Viral overexpression of human alpha-synuclein in mouse substantia nigra dopamine neurons results in hyperdopaminergia but no neurodegeneration.

Loss of select neuronal populations such as midbrain dopamine (DA) neurons is a pathological hallmark of Parkinson's disease (PD). The small neuronal protein α-synuclein has been related both genetically and neuropathologically to PD, yet how and if it contributes to selective vulnerability remains elusive. Here, we describe the generation of a novel adeno-associated viral vector (AAV) for Cre-dependent overexpression of wild-type human α-synuclein. Our strategy allows us to restrict α-synuclein to select neuronal populations and hence investigate the cell-autonomous effects of elevated α-synuclein in genetically-defined cell types. Since DA neurons in the substantia nigra pars compacta (SNc) are particularly vulnerable in PD, we investigated in more detail the effects of increased α-synuclein in these cells. AAV-mediated overexpression of wildtype human α-synuclein in SNc DA neurons increased the levels of α-synuclein within these cells and augmented phosphorylation of α-synuclein at serine-129, which is considered a pathological feature of PD and other synucleinopathies. However, despite abundant α-synuclein overexpression and hyperphosphorylation we did not observe any dopaminergic neurodegeneration up to 90 days post virus infusion. In contrast, we noticed that overexpression of α-synuclein resulted in increased locomotor activity and elevated striatal DA levels suggesting that α-synuclein enhanced dopaminergic activity. We therefore conclude that cell-autonomous effects of elevated α-synuclein are not sufficient to trigger acute dopaminergic neurodegeneration.

Viral overexpression of human alpha-synuclein in mouse substantia nigra dopamine neurons results in hyperdopaminergia but no neurodegeneration.

Loss of select neuronal populations such as midbrain dopamine (DA) neurons is a pathological hallmark of Parkinson's disease (PD). The small neuronal protein α-synuclein has been related both genetically and neuropathologically to PD, yet how it contributes to selective vulnerability remains elusive. Here, we describe the generation of a novel adeno-associated viral vector (AAV) for Cre-dependent overexpression of wild-type human α-synuclein. Our strategy allows us to restrict α-synuclein to select neuronal populations and hence investigate the cell-autonomous effects of elevated α-synuclein in genetically-defined cell types. Since DA neurons in the substantia nigra pars compacta (SNc) are particularly vulnerable in PD, we investigated in more detail the effects of increased α-synuclein in these cells. AAV-mediated overexpression of wildtype human α-synuclein in SNc DA neurons increased the levels of α-synuclein within these cells and augmented phosphorylation of α-synuclein at serine-129, which is considered a pathological feature of PD and other synucleinopathies. However, despite abundant α-synuclein overexpression and hyperphosphorylation we did not observe any DA neurodegeneration up to 90 days post virus infusion. In contrast, we noticed that overexpression of α-synuclein resulted in increased locomotor activity and elevated striatal DA levels suggesting that α-synuclein enhanced dopaminergic activity. We therefore conclude that cell-autonomous effects of elevated α-synuclein are not sufficient to trigger acute DA neurodegeneration.

Using agro-ecological zones to improve the representation of a multi-environment trial of soybean varieties.

This research introduces a novel framework for enhancing soybean cultivation in North America by categorizing growing environments into distinct ecological and maturity-based zones. Using an integrated analysis of long-term climatic data and records of soybean varietal trials, this research generates a zonal environmental characterization which captures major components of the growing environment which affect the range of adaptation of soybean varieties. These findings have immediate applications for optimizing multi-environment soybean trials. This characterization allows breeders to assess the environmental representation of a multi-environmental trial of soybean varieties, and to strategize the distribution of testing and the placement of test sites accordingly. This application is demonstrated with a historical scenario of a soybean multi-environment trial, using two resource allocation models: one targeted towards improving the general adaptation of soybean varieties, which focuses on widely cultivated areas, and one targeted towards specific adaptation, which captures diverse environmental conditions. Ultimately, the study aims to improve the efficiency and impact of soybean breeding programs, leading to the development of cultivars resilient to variable and changing climates.

