Establishment of a Patient-Derived Orthotopic Xenograft (PDOX) Model of HER-2-Positive Cervical Cancer Expressing the Clinical Metastatic Pattern

Squamous cell carcinoma of the cervix, highly prevalent in the developing world, is often metastatic and treatment resistant with no standard treatment protocol. Our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) nude mouse model with the technique of surgical orthotopic implantation (SOI). Unlike subcutaneous transplant patient-derived xenograft (PDX) models, PDOX models metastasize. Most importantly, the metastasis pattern correlates to the patient. In the present report, we describe the development of a PDOX model of HER-2-positive cervical cancer. Metastasis after SOI in nude mice included peritoneal dissemination, liver metastasis, lung metastasis as well as lymph node metastasis reflecting the metastatic pattern in the donor patient. Metastasis was detected in 4 of 6 nude mice with primary tumors. Primary tumors and metastases in the nude mice had histological structures similar to the original tumor and were stained by an anti-HER-2 antibody in the same pattern as the patient’s cancer. The metastatic pattern, histology and HER-2 tumor expression of the patient were thus preserved in the PDOX model. In contrast, subcutaneous transplantation of the patient’s cervical tumors resulted in primary growth but not metastasis.     

Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress

Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration—defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise.     

A Voltage-Gated Calcium Channel Regulates Lysosomal Fusion with Endosomes and Autophagosomes and Is Required for Neuronal Homeostasis

Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.     

A Low-Dose β1-Blocker in Combination with Milrinone Improves Intracellular Ca2+ Handling in Failing Cardiomyocytes by Inhibition of Milrinone-Induced Diastolic Ca2+ Leakage from the Sarcoplasmic Reticulum

Objectives

The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism.

Background

The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca²⁺ handling in heart failure remains unclear.

Methods

We investigated the effect of milrinone plus landiolol on intracellular Ca²⁺ transient (CaT), cell shortening (CS), the frequency of diastolic Ca²⁺ sparks (CaSF), and sarcoplasmic reticulum Ca²⁺ concentration ({Ca²⁺}_SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB).

Results

In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca²⁺}_SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca²⁺}_SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes.

Conclusion

A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca²⁺ leak.     

Risk Score to Predict 1-Year Mortality after Haemodialysis Initiation in Patients with Stage 5 Chronic Kidney Disease under Predialysis Nephrology Care

Background

Few risk scores are available for predicting mortality in chronic kidney disease (CKD) patients undergoing predialysis nephrology care. Here, we developed a risk score using predialysis nephrology practice data to predict 1-year mortality following the initiation of haemodialysis (HD) for CKD patients.

Methods

This was a multicenter cohort study involving CKD patients who started HD between April 2006 and March 2011 at 21 institutions with nephrology care services. Patients who had not received predialysis nephrology care at an estimated glomerular filtration rate (eGFR) of approximately 10 mL/min per 1.73 m² were excluded. Twenty-nine candidate predictors were selected, and the final model for 1-year mortality was developed via multivariate logistic regression and was internally validated by a bootstrapping technique.

Results

A total of 688 patients were enrolled, and 62 (9.0%) patients died within one year of HD initiation. The following variables were retained in the final model: eGFR, serum albumin, calcium, Charlson Comorbidity Index excluding diabetes and renal disease (modified CCI), performance status (PS), and usage of erythropoiesis-stimulating agent (ESA). Their β-coefficients were transformed into integer scores: three points were assigned to modified CCI≥3 and PS 3–4; two to calcium>8.5 mg/dL, modified CCI 1–2, and no use of ESA; and one to albumin<3.5 g/dL, eGFR>7 mL/min per 1.73 m², and PS 1–2. Predicted 1-year mortality risk was 2.5% (score 0–4), 5.5% (score 5–6), 15.2% (score 7–8), and 28.9% (score 9–12). The area under the receiver operating characteristic curve was 0.83 (95% confidence interval, 0.79–0.89).

Conclusions

We developed a simple 6-item risk score predicting 1-year mortality after the initiation of HD that might help nephrologists make a shared decision with patients and families regarding the initiation of HD.     

