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Background Hospitalists value mentorship and scholarly work, yet often struggle to find time and mentors amid busy clinical workloads. Objective To help catalyze writing for hospitalists nationally, we created a Writing Challenge, where we asked hospitalists to commit to the goal of writing 400 words a day, four days a week, for four weeks. Methods Prospective, programmatic evaluation with daily logs followed by a survey at the completion of the project. The four-week Writing Challenge occurred between June 7 and July 5, 2021. Email invitations to participate in the challenge were disseminated to peer networks, and the challenge was promoted using social media. Participants agreed to attempt to write 400 words per day, four days per week, for four weeks. Results Seventy-four individuals from 28 institutions registered for the Writing Challenge, with 36 (49%) participating in the challenge by logging their writing. Participants wrote an average of 4,372 +/- 4,324 words during the challenge. Sixty-eight percent of the participants reported that their amount of writing increased during the challenge and 50% of the participants stated they planned to publish their work, though many participants (46%) reported struggling to write each day. Conclusions The Writing Challenge is one way to generate increased writing and may result in increased scholarly output for academic hospitalists.

In reproductive medicine, the technique of rapid cooling becomes increasingly important for the preservation of tissue and cells. In order to protect the cells, incubation in different cryopreservation solutions is essential. The speed of the cooling process also makes a pivotal contribution to the success of this method. Using Flourescence Activated Cell Sorting (FACS), we investigated the impact of an open rapid and a closed rapid cooling technique on the vitality of human granulosa cells. Furthermore, we examined effects of the different solutions used for rapid cooling and warming before and after rapid cooling. We found a significant lower proportion of vital cells after rapid cooling compared to untreated controls independently of the technique and the tube size. However, we did not find any significant differences between open and closed rapid cooling. In both, a lower proportion of vital granulosa cells were found after incubation in rapid cooling solution only compared to warming solution only. Our results lend support to the conclusion that the difference of cooling-speed between open and closed rapid cooling is, in our settings, not crucial for the success of the procedure and that cryoprotective agents in the rapid cooling solutions have a higher potential to cause severe cell damage than agents used for warming.

Background Vitrification has superseded the slow freezing method for cryopreservation of oocytes, embryos, and sperm, but there are as yet no standard protocols for its use in ovarian tissue cryopreservation (OTC). Published protocols diverge mainly with regard to the extent of supplementation of dimethyl sulfoxide (DMSO) to the vitrification medium, and to the use of an open or closed vitrification system. We investigated the viability of cells after vitrification/warming, using ovarian tissue of transgender patients, by means of Fluorescence Activated Cells Sorting (FACS), and histomorphological analyses using a DMSO-containing (P1) and a DMSO-free protocol (P2) in an open or closed vitrification setting. Results Twelve ovarian samples were donated from female-to-male transgender patients: 6 were vitrified according to protocol 1, the other 6 according to protocol 2. The amount of viable cells was 90.1% (P1) and 88.4% (P2) before vitrification. After vitrification and subsequent warming, viable cells were reduced to 82.9% (P1, p = 0.093) and 72.4% (P2, p = 0.019). When comparing the closed and the open systems, the decline in cell viability from pre- to post-vitrification was significant only for the latter (p = 0.037). Histological examination reveals no significant differences with respect to degenerated follicles before or after vitrification. Conclusion These results led us to conclude that a protocol containing DMSO results in a higher viability of ovarian cells than a protocol that uses ethylene glycol as cryoprotective agent in vitrification. The use of an open vitrification system led to significant decline in the rate of viable cells. Trial registration NCT03649087, retrospectively registered 28.08.2018.

Latinx in the USA experience disparities in morbidity and mortality when compared to their non-Hispanic White counterparts. Patient-centered culturally sensitive health care (PC-CSHC) has been deemed a best practice approach to alleviate and eliminate these disparities. However, literature on how Latinx patients perceive their care and what indicators of PC-CSHC may be most related to treatment outcomes is limited. This study collected data from 81 adult Latinx participants who had been admitted to an inpatient care unit to understand the following: (a) their perception of their providers' PC-CSHC in three different areas: Competence/Confidence, Sensitivity/Interpersonal, and Respect/Communication; (b) whether there are differences between English- and Spanish-speaking Latinx patients in their perception of their providers' PC-CSHC; and (c) whether these PC-CSHC indicators were associated to patient satisfaction, patient-provider communication, and therapeutic alliance. Participants were mostly male, older than 55 years of age, and working or lower class, with English as their primary language. Results showed that patients rated their providers' Competence (M = 3.57, SD = .46) higher than both Sensitivity, t(68) = .04, p = .04, (M = 3.49, SD =.54), and Respect, t(53) = 2.765, p = .008, (M = 3.38, SD = .57). English-speaking Latinx were overall less satisfied with their providers than Spanish-speaking Latinx, in particular in their communication. Finally, higher provider cultural sensitivity appears to be a predictor of patient satisfaction, patient-provider communication, and working alliance. Implications for refining provider trainings to treat this vulnerable and understudied (i.e., Latinx) population are discussed.

