p21-activated kinase 4 controls the aggregation of α-synuclein by reducing the monomeric and aggregated forms of α-synuclein: involvement of the E3 ubiquitin ligase NEDD4-1.

Aggregation of misfolded alpha-synuclein (α-synuclein) is a central player in the pathogenesis of neurodegenerative diseases. Therefore, the regulatory mechanism underlying α-synuclein aggregation has been intensively studied in Parkinson's disease (PD) but remains poorly understood. Here, we report p21-activated kinase 4 (PAK4) as a key regulator of α-synuclein aggregation. Immunohistochemical analysis of human PD brain tissues revealed an inverse correlation between PAK4 activity and α-synuclein aggregation. To investigate their causal relationship, we performed loss-of-function and gain-of-function studies using conditional PAK4 depletion in nigral dopaminergic neurons and the introduction of lentivirus expressing a constitutively active form of PAK4 (caPAK4; PAK4S445N/S474E), respectively. For therapeutic relevance in the latter setup, we injected lentivirus into the striatum following the development of motor impairment and analyzed the effects 6 weeks later. In the loss-of-function study, Cre-driven PAK4 depletion in dopaminergic neurons enhanced α-synuclein aggregation, intracytoplasmic Lewy body-like inclusions and Lewy-like neurites, and reduced dopamine levels in PAK4DAT-CreER mice compared to controls. Conversely, caPAK4 reduced α-synuclein aggregation, as assessed by a marked decrease in both proteinase K-resistant and Triton X100-insoluble forms of α-synuclein in the AAV-α-synuclein-induced PD model. Mechanistically, PAK4 specifically interacted with the NEDD4-1 E3 ligase, whose pharmacological inhibition and knockdown suppressed the PAK4-mediated downregulation of α-synuclein. Collectively, these results provide new insights into the pathogenesis of PD and suggest PAK4-based gene therapy as a potential disease-modifying therapy in PD.

Health worker text messaging for blended learning, peer support, and mentoring in pediatric and adolescent HIV/AIDS care: a case study in Zimbabwe.

BACKGROUND: In sub-Saharan Africa, shortages of trained healthcare workers and limited resources necessitate innovative and cost-effective approaches for training, supervising, and mentoring. This qualitative case study describes participants' and trainers' perspectives and experiences with a text messaging component of a blended training course in HIV counseling and testing in Zimbabwe, using minimal resources in terms of staff time and equipment requirements. This component included a whole-group discussion forum as well as two-person partner discussions designed to promote reflection and analysis, teamwork, and active learning. CASE PRESENTATION: The Ministry of Health and Child Care (MoHCC) of Zimbabwe collaborated with the International Training and Education Center for Health (I-TECH) on adaptation of a 5-day in-service training in HIV Testing Services for Children and Adolescents. The new 7-week blended format included in-person sessions, tablet-based self-study, and discussions using the text messaging application, WhatsApp. Between August 2016 and January 2017, 11 cohorts (293 participants in total) were trained with this new curriculum, incorporating text messaging to support peer-to-peer and work-based education. Data collected included training participants' feedback, key informant interviews with the training team, and thematic analysis of WhatsApp messages from full-cohort discussions and a sampling of one-to-one partner discussions. A total of 293 healthcare workers from 233 health facilities across all provinces in Zimbabwe completed the blended learning course. Participants strongly endorsed using WhatsApp groups as part of the training. In the whole-group discussions, the combined cohorts generated over 6300 text messages. Several categories of communication emerged in analysis of group discussions: (1) participants' case experiences and questions; (2) feedback and recommendations for work issues raised; (3) inquiries, comments, and responses about course assignments and specific course content; (4) encouragement; and (5) technical challenges encountered using the blended learning methodology. Case discussions were complex, including patient history, symptoms, medications, and psychosocial issues-child abuse, adherence, and disclosure. CONCLUSIONS: Using text messaging in a communication platform that is an ongoing part of healthcare workers' daily lives can be an effective adjunct to in-service training, minimizing isolation and providing interactivity, supporting students' ability to fully integrate content into new skill attainment.

Iron is Responsible for Production of Reactive Oxygen Species Regulating Vasopressin Expression in the Mouse Paraventricular Nucleus.

