GTP-stimulated membrane fission by the N-BAR protein AMPH-1.

Membrane-enclosed transport carriers sort biological molecules between stations in the cell in a dynamic process that is fundamental to the physiology of eukaryotic organisms. While much is known about the formation and release of carriers from specific intracellular membranes, the mechanism of carrier formation from the recycling endosome, a compartment central to cellular signaling, remains to be resolved. In Caenorhabditis elegans, formation of transport carriers from the recycling endosome requires the dynamin-like, Eps15-homology domain (EHD) protein, RME-1, functioning with the Bin/Amphiphysin/Rvs (N-BAR) domain protein, AMPH-1. Here we show, using a free-solution single-particle technique known as burst analysis spectroscopy (BAS), that AMPH-1 alone creates small, tubular-vesicular products from large, unilamellar vesicles by membrane fission. Membrane fission requires the amphipathic H0 helix of AMPH-1 and is slowed in the presence of RME-1. Unexpectedly, AMPH-1-induced membrane fission is stimulated in the presence of GTP. Furthermore, the GTP-stimulated membrane fission activity seen for AMPH-1 is recapitulated by the heterodimeric N-BAR amphiphysin protein from yeast, Rvs161/167p, strongly suggesting that GTP-stimulated membrane fission is a general property of this important class of N-BAR proteins.

Diagnostic value of plasma and blood cells metagenomic next-generation sequencing in patients with sepsis.

BACKGROUND: Several studies have investigated the detection of plasma cell-free DNA (cfDNA) using metagenomic next-generation sequencing (mNGS). However, to our knowledge, no study has evaluated the diagnostic value of mNGS detection using blood cells. In this study, we aimed to evaluate the performance of a whole blood mNGS assay which includes the results of plasma and blood cells mNGS detection. METHODS: We selected a panel of seven microorganisms to validate both the plasma and blood cells assay for their limits of detection (LoD), linearity, precision, and robustness to interference. In a multicentered prospective study conducted from January 2021 to April 2022, we tested 253 septic patients with plasma and blood cells mNGS and compared it with blood cultures (BCs). The performance of pathogen detection was compared between mNGS and BCs. RESULTS: The LoD for plasma and blood cells mNGS was 8.3-140 genome equivalents (GE)/mL and 26 to 534 colony-forming units (CFU) or copies/mL, respectively. The inter- and intra-assay reproducibility of both plasma and blood cells mNGS was 100%. Compared to plasma mNGS alone, the sensitivity of whole blood mNGS was increased by 18.04% when using BCs as the standard (67.21% vs 85.25%). Furthermore, the sensitivity of whole blood mNGS in diagnosing bloodstream infections (BSIs) was 85.21%, which was significantly higher than that of BCs (36.09%, P＜0.0001) and plasma mNGS (69.82%; P = 0.0007). Additional analysis showed that blood cells mNGS was able to detect bacteria missed by plasma mNGS, while plasma mNGS was effective at detecting viruses. CONCLUSIONS: Our findings indicate that whole blood mNGS shows great potential as a promising diagnostic technique for BSIs owing to its ability to identify pathogens with higher sensitivity.

Parasite Circulating Cell-free DNA in the Blood of Alveolar Echinococcosis Patients as a Diagnostic and Treatment-Status Indicator.

BACKGROUND: Alveolar echinococcosis (AE) is a serious parasitic disease caused by the larvae of Echinococcus multilocularis. It is the less common but substantially more deadly of the 2 major echinococcosis diseases that can occur globally but are concentrated in central Asia. METHODS: We analyzed parasite circulating cell-free DNA (cfDNA) in 149 plasma samples using a DNA sequencing-based method (105 AE, 16 cystic echinococcosis, 4 liver cancer, 4 gallstones, and 20 healthy volunteers). After identifying the Echinococcus-specific cfDNA (Em-cfDNA) sequences in the samples, we determined whether Em-cfDNA could be used for AE diagnosis and as a potential indicator of the effectiveness of surgical treatment. We also examined potential associations between Em-cfDNA levels and clinical features of AE patients. RESULTS: Our work demonstrates that varying reads of Em-cfDNA were detectable in the plasma of 100% of preoperative AE patients and that all of the non-AE patients and healthy volunteers were negative. Em-cfDNA has good sensitivity and specificity for the diagnosis of AE. We also found that Em-cfDNA levels apparently have reference value for evaluating the therapeutic efficacy of surgery interventions for AE lesions. Finally, our analysis revealed that Em-cfDNA levels can reflect meaningful information about lesion size in preoperative AE patients. CONCLUSIONS: We demonstrate that sequencing-based monitoring of Em-cfDNA can be used in the clinic as a powerful diagnostic indicator for AE. We also note that there is a strong potential for use of this liquid-biopsy method to monitor ongoing disease status in postintervention AE patients.

