Preexposure chemoprophylaxis for HIV prevention in men who have sex with men.

BACKGROUND: Antiretroviral chemoprophylaxis before exposure is a promising approach for the prevention of human immunodeficiency virus (HIV) acquisition. METHODS: We randomly assigned 2499 HIV-seronegative men or transgender women who have sex with men to receive a combination of two oral antiretroviral drugs, emtricitabine and tenofovir disoproxil fumarate (FTC-TDF), or placebo once daily. All subjects received HIV testing, risk-reduction counseling, condoms, and management of sexually transmitted infections. RESULTS: The study subjects were followed for 3324 person-years (median, 1.2 years; maximum, 2.8 years). Of these subjects, 10 were found to have been infected with HIV at enrollment, and 100 became infected during follow-up (36 in the FTC-TDF group and 64 in the placebo group), indicating a 44% reduction in the incidence of HIV (95% confidence interval, 15 to 63; P=0.005). In the FTC-TDF group, the study drug was detected in 22 of 43 of seronegative subjects (51%) and in 3 of 34 HIV-infected subjects (9%) (P<0.001). Nausea was reported more frequently during the first 4 weeks in the FTC-TDF group than in the placebo group (P<0.001). The two groups had similar rates of serious adverse events (P=0.57). CONCLUSIONS: Oral FTC-TDF provided protection against the acquisition of HIV infection among the subjects. Detectable blood levels strongly correlated with the prophylactic effect. (Funded by the National Institutes of Health and the Bill and Melinda Gates Foundation; ClinicalTrials.gov number, NCT00458393.).

The G2385R variant of leucine-rich repeat kinase 2 associated with Parkinson's disease is a partial loss-of-function mutation.

Autosomal-dominant missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a common genetic cause of PD (Parkinson's disease). LRRK2 is a multidomain protein with kinase and GTPase activities. Dominant mutations are found in the domains that have these two enzyme activities, including the common G2019S mutation that increases kinase activity 2-3-fold. However, there is also a genetic variant in some populations, G2385R, that lies in a C-terminal WD40 domain of LRRK2 and acts as a risk factor for PD. In the present study we show that the G2385R mutation causes a partial loss of the kinase function of LRRK2 and deletion of the C-terminus completely abolishes kinase activity. This effect is strong enough to overcome the kinase-activating effects of the G2019S mutation in the kinase domain. Hsp90 (heat-shock protein of 90 kDa) has an increased affinity for the G2385R variant compared with WT (wild-type) LRRK2, and inhibition of the chaperone binding combined with proteasome inhibition leads to association of mutant LRRK2 with high molecular mass native fractions that probably represent proteasome degradation pathways. The loss-of-function of G2385R correlates with several cellular phenotypes that have been proposed to be kinase-dependent. These results suggest that the C-terminus of LRRK2 plays an important role in maintaining enzymatic function of the protein and that G2385R may be associated with PD in a way that is different from kinase-activating mutations. These results may be important in understanding the differing mechanism(s) by which mutations in LRRK2 act and may also have implications for therapeutic strategies for PD.