Successive Protein Extraction Using Hydroxylamine to Increase the Depth of Proteome Coverage in Fresh, Forensic, and Archaeological Bones.

Proteomics is continually being applied to a wider range of applications, now including the analysis of archaeological samples and anatomical specimens, particularly collagen-containing tissues such as bones and teeth. Here, we present the application of a chemical digestion-based proteomics sample preparation protocol to the analysis of fresh, anatomical, and archaeological samples. We describe and discuss two protocols: one that uses hydroxylamine as an additional step of the proteomic workflow, applied to the insoluble fraction, and another that applies hydroxylamine directly on demineralized bones and teeth. We demonstrate the additional information that can be extracted using both protocols, including an increase in the sequence coverage and number of peptides detected in modern and archaeological samples and an increase in the number of proteins identified in archaeological samples. By targeting research related to collagens or extracellular matrix proteins, the use of this protocol will open new insights, considering both fresh and ancient mineralized samples.

Species identification of ivory and bone museum objects using minimally invasive proteomics.

Ivory is a highly prized material in many cultures since it can be carved into intricate designs and have a highly polished surface. Due to its popularity, the animals from which ivory can be sourced are under threat of extinction. Identification of ivory species is not only important for CITES compliance, it can also provide information about the context in which a work was created. Here, we have developed a minimally invasive workflow to remove minimal amounts of material from precious objects and, using high-resolution mass spectrometry-based proteomics, identified the taxonomy of ivory and bone objects from The Metropolitan Museum of Art collection dating from as early as 4000 B.C. We built a proteomic database of underrepresented species based on exemplars from the American Museum of Natural History, and proposed alternative data analysis workflows for samples containing inconsistently preserved organic material. This application demonstrates extensive ivory species identification using proteomics to unlock sequence uncertainties, e.g., Leu/Ile discrimination.

Dyke-Davidoff-Masson syndrome: Imaging diagnosis in an asymptomatic adult.

Dyke-Davidoff-Masson syndrome (DDMS) was first described in 1933 as a cerebral condition of hemispheric atrophy characterized clinically by contralateral hemiparesis, facial-asymmetry, seizures, and mental retardation. Neuroimaging findings include asymmetric thickening of the calvarium and enlargement of frontal and ethmoid sinuses. There have been 21 reported cases described in the literature with the syndrome undiagnosed until adult age, likely due to less severe or absent clinical findings or symptoms as described in the case presented in this report. This article describes a case where the Dyke-Davidoff-Masson imaging features were identified as an incidental finding on a CT scan of the brain performed for non-seizure related symptoms. A 54-year-old woman presented with weakness and gait difficulty and only upon further evaluation was she found to have cranial deformities. CT and MRI demonstrate encephalomalacia in the right frontal lobe anteriorly with gliosis and moderate unilateral cerebral atrophy, and extensive hypertrophy of the right frontal calvarium, right ethmoid cells and frontal sinuses.

Novel 3D printable powered air purifying respirator for emergency use during PPE shortage of the COVID-19 pandemic: a study protocol and device safety analysis.

OBJECTIVES: To design a low-cost 3D printable powered air-purifying respirator (PAPR) that meets National Institute for Occupational Safety and Health (NIOSH) standard for flow rate and Occupational Safety and Health Administration (OSHA) standard for particle filtration for loose-fitting PAPRs and that can be made with a 3D printer and widely available materials. DESIGN: Detailed description of components, assembly instructions and testing of a novel PAPR design in an academic laboratory following respective protocols. The assembled PAPR must meet NIOSH standards of flow rate, 170 L/min; OSHA fit factor for particle filtration, ≥250 and maintain positive pressure during regular and deep breathing. MAIN OUTCOME MEASURES: The PAPR design was run through a series of tests: air flow (L/min), particle filtration (quantitative and qualitative) and positive pressure measured inside the helmet (mm Hg). RESULTS: Flow rate was 443.32 L/min (NIOSH standard: minimum 170 L/min) and overall fit factor for particle filtration was 1362 (OSHA pass level: ≥500), n=1. The device passed qualitative particle filtration, n=2, and measured peak pressure of 6mm Hg (>0 mm Hg indicates positive pressure) in the helmet, n=1. CONCLUSIONS: The Hygieia PAPR is a low-cost, easily accessible, just-in-time 3D printable PAPR design that meets minimum NIOSH and OSHA standards for flow-rate and particle filtration for loose-fitting PAPR devices to be made and used when industry-made designs are unavailable.