In a global economy, online meeting participants often have different backgrounds, making it difficult to know their mental and physical states. The Mental Motion State Model, trained by applying the Gaussian mixture model to electroencephalogram data, can indicate degrees of engagement and fatigue levels, providing a feedback channel for self-regulation. In the web extra at https://youtu.be/-lO3jsMUJVU, guest editor Katarzyna Wac interviews author Renate Fruchter, founding director of the Project Based Learning Laboratory at Stanford University.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to LDL receptors, leading to their degradation. Genetics studies have shown that loss-of-function mutations in PCSK9 result in reduced plasma LDL cholesterol and decreased risk of coronary heart disease. We aimed to investigate the safety and efficacy of ALN-PCS, a small interfering RNA that inhibits PCSK9 synthesis, in healthy volunteers with raised cholesterol who were not on lipid-lowering treatment.We did a randomised, single-blind, placebo-controlled, phase 1 dose-escalation study in healthy adult volunteers with serum LDL cholesterol of 3·00 mmol/L or higher. Participants were randomly assigned in a 3:1 ratio by computer algorithm to receive one dose of intravenous ALN-PCS (with doses ranging from 0·015 to 0·400 mg/kg) or placebo. The primary endpoint was safety and tolerability of ALN-PCS. Secondary endpoints were the pharmacokinetic characteristics of ALN-PCS and its pharmacodynamic effects on PCSK9 and LDL cholesterol. Study participants were masked to treatment assignment. Analysis was per protocol and we used ANCOVA to analyse pharmacodynamic endpoint data. This trial is registered with ClinicalTrials.gov, number NCT01437059.Of 32 participants, 24 were randomly allocated to receive a single dose of ALN-PCS (0·015 mg/kg [n=3], 0·045 mg/kg [n=3], 0·090 mg/kg [n=3], 0·150 mg/kg [n=3], 0·250 mg/kg [n=6], or 0·400 mg/kg [n=6]) and eight to placebo. The proportions of patients affected by treatment-emergent adverse events were similar in the ALN-PCS and placebo groups (19 [79%] vs seven [88%]). ALN-PCS was rapidly distributed, with peak concentration and area under the curve (0 to last measurement) increasing in a roughly dose-proportional way across the dose range tested. In the group given 0·400 mg/kg of ALN-PCS, treatment resulted in a mean 70% reduction in circulating PCSK9 plasma protein (p0·0001) and a mean 40% reduction in LDL cholesterol from baseline relative to placebo (p0·0001).Our results suggest that inhibition of PCSK9 synthesis by RNA interference (RNAi) provides a potentially safe mechanism to reduce LDL cholesterol concentration in healthy individuals with raised cholesterol. These results support the further assessment of ALN-PCS in patients with hypercholesterolaemia, including those being treated with statins. This study is the first to show an RNAi drug being used to affect a clinically validated endpoint (ie, LDL cholesterol) in human beings.Alnylam Pharmaceuticals.

Background: Current standard medications for the treatment of allergic inflammation consist primarily of glucocorticoids and anti-histamines, but adverse side effects or insufficient responsiveness by patient subpopulations illustrate the need for safe and novel alternatives. Thus, there is a demand to develop a porcine model that is able to mimic mast cell-mediated type I hypersensitivity. Previously, we found that escin, a pharmacologically active mix of triterpene saponins from horse chestnut extracts, exerts anti-allergic effects in murine models and merits further investigation as an anti-allergic therapeutic. Methods: We developed a new porcine model of allergic dermatitis based on a clinical prick test protocol. Histamine clearly provoked erythema and swelling at the prick site, whereas the mast cell-degranulating compound 48/80 even more pronounced caused wheal and flare reactions known from the human prick response. This model was used to test the anti-allergic efficacy of orally applied escin. Results: Oral pretreatment of animals with escin strongly inhibited the allergic skin response induced by compound 48/80 in a dose-dependent manner. Additional in vitro data from murine mast cells indicate an engagement of the glucocorticoid receptor pathway upon treatment with escin. Conclusions: This model provides a valuable and easy-to-set-up tool for preclinical studies of mast cell-inhibiting compounds. The successful implementation of this model supports the development of oral escin applications as a novel anti-allergic therapy.

