α-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity.

α-Synuclein (α-syn) phosphorylation at serine 129 (pS129­α-syn) is substantially increased in Lewy body disease, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129­α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129­α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129­α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129­α-syn (WT­α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129­α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129­α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129­α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129­α-syn as a measure of efficacy in clinical trials.

Evaluation of modified frailty index for predicting post-operative outcomes after lower-extremity free-flap reconstruction.

BACKGROUND: Risk stratification for complex procedures such as microsurgical reconstruction of the lower extremities is an important part of preoperative planning and counseling. The aim of this study was to determine the effectiveness of the modified five-item frailty index (5-mFI) score, a validated tool for assessing risk in surgical patients, in predicting postoperative complications after lower extremity (LE) free flap reconstruction. METHODS: A retrospective review of the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database was conducted from 2010 to 2020 on patients who underwent LE free-flap reconstruction. 5-mFI scores were calculated and patients were categorized as 5-mFI ≥2 or <2. The primary endpoint was the presence of 30-day overall complications. The secondary endpoints included 30-day readmission, need for reoperation, and need to discharge to a care facility. Comparisons were made using one-way analysis of variances, Pearson's chi-squared test, or Fisher's extract test. Multivariable logistic regression models were performed for sex, age, BMI, smoker status, operative time, and ASA classification. RESULTS: Total of 294 (61.6% males) patients were identified. Univariate analysis showed 5-mFI ≥2 had higher rates of overall complications (p = .043) and hematologic complications (p = .033). In this population, there were also higher rates of reoperation (p = .003) and discharge to care facility (p < .001). Multivariable regression models further substantiated that 5-mFI ≥2 was independently associated with increased overall complications [2.46, CI: 1.10-5.59, p = .031], hematologic complications [2.55, 1.02-6.35, p = .046], reoperation [4.55, 1.54-13.3, p = .006], and discharge to facility [2.86, 1.27-6.45, p = .011]. CONCLUSIONS: There is a strong association of 5-mFI ≥2 with adverse post-operative outcomes in male patients undergoing LE free-flap reconstruction. This can be a valuable adjunct in the counsel of patients for whom lower extremity salvage is feasible.

History of Parkinson's Disease-Associated Gene, Parkin: Research over a Quarter Century in Quest of Finding the Physiological Substrate.

Parkin, the gene responsible for hereditary Parkinson's disease (PD) called "Autosomal Recessive Juvenile Parkinsonism (AR-JP)" was discovered a quarter of a century ago. Owing to its huge gene structure and unique protein functions, parkin has become a subject of interest to those involved in PD research and researchers and clinicians in various fields and is being vigorously studied worldwide in relation to its nature and disease. The gene structure was registered under the gene name "parkin" in the GenBank in 1997. In 1998, deletion and point mutations in the parkin gene were reported, thereby demonstrating parkin is the causative gene for hereditary PD. Although 25 years have passed since the gene's discovery and many researchers have worked tirelessly to elucidate the function of the Parkin protein and the mechanism of its role against neuronal cell death and pathogenesis remain unknown, which raises a major question concerning the current leading hypothesis. In this review, we present the results of related research on the parkin gene in chronological order and discuss unresolved problems concerning its function and pathology as well as new trends in the research conducted to solve them. The relationship between parkin and tumorigenesis has also been addressed from the perspective of Parkin's redox molecule.

ATP13A2 Gene Silencing in Drosophila Affects Autophagic Degradation of A53T Mutant α-Synuclein.