Direct evidence of phosphorylated neuronal intermediate filament proteins in neurofibrillary tangles (NFTs): phosphoproteomics of Alzheimer's NFTs.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by brain pathology of intracellular neurofibrillary tangles (NFTs) and extracellular amyloid plaques. NFTs contain aberrantly hyperphosphorylated Tau as paired helical filaments (PHFs). Although NFs have been shown immunohistologically to be part of NFTs, there has been debate that the identity of NF proteins in NFTs is due to the cross-reactivity of phosphorylated NF antibodies with phospho-Tau. Here, we provide direct evidence on the identity of NFs in NFTs by immunochemical and mass spectrometric analysis. We have purified sarkosyl-insoluble NFTs and performed liquid chromatography/tandem mass spectrometry of NFT tryptic digests. The phosphoproteomics of NFTs clearly identified NF-M phosphopeptides SPVPKS*PVEEAK, corresponding to Ser685, and KAES*PVKEEAVAEVVTITK, corresponding to Ser736, and an NF-H phosphopeptide, EPDDAKAKEPS*KP, corresponding to Ser942. Western blotting of purified tangles with SMI31 showed a 150-kDa band corresponding to phospho-NF-M, while RT97 antibodies detected phospho-NF-H. The proteomics analysis also identified an NF-L peptide (ALYEQEIR, EAEEEKKVEGAGEEQAAAK) and another intermediate filament protein, vimentin (FADLSEAANR). Mass spectrometry revealed Tau phosphopeptides corresponding to Thr231, Ser235, Thr181, Ser184, Ser185, Thr212, Thr217, Ser396, and Ser403. And finally, phosphopeptides corresponding to MAP1B (corresponding to Ser1270, Ser1274, and Ser1779) and MAP2 (corresponding to Thr350, Ser1702, and Ser1706) were identified. In corresponding matched control preparations of PHF/NFTs, none of these phosphorylated neuronal cytoskeletal proteins were found. These studies independently demonstrate that NF proteins are an integral part of NFTs in AD brains.

Differential regulation of kainate receptor trafficking by phosphorylation of distinct sites on GluR6.

Kainate receptors are widely expressed in the brain, and are present at pre- and postsynaptic sites where they play a prominent role in synaptic plasticity and the regulation of network activity. Within individual neurons, kainate receptors of different subunit compositions are targeted to various locations where they serve distinct functional roles. Despite this complex targeting, relatively little is known about the molecular mechanisms regulating kainate receptor subunit trafficking. Here we investigate the role of phosphorylation in the trafficking of the GluR6 kainate receptor subunit. We identify two specific residues on the GluR6 C terminus, Ser(846) and Ser(868), which are phosphorylated by protein kinase C (PKC) and dramatically regulate GluR6 surface expression. By using GluR6 containing phosphomimetic and nonphosphorylatable mutations for these sites expressed in heterologous cells or in neurons lacking endogenous GluR6, we show that phosphorylation of Ser(846) or Ser(868) regulates receptor trafficking through the biosynthetic pathway. Additionally, Ser(846) phosphorylation dynamically regulates endocytosis of GluR6 at the plasma membrane. Our findings thus demonstrate that phosphorylation of PKC sites on GluR6 regulates surface expression of GluR6 at distinct intracellular trafficking pathways, providing potential molecular mechanisms for the PKC-dependent regulation of synaptic kainate receptor function observed during various forms of synaptic plasticity.

Quantitative phosphoproteomic analysis of neuronal intermediate filament proteins (NF-M/H) in Alzheimer's disease by iTRAQ.

Aberrant hyperphosphorylation of neuronal cytoskeletal proteins is one of the major pathological hallmarks of neurodegenerative disorders such as Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). Human NF-M/H display a large number of multiple KSP repeats in the carboxy-terminal tail domain, which are phosphorylation sites of proline-directed serine/threonine (pSer/Thr-Pro, KS/T-P) kinases. The phosphorylation sites of NF-M/H have not been characterized in AD brain. Here, we use quantitative phosphoproteomic methodology, isobaric tag for relative and absolute quantitation (iTRAQ), for the characterization of NF-M/H phosphorylation sites in AD brain. We identified 13 hyperphosphorylated sites of NF-M; 9 Lys-Ser-Pro (KSP) sites; 2 variant motifs, Glu-Ser-Pro (ESP) Ser-736 and Leu-Ser-Pro (LSP) Ser-837; and 2 non-S/T-P motifs, Ser-783 and Ser-788. All the Ser/Thr residues are phosphorylated at significantly greater abundance in AD brain compared with control brain. Ten hyperphosphorylated KSP sites have been identified on the C-terminal tail domain of NF-H, with greater abundance of phosphorylation in AD brain compared with control brain. Our data provide the direct evidence that NF-M/H are hyperphosphorylated in AD compared with control brain and suggest the role of both proline-directed and non-proline-directed protein kinases in AD. This study represents the first comprehensive iTRAQ analyses and quantification of phosphorylation sites of human NF-M and NF-H from AD brain and suggests that aberrant hyperphosphorylation of neuronal intermediate filament proteins is involved in AD.