SummaryXBP1 is a key regulator of the unfolded protein response (UPR), which is involved in a wide range of physiological and pathological processes. XBP1 ablation in liver causes profound hypolipidemia in mice, highlighting its critical role in lipid metabolism. XBP1 deficiency triggers feedback activation of its upstream enzyme IRE1α, instigating regulated IRE1-dependent decay (RIDD) of cytosolic mRNAs. Here, we identify RIDD as a crucial control mechanism of lipid homeostasis. Suppression of RIDD by RNA interference or genetic ablation of IRE1α reversed hypolipidemia in XBP1-deficient mice. Comprehensive microarray analysis of XBP1 and/or IRE1α-deficient liver identified genes involved in lipogenesis and lipoprotein metabolism as RIDD substrates, which might contribute to the suppression of plasma lipid levels by activated IRE1α. Ablation of XBP1 ameliorated hepatosteatosis, liver damage, and hypercholesterolemia in dyslipidemic animal models, suggesting that direct targeting of either IRE1α or XBP1 might be a feasible strategy to treat dyslipidemias.

Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that Sp-configured PS linkages are stabilized relative to Rp, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3'-SpSpRp, that promotes target RNA cleavage by RNase H1 in vitro and provides a more durable response in mice than stereorandom ASOs.

Recent GWAS have identified SNPs at a human chromosom1 locus associated with coronary artery disease risk and LDL cholesterol levels. The SNPs are also associated with altered expression of hepatic sortilin-1 (SORT1), which encodes a protein thought to be involved in apoB trafficking and degradation. Here, we investigated the regulation of Sort1 expression in mouse models of obesity. Sort1 expression was markedly repressed in both genetic (ob/ob) and high-fat diet models of obesity; restoration of hepatic sortilin-1 levels resulted in reduced triglyceride and apoB secretion. Mouse models of obesity also exhibit increased hepatic activity of mammalian target of rapamycin complex 1 (mTORC1) and ER stress, and we found that administration of the mTOR inhibitor rapamycin to ob/ob mice reduced ER stress and increased hepatic sortilin-1 levels. Conversely, genetically increased hepatic mTORC1 activity was associated with repressed Sort1 and increased apoB secretion. Treating WT mice with the ER stressor tunicamycin led to marked repression of hepatic sortilin-1 expression, while administration of the chemical chaperone PBA to ob/ob mice led to amelioration of ER stress, increased sortilin-1 expression, and reduced apoB and triglyceride secretion. Moreover, the ER stress target Atf3 acted at the SORT1 promoter region as a transcriptional repressor, whereas knockdown of Atf3 mRNA in ob/ob mice led to increased hepatic sortilin-1 levels and decreased apoB and triglyceride secretion. Thus, in mouse models of obesity, induction of mTORC1 and ER stress led to repression of hepatic Sort1 and increased VLDL secretion via Atf3. This pathway may contribute to dyslipidemia in metabolic disease.

SummaryInsulin resistance leads to hypertriglyceridemia and hepatic steatosis and is associated with increased SREBP-1c, a transcription factor that activates fatty acid synthesis. Here, we show that steatosis in insulin-resistant ob/ob mice was abolished by deletion of Scap, an escort protein necessary for generating nuclear isoforms of all three SREBPs. Scap deletion reduced lipid synthesis and prevented fatty livers despite persistent obesity, hyperinsulinemia, and hyperglycemia. Scap deficiency also prevented steatosis in mice fed high-fat diets. Steatosis was also prevented when siRNAs were used to silence Scap in livers of sucrose-fed hamsters, a model of diet-induced steatosis and hypertriglyceridemia. This silencing reduced all three nuclear SREBPs, decreasing lipid biosynthesis and abolishing sucrose-induced hypertriglyceridemia. These results demonstrate that SREBP activation is essential for development of diabetic hepatic steatosis and carbohydrate-induced hypertriglyceridemia, but not insulin resistance. Inhibition of SREBP activation has therapeutic potential for treatment of hypertriglyceridemia and fatty liver disease.