Common Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Epitopes Mediate Multiple Routes for Internalization and Function

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a soluble protein that directs membrane-bound receptors to lysosomes for degradation. In the most studied example of this, PCSK9 binding leads to the degradation of low density lipoprotein receptor (LDLR), significantly affecting circulating LDL-C levels. The mechanism mediating this degradation, however, is not completely understood. We show here that LDLR facilitates PCSK9 interactions with amyloid precursor like protein 2 (APLP2) at neutral pH leading to PCSK9 internalization, although direct binding between PCSK9 and LDLR is not required. Moreover, binding to APLP2 or LDLR is independently sufficient for PCSK9 endocytosis in hepatocytes, while LDL can compete with APLP2 for PCSK9 binding to indirectly mediate PCSK9 endocytosis. Finally, we show that APLP2 and LDLR are also required for the degradation of another PCSK9 target, APOER2, necessitating a general role for LDLR and APLP2 in PCSK9 function. Together, these findings provide evidence that PCSK9 has at least two endocytic epitopes that are utilized by a variety of internalization mechanisms and clarifies how PCSK9 may direct proteins to lysosomes.     

Gender Differences in Patients with Takotsubo Cardiomyopathy: Multi-Center Registry from Tokyo CCU Network

Background

The clinical features of gender differences in takotsubo cardiomyopathy (TC) remain to be determined. The aim of this study was to evaluate the differences in clinical characteristics of male and female patients with TC.

Methods

We obtained the clinical information of 368 patients diagnosed with TC (84 male, 284 female) from the Tokyo CCU Network database collected from 1 January 2010 to 31 December 2012; the Network is comprised of 71 cardiovascular centers in the Tokyo (Japan) metropolitan area. We attempted to characterize clinical differences during hospitalization, comparing male and female patients with TC.

Results

There were no significant differences in apical ballooning type, median echocardiography ejection fraction, serious ventricular arrhythmias (such as ventricular tachycardia or fibrillation), or cardiovascular death between male and female patients. Male patients were younger than female patients (median age at hospitalization for male patients was 72 years vs. 76 years for female patients; p = 0.040). Prior physical stress was more common in male than female patients (50.0% vs.31.3%; p = 0.002), while emotional stress was more common in female patients (19.0% vs. 31.0%; p = 0.039). Severe pump failure (defined as Killip Class > III) (20.2% vs. 10.6%; p = 0.020) and cardiopulmonary supportive therapies (28.6% vs. 12.7%, p < 0.001) were more common in male than female patients. Multivariate analysis revealed that male gender (odds ratio = 4.32, 95% CI = 1.41–13.6, p = 0.011) was an independent predictor of adverse composite cardiac events, including cardiovascular death, severe pump failure, and serious ventricular arrhythmia.

Conclusions

Cardiac complications in our dataset appeared to be more common in male than female patients with TC during their hospitalization. Further investigation is required to clarify the underlying mechanisms responsible for the observed gender differences.     

The Relationship between Processing Speed and Regional White Matter Volume in Healthy Young People

Processing speed is considered a key cognitive resource and it has a crucial role in all types of cognitive performance. Some researchers have hypothesised the importance of white matter integrity in the brain for processing speed; however, the relationship at the whole-brain level between white matter volume (WMV) and processing speed relevant to the modality or problem used in the task has never been clearly evaluated in healthy people. In this study, we used various tests of processing speed and Voxel-Based Morphometry (VBM) analyses, it is involves a voxel-wise comparison of the local volume of gray and white, to assess the relationship between processing speed and regional WMV (rWMV). We examined the association between processing speed and WMV in 887 healthy young adults (504 men and 383 women; mean age, 20.7 years, SD, 1.85). We performed three different multiple regression analyses: we evaluated rWMV associated with individual differences in the simple processing speed task, word–colour and colour–word tasks (processing speed tasks with words) and the simple arithmetic task, after adjusting for age and sex. The results showed a positive relationship at the whole-brain level between rWMV and processing speed performance. In contrast, the processing speed performance did not correlate with rWMV in any of the regions examined. Our results support the idea that WMV is associated globally with processing speed performance regardless of the type of processing speed task.