Reactive oxygen species (ROS) act as signaling molecules for maintaining homeostasis, particularly in the regulation of body-fluid balance in the paraventricular nucleus (PVN) of the hypothalamus. However, there has been little discussion regarding the source of ROS generation in this hypothalamic region. Because iron is the most abundant metal in the brain, we hypothesized that iron may act as a source of ROS, which regulate vasopressin (VP) expression. In the present study, we compared the amount of iron in the PVN to that in other forebrain regions of normal ICR mice, and examined the relationship among iron, ROS, and VP in the PVN of the iron-overloaded with iron dextran and iron-chelated mice with deferoxamine. The amount of iron in the PVN was significantly higher than in any of the forebrain regions we examined. The amount of iron in the PVN was significantly increased in iron-overloaded mice, although not in iron-chelated mice. These results suggest that the PVN exhibits high iron affinity. Both ROS production and VP expression in the PVN of iron-overloaded mice were significantly increased relative to levels observed in control mice. VP concentration in blood of iron-overloaded mice was also significantly higher than that of control mice. Interestingly, iron overload did not alter the expression of nitric oxide synthase, another modulator of VP expression. Taken together, our results suggest that high levels of iron in the PVN induce the production of ROS, which regulate VP expression, independent of nitric oxide signaling.

Assessment of Formaldehyde Concentrations in an Anatomy Laboratory Equipped Dissecting Tables with Inbuilt Exhaust and an Air Diffuser/Return System / 체질인류학회지

Formaldehyde (FA) used for preserving the body for anatomy dissection is harmful to the human body. In many countries, for the purpose of protecting the health of workers in the industrial field, the maximal allowable air concentration of FA has been set. The threshold limit values of time weighted average (TLV-TWA) and short-term exposure limit (TLV-STEL) of FA recommended by Ministry of Employment and Labor (MOEL) of Korea are less than 0.5 and 1 ppm, respectively. In the United States and Europe, TLV-TWAs of FA are recommended at between 0.3 and 2 ppm. In this study, we compared the air concentration of FA to domestic and foreign standards of FA in an anatomy laboratory equipped dissecting tables with inbuilt exhaust and an air diffuser/return system. We installed ten elevated dissection tables, 18 air diffusers on the ceiling, and 10 air returns at the bottom of both side walls. The concentration of FA was measured at five sites in the anatomy laboratory and above the cadavers on the dissecting tables at a height of 1.5 m from the floor using a Formaldemeter. The average concentration of FA in the anatomy laboratory (five sites) was 0.31 ppm (0.45 mg/m³), range 0.21 to 0.41 ppm (0.26~0.51 mg/m³). The average concentration of FA above the cadavers was 0.45 ppm (0.56 mg/m³), range 0.31 to 0.64 ppm (0.39~0.80 mg/m³). The average TWA of FA in the anatomy laboratory was 0.19 ppm (0.24 mg/m³), range 0.13 to 0.26 ppm. The average TWA of FA above the cadavers was 0.28 ppm (0.35 mg/m³), range 0.19 to 0.40 ppm. The anatomy laboratory dissecting tables equipped with inbuilt exhaust and air diffuser/return system met the criteria of the FA concentration recommended by MOEL of Korea and most foreign countries. This study was the first evaluation of the air concentration of FA in an anatomy laboratory equipped dissecting tables with inbuilt exhaust and an air diffuser/return system in Korea. We expect it will be not only used as a standard of comparison for anatomy laboratories, but as a reference for design and construction to improve air quality in Korean Medical Colleges.

Physiological function of NbRanBP1 in Nicotiana benthamiana.

This study addresses the physiological functions of the Ran-binding protein homolog NbRanBP1 in Nicotiana benthamiana. Virus-induced gene silencing (VIGS) of NbRanBP1 caused stunted growth, leaf yellowing, and abnormal leaf morphology. The NbRanBP1 gene was constitutively expressed in diverse tissues and an NbRanBP1:GFP fusion protein was primarily localized to the nuclear rim and the cytosol. BiFC analysis revealed in vivo interaction between NbRanBP1 and NbRan1 in the nuclear envelope and the cytosol. Depletion of NbRanBP1 or NbRan1 reduced nuclear accumulation of a NbBTF3:GFP marker protein. In the later stages of development, NbRanBP1 VIGS plants showed stress responses such as reduced mitochondrial membrane potential, excessive production of reactive oxygen species, and induction of defense-related genes. The molecular role of RanBP1 in plants is discussed in comparison with RanBP1 function in yeast and mammals.

Silencing of NbBTF3 results in developmental defects and disturbed gene expression in chloroplasts and mitochondria of higher plants.

