Mechanisms of Neonatal Brain Injury in a Rat Pup Hypoxic-Ischemic Model

Perinatal hypoxic-ischemic (HI) brain injury is a major cause of acute morbidity and chronic nemological mortality in infants and children, with a reported incidence of 2- 9 per 1000 births. The pathophyiological mechanisms leading to immature brain damage after HI are complex and relate to the developmental stage of the brain. The objective of our study is to understand the neonatal hypoxic-ischemic brain injury and to aid in the development of pharmacological treatments. Hypoxic inducible factor 1 (HIF-1) is a transcription factor which is stabilized and activated by hypoxia. It is the most important factor involved in the cellular response to hypoxia. However, the role of HIF-1 in the central nervous system is unclear, which has been considered as a regulator of both prosurvival and prodeath pathways after brain injury. Here we tested the hypothesis that acute HIF-1 inhibition early after neonatal HI injiuy provides neuroprotection in the developing brain by preserving blood-brain barrier (BBB), ameliorating brain edema, and reducing infarct volume via VEGF-dependent pathway. We also investigated if matrix metalloproteinases (MMPs), one of HIF-1 downstream factors, are involved in the process of neonatal HI brain injury. We hypothesized that broad-spectrum MMPs inhibition confers neuroprotection by reducing tight junction proteins (TJPs) degradation and preserve BBB integrity. Moreover, acute MMPs inhibition provides long term neuroprotection and neurological function recovery against neonatal HI brain injury. Rice-Vannucci model of neonatal HI brain injury was used in seven-day-old rat pups, by subjecting unilateral carotid artery ligation followed by 2 h of hypoxia (8% O2 at 37°C). Following insult, brain infarction, neuronal cell death, brain edema, elevation of HIF-1 and MMP-2/-9 activity, elevation of VEGF and TIMP-1/-2 expression, degradation of TJPs, along with severe brain atrophy was observed in the ipsilateral hemisphere, which led to significant neurological deficits. HIF-1 a activity was inhibited by 2-methoxyestradiol (2ME2,1.5, 15 or 150 mg/kg) or enhanced by dimethyloxalylglycine (DMOG, 250 mg/kg). 2ME2 treatment exhibited dose-dependent neuroprotection by decreasing infarct volume and reducing brain edema at 48 h post HI. The neuroprotection was lost when 2ME2 was administered 3 h post HI. HIF-1 a upregulation by DMOG increased the permeability of the BBB and brain edema compared with HI group. 2ME2 (15 mg/kg) attenuated the elevation of HIF-1 a and VEGF at 24 h after HI. 2ME2 (15 mg/kg) also had a long-term effect of protecting against the loss of brain tissue. To investigate the role of MMPs, GM6001 (50 mg/kg or 100 mg/kg) or doxycycline (10 mg/kg or 30 mg/kg), a broad spectrum MMPs inhibitor, was injected intraperitoneally at 2 h and 24 h after HI injury. Either GM6001 (100 mg/kg) or doxycycline (30 mg/kg) treatment attenuated brain edema and BBB disruption. GM6001 (100 gm/kg) treatment also attenuated MMP-9/-2 activities, preserved the degradation of tight junction proteins (TJPs), protected BBB, and provided a long term neuroprotection morphologically and functionally. These results suggest that the early inhibition of HIF-1 acutely after injury provided neuroprotection after neonatal hypoxia-ischemia which was associated with preservation of BBB integrity, attenuation of brain edema, and neuronal death. Meanwhile, MMPs are also involved in the neonatal HI brain injury. Early MMPs inhibition affords both acute and long-term neuroprotection. Thus, we propose that either early HIF-1 inhibition or a short duration of MMPs inhibition in the early stage after neonatal HI injury may offer a promising therapeutic strategy for the prevention of the brain injury in the children who suffered a hypoxic-ischemic insult

