Immunomodulatory and Antibacterial Effects of Cystatin 9 against Francisella tularensis

Cystatin 9 (CST9) is a member of the type 2 cysteine protease inhibitor family, which has been shown to have immunomodulatory effects that restrain inflammation, but its functions against bacterial infections are unknown. Here, we report that purified human recombinant (r)CST9 protects against the deadly bacterium Francisella tularensis (Ft) in vitro and in vivo. Macrophages infected with the Ft human pathogen Schu 4 (S4), then given 50 pg of rCST9 exhibited significantly decreased intracellular bacterial replication and increased killing via preventing the escape of S4 from the phagosome. Further, rCST9 induced autophagy in macrophages via the regulation of the mammalian target of rapamycin (mTOR) signaling pathways. rCST9 promoted the upregulation of macrophage proteins involved in antiinflammation and antiapoptosis, while restraining proinflammatory-associated proteins. Interestingly, the viability and virulence of S4 also was decreased directly by rCST9. In a mouse model of Ft inhalation, rCST9 significantly decreased organ bacterial burden and improved survival, which was not accompanied by excessive cytokine secretion or subsequent immune cell migration. The current report is the first to show the immunomodulatory and antimicrobial functions of rCST9 against Ft. We hypothesize that the attenuation of inflammation by rCST9 may be exploited for therapeutic purposes during infection.     

The mutational spectrum in a cohort of Charcot-Marie-Tooth disease type 2 among the Han Chinese in Taiwan

Background

Charcot-Marie-Tooth disease type 2 (CMT2) is a clinically and genetically heterogeneous group of inherited axonal neuropathies. The aim of this study was to extensively investigate the mutational spectrum of CMT2 in a cohort of patients of Han Chinese.

Methodology and Principal Findings

Genomic DNA from 36 unrelated Taiwanese CMT2 patients of Han Chinese descent was screened for mutations in the coding regions of the MFN2, RAB7, TRPV4, GARS, NEFL, HSPB1, MPZ, GDAP1, HSPB8, DNM2, AARS and YARS genes. Ten disparate mutations were identified in 14 patients (38.9% of the cohort), including p.N71Y in AARS (2.8%), p.T164A in HSPB1 (2.8%), and p.[H256R]+[R282H] in GDAP1 (2.8%) in one patient each, three NEFL mutations in six patients (16.7%) and four MFN2 mutations in five patients (13.9%). The following six mutations were novel: the individual AARS, HSPB1 and GDAP1 mutations and c.475-1G>T, p.L233V and p.E744M mutations in MFN2. An in vitro splicing assay revealed that the MFN2 c.475-1G>T mutation causes a 4 amino acid deletion (p.T159_Q162del). Despite an extensive survey, the genetic causes of CMT2 remained elusive in the remaining 22 CMT2 patients (61.1%).

Conclusions and Significance

This study illustrates the spectrum of CMT2 mutations in a Taiwanese CMT2 cohort and expands the number of CMT2-associated mutations. The relevance of the AARS and HSPB1 mutations in the pathogenesis of CMT2 is further highlighted. Moreover, the frequency of the NEFL mutations in this study cohort was unexpectedly high. Genetic testing for NEFL and MFN2 mutations should, therefore, be the first step in the molecular diagnosis of CMT2 in ethnic Chinese.     

Endophilin,Lamellipodin, and Mena cooperate to regulate F‐actin‐dependent EGF‐receptor endocytosis

The epidermal growth factor receptor (EGFR) plays an essential role during development and diseases including cancer. Lamellipodin (Lpd) is known to control lamellipodia protrusion by regulating actin filament elongation via Ena/VASP proteins. However, it is unknown whether this mechanism supports endocytosis of the EGFR. Here, we have identified a novel role for Lpd and Mena in clathrin‐mediated endocytosis (CME) of the EGFR. We have discovered that endogenous Lpd is in a complex with the EGFR and Lpd and Mena knockdown impairs EGFR endocytosis. Conversely, overexpressing Lpd substantially increases the EGFR uptake in an F‐actin‐dependent manner, suggesting that F‐actin polymerization is limiting for EGFR uptake. Furthermore, we found that Lpd directly interacts with endophilin, a BAR domain containing protein implicated in vesicle fission. We identified a role for endophilin in EGFR endocytosis, which is mediated by Lpd. Consistently, Lpd localizes to clathrin‐coated pits (CCPs) just before vesicle scission and regulates vesicle scission. Our findings suggest a novel mechanism in which Lpd mediates EGFR endocytosis via Mena downstream of endophilin.     