Mutations in ATP13A2 (PARK9), an autophagy-related protein, cause Kufor-Rakeb syndrome, an autosomal recessive, juvenile-onset form of parkinsonism. α-Synuclein (α-syn) is a presynaptic neuronal protein that forms toxic aggregates in Parkinson's disease (PD). We studied α-syn aggregation and autophagic flux in ATP13A2-knockdown Drosophila expressing either wild-type (WT) or mutant α-syn. Dopaminergic (DA) neuron loss was studied by confocal microscopy. Sleep and circadian activity were evaluated in young and old flies using a Drosophila activity monitor. Thirty-day-old ATP13A2-RNAi A53T-α-syn flies had increased Triton-insoluble α-syn levels, compared to control A53T-α-syn flies without ATP13A2-RNAi. Whole-brain staining revealed significantly fewer dopaminergic (DA) neurons in the PPL2 cluster of 30-day-old ATP13A2-RNAi flies expressing WT-, A30P-, and A53T-α-syn than in that of controls. In ATP13A2-RNAi A53T-α-syn flies, autophagic flux was decreased, as indicated by increased accumulation of Ref(2)P, the Drosophila p62 homologue. ATP13A2 silencing decreased total locomotor activity in young, and enhanced sleep features, similar to PD (decreasing bout length), in old flies expressing A53T-α-syn. ATP13A2 silencing also altered the circadian locomotor activity of A30P- and A53T-α-syn flies. Thus, ATP13A2 may play a role in the autophagic degradation of A53T-α-syn.

Parkin Precipitates on Mitochondria via Aggregation and Autoubiquitination.

The loss of the E3 ligase Parkin, in a familial form of Parkinson's disease, is thought to cause the failure of both the polyubiquitination of abnormal mitochondria and the consequent induction of mitophagy, resulting in abnormal mitochondrial accumulation. However, this has not been confirmed in patient autopsy cases or animal models. More recently, the function of Parkin as a redox molecule that directly scavenges hydrogen peroxide has attracted much attention. To determine the role of Parkin as a redox molecule in the mitochondria, we overexpressed various combinations of Parkin, along with its substrates FAF1, PINK1, and ubiquitin in cell culture systems. Here, we observed that the E3 Parkin monomer was surprisingly not recruited to abnormal mitochondria but self-aggregated with or without self-ubiquitination into the inner and outer membranes, becoming insoluble. Parkin overexpression alone generated aggregates without self-ubiquitination, but it activated autophagy. These results suggest that for damaged mitochondria, the polyubiquitination of Parkin substrates on the mitochondria is not indispensable for mitophagy.

Literature Review: Robotic-Assisted Harvest of Deep Inferior Epigastric Flap for Breast Reconstruction.

INTRODUCTION: Robotic-assisted surgery is gaining popularity because of reported improvement in aesthetic outcomes while reducing the occurrence of complications compared with conventional surgical methods. Deep inferior epigastric perforator (DIEP) flap harvesting has a long track record as a viable procedure for autologous reconstruction of the breast. In this literature review, we describe the feasibility of using the robotic platform in DIEP flap harvest. METHODS: The Preferred Reporting Items for Systemic Reviews and Meta-Analysis methodology was to guide the literature review. PubMed and Scopus databases were searched from inception to June 6, 2022. The Medical Subject Heading terms and keywords used to conduct this search are as described: "Robotic AND deep inferior epigastric perforator AND Breast reconstruction." RESULTS: Seven publications, detailing a total of 56 robotic-assisted DIEP flap harvest procedures, were selected for review. Four publications used the transabdominal preperitoneal approach, whereas 2 exclusively used a totally extraperitoneal approach, and 1 compared the 2 approaches. The measured outcomes included technical feasibility of flap harvest in cadavers, viable flap harvest in live patients, harvest time and pedicle dissection time, pedicle length, fascial incision length, donor site pain, need for postoperative narcotic, donor site morbidity, and hernia formation. Overall, the reviewed articles demonstrated successful DIEP flap harvesting without the need for conversion to the conventional open procedure. Postoperative complications were minimal. Robotic DIEP flap harvest was shown to be safe and there were no reports of donor-site morbidity in the studies reviewed. The main advantages of the robotic approach include decreased postoperative pain and length of hospital stay, along with improved aesthetic outcomes. The main disadvantages are increased operative time and cost. CONCLUSIONS: Although at its current iteration, the robotic-assisted DIEP flap is feasible, it may not be practical in all settings. Furthermore, the true benefit of the robotic platform is yet to be determined, as more long-term studies are necessary.

Short-term physical exercise impacts on the human holobiont obtained by a randomised intervention study.