Confirmation of Rickettsia conorii Subspecies indica Infection by Next-Generation Sequencing, Shandong, China.

We describe 3 similar cases of rickettsial disease that occurred after tick bites in a mountainous rural area of Shandong Province, China. Next-generation sequencing indicated the etiologic agent of 1 patient was Rickettsia conorii subspecies indica. This agent may be more widely distributed across China than previously thought.

Alpha-synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation.

Aggregation of α-synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca2+ and α-synuclein aggregation. Analyses of cell lines and primary culture models of α-synuclein cytopathology reveal an early phase with reduced cytosolic Ca2+ levels followed by a later Ca2+ increase. Aggregated but not monomeric α-synuclein binds to and activates SERCA in vitro, and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca2+ CPA protects the cells against α-synuclein-aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo Proximity ligation assays also reveal an increased interaction between α-synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α-synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down-stream processes may be therapeutic targets for treating α-synucleinopathies.

Employing Shadows for Multi-Person Tracking Based on a Single RGB-D Camera.

Although there are many algorithms to track people that are walking, existing methods mostly fail to cope with occluded bodies in the setting of multi-person tracking with one camera. In this paper, we propose a method to use people's shadows as a clue to track them instead of treating shadows as mere noise. We introduce a novel method to track multiple people by fusing shadow data from the RGB image with skeleton data, both of which are captured by a single RGB Depth (RGB-D) camera. Skeletal tracking provides the positions of people that can be captured directly, while their shadows are used to track them when they are no longer visible. Our experiments confirm that this method can efficiently handle full occlusions. It thus has substantial value in resolving the occlusion problem in multi-person tracking, even with other kinds of cameras.

Multiband mucosectomy versus endoscopic submucosal dissection and endoscopic submucosal excavation for GI submucosal tumors: short and long term follow-up.

AIMS: To evaluate the short- and long-term outcomes of 3 different endoscopic dissection techniques for upper gastrointestinal (GI) submucosal tumours (SMTs). METHODS: Data for 135 patients withGI SMTs who underwent multiband mucosectomy (MBM), endoscopic submucosal dissection (ESD), or endoscopic submucosal excavation (ESE) were retrospectively assessed. The en bloc resection rate, endoscopic complete resection rate, operation time, potential complications and local recurrence rate were compared. RESULTS: No significant differences were observed in the rate of endoscopic complete resections and pathologic complete resections among the three groups. For SMTs > 15 mm in width, the lowest en bloc resection rate was found for MBM (P = 0.000). MBM was also associated with the shortest procedure time, lowest perforation rate and lowest rate of major bleeding. ESE was the most effective procedure for muscularis propria (MP) lesions but was associated with the longest operation time (P < 0.01). The ESD and ESE groups had similar perforation rates (P > 0.05). No differences were observed in 4-year local recurrence rates among the groups (P = 0.945). CONCLUSIONS: MBM is a simple and effective method for the treatment of small SMTs and achieves clinical success rates similar to those of ESD and ESE. However, ESD and ESE are preferable for larger and deep lesions and are associated with a longer operation time. Nonetheless, all 3 techniques resulted in a low 4-year local recurrence rate. Large-scale randomized clinical trials are needed to further investigate these results.

MiR-375 Mediates Chondrocyte Metabolism and Oxidative Stress in Osteoarthritis Mouse Models through the JAK2/STAT3 Signaling Pathway.