Hydrogen is neuroprotective against surgically induced brain injury

<p>Abstract</p> <p>Background</p> <p>Neurosurgical operations cause unavoidable damage to healthy brain tissues. Direct surgical injury as well as surgically induced oxidative stress contributes to the subsequent formation of brain edema. Therefore, we tested the neuroprotective effects of hydrogen (H₂) in an established surgical brain injury (SBI) model in rats.</p> <p>Materials and methods</p> <p>Adult male Sprague - Dawley rats (weight 300-350g) were divided into three groups to serve as sham operated, SBI without treatment, and SBI treated with H₂(2.9%). Brain water content, myeloperoxidase (MPO) assay, lipid peroxidation (LPO), and neurological function were measured at 24 hrs after SBI.</p> <p>Results</p> <p>SBI resulted in localized brain edema (p = < 0.001). Hydrogen (2.9%) administered concurrently with surgery significantly decreased the formation of cerebral edema (p = 0.028) and improved neurobehavioral score (p = 0.022). However, hydrogen treatment failed to reduce oxidative stress (LPO assay) or inflammation (MPO assay) in brain tissues.</p> <p>Conclusions</p> <p>Hydrogen appears to be promising as an effective, yet inexpensive way to reduce cerebral edema caused by surgical procedures. Hydrogen has the potential to improve clinical outcome, decrease hospital stay, and reduce overall cost to patients and the health care system.</p

Dietary supplementation with Dendrobium officinale leaves improves growth, antioxidant status, immune function, and gut health in broilers

BackgroundThe Dendrobium officinale leaves (DOL) is an underutilized by-product with a large biomass, which have been shown to exhibit immunomodulatory and antioxidant functions. The purpose of this research was to investigate the effects of DOL on broiler growth performance, antioxidant status, immune function, and gut health.MethodsOne hundred and ninety-two 1-day-old chicks were selected and divided into 4 groups at random, 6 replicates for each group and 8 in each. Chicks were given a basal diet supplemented with different amounts of DOL: 0% (control group, NC), 1% (LD), 5% (MD), or 10% (HD). During the feeding trial (70 days), broiler body weight, feed intake, and residual feeding were recorded. On d 70, 12 broilers from each group were sampled for serum antioxidant and immune indexes measurement, intestinal morphological analysis, as well as 16S rRNA sequencing of cecal contents and short-chain fatty acid (SCFA) determination.ResultsIn comparison to the NC group, the LD group had greater final body weight and average daily gain, and a lower feed conversion ratio (p &lt; 0.05, d 1 to 70). However, in MD group, no significant change of growth performance occurred (p &gt; 0.05). Furthermore, DOL supplementation significantly improved the levels of serum total antioxidant capacity, glutathione peroxidase, superoxide dismutase, and catalase, but reduced the level of malondialdehyde (p &lt; 0.05). Higher serum immunoglobulin A (IgA) content and lower cytokine interleukin-2 (IL-2) and IL-6 contents were observed in DOL-fed broilers than in control chickens (p &lt;0.05). Compared to the NC group, duodenal villus height (VH) and villus height-to-crypt depth (VH:CD) ratio were considerably higher in three DOL supplementation groups (p &lt; 0.05). Further, 16S rRNA sequencing analysis revealed that DOL increased the diversity and the relative abundance of cecal bacteria, particularly helpful microbes like Faecalibacterium, Lactobacillus, and Oscillospira, which improved the production of SCFA in cecal content. According to Spearman correlation analysis, the increased butyric acid and acetic acid concentrations were positively related to serum antioxidant enzyme activities (T-AOC and GSH-Px) and immunoglobulin M (IgM) level (p &lt; 0.05).ConclusionOverall, the current study demonstrated that supplementing the dies with DOL in appropriate doses could enhance growth performance, antioxidant capacity, and immune response, as well as gut health by promoting intestinal integrity and modulating the cecal microbiota in broilers. Our research may serve as a preliminary foundation for the future development and application of DOL as feed additive in broiler chicken diets

Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse

BACKGROUND: Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%). RESULTS: We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects. CONCLUSIONS: These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components

HPV prevalence and genotype distribution in 2,306 patients with cervical squamous cell carcinoma in central and eastern China