BTF3 (betaNAC) was originally isolated as a general transcription factor required for RNA polymerase II-dependent transcription, and later found to be a beta-subunit of nascent-polypeptide-associated complex that has been implicated in regulating protein localization during translation. In this study, virus-induced gene silencing of NbBTF3 encoding a Nicotiana benthamiana homolog of human BTF3 caused leaf yellowing and abnormal leaf morphology without altering the overall growth of the plant. The NbBTF3 gene is constitutively expressed and the NbBTF3-GFP fusion protein is primarily targeted to the nucleus. At the cellular level, downregulation of NbBTF3 expression reduced the chloroplast sizes and chlorophyll contents. The affected cells produced excessive amounts of reactive oxygen species. Furthermore, the transcript level of various plastid- and mitochondria-encoded genes was severely reduced in the NbBTF3-depleted leaf cells. These findings indicate that depletion of NbBTF3 activity preferentially affected development and/or physiology of chloroplasts and mitochondria in plants, possibly by hampering efficient translocation of the nascent organellar proteins into the organelles.

Constitutive expression of abiotic stress-inducible hot pepper CaXTH3, which encodes a xyloglucan endotransglucosylase/hydrolase homolog, improves drought and salt tolerance in transgenic Arabidopsis plants.

Xyloglucan endotransglucosylase/hydrolase (XTH) has been recognized as a cell wall-modifying enzyme, participating in the diverse physiological roles. From water-stressed hot pepper plants, we isolated three different cDNA clones (pCaXTH1, pCaXTH2, and pCaXTH3) that encode XTH homologs. RT-PCR analysis showed that three CaXTH mRNAs were concomitantly induced by a broad spectrum of abiotic stresses, including drought, high salinity and cold temperature, and in response to stress hormone ethylene, suggesting their role in the early events in the abiotic-related defense response. Transgenic Arabidopsis plants that constitutively expressed the CaXTH3 gene under the control of the CaMV 35S promoter exhibited abnormal leaf morphology; the transgenic leaves showed variable degrees of twisting and bending along the edges, resulting in a severely wrinkled leaf shape. Microscopic analysis showed that 35S-CaXTH3 leaves had increased numbers of small-sized cells, resulting in disordered, highly populated mesophyll cells in each dorsoventral layer, and appeared to contain a limited amount of starch. In addition, the 35S-CaXTH3 transgenic plants displayed markedly improved tolerance to severe water deficit, and to lesser extent to high salinity in comparison with the wild-type plants. These results indicate that CaXTH3 is functional in heterologous Arabidopsis cells, thereby effectively altering cell growth and also the response to abiotic stresses. Although the physiological function of CaXTHs is not yet clear, there are several possibilities for their involvement in a subset of physiological responses to counteract dehydration and high salinity stresses in transgenic Arabidopsis plants.

Silencing of NbECR encoding a putative enoyl-CoA reductase results in disorganized membrane structures and epidermal cell ablation in Nicotiana benthamiana.

The very long chain fatty acids (VLCFAs) are synthesized by the microsomal fatty acid elongation system in plants. We investigated cellular function of NbECR putatively encoding enoyl-CoA reductase that catalyzes the last step of VLCFA elongation in Nicotiana benthamiana. Virus-induced gene silencing of NbECR produced necrotic lesions with typical cell death symptoms in leaves. In the affected tissues, ablation of the epidermal cell layer preceded disintegration of the whole leaf cell layers, and disorganized cellular membrane structure was evident. The amount of VLCFAs was reduced in the NbECR VIGS lines, suggesting NbECR function in elongation of VLCFAs. The results demonstrate that NbECR encodes a putative enoyl-CoA reductase and that the NbECR activity is essential for membrane biogenesis in N. benthamiana.

Retinoblastoma protein regulates cell proliferation, differentiation, and endoreduplication in plants.

Retinoblastoma protein (Rb) plays a key role in cell cycle control, cell differentiation, and apoptosis in animals. In this study, we used virus-induced gene silencing (VIGS) to investigate the cellular functions of Rb in higher plants. VIGS of NbRBR1, which encodes the Nicotiana benthamiana Rb homolog, resulted in growth retardation and abnormal organ development. At the cellular level, Rb suppression caused prolonged cell proliferation in tissues that are normally differentiated, which indicates that Rb is a negative regulator of plant cell division. Furthermore, differentiation of the epidermal pavement cells and trichomes was partially retarded, and stomatal clusters formed in the epidermis, likely due to uncontrolled cell division of stomata precursor cells. Rb suppression also caused extra DNA replication in endoreduplicating leaf cells, suggesting a role of Rb in the endocycle. These Rb phenotypes were accompanied by stimulated transcription of E2F and E2F-regulated S-phase genes. Thus, disruption of Rb function in plants leads to ectopic cell division in major organs that correlates with a delay in cell differentiation as well as increased endoreduplication, which indicates that Rb coordinates these processes in plant organ development.