OBJECTIVE: The aim of this work was to determine the effect of miR-375 on chondrocyte metabolism and oxidative stress in osteoarthritis (OA) mouse models through the JAK2/STAT3 signaling pathway. METHODS: Chondrocytes were divided into control, IL-1ß, IL-1ß + miR-375 mimic, IL-1ß + miR-375 inhibitor, IL-1ß + miR-NC (negative control), and IL-1ß + miR-375 inhibitor + siJAK2 groups. The chondrocyte proliferation was determined by MTT assay, the superoxide dismutase (SOD) and malondialdehyde (MDA) levels by corresponding kits, and the chondrocyte apoptosis by TUNEL staining. Furthermore, OA mouse models were divided into Sham, OA + miR-NC, and OA + miRNA-375 antagomir groups. The pathological changes were observed, and the expressions of miR-375 and the JAK2/STAT3 pathway were determined by qRT-PCR and Western blotting, respectively. RESULTS: IL-1ß-induced chondrocytes had significant increases in miR-375 and MDA, with decreased proliferation and SOD levels, as compared to the control group. Meanwhile, they also exhibited elevated apoptosis, with upregulations of ADAMTS-5 and MMP-13 and downregulations of COL2A1 and ACAN, as well as decreased p-JAK2/JAK2, p-STAT3/STAT3, and Bcl-2/Bax. However, these changes were significantly improved after transfection with miR-375 inhibitor, but transfection with miR-375 mimic resulted in severer exacerbation. Notably, the improvement of miR-375 inhibitor could be abolished by transfection with siJAK2. Furthermore, miR-375 antagomir significantly alleviated OA progression in OA mice in vivo. CONCLUSION: MiR-375 suppression enhanced the ability of chondrocyte to antagonize the oxidative stress and maintained the homeostasis of extracellular matrix metabolism to protect chondrocytes from OA via activation of the JAK2/STAT3 pathway, indicating that miR-375 is a potential molecular target for OA treatment.

Aging, memory, and nonhierarchical energy landscape of spin jam.

The notion of complex energy landscape underpins the intriguing dynamical behaviors in many complex systems ranging from polymers, to brain activity, to social networks and glass transitions. The spin glass state found in dilute magnetic alloys has been an exceptionally convenient laboratory frame for studying complex dynamics resulting from a hierarchical energy landscape with rugged funnels. Here, we show, by a bulk susceptibility and Monte Carlo simulation study, that densely populated frustrated magnets in a spin jam state exhibit much weaker memory effects than spin glasses, and the characteristic properties can be reproduced by a nonhierarchical landscape with a wide and nearly flat but rough bottom. Our results illustrate that the memory effects can be used to probe different slow dynamics of glassy materials, hence opening a window to explore their distinct energy landscapes.

Caspase-1 causes truncation and aggregation of the Parkinson's disease-associated protein α-synuclein.

The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson's disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well.

Comparison of Endoscopic Resection and Minimally Invasive Esophagectomy in Patients With Early Esophageal Cancer.

OBJECTIVE: To determine whether endoscopic resection (ER) and minimally invasive esophagectomy (MIE) are safe and effective for treating squamous intraepithelial neoplasia of the esophagus. MATERIALS AND METHODS: This study retrospectively analyzed a total of 99 consecutive patients with pathologically confirmed early esophageal cancer between December 2007 and 2011. ER was performed in 59 patients, whereas MIE was performed in 40 patients. We compared the 2 groups according to R0 resection rates, treatment-related complications, mean hospital stay, local recurrence rates, and 3- and 4-year overall survival. RESULTS: No significant differences were found in the R0 resection rates between ER and MIE (94.9% vs. 97.5%, P>0.05). The occurrence rate of minor complications in the ER group was significantly lower than that in the thoracoscopic esophagectomy group (11.8% vs. 32.5%, P>0.05). The mean operative time in the ER group was 74±23 minutes, which was significantly shorter than that in the MIE group (298±46 min). The average length of hospital stay in the ER group was significantly shorter than that in the MIE group (P<0.001). No significant differences were observed in the local recurrence rates between the 2 groups (P>0.05). Similarly, no differences were found in the 3-year survival rate (ER: 96.6%, vs. MIE: 97.5%, P>0.05) and 4-year survival rate (ER: 91.5% vs. MIE: 90%, P>0.05) between the 2 groups. CONCLUSIONS: ER achieves the same positive results as MIE in the treatment of early esophageal cancer and is associated with a lower complication rate, a shorter recovery time, and a similar survival rate. However, multiple ER procedures were required for several patients in this study.