BACKGROUND: Human well-being has been linked to the composition and functional capacity of the intestinal microbiota. As regular exercise is known to improve human health, it is not surprising that exercise was previously described to positively modulate the gut microbiota, too. However, most previous studies mainly focused on either elite athletes or animal models. Thus, we conducted a randomised intervention study that focused on the effects of different types of training (endurance and strength) in previously physically inactive, healthy adults in comparison to controls that did not perform regular exercise. Overall study duration was ten weeks including six weeks of intervention period. In addition to 16S rRNA gene amplicon sequencing of longitudinally sampled faecal material of participants (six time points), detailed body composition measurements and analysis of blood samples (at baseline and after the intervention) were performed to obtain overall physiological changes within the intervention period. Activity tracker devices (wrist-band wearables) provided activity status and sleeping patterns of participants as well as exercise intensity and heart measurements. RESULTS: Different biometric responses between endurance and strength activities were identified, such as a significant increase of lymphocytes and decrease of mean corpuscular haemoglobin concentration (MCHC) only within the strength intervention group. In the endurance group, we observed a significant reduction in hip circumference and an increase in physical working capacity (PWC). Though a large variation of microbiota changes were observed between individuals of the same group, we did not find specific collective alterations in the endurance nor the strength groups, arguing for microbiome variations specific to individuals, and therefore, were not captured in our analysis. CONCLUSIONS: We could show that different types of exercise have distinct but moderate effects on the overall physiology of humans and very distinct microbial changes in the gut. The observed overall changes during the intervention highlight the importance of physical activity on well-being. Future studies should investigate the effect of exercise on a longer timescale, investigate different training intensities and consider high-resolution shotgun metagenomics technology. TRIAL REGISTRATION: DRKS, DRKS00015873 . Registered 12 December 2018; Retrospectively registered.

Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of dopamine neurons and the deposition of misfolded proteins known as Lewy bodies (LBs), which contain α-synuclein (α-syn). The causes and molecular mechanisms of PD are not clearly understood to date. However, misfolded proteins, oxidative stress, and impaired autophagy are believed to play important roles in the pathogenesis of PD. Importantly, α-syn is considered a key player in the development of PD. The present study aimed to assess the role of Ellagic acid (EA), a polyphenol found in many fruits, on α-syn aggregation and toxicity. Using thioflavin and seeding polymerization assays, in addition to electron microscopy, we found that EA could dramatically reduce α-syn aggregation. Moreover, EA significantly mitigated the aggregated α-syn-induced toxicity in SH-SY5Y cells and thus enhanced their viability. Mechanistically, these cytoprotective effects of EA are mediated by the suppression of apoptotic proteins BAX and p53 and a concomitant increase in the anti-apoptotic protein, BCL-2. Interestingly, EA was able to activate autophagy in SH-SY5Y cells, as evidenced by normalized/enhanced expression of LC3-II, p62, and pAKT. Together, our findings suggest that EA may attenuate α-syn toxicity by preventing aggregation and improving viability by restoring autophagy and suppressing apoptosis.

Development of Nonviral Vectors Targeting the Brain as a Therapeutic Approach For Parkinson's Disease and Other Brain Disorders.

Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by tremor, rigidity, bradykinesia, and postural instability, for which there is no effective treatment available till date. Here, we report the development of nonviral vectors specific for neuronal cells that can deliver short interfering RNA (siRNA) against the α-synuclein gene (SNCA), and prevent PD-like symptoms both in vitro and in vivo. These vectors not only help siRNA duplexes cross the blood-brain barrier in mice, but also stabilize these siRNAs leading to a sustainable 60-90% knockdown of α-synuclein protein. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine rapidly develop PD-like symptoms which were significantly alleviated when SNCA was knocked down using our vectors. Together, our data not only confirm the central role of α-synuclein in the onset of PD, but also provide a proof of principle that these nonviral vectors can be used as novel tools to design effective strategies to combat central nervous system diseases.

Prospective analysis of the sealing ability of the ENSEAL® G2 Articulating Tissue Sealer and transector on human mesenteric vessels in colorectal surgery.