Left Ventricular Systolic Function by Tissue Doppler Imaging and Strain Rate Imaging in Obese Adolescents

PURPOSE: Obesity is a risk factor for cardiovascular morbidity and is frequently associated with coronary artery disease, hypertension, and diabetes mellitus. Conventional Doppler technique is limited by the absence of an adequate apical window to assess the transmitral flow in obese patients. Tissue Doppler imaging(TDI) and strain rate imaging(SRI) were performed to assess the influence of obesity on left ventricular systolic function. METHODS: In 13 obese and 15 normal adolescents aged 16 to 17 years, height, weight, body mass index(BMI), and obesity index(OI) were measured. Fat mass, body fat percent, and abdominal fat percent were estimated by bioelectrical impedance. Ejection fraction(EF) and myocardial performance index(MPI) were estimated by conventional echocardiography to evaluate left ventricular systolic function. Systolic myocardial velocity and strain rate were estimated by TDI and SRI. RESULTS: EF(63.8+/-6.4% vs 55.7+/-3.4%) was significantly lower in obese adolescents than normal controls. MPI(0.34+/-0.03 vs 0.48+/-0.06) was significantly higher in obese adolescents than normal controls. Systolic myocardial velocity and strain rate were significantly lower in obese adolescents than normal controls. Strain rate showed a negative correlation with arm circumference(r=-0.558, P<0.05) and BMI(r=-0.332, P<0.05). Strain rate was positively correlated with EF(r=0.557, P<0.05) at the base of left ventricle by SRI CONCLUSION: Significant decrease in left ventricular systolic function was noted in the moderate degree of obesity. SRI may be a more useful diagnostic tool in evaluating systolic dysfunction in patients with moderate degree of obesity.

The CaPRP1 gene encoding a putative proline-rich glycoprotein is highly expressed in rapidly elongating early roots and leaves in hot pepper (Capsicum annuum L. cv. Pukang).

Most of the proline-rich cell wall glycoprotein genes isolated from higher plants are preferentially expressed in the transmitting tissues of the flower organ. In conducting expressed sequence tag (EST) analysis, which was prepared from 5-day-old early roots of hot pepper (Capsicum annuum L. cv. Pukang), we identified a cDNA clone, pCaPRP1, encoding a putative cell wall proline-rich glycoprotein. CaPRP1 (Mr=28 kDa, pI=9.98) was most closely related to Nicotiana alata NaPRP4 (71%), while most distantly related to soybean PvPRP (37%). The predicted primary structure of CaPRP1 contains a putative N-terminal signal peptide, six repeats of the Lys-Pro-Pro tripeptide, four repeats of a five-amino acid sequence [Pro-(Ser/The)-Pro-Pro-Pro] and one potential N-glycosylation site (Asn-Asn-Ser). In contrast to most proline-rich cell wall glycoprotein genes, CaPRP1 was highly expressed in rapidly elongating very early roots and young leaves as well as developing flower tissues. Although the physiological function of CaPRP1 is not yet clear, there are several possibilities for its role in cell expansion and elongation during early development of hot pepper plants.

Granulocyte colony stimulation factor (G-CSF) suppresses interleukin (IL)-12 and/or IL-2 induced interferon (IFN)-gamma production and cytotoxicity of decidual mononuclear cells.

PROBLEM: The placenta is one of the few non-hematopoietic tissues to express granulocyte colony stimulation factor (G-CSF). Placental G-CSF production is considered to be one of the major causes of granulocytosis during pregnancy although its physiological role in pregnancy has not yet been examined. METHOD OF STUDY: The effects of G-CSF on interleukin (IL)-2 and/or IL-12 induced interferon (IFN)-gamma production of magnetic cell sorting (MACS) sorted decidual lymphocytes was examined by enzyme-linked immunosorbent spot-forming cell assay (ELISPOT). The effect of G-CSF on cytotoxicity of decidual lymphocytes against the choriocarcinoma cell line JEG-3 was examined by lactate dehydrogenase (LDH) release assay. RESULTS: As previously reported by us, IL-2 and/or IL-12 activated decidual mononuclear cells were capable of killing choriocarcinoma cells. We observed that G-CSF abolished IFN-gamma production and cytotoxicity of decidual mononuclear cells and MACS sorted CD56+ cells. CONCLUSIONS: In addition to its well-known trophic effects on hematopoiesis, our results suggest about new roles of G-CSF in reproductive immunology.