Multiband Mucosectomy Versus Endoscopic Submucosal Dissection for Treatment of Squamous Intraepithelial Neoplasia of the Esophagus.

BACKGROUND & AIMS: We compared the efficacy and safety of multiband mucosectomy (MBM) vs endoscopic submucosal dissection (ESD) for the treatment of squamous intraepithelial neoplasia of the esophagus. METHODS: We performed a retrospective study of 78 patients with squamous intraepithelial neoplasia of the esophagus who received either ESD or MBM between January 2009 and January 2011 at the Tengzhou Central People's Hospital in China. We compared rates of bloc resection and curative resection, as well as complications and local recurrence, between groups. RESULTS: Overall, there was no statistical difference in the rate of complete resection between patients who received ESD (95.8%) vs MBM (93%) (P > .05). For tumors less than 15 mm in width, ESD produced a significantly higher rate of en bloc resection (100%) and curative resection (92.3%) than MBM (44.8% and 41%; P < .05). No significant differences were found between lesions less than 15 mm. MBM had a significantly shorter procedure time (38 ± 11 min) than ESD (84 ± 35 min) (P < .05). Major bleeding occurred in 1.85% of MBM procedures and in 16.7% of ESD procedures (P > .05). ESD led to perforations in 8.3% of cases, whereas MBM did not lead to any perforations (P < .05). No significant differences were found between groups in proportions of cases with postoperative esophageal strictures (16.7% vs 14.8%; P > .05) or the 3-year rate of local recurrence (P > .05). CONCLUSIONS: Based on a retrospective comparison of patients who underwent ESD vs MBM for squamous intraepithelial neoplasia of the esophagus, ESD should be reserved for patients with larger neoplastic lesions (>15 mm), with respect to the success of attempted en bloc resection and the number of curative resections achieved. However, ESD has longer procedure times and higher rates of complication. MBM allows for safe and easy piecemeal resections, and is associated with similar levels of clinical success as ESD for lesions less than 15 mm. Large, randomized, controlled studies are needed to determine which endoscopic resection modality is superior for patients with high-grade intraepithelia neoplasms.

In Situ Observation of Dark Current Emission in a High Gradient rf Photocathode Gun.

Undesirable electron field emission (also known as dark current) in high gradient rf photocathode guns deteriorates the quality of the photoemission current and limits the operational gradient. To improve the understanding of dark current emission, a high-resolution (∼100 µm) dark current imaging experiment has been performed in an L-band photocathode gun operating at ∼100 MV/m of surface gradient. Scattered strong emission areas with high current have been observed on the cathode. The field enhancement factor ß of selected regions on the cathode has been measured. The postexaminations with scanning electron microscopy and white light interferometry reveal the origins of ∼75% strong emission areas overlap with the spots where rf breakdown has occurred.

Tunable High-Intensity Electron Bunch Train Production Based on Nonlinear Longitudinal Space Charge Oscillation.

High-intensity trains of electron bunches with tunable picosecond spacing are produced and measured experimentally with the goal of generating terahertz (THz) radiation. By imposing an initial density modulation on a relativistic electron beam and controlling the charge density over the beam propagation, density spikes of several-hundred-ampere peak current in the temporal profile, which are several times higher than the initial amplitudes, have been observed for the first time. We also demonstrate that the periodic spacing of the bunch train can be varied continuously either by tuning launching phase of a radio-frequency gun or by tuning the compression of a downstream magnetic chicane. Narrow-band coherent THz radiation from the bunch train was also measured with µJ-level energies and tunable central frequency of the spectrum in the range of ∼0.5 to 1.6 THz. Our results pave the way towards generating mJ-level narrow-band coherent THz radiation and driving high-gradient wakefield-based acceleration.

The degree of astrocyte activation in multiple system atrophy is inversely proportional to the distance to α-synuclein inclusions.