Comparative Genomic Screen in Two Yeasts Reveals Conserved Pathways in the Response Network to Phenol Stress.

Living organisms encounter various perturbations, and response mechanisms to such perturbations are vital for species survival. Defective stress responses are implicated in many human diseases including cancer and neurodegenerative disorders. Phenol derivatives, naturally occurring and synthetic, display beneficial as well as detrimental effects. The phenol derivatives in this study, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and bisphenol A (BPA), are widely used as food preservatives and industrial chemicals. Conflicting results have been reported regarding their biological activity and correlation with disease development; understanding the molecular basis of phenol action is a key step for addressing issues relevant to human health. This work presents the first comparative genomic analysis of the genetic networks for phenol stress response in an evolutionary context of two divergent yeasts, Schizosaccharomyces pombe and Saccharomyces cerevisiae Genomic screening of deletion strain libraries of the two yeasts identified genes required for cellular response to phenol stress, which are enriched in human orthologs. Functional analysis of these genes uncovered the major signaling pathways involved. The results provide a global view of the biological events constituting the defense process, including cell cycle arrest, DNA repair, phenol detoxification by V-ATPases, reactive oxygen species alleviation, and endoplasmic reticulum stress relief through ergosterol and the unfolded protein response, revealing novel roles for these cellular pathways.

Student Factors That Influence Clerkship Grades and Matching Into a Surgical Residency.

OBJECTIVE: Evaluate the relationship between medical school factors (including preclinical mentorship, order of clerkships, and clerkship grades) and matching into surgical specialties. DESIGN: Clerkship information, match data, and data on structured preclinical research obtained from 2010 to 2015 for a single institution was obtained and analyzed using multivariate analysis. SETTING: University of Michigan Medical School. PARTICIPANTS: Seven hundred and forty-six students who took both the Internal Medicine and Surgery clerkships between 2010 and 2015 and have since participated in the match. RESULTS: Among 740 students studied, 243 matched into a surgical field. Higher Shelf scores were associated with higher clerkship grades in Surgery and Internal Medicine. Honors or High Pass in Surgery were associated with matching into a surgical field. Structured preclinical research in Surgery and order of clerkship were not associated with matching into a surgical field. CONCLUSIONS: Students who went into surgery were more likely to receive Honors or High Pass. Preclinical choices geared toward a surgical specialty (e.g., order of clerkship and structured research) were not associated with matching into a surgical field. These data may help guide school specific advice for students.

The cost and quality of life outcomes in developing a robotic lobectomy program.

The use of the robotic platform is increasingly being utilized for lung resections. Our aim was to compare outcomes of thoracoscopic (VATS) versus robotic-assisted thoracoscopic (RATS) lobectomy early in a program's adoption of robotic surgery, including perioperative outcomes, cost, and long-term quality of life. A prospective database was retrospectively reviewed for all patients undergoing minimally invasive lobectomy by either VATS or RATS techniques from 2010 to 2012. Patients' operative, post-operative complications, cost (operating room and hospital) and quality of life were compared between the two resection techniques. Long-term follow-up including assessment using the European Organization for Research and Treatment of Cancer quality of life questionnaire was documented. During the first 25 RATS lobectomies, there were 73 VATS lobectomies performed, for a total of 98 cases. There was no significant difference in cancer stage, operative time, estimated blood loss, lymph node count, or hospital length of stay. The RATS resections had significantly higher operative and total hospital cost (p < 0.0001 and p < 0.05). At a median of 65-month follow-up, 29 patients (9 robotic; 20 VATS) completed the EORTC questionnaire. The global health status and symptom scale median scores were similar to the general population and did not significantly differ between groups. While transitioning from thoracoscopic to robotic lobectomy incurs increased operative and total hospital cost, equivalent operative outcomes, length of hospitalization, and long-term quality of life can be maintained during this transition. With increasing patient and surgeon interest in robotic resection, it appears both safe and feasible to adopt this approach while maintaining outcomes.