Online supplemental material. Table S1 shows the expression of genes related to drug metabolism in XBP1-deficient liver. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20111298/DC1., The mammalian stress sensor IRE1α plays a central role in the unfolded protein, or endoplasmic reticulum (ER), stress response by activating its downstream transcription factor XBP1 via an unconventional splicing mechanism. IRE1α can also induce the degradation of a subset of mRNAs in a process termed regulated IRE1-dependent decay (RIDD). Although diverse mRNA species can be degraded by IRE1α in vitro, the pathophysiological functions of RIDD are only beginning to be explored. Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in young adults in the United States and is primarily caused by CYP1A2-, CYP2E1-, and CYP3A4-driven conversion of APAP into hepatotoxic metabolites. We demonstrate here that genetic ablation of XBP1 results in constitutive IRE1α activation in the liver, leading to RIDD of Cyp1a2 and Cyp2e1 mRNAs, reduced JNK activation, and protection of mice from APAP-induced hepatotoxicity. A pharmacological ER stress inducer that activated IRE1α suppressed the expression of Cyp1a2 and Cyp2e1 in WT, but not IRE1α-deficient mouse liver, indicating the essential role of IRE1α in the down-regulation of these mRNAs upon ER stress. Our study reveals an unexpected function of RIDD in drug metabolism., National Institutes of Health (U.S.) (grant AI32412), National Institutes of Health (U.S.) (grant DK082448), National Institutes of Health (U.S.) (grant DK089211), American Heart Association

Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.

Triethylene glycol dimethacrylate (TEGDMA) is a resin monomer which is released from polymerized dental composite materials. It induced apoptosis in various target cells or inhibition of LPS-induced cytokine production in cells of the immune system after prolonged exposure. In these tissues, mitogen-activated protein kinases (MAPK) regulate signal transduction pathways that support cell survival and cytokine synthesis. The time-dependent regulation of MAPK as well as their linkage to the induction of apoptosis and cytokine release under the influence of resin monomers is unknown. It was the aim of the present study to investigate the kinetics of the up- or down-regulation of the MAPK p38, JNK, and ERK1/2, the induction of apoptosis and cytokine release in RAW264.7 mouse macrophages and human pulp-derived cells. ERK1/2, p38 and JNK were differentially activated by phosphorylation in the presence of lipopolysaccharide (0.1 microg/ml; LPS), a known inducer of MAPK activity, and TEGDMA (3 mM) as detected by Western blotting. In macrophages, ERK1/2 was activated about 6-fold by LPS, while no activation was observed in the presence of TEGDMA after 15 and 30 min. A slight activation of p38 was detected in cell cultures after short exposure to TEGDMA (30 min), but activated JNK was identified after LPS stimulation only. After a long 24 h exposure period, ERK1/2 and p38 were strongly activated by LPS, a combination of LPS/TEGDMA, and TEGDMA alone (15-20-fold). In human pulp-derived cells, ERK1/2 was phosphorylated after exposure to TEGDMA up to 2 h, and sustained activation of ERK1/2 as well as p38 (12-15-fold) was detected after prolonged exposure for 24 h. The LPS-induced, time-related increase in the secretion of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) as well as the anti-inflammatory IL-10 was instantaneously inhibited by TEGDMA in mouse macrophages. In parallel, the percentage of cells in macrophage cultures in the stage of apoptosis and necrosis increased with exposure period. Yet, in contrast to the inhibition of cytokine release, apoptosis and necrosis caused by LPS and TEGDMA was a late response in both mouse macrophages and human pulp-derived cells. From these data it appears as if MAPK activation, inhibition of cytokine release and the induction of apoptosis and necrosis by TEGDMA are tightly related. The direct causal correlation of these phenomena, however, requires further investigation.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50–70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5′-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia.