Retinoschisin (RS1) interacts with negatively charged lipid bilayers in the presence of Ca2+: an atomic force microscopy study.

Retinoschisin (RS1) is a retina-specific secreted protein encoding a conserved discoidin domain sequence. As an adhesion molecule, RS1 preserves the retinal cell architecture and promotes visual signal transduction. In young males, loss-of-function mutations in the X-linked retinoschisis gene (RS1) cause X-linked retinoschisis, a form of progressive blindness. Neither the structure of RS1 nor the nature of its anchoring and organization on the plasma membranes is fully understood. The discoidin C2 domains of coagulation factors V and VIII are known to interact with extracellular phosphatidylserine (PS). In this study we have used atomic force microscopy (AFM) to study the interactions of murine retinoschisin (Rs1) with supported anionic lipid bilayers in the presence of Ca(2+). The bilayers consisting of a single lipid, PS, and mixtures of lipids with or without PS were used. Consistent with previous X-ray diffraction studies, AFM imaging showed two distinct domains in pure PS bilayers when Ca(2+) was present. Upon Rs1 adsorption, these PS and PS-containing mixed bilayers underwent fast and extensive reorganization. Protein localization was ascertained by immunolabeling. AFM imaging showed the Rs1 antibody bound exclusively to the calcium-rich ordered phase of the bilayers pointing to the sequestration of Rs1 within those domains. This was further supported by the increased mechanical strength of these domains after Rs1 binding. Besides, changes in bilayer thickness suggested that Rs1 was partially embedded into the bilayer. These findings support a model whereby the Rs1 protein binds to PS in the retinal cell plasma membranes in a calcium-dependent manner.

Regulation of phosphorylation at the postsynaptic density during different activity states of Ca2+/calmodulin-dependent protein kinase II.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), the most abundant kinase at the postsynaptic density (PSD), is expected to be involved in activity-induced regulation of synaptic properties. CaMKII is activated when it binds calmodulin in the presence of Ca(2+) and, once autophosphorylated on T-286/7, remains active in the absence of Ca(2+) (autonomous form). In the present study we used a quantitative mass spectrometric strategy (iTRAQ) to identify sites on PSD components phosphorylated upon CaMKII activation. Phosphorylation in isolated PSDs was monitored under conditions where CaMKII is: (1) mostly inactive (basal state), (2) active in the presence of Ca(2+), and (3) active in the absence of Ca(2+). The quantification strategy was validated through confirmation of previously described autophosphorylation characteristics of CaMKII. The effectiveness of phosphorylation of major PSD components by the activated CaMKII in the presence and absence of Ca(2+) varied. Most notably, autonomous activity in the absence of Ca(2+) was more effective in the phosphorylation of three residues on SynGAP. Several PSD scaffold proteins were phosphorylated upon activation of CaMKII. The strategy adopted allowed the identification, for the first time, of CaMKII-regulated sites on SAPAPs and Shanks, including three conserved serine residues near the C-termini of SAPAP1, SAPAP2, and SAPAP3. Involvement of CaMKII in the phosphorylation of PSD scaffold proteins suggests a role in activity-induced structural re-organization of the PSD.

The familial Mediterranean fever protein, pyrin, is cleaved by caspase-1 and activates NF-kappaB through its N-terminal fragment.

Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by mutations in MEFV, which encodes a 781-amino acid protein denoted pyrin. We have previously shown that pyrin regulates caspase-1 activation and IL-1beta production through interaction of its N-terminal PYD motif with the ASC adapter protein, and also modulates IL-1beta production by interaction of its C-terminal B30.2 domain with the catalytic domains of caspase-1. We now asked whether pyrin might itself be a caspase-1 substrate, and found that pyrin is cleaved by caspase-1 at Asp330, a site remote from the B30.2 domain. Pyrin variants harboring FMF-associated B30.2 mutations were cleaved more efficiently than wild-type pyrin. The N-terminal cleaved fragment interacted with the p65 subunit of NF-kappaB and with IkappaB-alpha through its 15-aa bZIP basic domain and adjacent sequences, respectively, and translocated to the nucleus. The interaction of the N-terminal fragment with p65 enhanced entrance of p65 into the nucleus. The interaction of N-terminal pyrin with IkappaB-alpha induced calpain-mediated degradation of IkappaB-alpha, thus potentiating NF-kappaB activation. Absolute and relative quantities of cleaved pyrin and IkappaB-alpha degradation products were substantially increased in leukocytes from FMF patients compared with healthy controls. Our data support a new pyrin/caspase-1 pathway for NF-kappaB activation.

Inhibition of Pin1 reduces glutamate-induced perikaryal accumulation of phosphorylated neurofilament-H in neurons.

Under normal conditions, the proline-directed serine/threonine residues of neurofilament tail-domain repeats are exclusively phosphorylated in axons. In pathological conditions such as amyotrophic lateral sclerosis (ALS), motor neurons contain abnormal perikaryal accumulations of phosphorylated neurofilament proteins. The precise mechanisms for this compartment-specific phosphorylation of neurofilaments are not completely understood. Although localization of kinases and phosphatases is certainly implicated, another possibility involves Pin1 modulation of phosphorylation of the proline-directed serine/threonine residues. Pin1, a prolyl isomerase, selectively binds to phosphorylated proline-directed serine/threonine residues in target proteins and isomerizes cis isomers to more stable trans configurations. In this study we show that Pin1 associates with phosphorylated neurofilament-H (p-NF-H) in neurons and is colocalized in ALS-affected spinal cord neuronal inclusions. To mimic the pathology of neurodegeneration, we studied glutamate-stressed neurons that displayed increased p-NF-H in perikaryal accumulations that colocalized with Pin1 and led to cell death. Both effects were reduced upon inhibition of Pin1 activity by the use of an inhibitor juglone and down-regulating Pin1 levels through the use of Pin1 small interfering RNA. Thus, isomerization of lys-ser-pro repeat residues that are abundant in NF-H tail domains by Pin1 can regulate NF-H phosphorylation, which suggests that Pin1 inhibition may be an attractive therapeutic target to reduce pathological accumulations of p-NF-H.

The Parkinson's disease kinase LRRK2 autophosphorylates its GTPase domain at multiple sites.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited Parkinson's disease (PD). The protein is large and complex, but pathogenic mutations cluster in a region containing GTPase and kinase domains. LRRK2 can autophosphorylate in vitro within a dimer pair, although the significance of this reaction is unclear. Here, we mapped the sites of autophosphorylation within LRRK2 and found several potential phosphorylation sites within the GTPase domain. Using mass spectrometry, we found that Thr1343 is phosphorylated and, using kinase dead versions of LRRK2, show that this is an autophosphorylation site. However, we also find evidence for additional sites in the GTPase domain and in other regions of the protein suggesting that there may be multiple autophosphorylation sites within LRRK2. These data suggest that the kinase and GTPase activities of LRRK2 may exhibit complex autoregulatory interdependence.

Comparative proteomic profiles of meningioma subtypes.

Meningiomas are classified into three groups (benign, atypical, and anaplastic) based on morphologic characteristics. Atypical meningiomas, which are WHO grade 2 tumors, and anaplastic meningiomas, which are WHO grade 3 tumors, exhibit an increased risk of recurrence and premature death compared with benign WHO grade 1 tumors. Although atypical and anaplastic meningiomas account for <10% of all of meningiomas, it can be difficult to distinguish them from benign meningiomas by morphologic criteria alone. We used selective tissue microdissection to examine 24 human meningiomas and did two-dimensional gel electrophoresis to determine protein expression patterns. Proteins expressed differentially by meningiomas of each WHO grade were identified and sequenced. Proteomic analysis revealed protein expression patterns unique to WHO grade 1, 2, and 3 meningiomas and identified 24 proteins that distinguish each subtype. Fifteen proteins showed significant changes in expression level between benign and atypical meningiomas, whereas nine distinguished atypical from anaplastic meningiomas. Differential protein expression was confirmed by Western blotting and immunohistochemistry. We established differential proteomic profiles that characterize and distinguish meningiomas of increasing grades. The proteins and proteomic profiles enhance understanding of the pathogenesis of meningiomas and have implications for diagnosis, prognosis, and treatment.