Understanding Interconnectedness From the Military Nurse Perspective.

BACKGROUND: Nurses who serve in the military have a unique perspective on nursing and health care delivery that nurtures wholeness and inspires peace and healing on a global scale. PURPOSE: The purpose of this study was to explore health promotion and healing from the military nurse perspective. DESIGN: Video-recorded interviews were conducted with 10 military nurses who represented various branches and times of service. Participants were asked to share their experiences as military nurses and discuss the challenges and rewards. FINDINGS: Thematic analysis of the recorded interviews revealed two major themes: interconnectedness and human potential. CONCLUSION: This study showed that military nurses have unique experiences that influenced their way of promoting health and healing. Interconnectedness with family (personal and military) had many positive and negative factors. Interconnectedness with the health care team was more prominent for the nurses during military service than in the civilian arena. Global interconnectedness included working with teams from around the world, helping children of detainees see that Americans were not evil, and caring for international communities. Military service strengthened the three human qualities of mind, body, and spirit, which resulted in increasing each military nurse's human potential by enabling them to serve as instruments of healing on a global scale.

Commonly used trafficking blocks disrupt ARF1 activation and the localization and function of specific Golgi proteins.

Cold temperature blocks used to synchronize protein trafficking inhibit GBF1 function, leading to a decrease in ARF1-GTP levels and mislocalization of the ARF1 effector golgin-160. Several other, but not all, Golgi proteins including ARL1 also mislocalize. ARF1 activity and golgin-160 localization require more than 30 min to recover from these blocks.

Dynamic modulation of Cyp21a1 (21-hydroxylase) expression sites in the mouse developing lung.

21-hydroxylase is expressed in the developing lung where it is proposed as a local source of glucocorticoids playing important roles in lung development. We have studied the precise sites of Cyp21a1 expression in the developing mouse lung from the pseudoglandular stage (gestation day (GD) 15.5) to the alveolar stage (postnatal day (PND) 15) by in situ hybridization. Cyp21a1-mRNA was found mainly in epithelial cells from GD 15.5 to PND 5, but the precise site of expression shifted from the distal epithelium during the pseudoglandular and the canalicular stages including the distal epithelium without lumina, to the proximal epithelium and the wall of developing saccules during the perinatal period (GD 19.5 and PND 0), and to the wall of developing saccules and septa, most probably in type I pneumonocytes (PTI), on PND 5. Cyp21a1 expression changed from PTI cells to capillary endothelial cells of the same distal structures during alveolarization. The mesenchyme was generally negative. Endothelial cells forming large vessels were negative. However the tunica adventitia surrounding arteries was Cyp21a1-positive, while several veins were surrounded by a Cyp21a1-positive layer. In conclusion, Cyp21a1 remains expressed in the most distal structure of the developing lung even though these structures are changing, but its expression is not restricted to these areas. Taken together, our data show the highly dynamic modulation of Cyp21a1 expression sites, consistent with the evolving structures of the developing lung.

Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons.

The LIM-homeodomain transcription factors Lmx1a and Lmx1b play critical roles during the development of midbrain dopaminergic progenitors, but their functions in the adult brain remain poorly understood. We show here that sustained expression of Lmx1a and Lmx1b is required for the survival of adult midbrain dopaminergic neurons. Strikingly, inactivation of Lmx1a and Lmx1b recreates cellular features observed in Parkinson's disease. We found that Lmx1a/b control the expression of key genes involved in mitochondrial functions, and their ablation results in impaired respiratory chain activity, increased oxidative stress, and mitochondrial DNA damage. Lmx1a/b deficiency caused axonal pathology characterized by α-synuclein(+) inclusions, followed by a progressive loss of dopaminergic neurons. These results reveal the key role of these transcription factors beyond the early developmental stages and provide mechanistic links between mitochondrial dysfunctions, α-synuclein aggregation, and the survival of dopaminergic neurons.