To identify the clinical and epidemiological profile of cryptococcosis diagnosed at the University Hospital of the Federal University of Mato Grosso do Sul, Brazil, medical records of 123 patients admitted from January 1995 to December 2005 were analyzed. One hundred and four cases (84.5%) had HIV infection, six (4.9%) had other predisposing conditions and 13 (10.6%) were immunocompetent. Male patients predominated (68.3%) and their age ranged from 19 to 69 years (mean: 35.9). Most patients (73.2%) were born and lived lifelong in the state of Mato Grosso do Sul. Involvement of the central nervous system occurred in 103 patients (83.7%) and headache and vomiting were the most frequent symptoms. In 77 cases it was possible to identify the Cryptococcus species: 69 (89.6%) C. neoformans and eight (10.4%) C. gattii. Amphotericin B was the drug of choice for treatment (106/123), followed by fluconazole in 60% of cases. The overall lethality rate was 49.6%, being 51% among the HIV infected patients and 41.2% among the non-HIV infected (p > 0.05). Although cryptococcosis exhibited in our region a similar behavior to that described in the literature, the detection of an important rate of immunocompetent individuals and five C. gattii cryptococcosis in HIV-infected patients is noteworthy.O perfil clínico-epidemiológico de 123 casos de criptococose diagnosticados no Hospital Universitário da Universidade Federal de Mato Grosso do Sul, no período de janeiro de 1995 até dezembro de 2005, foi estudado retrospectivamente. Cento e quatro (84,9%) casos tinham associação com HIV, seis (4,9%) tinham outra condição predisponente e 13 (10,6%) eram imunocompetentes. Houve predomínio do sexo masculino (68,3%) e a idade variou de 19 a 69 anos (média de 35,9 anos). A maioria (73,2%) era natural e procedente de Mato Grosso do Sul. O envolvimento do sistema nervoso central ocorreu em 103 (83,7%) pacientes e os sintomas mais freqüentes foram cefaléia e vômitos. Em 77 casos foi possível identificar a espécie do agente, sendo 69 (89,6%) C. neoformans e oito (10,4%) C. gattii. O antifúngico mais utilizado foi anfotericina B (106/123) seguido de fluconazol em aproximadamente 60% dos casos. A taxa de letalidade foi de 49,6%, sendo 51% entre os pacientes infectados pelo HIV e 41,2% entre os não infectados pelo HIV (p > 0,005). Apesar da criptococose observada em nossa região apresentar comportamento semelhante ao descrito na literatura, chama a atenção a importante taxa da micose em imunocompetentes e cinco casos de infecção por C. gattii em pacientes HIV-positivos.

Recently, chemically synthesized short interfering RNA (siRNA) duplexes have been used with success for gene silencing. Chemical modification is desired for therapeutic applications to improve biostability and pharmacokinetic properties; chemical modification may also provide insight into the mechanism of silencing. siRNA duplexes containing the 2,4-difluorotoluyl ribonucleoside (rF) were synthesized to evaluate the effect of noncanonical nucleoside mimetics on RNA interference. 5'-Modification of the guide strand with rF did not alter silencing relative to unmodified control. Internal uridine to rF substitutions were well-tolerated. Thermal melting analysis showed that the base pair between rF and adenosine (A) was destabilizing relative to a uridine-adenosine pair, although it was slightly less destabilizing than other mismatches. The crystal structure of a duplex containing rFoA pairs showed local structural variations relative to a canonical RNA helix. As the fluorine atoms cannot act as hydrogen bond acceptors and are more hydrophobic than uridine, there was an absence of a well-ordered water structure around the rF residues in both grooves. siRNAs with the rF modification effectively silenced gene expression and offered improved nuclease resistance in serum; therefore, evaluation of this modification in therapeutic siRNAs is warranted.

Cross-cultural interaction in global business has become standard in design and construction industry. This paper presents lessons learned from a study aimed to determine how different multi-cultural team settings and cultural awareness trigger different conflict situations and influence team dynamics and processes. We build on extant classic mono-cultural studies and extend it to focus on analyzing the influence of different multicultural team settings on global project product and process success. We used empirical data from the 2012 generation of the 19 th AEC Global Teamwork course offered by the PBL Lab at Stanford with partners worldwide. We took an inductive approach integrating qualitative and quantitative methods for date analysis. The paper presents cultural team compilation cases of AEC global student teams that were successful or challenged in producing high quality solutions and achieving effective team processes. The paper discusses findings related to: the relation between nationality and migration backgrounds indicating that mono-culture studies do not suffice to analyze global teamwork; correlation of project outcome and the multicultural team compilation; correlation of cultural awareness and diversity of global team processes towards successful multi-cultural team interaction.