BACKGROUND: The sealing and transection of mesenteric vessels is a crucial step in minimally invasive colorectal surgery. We examined the sealing quality of the ENSEAL® G2 Articulating Tissue Sealer in three different articulations in mesenteric vessels. METHODS: This was a prospective experimental study within a tertiary healthcare center, and 30 patients were recruited. Burst pressures for each specimen were measured as the primary outcome. Ten specimens at each of the three articulations were also histologically assessed for the quality of seal. RESULTS: We evaluated 54 sets of specimens from 30 patients for bursting pressure, all of which were harvested and sealed in the operating room. No statistical difference was seen in burst pressures from seals recorded at no angulation, half-maximal angulation, or maximal angulation (1604, 1507, 1478 mmHg; p = 0.07). Histological analysis showed no statistical differences in the average vessel diameter (p = 0.57), lateral extent of thermal injury (p = 0.48), degree of vascular sclerosis, or the integrity of seal at the three articulations. No cases of intraoperative or postoperative bleeding were observed in any of the patients. Five (16.7%) of the ENSEAL® devices developed breaks in the black, heat-shrink, polyethylene covering as a result of repeated articulation and disarticulation. Electrical arcing did not appear to have occurred as a result of the break, although this was not formally examined. CONCLUSIONS: The maximum sustainable pressure in mesenteric vessels sealed with a bipolar electrothermal device is supraphysiological, and consequently, the device can be safely used at various articulations to seal vessels during colorectal surgery.

Relation of the Allelic Variants of Multidrug Resistance Gene to Agranulocytosis Associated With Clozapine.

Clozapine use is associated with leukopenia and more rarely agranulocytosis, which may be lethal. The drug and its metabolites are proposed to interact with the multidrug resistance transporter (ABCB1/MDR1) gene product, P-glycoprotein (P-gp). Among various P-glycoprotein genetic polymorphisms, nucleotide changes in exons 26 (C3435T), 21 (G2677T), and 12 (C1236T) have been implicated for changes in pharmacokinetics and pharmacodynamics of many substrate drugs. In this study, we aimed to investigate the association between these specific ABCB1 polymorphisms and clozapine-associated agranulocytosis (CAA). Ten patients with a history of CAA and 91 control patients without a history of CAA, despite 10 years of continuous clozapine use, were included. Patient recruitment and blood sample collection were conducted at the Hacettepe University Faculty of Medicine, Department of Psychiatry, in collaboration with the members of the Schizophrenia and Other Psychotic Disorders Section of the Psychiatric Association of Turkey, working in various psychiatry clinics. After DNA extraction from peripheral blood lymphocytes, genotyping was performed using polymerase chain reaction and endonuclease digestion. Patients with CAA had shorter duration of clozapine use but did not show any significant difference in other clinical, sociodemographic characteristics and in genotypic or allelic distributions of ABCB1 variants and haplotypes compared with control patients. Among the 10 patients with CAA, none carried the ABCB1 all-variant haplotype (TT-TT-TT), whereas the frequency of this haplotype was approximately 12% among the controls. Larger sample size studies and thorough genetic analyses may reveal both genetic risk and protective factors for this serious adverse event.

Ginsenoside Rb1 inhibits fibrillation and toxicity of alpha-synuclein and disaggregates preformed fibrils.

Compelling evidence indicates that α-synuclein (α-syn) aggregation plays a central role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies. Identification of compounds that inhibit or reverse the aggregation process may thus represent a viable therapeutic strategy against PD and related disorders. Ginseng is a well-known medicinal plant that has been used in East Asia for more than two thousand years to treat several conditions. It is now understood that the pharmacological properties of ginseng can be attributed to its biologically active components, the ginsenosides, which in turn have been shown to have neuroprotective properties. We therefore sought to determine for the first time, the potential of the most frequently used and studied ginsenosides, namely Rg1, Rg3 and Rb1, as anti-amyloidogenic agents. The effect of Rg1, Rg3 and Rb1 on α-syn aggregation and toxicity was determined by an array of biophysical, biochemical and cell-culture-based techniques. Among the screened ginsenosides, only Rb1 was shown to be a potent inhibitor of α-syn fibrillation and toxicity. Additionally, Rb1 exhibited a strong ability to disaggregate preformed fibrils and to inhibit the seeded polymerization of α-syn. Interestingly, Rb1 was found to stabilize soluble non-toxic oligomers with no ß-sheet content, that were susceptible to proteinase K digestion, and the binding of Rb1 to those oligomers may represent a potential mechanism of action. Thus, Rb1 could represent the starting point for designing new molecules that could be utilized as drugs for the treatment of PD and related disorders.