Proteomic comparison of oligodendrogliomas with and without 1pLOH.

OBJECTIVE: Chemoresistance is a widespread therapeutic challenge in glial tumors. The molecular basis of chemoresistance is poorly understood, precluding advances in glioma treatment and leaving gliomas among the most lethal tumors. Oligodendrogliomas provide a unique model to study the molecular basis of chemoresistance, as there are two distinct genetic subtypes with significant differences in chemosensitivity. Despite a high morphological similarity, tumors with allelic loss on the short arm of chromosome 1 (1pLOH) are more chemosensitive than those without 1pLOH. METHODS: In order to identify candidate proteins potentially responsible for glioma chemosensitivity, we compared the proteome of four oligodendrogliomas with and five without 1pLOH using comparative proteomic profiling. Proteomic analysis was performed by two-dimensional protein gel electrophoresis and subsequent computerized gel analysis for detection of distinguishing patterns of protein expression. Differentially expressed proteins were identified using Liquid Chromatography/Mass Spectrometry. Differential expression of select proteins was confirmed by Western blotting. RESULTS: We identified seven candidate proteins that are overexpressed in oligodendrogliomas without 1pLOH. Two of these proteins (glyoxalase I and Rho GDP dissociation inhibitor) have previously been shown to enhance chemoresistance in other tumors. In turn, we identified twelve overexpressed proteins in tumors with 1pLOH that have previously been reported to induce chemosensitivity in other forms of human neoplasia. CONCLUSIONS: These identified proteins are potential targets for pharmacological therapy and may also be useful as biomarkers for differentiation of chemoresistant and chemosensitive oligodendroglioma.

Thoracic splenosis: History is the key.

Splenosis is an acquired ectopic autotransplantation of splenic tissue; that occurs after traumatic splenic rupture and splenectomy [1]. Splenosis is a rare but benign disease, and the diagnosis can be challenging as the multiple incidentally found nodules could mimic malignancy [2]. Abdominopelvic Splenosis is thought to occur in as many as 65% of cases of splenic rupture [1]. However, Thoracic Splenosis is rare and usually involve the left parietal and visceral pleura [1,2]. Intraparenchymal lesions are less common but have been reported in cases of parenchymal and diaphragm laceration [1,2]. Taking a thorough history is of utmost importance, as these patients usually present more than two decades after the splenic traumatic rupture. The use of commonly available nuclear studies will further confirm the diagnosis [3]. This will help to avoid unnecessary procedures, like biopsies; and prevent the potential complications. We present a case of Thoracic Splenosis that highlights the importance of taking a detailed history; and the importance of using nuclear studies for the diagnosis. Further adding to its uniqueness, this case showed with multiple intraparenchymal nodules which is a less common presentation of Splenosis.

Proteomic analysis of inclusion body myositis.

Sporadic inclusion body myositis (IBM) is the most frequently acquired inflammatory myopathy of late adult life, yet its diagnostic criteria and pathogenesis remain poorly defined. Because effective treatment is lacking, research efforts have intensified to identify specific markers for this debilitating disorder. In this study, proteomic analysis of 4 cases of sporadic IBM was compared with 5 cases of inflammatory myopathy without clinicopathologic features of IBM to distinguish the IBM-specific proteome. Proteins were separated by 2-dimensional polyacrylamide gel electrophoresis and profiled by mass spectrometric sequencing. Expression of most proteins remained unchanged; however, 16 proteins were upregulated and 6 proteins were downregulated in IBM compared with cases of non-IBM inflammatory myopathy. These IBM-specific proteins included apolipoprotein A-I, amyloid beta precursor protein, and transthyretin, which have been associated with amyloidosis; superoxide dismutase, enolase, and various molecular chaperones indicate perturbations in detoxification, energy metabolism, and protein folding, respectively. The IBM-downregulated proteins mainly serve as carriers for muscle contraction and other normal muscle functions. We further applied Western blot and immunohistochemistry to verify the proteomic findings. This study validates proteomics as a powerful tool in the study of muscle disease and indicates a unique pattern of protein expression in IBM.