Genome-wide association studies (GWAS) have identified 95 loci in the human genome that harbor common variants associated with plasma lipid traits. Of the 95 loci, 17 harbor genes known to cause monogenic lipid disorders and collectively a third of them contain genes with characterized roles in lipid metabolism. Therefore in the majority of loci the causal genes are unknown. We selected 32 genes, not previously implicated in lipid metabolism and representing a total of 26 loci, to test for their ability to modify plasma lipid concentrations upon somatic overexpression in vivo. We utilized adeno-associated virus serotype 8 (AAV8) to overexpress the selected genes specifically in the livers of both C57BL/6 mice and in an appropriate humanized mouse model (either mice expressing human apolipoprotein A-I for HDL loci or Apobec1-knockout, Ldlr haploinsufficient mice expressing human apolipoprotein B-100 for triglyceride and LDL loci). Approximately half of the genes tested reproducibly affected plasma lipids. For 13 of the interrogated loci the lipid-associated variants also correlated with expression variations of the respective genes in liver (liver expression quantitative trait loci-eQTLs). We demonstrate a causal role for 7 of these 13 genes. The overexpression of these 7 genes not only affected the predicted lipid class, but additionally exerted its effect in the predicted direction in 6 of 7 cases (Tmem57, Slc39a8, Ppp1r3b, Vkorc1, Tbkbp1 and Ube2l3). Additionally for a subset of the examined genes we proceeded to develop small interfering RNA (siRNA) nanoparticles that were particularly targeted to the liver. We were able to obtain robust knockdown for a significant number of genes and, in several cases, observe reciprocal effects on plasma lipids from our overexpression and knockdown studies. This work has identified several novel lipid regulators, whose further investigation can uncover novel mechanisms and pathways controlling plasma lipids.

Proprotein convertases subtilisin/kexin type 9 (PCSK9) is a member of the proprotein convertase (PC) family of subtilisin-like serine endoproteases that regulates low density lipoprotein receptor (LDLR) levels and function. Murine models and human genetic studies indicate that loss of PCSK9 protein increases LDLR levels while excess PCSK9 decreases LDLR levels. These changes in LDLR protein levels coincide with reciprocal changes in circulating levels of plasma LDL cholesterol (LDL-C). We have developed a highly potent RNA interference (RNAi) therapeutic, ALN-PCS, targeting both intra and extracellular PCSK9 for inhibition through an RNAi mechanism. Pre-clinical data in non-human primate models, indicate that a single intravenous dose of ALN-PCS results in rapid, dose dependent, and significant lowering of liver PCSK9 transcript, plasma PCSK9 protein and subsequently serum LDL-C and ApoB levels, without impacting serum HDL-C. Here we report on interim data from an ongoing Phase 1 trial of ALN-PCS being conducted as a randomized, single-blind, placebo-controlled, single-ascending dose study in healthy volunteer subjects with elevated baseline LDL-C (>116mg/dL) who are not on any lipid lowering therapy. The primary objective of the study is to evaluate the safety and tolerability of a single dose of ALN-PCS, with subjects being enrolled into sequential cohorts of increasing doses. Secondary objectives of the study include characterization of plasma and urine pharmacokinetics of ALN-PCS, assessment of pharmacodynamic effects of the drug on plasma PCSK9 protein levels, and evaluation of clinical efficacy as measured by LDL-C levels. Data from 20 subjects enrolled in five sequential dose cohorts ranging from 0.015 to 0.250 mg/kg in a 3:1 randomization of drug to placebo will be presented. ALN-PCS was safe and well tolerated in this study and there have been no serious adverse events related to study drug administration to date. There have been no drug-related discontinuations from the study and no liver enzyme elevations. A mild, transient rash that resolved spontaneously was observed in three subjects that received ALN-PCS, and in two that received placebo. To date, administration of ALN-PCS resulted in a rapid, dose-dependent, and durable silencing of PCSK9 protein levels in plasma of up to 66% relative to baseline, with a statistically significant mean reduction of 60% at day four in the current high dose group of 0.250 mg/kg (p

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proprotein convertase (PC) family of subtilisin-like serine endoproteases that regulates low density lipoprotein receptor (LDLR) levels and function. Loss of PCSK9 protein (in murine models, as well as, in human individuals) increases LDLR levels while excess PCSK9 decreases LDLR levels. These changes in LDLR protein levels coincide with reciprocal changes in circulating levels of plasma LDL cholesterol. Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-like family of secreted proteins. Similar to PCSK9, it is predominantly expressed in the liver. ANGPTL3 acts as dual inhibitor of both lipoprotein and endothelial lipases. Animal studies as well as human genetic studies suggest that siRNA silencing of PCSK9 should result in substantial decrease in LDLc. Similar studies have suggested that liver silencing of ANGPTL3 should result in profound lowering of LDLc, Triglycerides and HDLc, while maintaining the HDLc/LDLc ratio. We have developed highly potent RNAi therapeutics targeting both PCSK9 and ANGPTL3. Pre-clinical data utilizing intravenous infusion of siRNA formulated in lipid nanoparticles, and subcutaneous delivery of siRNA-GalNAc conjugates will be discussed.