Regulation of oocyte and cumulus cell interactions by intermedin/adrenomedullin 2

Ovarian folliculogenesis has been studied as a model of hormonal regulation of development and differentiation, cell death, and cell-cell communication. In addition to gonadotropins from the pituitary and follicular paracrine factors, oocyte secreted factors have been shown to play critical roles in the regulation of follicular cell functions. Except for the well characterized BMP family proteins, including GDF9 and BMP15, oocytes are known to secrete oocyte secreted factors that are important for the regulation of cumulus cell survival and the maintenance of tertiary structure of cumulus cell-enclosed oocyte complexes (COCs). Based on genomic screening and studies of COCs cultured in vitro, we showed that intermedin (IMD)/adrenomedullin 2 (ADM2) is a novel oocyte-derived ligand important for the regulation of cell interactions in COCs that functions, in part, by suppressing cumulus cell apoptosis. Consistently, we showed that suppression of IMD/ADM2 signaling in growing rat ovaries in vivo leads to oocyte atresia and aberrant cell cycle progression in follicular cells. Together, our studies indicated that mammalian oocytes deploy a G protein-coupled receptor ligand to coordinate normal interactions of oocytes and cumulus cells and provided a better understanding of how the tertiary structure of a COC is maintained as follicles undergo exponential growth during the late stages of folliculogenesis.     

Alternative Splicing Generates a Novel Truncated Cav1.2 Channel in Neonatal Rat Heart

L-type Ca_v1.2 Ca²⁺ channel undergoes extensive alternative splicing, generating functionally different channels. Alternatively spliced Ca_v1.2 Ca²⁺ channels have been found to be expressed in a tissue-specific manner or under pathological conditions. To provide a more comprehensive understanding of alternative splicing in Ca_v1.2 channel, we systematically investigated the splicing patterns in the neonatal and adult rat hearts. The neonatal heart expresses a novel 104-bp exon 33L at the IVS3-4 linker that is generated by the use of an alternative acceptor site. Inclusion of exon 33L causes frameshift and C-terminal truncation. Whole-cell electrophysiological recordings of Ca_v1.2_33L channels expressed in HEK 293 cells did not detect any current. However, when co-expressed with wild type Ca_v1.2 channels, Ca_v1.2_33L channels reduced the current density and altered the electrophysiological properties of the wild type Ca_v1.2 channels. Interestingly, the truncated 3.5-domain Ca_v1.2_33L channels also yielded a dominant negative effect on Ca_v1.3 channels, but not on Ca_v3.2 channels, suggesting that Ca_vβ subunits is required for Ca_v1.2_33L regulation. A biochemical study provided evidence that Ca_v1.2_33L channels enhanced protein degradation of wild type channels via the ubiquitin-proteasome system. Although the physiological significance of the Ca_v1.2_33L channels in neonatal heart is still unknown, our report demonstrates the ability of this novel truncated channel to modulate the activity of the functional Ca_v1.2 channels. Moreover, the human Ca_v1.2 channel also contains exon 33L that is developmentally regulated in heart. Unexpectedly, human exon 33L has a one-nucleotide insertion that allowed in-frame translation of a full Ca_v1.2 channel. An electrophysiological study showed that human Ca_v1.2_33L channel is a functional channel but conducts Ca²⁺ ions at a much lower level.     

A Possible Case of Cherubism in a 17th-Century Korean Mummy

Cherubism is a benign fibro-osseous disease of childhood limited specifically to the maxilla and mandible. The progressive replacement of the jaw bones with expansile multilocular cystic lesions causes eventual prominence of the lower face, and hence the classic “cherubic” phenotype reflecting variable extents of jaw hypertrophy. Histologically, this condition has been characterized as replacement of the normal bone matrix with multicystic pockets of fibrous stroma and osteoclastic giant cells. Because of radiographic features common to both, primarily the presence of multiloculated lucencies with heterogeneous “ground-glass” sclerosis on CT imaging, cherubism was long mistaken for a craniofacial subtype of fibrous dysplasia. In 1999, however, the distinct genetic basis for cherubism was mapped to chromosome 4p16.3 and the SH-3 binding protein SH3BP2. But while there are already three suspected cases of fibrous dysplasia amongst archaeological populations, no definitive cases of cherubism have yet been reported in historical populations. In the current study we describe micro- and macro-structural changes in the face of a 17^th century Joseon Dynasty Korean mummy which may coincide with the clinic-pathologic and radiologic features of cherubism.     

Do Post Discharge Phone Calls Improve Care Transitions? A Cluster-Randomized Trial

Importance

The transition from hospital to home can expose patients to adverse events during the post discharge period. Post discharge care including phone calls may provide support for patients returning home but the impact on care transitions is unknown.