Multiple system atrophy (MSA) exhibits widespread astrogliosis together with α-synuclein (α-syn) glial cytoplasmic inclusions (GCIs) in mature oligodendrocytes. We quantified astrocyte activation by morphometric analysis of MSA cases, and investigated the correlation to GCI proximity. Using Imaris software, we obtained "skinned" three-dimensional models of GFAP-positive astrocytes in MSA and control tissue (n=75) from confocal z-stacks and measured the astrocyte process length and thickness and radial distance to the GCI. Astrocytes proximal to GCI-containing oligodendrocytes (r<25µm) had significantly (p, 0.05) longer and thicker processes characteristic of activation than distal astrocytes (r>25µm), with a reciprocal linear correlation (m, 90µm(2)) between mean process length and radial distance to the nearest GCI (R(2), 0.7). In primary cell culture studies, α-syn addition caused ERK-dependent activation of rat astrocytes and perinuclear α-syn inclusions in mature (MOSP-positive) rat oligodendrocytes. Activated astrocytes were also observed in close proximity to α-syn deposits in a unilateral rotenone-lesion mouse model. Moreover, unilateral injection of MSA tissue-derived α-syn into the mouse medial forebrain bundle resulted in widespread neuroinflammation in the α-syn-injected, but not sham-injected hemisphere. Taken together, our data suggests that the action of localized concentrations of α-syn may underlie both astrocyte and oligodendrocyte MSA pathological features.

Generation and characterization of novel conformation-specific monoclonal antibodies for α-synuclein pathology.

α-Synuclein (α-syn), a small protein that has the intrinsic propensity to aggregate, is implicated in several neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), which are collectively known as synucleinopathies. Genetic, pathological, biochemical, and animal modeling studies provided compelling evidence that α-syn aggregation plays a key role in the pathogenesis of PD and related synucleinopathies. It is therefore of utmost importance to develop reliable tools that can detect the aggregated forms of α-syn. We describe here the generation and characterization of six novel conformation-specific monoclonal antibodies that recognize specifically α-syn aggregates but not the soluble, monomeric form of the protein. The antibodies described herein did not recognize monomers or fibrils generated from other amyloidogenic proteins including ß-syn, γ-syn, ß-amyloid, tau protein, islet amyloid polypeptide and ABri. Interestingly, the antibodies did not react to overlapping linear peptides spanning the entire sequence of α-syn, confirming further that they only detect α-syn aggregates. In immunohistochemical studies, the new conformation-specific monoclonal antibodies showed underappreciated small micro-aggregates and very thin neurites in PD and DLB cases that were not observed with generic pan antibodies that recognize linear epitope. Furthermore, employing one of our conformation-specific antibodies in a sandwich based ELISA, we observed an increase in levels of α-syn oligomers in brain lysates from DLB compared to Alzheimer's disease and control samples. Therefore, the conformation-specific antibodies portrayed herein represent useful tools for research, biomarkers development, diagnosis and even immunotherapy for PD and related pathologies.

Observation of Field-Emission Dependence on Stored Energy.

Field emission from a solid metal surface has been continuously studied for a century over macroscopic to atomic scales. It is general knowledge that, other than the surface properties, the emitted current is governed solely by the applied electric field. A pin cathode has been used to study the dependence of field emission on stored energy in an L-band rf gun. The stored energy was changed by adjusting the axial position (distance between the cathode base and the gun back surface) of the cathode while the applied electric field on the cathode tip is kept constant. A very strong correlation of the field-emission current with the stored energy has been observed. While eliminating all possible interfering sources, an enhancement of the current by a factor of 5 was obtained as the stored energy was increased by a factor of 3. It implies that under certain circumstances a localized field emission may be significantly altered by the global parameters in a system.

Non-neuronal and neuronal BACE1 elevation in association with angiopathic and leptomeningeal ß-amyloid deposition in the human brain.