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.

Oral Candida carriage and prevalence of Candida species among Maras powder users and non-users.

INTRODUCTION: The aim of this study was to investigate Candida carriage and species in Maras powder users and non-users. MATERIAL AND METHODS: This study included 100 volunteering men in 12 cafés in the city of Kahramanmaras, Turkey. A questionnaire composed of questions about socio-demographic features and Maras powder use was filled in by the participants. Culture specimens were obtained from bilateral buccal mucosa and dorsum of the tongue with a sterile cotton-tipped swap. The specimens were inoculated in Sabouraud Dextrose Agar (SDA). RESULTS: The mean age of the participants was 48.0 ± 12.5 years (min = 20, max = 70). Fifty-four percent of the Maras powder users and 22% of the non-users were Candida carriers. The difference between the groups was significant (P = 0.001). The most frequently isolated species was Candida albicans at a rate of 44% in the Maras powder users and at a rate of 18% in the non-users. Other frequent species were Candida glabrata at a rate of 6% in the Maras powder users and 2% in the control group and Candida tropicalis at a rate of 4% in the Maras powder users and 2% in the nonusers. CONCLUSION: We found that a significantly high rate of the Maras powder users was Candida carriers. It should be kept in mind that opportunistic infections may be caused by Candida species in Maras powder users especially with immunosuppressive conditions since Candida species are likely to lead to such infections in cases of immunosuppressive diseases.

Effect of Cyantraniliprole on Probing Behavior of the Potato Psyllid (Hemiptera: Triozidae) as Measured by the Electrical Penetration Graph Technique.

Zebra chip (ZC), an economically important disease of potato, is caused by 'Candidatus Liberibacter solanacearum' (Lso) transmitted by the potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae). Currently, using insecticides against potato psyllid is the only means to manage ZC. However, the ability of the potato psyllid to rapidly transmit Lso represents a substantial challenge in preventing the spread of ZC. Cyantraniliprole, a novel second-generation anthranilic diamide insecticide has been shown to deter insect feeding and reduce disease transmission. During this study, the effect of cyantraniliprole on potato psyllid probing behavior was assessed using electrical penetration graph technology and compared with abamectin, a commonly used insecticide to control potato psyllid. Results showed that both cyantraniliprole and abamectin significantly deterred probing behavior of the potato psyllid. Average duration of intercellular stylet penetration on cyantraniliprole- and abamectin-treated and untreated control plants was 2.36, 1.80, and 9.15 h, respectively. It took psyllids 1.82, 1.10, and 2.42 h to reach the xylem of cyantraniliprole- and abamectin-treated and untreated plants, respectively. Xylem sap ingestion duration averaged 0.53, 0.57, and 3.66 h on cyantraniliprole- and abamectin-treated and untreated controls, respectively. None of the psyllids exposed to insecticide-treated plants reached the phloem tissue, except one that bypassed the xylem. The insects completely ceased probing after 4.44 and 3.64 h on cyantraniliprole- and abamectin-treated plants, respectively, in contrast with those on untreated plants that probed throughout the entire 24-h experiment duration. These results indicate that cyantraniliprole is as effective as abamectin in deterring potato psyllid feeding and could significantly reduce transmission of Lso and the spread of ZC.

What Do Anticoagulants Say about Microemboli?