Significant effort has been applied to discover and develop vehicles which can guide small interfering RNAs (siRNA) through the many barriers guarding the interior of target cells. While studies have demonstrated the potential of gene silencing in vivo, improvements in delivery efficacy are required to fulfill the broadest potential of RNA interference therapeutics. Through the combinatorial synthesis and screening of a different class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in mice at doses below 0.01 mg/kg. This formulation was also shown to specifically inhibit expression of five hepatic genes simultaneously, after a single injection. The potential of this formulation was further validated in nonhuman primates, where high levels of knockdown of the clinically relevant gene transthyretin was observed at doses as low as 0.03 mg/kg. To our knowledge, this formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously described siRNA liver delivery system.

To identify the clinical and epidemiological profile of cryptococcosis diagnosed at the University Hospital of the Federal University of Mato Grosso do Sul, Brazil, medical records of 123 patients admitted from January 1995 to December 2005 were analyzed. One hundred and four cases (84.5%) had HIV infection, six (4.9%) had other predisposing conditions and 13 (10.6%) were immunocompetent. Male patients predominated (68.3%) and their age ranged from 19 to 69 years (mean: 35.9). Most patients (73.2%) were born and lived lifelong in the state of Mato Grosso do Sul. Involvement of the central nervous system occurred in 103 patients (83.7%) and headache and vomiting were the most frequent symptoms. In 77 cases it was possible to identify the Cryptococcus species: 69 (89.6%) C. neoformans and eight (10.4%) C. gattii. Amphotericin B was the drug of choice for treatment (106/123), followed by fluconazole in 60% of cases. The overall lethality rate was 49.6%, being 51% among the HIV infected patients and 41.2% among the non-HIV infected (p > 0.05). Although cryptococcosis exhibited in our region a similar behavior to that described in the literature, the detection of an important rate of immunocompetent individuals and five C. gattii cryptococcosis in HIV-infected patients is noteworthy. O perfil clínico-epidemiológico de 123 casos de criptococose diagnosticados no Hospital Universitário da Universidade Federal de Mato Grosso do Sul, no período de janeiro de 1995 até dezembro de 2005, foi estudado retrospectivamente. Cento e quatro (84,9%) casos tinham associação com HIV, seis (4,9%) tinham outra condição predisponente e 13 (10,6%) eram imunocompetentes. Houve predomínio do sexo masculino (68,3%) e a idade variou de 19 a 69 anos (média de 35,9 anos). A maioria (73,2%) era natural e procedente de Mato Grosso do Sul. O envolvimento do sistema nervoso central ocorreu em 103 (83,7%) pacientes e os sintomas mais freqüentes foram cefaléia e vômitos. Em 77 casos foi possível identificar a espécie do agente, sendo 69 (89,6%) C. neoformans e oito (10,4%) C. gattii. O antifúngico mais utilizado foi anfotericina B (106/123) seguido de fluconazol em aproximadamente 60% dos casos. A taxa de letalidade foi de 49,6%, sendo 51% entre os pacientes infectados pelo HIV e 41,2% entre os não infectados pelo HIV (p > 0,005). Apesar da criptococose observada em nossa região apresentar comportamento semelhante ao descrito na literatura, chama a atenção a importante taxa da micose em imunocompetentes e cinco casos de infecção por C. gattii em pacientes HIV-positivos.

Delivery of small interfering RNAs (siRNAs) in vivo , using clinically relevant modes of administration, is critical for the advancement of RNA interference (RNAi) therapeutics. In this work, we demonstrate systemic delivery of siRNAs and potent in vivo down-modulation of two important disease targets, apolipoprotein B (apoB) and proprotein convertase subtilisin kexin 9 (PCSK9). A single injection of liposomal siRNA resulted in >90% silencing of apoB mRNA expression in the liver 48 h after administration. The effect was demonstrated to occur through cleavage of the apoB mRNA at precisely the site predicted for the RNAi mechanism. Reductions in apoB protein, cholesterol, and low-density lipoprotein (LDLc) levels were observed in 48 hours that lasted for at least 23 days, thus demonstrating an immediate, potent and durable biological effect. In addition to apoB we have also demonstrated the ability to down-modulate other important liver targets such as PCSK9. PCSK9 has been closely implicated in LDLc regulation. We have demonstrated PCSK9 down-modulation in several animal models including, mouse, humanized mouse, rat, and non-human primate. Down-modulation of PCSK9 levels resulted in significant lowering of cholesterol (20 – 60%) in all animal models tested. These findings strongly support the potential of RNAi therapeutics as a new class of drug for metabolic and cardiovascular diseases. Our next steps include selecting the most potent lead molecule and moving it into GLP safety studies.