BACKGROUND: Cerebral amyloid angiopathy (CAA) refers to the deposition of ß-amyloid (Aß) peptides in the wall of brain vasculature, commonly involving capillaries and arterioles. Also being considered a part of CAA is the Aß deposition in leptomeninge. The cellular origin of angiopathic Aß and the pathogenic course of CAA remain incompletely understood. METHODS: The present study was aimed to explore the pathogenic course of CAA in the human cerebrum via examination of changes in ß-secretase-1 (BACE1), the obligatory Aß producing enzyme, relative to Aß and other cellular markers, by neuroanatomical and biochemical characterizations with postmortem brain samples and primary cell cultures. RESULTS: Immunoreactivity (IR) for BACE1 was essentially not visible at vasculature in cases without cerebral amyloidosis (control group, n = 15, age = 86.1 ± 10.3 year). In cases with brain amyloid pathology (n = 15, age = 78.7 ± 12.7 year), increased BACE1 IR was identified locally at capillaries, arterioles and along the pia, localizing to endothelia, perivascular dystrophic neurites and meningeal cells, and often coexisting with vascular iron deposition. Double immunofluorescence with densitometric analysis confirmed a site-specific BACE1 elevation at cerebral arterioles in the development of vascular Aß deposition. Levels of BACE1 protein, activity and its immediate product (C99) were elevated in leptomeningeal lysates from cases with CAA relative to controls. The expression of BACE1 and other amyloidogenic proteins in the endothelial and meningeal cells was confirmed in primary cultures prepared from human leptomeningeal and arteriolar biopsies. CONCLUSION: These results suggest that BACE1 elevation in the endothelia and perivascular neurites may be involved in angiopathic Aß deposition, while BACE1 elevation in meningeal cells might contribute Aß to leptomeningeal amyloidosis.

A dual-site simultaneous binding mode in the interaction between parallel-stranded G-quadruplex [d(TGGGGT)]4 and cyanine dye 2,2'-diethyl-9-methyl-selenacarbocyanine bromide.

G-quadruplexes have attracted growing attention as a potential cancer-associated target for both treatment and detection in recent years. For detection purpose, high specificity is one of the most important factors to be considered in G-quadruplex probe design. It is well known that end stacking and groove binding are two dominated quadruplex-ligand binding modes, and currently most reported G-quadruplex probes are designed based on the former, which has been proven to show good selectivity between quadruplexes and non-quadruplexes. Because groove of G-quadruplex also has some unique chemical properties, it could be inferred that probes that can interact with both the groove and G-tetrad site of certain G-quadruplexes simultaneously might possess higher specificity in aspects of discriminating different quadruplexes. In this article, we report a cyanine dye as a potential novel probe scaffold that could occupy both the 5'-end external G-tetrad and the corresponding groove of the G-quadruplex simultaneously. By using various spectrum and nuclear magnetic resonance techniques, we give a detailed binding characterization for this dual-site simultaneous binding mode. A preliminary result suggests that this mode might provide highly specific recognition to a parallel-stranded G-quadruplex. These findings and the structural elucidation might give some clues in aspects of developing highly specific G-quadruplex probes.

Evidence that the LRRK2 ROC domain Parkinson's disease-associated mutants A1442P and R1441C exhibit increased intracellular degradation.

Mutations in the leucine-rich repeat kinase 2 (lrrk2) gene are the leading genetic cause of Parkinson's disease (PD). In characterizing the novel ROC domain mutant A1442P, we compared its steady-state protein levels, propensity to aggregate, and toxicity with the pathogenic R1441C mutant and wild-type (WT) LRRK2. Mutant (R1441C and A1442P) and WT LRRK2 fused to green fluorescent protein (GFP) and FLAG were transiently expressed in HEK293 cells using plasmid constructs. Western analysis and fluorescence microscopy consistently demonstrated lower mutant LRRK2 protein levels compared with WT. A time-course expression study using flow cytometry showed that WT LRRK2 expression increased initially but then plateaued by 72 hr. Conversely, R1441C and A1442P mutant expression attained 85% and 74% of WT levels at 24 hr but fell to 68% and 55% of WT levels by 72 hr, respectively. We found that proteasome inhibition markedly increased mutant LRRK2 to levels approaching those of WT. Taken together, our findings reveal increased intracellular degradation for both mutants. Furthermore, the impact of mutant and WT LRRK2 expression on HEK293 cell viability was assessed under normative and oxidative (hydrogen peroxide) conditions and found not to differ. Expression of WT and mutant LRRK2 protein gave rise to intracellular aggregates of similar appearance and cellular localization. In summary, we provide evidence that the novel A1442P mutant and the previously investigated R1441C pathogenic mutant exhibit increased intracellular degradation, a property reportedly demonstrated for the pathogenic LRRK2 kinase domain mutant I2020T.