Discovery and Characterization of New Hydroxamate Siderophores, Baumannoferrin A and B, produced by Acinetobacter baumannii.

Acinetobacter baumannii AYE does not produce acinetobactin but grows under iron limitation. Accordingly, analyses of AYE iron-restricted culture supernatants resulted in the isolation of two fractions, which contained only hydroxamates and showed siderophore activity. Structural analyses identified baumannoferrin A and baumannoferrin B, which differ only by a double bond. These siderophores are composed of citrate, 1,3-diaminopropane, 2,4-diaminobutyrate, decenoic acid, and α-ketoglutarate. Analysis of the AYE genome showed the presence of a 12-gene cluster coding for proteins similar to those involved in the production and utilization of the hydroxamate siderophores acinetoferrin and achromobactin. As A. baumannii AYE does not produce acinetobactin and harbors only one gene cluster encoding the production and utilization of a siderophore, this strain's growth under iron limitation depends on baumannoferrin, a novel hydroxamate that could play a role in its virulence.

Three basic residues of intracellular loop 3 of the beta-1 adrenergic receptor are required for golgin-160-dependent trafficking.

Golgin-160 is a member of the golgin family of proteins, which have been implicated in the maintenance of Golgi structure and in vesicle tethering. Golgin-160 is atypical; it promotes post-Golgi trafficking of specific cargo proteins, including the ß-1 adrenergic receptor (ß1AR), a G protein-coupled receptor. Here we show that golgin-160 binds directly to the third intracellular loop of ß1AR and that this binding depends on three basic residues in this loop. Mutation of the basic residues does not affect trafficking of ß1AR from the endoplasmic reticulum through the Golgi complex, but results in reduced steady-state levels at the plasma membrane. We hypothesize that golgin-160 promotes incorporation of ß1AR into specific transport carriers at the trans-Golgi network to ensure efficient delivery to the cell surface. These results add to our understanding of the biogenesis of ß1AR, and suggest a novel point of regulation for its delivery to the plasma membrane.

Genome-wide consequences of deleting any single gene.

Loss or duplication of chromosome segments can lead to further genomic changes associated with cancer. However, it is not known whether only a select subset of genes is responsible for driving further changes. To determine whether perturbation of any given gene in a genome suffices to drive subsequent genetic changes, we analyzed the yeast knockout collection for secondary mutations of functional consequence. Unlike wild-type, most gene knockout strains were found to have one additional mutant gene affecting nutrient responses and/or heat-stress-induced cell death. Moreover, independent knockouts of the same gene often evolved mutations in the same secondary gene. Genome sequencing identified acquired mutations in several human tumor suppressor homologs. Thus, mutation of any single gene may cause a genomic imbalance, with consequences sufficient to drive adaptive genetic changes. This complicates genetic analyses but is a logical consequence of losing a functional unit originally acquired under pressure during evolution.

Simultaneous liquid chromatographic determination of doxorubicin and its major metabolite doxorubicinol in parrot plasma.

A new microscale method is reported for the determination of doxorubicin and its active metabolite, doxorubicinol, in parrot plasma. Sample workup involved acetonitrile protein precipitation, ethyl acetate extraction, followed by back extraction into HCl. Separations were achieved on a phenyl-hexyl column at 30 degrees C using acetonitrile (17%, v/v) in 0.01 M orthophosphoric acid (83%, v/v) delivered via a linear flow program. Fluorometric detection wavelengths were 235 nm (excitation) and 550 nm (emission). Calibration plots were linear (r2>0.999), and recoveries were 71-87% from 20 to 400 ng/mL. Assay imprecision was