BackgroundTo explore the positivity rate and genotype distribution of human papillomavirus (HPV) in cervical squamous cell carcinoma (CSCC) tissues in central and eastern China and to provide theoretical basis for cervical cancer screening and prophylactic HPV vaccine development in China.MethodsDNA was extracted from paraffin-embedded tissues of CSCC samples and exfoliated cervical cells of cervical cancer screening populations. 23 HPV genotypes were detected by combining polymerase chain reaction (PCR) and reverse dot hybridized gene chip detection technology in 2,306 CSCC tissues and 10,245 cervical cancer screening populations. The genotype distribution of HPV infection was analyzed.ResultsThe overall infection rate of HPVs in 2,306 CSCC patients was 92.71%. The frequency of single-type HPV infection and multiple-type HPV infection were 86.48% and 13.51%, respectively. The most common HPV genotypes detected in Chinese CSCC tissues were HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, HPV-58, and HPV-59. The overall positivity rate of these eight high-risk HPV (HR-HPV) genotypes in HPV-positive CSCC was as high as 96.91%. Of which the positivity rate of seven HR-HPV genotypes related to nine-valent HPV vaccines in HPV-positive CSCC was 95.09%. Meanwhile, the overall infection rates of HR-HPV and low-risk HPV (LR-HPV) in female aged 35–64 years who underwent cervical cancer screening were 13.16% and 1.32%, respectively. The high-frequency HR-HPV genotypes in cervical cancer screening women were HPV-52, HPV-58, HPV-16, HPV-53, HPV-68, HPV-39, HPV-51, and HPV-56, with positivity rates of 2.25%, 1.60%, 1.31%, 1.22%, 0.93%, 0.92%, 0.78%, and 0.74%, respectively.ConclusionAmong women screened for cervical cancer in China, detecting the 8 high-frequency HR-HPV genotypes can reduce technical difficulty and reagent costs, while also improving the efficiency and effectiveness of cervical cancer screening. HPV genotyping assists gynecologists in assessing the risk of HR-HPV-positive cervical intraepithelial neoplasia and guiding them in implementing appropriate interventions. Furthermore, HPV genotyping is helpful for doctors to follow up HR-HPV-positive women and to evaluate the protective effect of HPV vaccine

Combination of Decitabine and a Modified Regimen of Cisplatin, Cytarabine and Dexamethasone: A Potential Salvage Regimen for Relapsed or Refractory Diffuse Large B-Cell Lymphoma After Second-Line Treatment Failure

ObjectiveThe prognosis for patients with relapsed or refractory diffuse large B-cell lymphoma (R/R-DLBCL) after second-line treatment failure is extremely poor. This study prospectively observed the efficacy and safety of decitabine with a modified cisplatin, cytarabine, and dexamethasone (DHAP) regimen in R/R-DLBCL patients who failed second-line treatment.MethodsTwenty-one R/R-DLBCL patients were enrolled and treated with decitabine and a modified DHAP regimen. The primary endpoints were overall response rate (ORR) and safety. The secondary endpoints were progression-free survival (PFS) and overall survival (OS).ResultsORR reached 50% (complete response rate, 35%), five patients (25%) had stable disease (SD) with disease control rate (DCR) of 75%. Subgroup analysis revealed patients over fifty years old had a higher complete response rate compared to younger patients (P = 0.005), and relapsed patients had a better complete response rate than refractory patients (P = 0.031). Median PFS was 7 months (95% confidence interval, 5.1-8.9 months). Median OS was not achieved. One-year OS was 59.0% (95% CI, 35.5%-82.5%), and two-year OS was 51.6% (95% confidence interval, 26.9%-76.3%). The main adverse events (AEs) were grade 3/4 hematologic toxicities such as neutropenia (90%), anemia (50%), and thrombocytopenia (70%). Other main non-hematologic AEs were grade 1/2 nausea/vomiting (40%) and infection (50%). No renal toxicity or treatment-related death occurred.ConclusionDecitabine with a modified DHAP regimen can improve the treatment response and prognosis of R/R-DLBCL patients with good tolerance to AEs, suggesting this regimen has potential as a possible new treatment option for R/R-DLBCL patients after second-line treatment failure.Clinical Trial RegistrationClinicalTrials.gov, identifier: NCT03579082

Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein

The Beclin 1 gene is a haplo-insufficient tumor suppressor and plays an essential role in autophagy. However, the molecular mechanism by which Beclin 1 functions remains largely unknown. Here we report the crystal structure of the evolutionarily conserved domain (ECD) of Beclin 1 at 1.6 Å resolution. Beclin 1 ECD exhibits a previously unreported fold, with three structural repeats arranged symmetrically around a central axis. Beclin 1 ECD defines a novel class of membrane-binding domain, with a strong preference for lipid membrane enriched with cardiolipin. The tip of a surface loop in Beclin 1 ECD, comprising three aromatic amino acids, acts as a hydrophobic finger to associate with lipid membrane, consequently resulting in the deformation of membrane and liposomes. Mutation of these aromatic residues rendered Beclin 1 unable to stably associate with lipid membrane in vitro and unable to fully rescue autophagy in Beclin 1-knockdown cells in vivo. These observations form an important framework for deciphering the biological functions of Beclin 1