BACKGROUND: In this study, we evaluated the microembolic signals (MES) frequency with transcranial Doppler ultrasound (TCD) in patients with atrial fibrillation (AF) under anticoagulant therapy, and we compared the treatment groups. METHODS: Ninety-nine patients with nonvalvular AF with a history of stroke using warfarin (46%), 67 patients using rivaroxaban (31%), and 49 patients using dabigatran (23%), that is, a total of 215 patients, who have been referred to the stroke outpatient section of our department from May 2013 to November 2014, were included in the study. CHA(2)DS(2)VASc scoring was made for all patients, and International Normalized Ratio (INR) value was evaluated in patients using warfarin. All patients were monitored with TCD on the middle cerebral arteries bilaterally for 30 minutes. Embolic signals were evaluated according to their density and the mean number of signals in 2 consecutive recordings. RESULTS: The incidence of emboli in the treatment group was 32 (32%) for warfarin, 24 (36%) for rivaroxaban, and 17 (35%) for dabigatran. The analysis of variance revealed that there was no statistically significant differences between the treatment groups in terms of patients' age (P = .145), CHA(2)DS(2)VASc scores (P = .968), and the number of emboli (P = .783). As CHA(2)DS(2)VASc score increases, number of emboli increase. A statistically significant negative correlation between the number of emboli and INR scores was found in the warfarin group. The number of emboli decreases as INR decreases. CONCLUSIONS: As we aim to reduce the risk of emboli to a minimum with anticoagulant therapy, this screening for MES can give us an idea for the risk of stroke.

The protective role of AMP-activated protein kinase in alpha-synuclein neurotoxicity in vitro.

In the present study, we investigated the role of the main intracellular energy sensor, AMP-activated protein kinase (AMPK), in the in vitro neurotoxicity of α-synuclein (ASYN), one of the key culprits in the pathogenesis of Parkinson's disease. The loss of viability in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells inducibly overexpressing wild-type ASYN was associated with the reduced activation of AMPK and its activator LKB1, as well as AMPK target Raptor. ASYN-overexpressing rat primary neurons also displayed lower activity of LKB1/AMPK/Raptor pathway. Restoration of AMPK activity by metformin or AICAR reduced the in vitro neurotoxicity of ASYN overexpression, acting independently of the prosurvival kinase Akt or the induction of autophagic response. The conditioned medium from ASYN-overexpressing cells, containing secreted ASYN, as well as dopamine-modified or nitrated recombinant ASYN oligomers, all inhibited AMPK activation in differentiated SH-SY5Y cells and reduced their viability, but not in the presence of metformin or AICAR. The RNA interference-mediated knockdown of AMPK increased the sensitivity of SH-SY5Y cells to the harmful effects of secreted ASYN. AMPK-dependent protection from extracellular ASYN was also observed in rat neuron-like pheochromocytoma cell line PC12. These data demonstrate the protective role of AMPK against the toxicity of both intracellular and extracellular ASYN, suggesting that modulation of AMPK activity may be a promising therapeutic strategy in Parkinson's disease.

Molecular and immunohistochemical study of APC exon 16 and its possible role in colorectal carcinoma development.

Background: Colorectal cancer ranks second as a cause of cancer deaths. Mutations in the adenomatous polyposis coli (APC) gene, especially in exon 16, could contribute to colorectal carcinoma development. This study explored the correlations between APC gene exon 16 variations/expression and colorectal carcinoma progression. Methods: In a case-control study, blood samples from 150 colorectal carcinoma patients and 50 healthy volunteers were analyzed by PCR and sequencing for APC exon 16 variations. The APC protein expression on tissue samples was evaluated by immunohistochemistry and statistical analyses were used to examine clinicopathological correlations. Results: The sequencing analysis revealed a mutation in exon 16 of the APC gene (rs459552) in 36 % of colorectal cancer cases while absent in all non-cancer controls. Subgroup analysis by tumor grade showed higher prevalence of mutant allele in Grade II and Grade III cases, with frequencies reaching 60.0 % and 69.2 %, respectively, compared to a substantially lower prevalence of 29.4 % in Grade I patients. Immunohistochemistry showed no significant correlation between this mutation and APC expression. APC positivity proportions were 25.5 % in Grade I tumors (n = 26/102) versus 17.1 % in Grade II (n = 6/35) and 46.2 % in Grade III (n = 6/13), showing a non-significant trend of reduced positivity in higher grade tumors (p>0.05). Conclusions: The frequency of APC exon 16 mutation (rs459552) rose significantly with increasing tumor grade. Similarly, although not statistically significant, the percentage of APC positive staining increased with poorer tumor differentiation, rather than declining. Therefore, the APC exon 16 mutation and expression analysis provides insights into colorectal cancer progression, with the rs459552 mutation correlating with grade and may promoting aggression.