To identify the clinical and epidemiological profile of cryptococcosis diagnosed at the University Hospital of the Federal University of Mato Grosso do Sul, Brazil, medical records of 123 patients admitted from January 1995 to December 2005 were analyzed. One hundred and four cases (84.5%) had HIV infection, six (4.9%) had other predisposing conditions and 13 (10.6%) were immunocompetent. Male patients predominated (68.3%) and their age ranged from 19 to 69 years (mean: 35.9). Most patients (73.2%) were born and lived lifelong in the state of Mato Grosso do Sul. Involvement of the central nervous system occurred in 103 patients (83.7%) and headache and vomiting were the most frequent symptoms. In 77 cases it was possible to identify the Cryptococcus species: 69 (89.6%) C. neoformans and eight (10.4%) C. gattii. Amphotericin B was the drug of choice for treatment (106/123), followed by fluconazole in 60% of cases. The overall lethality rate was 49.6%, being 51% among the HIV infected patients and 41.2% among the non-HIV infected (p >; 0.05). Although cryptococcosis exhibited in our region a similar behavior to that described in the literature, the detection of an important rate of immunocompetent individuals and five C. gattii cryptococcosis in HIV-infected patients is noteworthy. O perfil clínico-epidemiológico de 123 casos de criptococose diagnosticados no Hospital Universitário da Universidade Federal de Mato Grosso do Sul, no período de janeiro de 1995 até dezembro de 2005, foi estudado retrospectivamente. Cento e quatro (84,9%) casos tinham associação com HIV, seis (4,9%) tinham outra condição predisponente e 13 (10,6%) eram imunocompetentes. Houve predomínio do sexo masculino (68,3%) e a idade variou de 19 a 69 anos (média de 35,9 anos). A maioria (73,2%) era natural e procedente de Mato Grosso do Sul. O envolvimento do sistema nervoso central ocorreu em 103 (83,7%) pacientes e os sintomas mais freqüentes foram cefaléia e vômitos. Em 77 casos foi possível identificar a espécie do agente, sendo 69 (89,6%) C. neoformans e oito (10,4%) C. gattii. O antifúngico mais utilizado foi anfotericina B (106/123) seguido de fluconazol em aproximadamente 60% dos casos. A taxa de letalidade foi de 49,6%, sendo 51% entre os pacientes infectados pelo HIV e 41,2% entre os não infectados pelo HIV (p >; 0,005). Apesar da criptococose observada em nossa região apresentar comportamento semelhante ao descrito na literatura, chama a atenção a importante taxa da micose em imunocompetentes e cinco casos de infecção por C. gattii em pacientes HIV-positivos.

The Hedgehog (Hh) signaling pathway is vital to animal development as it mediates the differentiation of multiple cell types during embryogenesis. In adults, Hh signaling can be activated to facilitate tissue maintenance and repair. Moreover, stimulation of the Hh pathway has shown therapeutic efficacy in models of neuropathy. The underlying mechanisms of Hh signal transduction remain obscure, however: little is known about the communication between the pathway suppressor Patched (Ptc), a multipass transmembrane protein that directly binds Hh, and the pathway activator Smoothened (Smo), a protein that is related to G-protein-coupled receptors and is capable of constitutive activation in the absence of Ptc. We have identified and characterized a synthetic non-peptidyl small molecule, Hh-Ag, that acts as an agonist of the Hh pathway. This Hh agonist promotes cell-type-specific proliferation and concentration-dependent differentiation in vitro, while in utero it rescues aspects of the Hh-signaling defect in Sonic hedgehog-null, but not Smo-null, mouse embryos. Biochemical studies with Hh-Ag, the Hh-signaling antagonist cyclopamine, and a novel Hh-signaling inhibitor Cur61414, reveal that the action of all these compounds is independent of Hh-protein ligand and of the Hh receptor Ptc, as each binds directly to Smo. Smo can have its activity modulated directly by synthetic small molecules. These studies raise the possibility that Hh signaling may be regulated by endogenous small molecules in vivo and provide potent compounds with which to test the therapeutic value of activating the Hh-signaling pathway in the treatment of traumatic and chronic degenerative conditions.

Wir Europaer konnen heutzutage das Leben ohne schriftliche Mitteilungen nicht meistern. Die Schrift stellt in unserer gesamten Kultur einen integrierenden Bestandteil dar. Gerade das Vorkommen derselben gilt als eines der Unterscheidungsmerkmale zwischen Natur- und Hochkulturvolkern. Den Primitiven fehlt die Schrift im eigentlichen Sinne des Wortes.