Regulation of ASAP1 by phospholipids is dependent on the interface between the PH and Arf GAP domains.

ASAP1 is an Arf GAP with a PH domain immediately N-terminal to the catalytic Arf GAP domain. PH domains are thought to regulate enzymes by binding to specific phosphoinositide lipids in membranes, thereby recruiting the enzyme to a site of action. Here, we have examined the functional relationship between the PH and Arf GAP domains. We found that GAP activity requires the cognate PH domain of ASAP1, leading us to hypothesize that the Arf GAP and PH domains directly interact to form the substrate binding site. This hypothesis was supported by the combined results of protection and hydrodynamic studies. We then examined the role of the PH domain in the regulation of Arf GAP activity. The results of saturation kinetics, limited proteolysis, FRET and fluorescence spectrometry support a model in which regulation of the GAP activity of ASAP1 involves a conformational change coincident with recruitment to a membrane surface, and a second conformational change following the specific binding of phosphatidylinositol 4,5-bisphosphate.

p35/cyclin-dependent kinase 5 phosphorylation of ras guanine nucleotide releasing factor 2 (RasGRF2) mediates Rac-dependent Extracellular Signal-regulated kinase 1/2 activity, altering RasGRF2 and microtubule-associated protein 1b distribution in neurons.

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed kinase the activity of which is dependent on association with its neuron-specific activators, p35 and p39. Cdk5 activity is critical for the proper formation of cortical structures and lamination during development. In the adult nervous system, Cdk5 function is implicated in cellular adhesion, dopamine signaling, neurotransmitter release, and synaptic activity. In addition, Cdk5 is also involved in "cross-talk" with other signal transduction pathways. To further examine its involvement in cross-talk with other pathways, we identified proteins that interacted with p35 using the yeast two-hybrid system. We report here that p35 associates with Ras guanine nucleotide releasing factor 2 (RasGRF2) in coimmunoprecipitation and colocalization studies using transfected cell lines as well as primary cortical neurons. Additionally, Cdk5 phosphorylates RasGRF2 both in vitro and in vivo, leading to a decrease in Rac-guanidine exchange factor activity and a subsequent reduction in extracellular signal-regulated kinase 1/2 activity. We show that p35/Cdk5 phosphorylates RasGRF2 on serine737, which leads to an accumulation of RasGRF2 in the neuronal cell bodies coinciding with an accumulation of microtubule-associated protein 1b. The membrane association of p35 and subsequent localization of Cdk5 activity toward RasGRF2 and Rac provide insights into important cellular signaling processes that occur at the membrane, resulting in downstream effects on signal transduction cascades.

Regulation of Sox6 by cyclin dependent kinase 5 in brain.

