three GnRH-Dox conjugates showed improved metabolic stability in human serum in comparison with AN-152. The antiproliferative effect of conjugate II ([d-Cys6 , desGly10 , Pro9 -NHEt]-GnRH-Dox) on MCF-7 cells was higher than that of conjugate I ([d-Cys6 , desGly10 , Pro9 -NH2 ]-GnRH-Dox) and conjugate III ([d-Cys6 , α-aza-Gly10 -NH2 ]-GnRH-Dox), and the cytotoxicity of conjugate II against GnRH receptor-negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d-Cys6 -des-Gly10 -Pro9 -NHEt]-GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d-Cys6 -des-Gly10 -Pro9 -NHEt]-GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.

Ipsilateral Ptosis and Contralateral Ataxic Hemiparesis as Initial Symptoms of Combined Tuberothalamic and Paramedian Artery Infarction.

Thalamic infarcts, accounting for approximately 14% of lacunar infarcts, exhibit varied clinical manifestations due to complex anatomy of nuclei and varying blood supply. Pure and combined types of thalamic infarctions have been summarized in some paper, but information of cerebral angiography was not mentioned. Here we report a rare case of combined tuberothalamic and paramedian artery occlusion presenting with ipsilateral ptosis and contralateral ataxic hemiparesis.

What influenced the lesion patterns and hemodynamic characteristics in patients with internal carotid artery stenosis? A retrospective study.

OBJECTIVES: This study aimed to explore the dynamic changes of lesion patterns and hemodynamic characteristics in patients with internal carotid artery stenosis (ICAS). PATIENTS AND METHODS: Patients who had suffered an acute ischemic stroke in the distribution of ipsilateral ICAS were included. Computed tomography (CT) and transcranial doppler ultrasound (TCD) were conducted to evaluate the degree of ICAS and the hemodynamic characteristics of the intracranial and extracranial arteries. RESULT: A total of 424 patients were included in the study. With the aggravation of ICAS, blood velocity in ipsilateral ICA was increased, while blood flow in the ipsilateral middle cerebral artery (MCA) was decreased. In the same degree of ICAS, patients with opened communicating arteries showed relatively higher blood perfusion in MCA compared with those without communicating arteries. In the average stage of ICAS, small lesions (D=0-1.5cm), middle lesions (1.5cm3.0cm) commonly existed. The number of small and large lesions significantly increased when the blood flow of ipsilateral MCA decreased. In the same degree of stenosis, the number of small lesions and large lesions, and the total area of all lesions, evidently increased with the decrease of ipsilateral MCA blood velocity. CONCLUSION: Hypoperfusion is an independent risk factor for ischemic lesions in patients with ICAS. Whether or not the communicating arteries are open influences the blood flow of the intracranial arteries. TCD was a convenient and rapid tool to assess intracranial perfusion and vascular compensatory status.

Deletions of Retinoblastoma 1 (Rb1) and Its Repressing Target S Phase Kinase-associated protein 2 (Skp2) Are Synthetic Lethal in Mouse Embryogenesis.

Tumor suppressor pRb represses Skp2, a substrate-recruiting subunit of the SCF(Skp2) ubiquitin ligase. Rb1(+/-) mice incur "two-hit" pituitary tumorigenesis; Skp2(-/-);Rb1(+/-) mice do not. Rb1(-/-) embryos die on embryonic day (E) 14.5-15.5. Here, we report that Skp2(-/-);Rb1(-/-) embryos died on E11.5, establishing an organismal level synthetic lethal relationship between Rb1 and Skp2 On E10.5, Rb1(-/-) placentas showed similarly active proliferation and similarly inactive apoptosis as WT placenta, whereas Rb1(-/-) embryos showed ectopic proliferation without increased apoptosis in the brain. Combining Skp2(-/-) did not reduce proliferation or increase apoptosis in the placentas but induced extensive apoptosis in the brain. We conditionally deleted Rb1 in neuronal lineage with Nes-Cre and reproduced the brain apoptosis in E13.5 Nes-Cre;Rb1(lox/lox);Skp2(-/-) embryos, demonstrating their synthetic lethal relationship at a cell autonomous level. Nes-Cre-mediated Rb1 deletion increased expression of proliferative E2F target genes in the brains of Skp2(+/+) embryos; the increases rose higher with activation of expression of apoptotic E2F target genes in Skp2(-/-) embryos. The brain apoptosis was independent of p53 but coincident with proliferation. The highly activated expression of proliferative and apoptotic E2F target genes subsided with gradually reduced roles of Skp2 in preventing p27 protein accumulation in the brain in late gestation, allowing the embryos to reach full term with normally sized brains. These findings establish that Rb1 and Skp2 deletions are synthetic lethal and suggest how this lethal relationship might be circumvented, which could help design better therapies for pRb-deficient cancer.

pRb is an obesity suppressor in hypothalamus and high-fat diet inhibits pRb in this location.

pRb is frequently inactivated in tumours by mutations or phosphorylation. Here, we investigated whether pRb plays a role in obesity. The Arcuate nucleus (ARC) in hypothalamus contains antagonizing POMC and AGRP/NPY neurons for negative and positive energy balance, respectively. Various aspects of ARC neurons are affected in high-fat diet (HFD)-induced obesity mouse model. Using this model, we show that HFD, as well as pharmacological activation of AMPK, induces pRb phosphorylation and E2F target gene de-repression in ARC neurons. Some affected neurons express POMC; and deleting Rb1 in POMC neurons induces E2F target gene de-repression, cell-cycle re-entry, apoptosis, and a hyperphagia-obesity-diabetes syndrome. These defects can be corrected by combined deletion of E2f1. In contrast, deleting Rb1 in the antagonizing AGRP/NPY neurons shows no effects. Thus, pRb-E2F1 is an obesity suppression mechanism in ARC POMC neurons and HFD-AMPK inhibits this mechanism by phosphorylating pRb in this location.