Objective

To examine the effect of a 72-hour post discharge phone call on the patient''s transition of care experience.

Design

Cluster-randomized control trial.

Setting

Urban, academic medical center.

Participants

General medical patients age 18 and older discharged home after hospitalization.

Main Outcomes and Measures

Primary outcome measure was the Care Transition Measure (CTM-3) score, a validated measure of the quality of care transitions. Secondary measures included self-reported adherence to medication and follow up plans, and 30-day composite of emergency department (ED) visits and hospital readmission.

Results

328 patients were included in the study over an 6-month period. 114 (69%) received a post discharge phone call, and 214 of all patients in the study completed the follow outcome survey (65% response rate). A small difference in CTM-3 scores was observed between the intervention and control groups (1.87 points, 95% CI 0.47–3.27, p = 0.01). Self-reported adherence to treatment plans, ED visits, and emergency readmission rates were similar between the two groups (odds ratio 0.57, 95% CI 0.13–2.45, 1.20, 95% CI 0.61–2.37, and 1.18, 95% CI 0.53–2.61, respectively).

Conclusions and Relevance

A single post discharge phone call had a small impact on the quality of care transitions and no effect on hospital utilization. Higher intensity post discharge support may be required to improve the patient experience upon returning home.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01580774","term_id":"NCT01580774"}}NCT01580774     

Mutations in the gene PRRT2 cause paroxysmal kinesigenic dyskinesia with infantile convulsions

Paroxysmal Kinesigenic Dyskinesia with Infantile Convulsions (PKD/IC) is an episodic movement disorder with autosomal dominant inheritance and high penetrance, but the causative gene is unknown. We have now identified four truncating mutations involving the PRRT2 gene in the vast majority (24/25) of well characterized families with PKD/IC. PRRT2 truncating mutations were also detected in 28 of 78 additional families. The PRRT2 gene encodes a proline-rich transmembrane protein of unknown function that has been reported to interact with the t-SNARE, SNAP25. PRRT2 localizes to axons but not to dendritic processes in primary neuronal culture and mutants associated with PKD/IC lead to dramatically reduced PRRT2 protein levels leading ultimately to neuronal hyperexcitability that manifests in vivo as PKD/IC.     

Differences between Spinocerebellar Ataxias and Multiple System Atrophy-Cerebellar Type on Proton Magnetic Resonance Spectroscopy

Purpose

A broad spectrum of diseases can manifest cerebellar ataxia. In this study, we investigated whether proton magnetic resonance spectroscopy (MRS) may help differentiate spinocerebellar ataxias (SCA) from multiple systemic atrophy- cerebellar type (MSA-C).

Material and Methods

This prospective study recruited 156 patients with ataxia, including spinocerebellar ataxia (SCA) types 1, 2, 3, 6 and 17 (N = 94) and MSA-C (N = 62), and 44 healthy controls. Single voxel proton MRS in the cerebellar hemispheres and vermis were measured. The differences were evaluated using nonparametric statistic tests.

Results

When compared with healthy controls, the cerebellar and vermis NAA/Cr and NAA/Cho were lower in all patients(p<0.002). The Cho/Cr was lower in SCA2 and MSA-C (p<0.0005). The NAA/Cr and Cho/Cr were lower in MSA-C or SCA2 comparing with SCA3 or SCA6. The MRS features of SCA1 were in between (p<0.018). The cerebellar NAA/Cho was lower in SCA2 than SCA1, SCA3 or SCA6 (p<0.04). The cerebellar NAA/Cho in MSA-C was lower than SCA3 (p<0.0005). In the early stages of diseases (SARA score<10), significant lower NAA/Cr and NAA/Cho in SCA2, SCA3, SCA6 or MSA-C were observed comparing with healthy controls (p<0.017). The Cho/Cr was lower in MSA-C or SCA2 (p<0.0005). Patients with MSA-C and SCA2 had lower NAA/Cr and Cho/Cr than SCA3 or SCA6 (p<0.016).

Conclusion

By using MRS, significantly lower NAA/Cr, Cho/Cr and NAA/Cho in the cerebellar hemispheres and vermis were found in patients with ataxia (SCAs and MSA-C). Rapid neuronal degeneration and impairment of membrane activities were observed more often in patients with MSA-C than those with SCA, even in early stages. MRS could also help distinguish between SCA2 and other subtypes of SCAs. MRS ratios may be of use as biomarkers in early stages of disease and should be further assessed in a longitudinal study.