Cyclin dependent kinase 5 (Cdk5) is a proline-directed Ser/Thr kinase involved in various biological functions during normal brain development and neurodegeneration. In brain, Cdk5 activity is specific to post-mitotic neurons, due to neuronal specific expression of its activator p35. The biological functions of Cdk5 have been ascribed to its cytoplasmic substrates, however not much is known in nucleus. Here, we show that nuclear transcription factor Sox6 is a direct nuclear target of Cdk5. Sox6 is expressed in Tuj1 positive neurons, suggesting that Sox6 is expressed in differentiating neurons. The expression of Sox6 is high in mitotic nuclei during embryonic day 12 (E12) and gradually decreases during development into adult. On the other hand, Cdk5 expression gradually increases during its development. We show that Sox6 is expressed in mitotic nuclei in embryonic day 12 (E12) and in migrating neurons of E16. Sox6 is phosphorylated in vivo. Sox6 was detected by phospho-Ser/Thr and phospho-Ser/Thr-Pro and MPM-2 (Mitotic protein #2) antibodies in brain. Furthermore, calf intestinal alkaline phosphatase (CIAP) digestion resulted in faster migration of Sox6 band. The GST-Sox6 was phosphorylated by Cdk5/p35. The mass spectrometry analysis revealed that Sox6 is phosphorylated at T119PER motif. We show that Sox6 steady state levels are regulated by Cdk5. Cdk5 knockout mice die in utero and Sox6 protein expression is remarkably high in Cdk5-/- brain, however, there is no change in mRNA expression, suggesting a post-translational regulation of Sox6 by Cdk5. Transfection of primary cortical neurons with WT Cdk5 reduced Sox6 levels, while dominant negative (DN) Cdk5 and p35 increased Sox6 levels. Thus, our results indicate that Cdk5 regulates Sox6 steady state protein level that has an important role in brain development and function.

Anti-lymphoproliferative activity of alpha-2-macroglobulin in the plasma of hibernating 13-lined ground squirrels and woodchucks.

Plasma from hibernating (HIB) woodchucks (Marmota monax) or 13-lined ground squirrels (Ictidomys tridecemlineatus) suppressed (3)H-thymidine uptake in mouse spleen cell cultures stimulated with Concanavalin A (ConA); plasma from non-hibernating animals were only slightly inhibitory. Maximum inhibition occurred when HIB plasma was added to the cultures prior to ConA. After HPLC size exclusion chromatography of the HIB ground squirrel plasma, a single fraction (fraction-14) demonstrated inhibitory activity. Assay of fraction-14 from 8 HIB squirrels showed inhibition ranging from 13 to 95%; inhibition was correlated to the time the squirrels were exposed to cold prior to hibernation. Western blot analysis showed the factor to be a large molecular weight protein (>300 kDa), and mass spectrometry identified sequences that were 100% homologous with alpha-2-macroglobulin from humans and other species. These findings indicate a hibernation-related protein that may be responsible for immune system down regulation.

Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice.

Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate.

Interventions to reduce rehospitalizations after chronic obstructive pulmonary disease exacerbations. A systematic review.

RATIONALE: Approximately 20% of patients hospitalized for COPD exacerbations in the United States will be readmitted within 30 days. The Centers for Medicare and Medicaid Services has recently proposed to revise the Hospital Readmissions Reduction Program to financially penalize hospitals with high all-cause 30-day rehospitalization rates after a hospitalization for COPD exacerbation on or after October 1, 2014. OBJECTIVES: To report the results of a systematic review of randomized clinical trials evaluating interventions to reduce the rehospitalizations after COPD exacerbations. METHODS: Multiple electronic databases were systematically searched to identify relevant studies published between January 1966 and June 2013. Titles, abstracts, and, subsequently, full-text articles were assessed for eligibility. Each study was appraised using predefined criteria. MEASUREMENTS AND MAIN RESULTS: Among 913 titles and abstracts screened, 5 studies (1,393 participants) met eligibility criteria. All studies had a primary outcome of rehospitalization at 6 or 12 months. No study examined 30-day rehospitalization as the primary outcome. Each study tested a different set of interventions. Two studies (one conducted in Canada and one conducted in Spain and Belgium) showed a decrease in all-cause rehospitalization over 12 months in the intervention group versus comparator group (mean number of hospitalizations per patient, 1.0 vs. 1.8; P = 0.01; percent hospitalized, 45 vs. 67%; P = 0.028; respectively). The only study conducted in the United States found a greater than twofold higher risk of mortality in the intervention group (17 vs. 7%, P = 0.003) but no significant difference in rehospitalizations. It was unclear which set of interventions was effective or harmful. CONCLUSIONS: The evidence base is inadequate to recommend specific interventions to reduce rehospitalizations in this population and does not justify penalizing hospitals for high 30-day rehospitalization rates after COPD exacerbations.