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Journal Club Presentation

Transcript: Presented by: Julio Vega Torres, Klondy Canales, and Maleeha Memon Mentor: Dr. Craig Hendrix, Clinical Pharmacology As of 2004, every eight hours someone in Baltimore becomes infected with HIV/AIDS. AIDS is the #1 cause of death among 25-44 year olds in Baltimore. Introduction According to UNAIDS, a total of 2.7 million new infections with HIV were diagnosed worldwide in 2008! This paper focuses on a combination drug which aids the prevention of HIV and STI's in men that have sex with men. Restore health, avoid transmission to uninfected partners, and to decrease mother-to-child transmission. Emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) A single tablet as FTC-TDF FTC and TDF- nucleoside (nucleobase binded to a ribose or deoxyribose) reverse transcriptase inhibitors (NRTI) and they work by inhibiting reverse transcriptase, the enzyme that copies HIV RNA into new viral DNA. Requires that people RECOGNIZE when they have been exposed to HIV and START Therapy within 72 hours (Both are challenging!!!). Although each drug separately has its own effect against HIV, the administration of both have a much bigger impact. TDF for Prevention of HIV Infection in Women: A Phase 2, Double-Blind, Randomized, Placebo-Controlled Trial TDF research was done with West African women and no difference was observed between the placebo and TDF group Evaluate the safety and efficacy of once-daily oral FTC-TDF as compared with placebo for the prevention of HIV acquisition among men and transgender women who have sex with men. Methods Protocol was approved by UNAIDS and the national government of Peru, Ecuador, South Africa, Brazil, Thailand, and USA. Sponsored by NIH Division of Acquired Immunodeficiency Syndrome (DAIDS). Study samples were MALE at birth and 18 years of age or older. HIV-negative status (comes from serostatus-finding of a substance in blood). Evidence of high risk for acquiring HIV. HBV test were performed at screening. Every Four Weeks: ~Pill count ~Counseling ~Testing for HIV antibodies ~Taking of medical history Chemical and hematological analyses performed at weeks 4, 8, 12, 16, 24, and then every 3 months after the 24th week. Purpose was to investigate whether drug levels correlated with protective effect. Subjects with HIV were matched with two control subjects, one from each group that were HIV-negative. At each visit patients received prevention info package containing: HIV testing, risk-reduction counseling, condoms, diagnosis and treatment for STDs Sexual partners were offered STD treatment if infection was found in subject Subjects were instructed to protect themselves from HIV using conventional methods If a patient was infected with an HIV positive partner by unprotected sex, they were sent to POSTEXPOSURE treatment and study was suspended Testing for drug resistance genotyping and phenotyping were performed with assays on the basis of the viral load of the seroconversion data Plasma tests for presence of FTC and TFV Pheripheral-blood mononuclear cells were tested for FTC-TP and TFV-DP (the active intracellular metabolite of FTC and TFV) Results 4905 subjects were screened, 2499 were enrolled in the study Dates of study: July 10, 2007 through December 17, 2009 Location of study: 11 sites in 6 countries Subjects were compared for baseline characteristics. Socio-demographic and behavioral characteristics were shown and only age is significantly different. All subjects born male except 29 (1%) reported their current gender identity as female. Age: 18 to 67 years (mean age, 27.5 vs. 26.8 years; P=0.04 in both study groups). Study Subjects were followed for (Median, 1.2 years; maximum, 2.8 years). 69-70 % had side effects They were not statistically significant and only nausea and weight loss was common Nausea was reported more frequently (2%) during the first 4 weeks in the FTC-TDF group than in the placebo group (P<.001) Weight loss had a 2:1 ratio between FTC-TDF and placebo groups The two groups had similar rates of serious adverse events (P=.57) Similar in the two groups at all time points (P=.97). Number of sexual partners during Intercourse decreased and percentage of partners who used condoms increased after subjects enrolled in the study. The efficacy of preexposure Prophylaxis with emtricitabine and tenofovir disoproxil fumarate (FTC-TDF) was 44%, as compared with placebo (P=.005). Figure 4. Levels of Study-Drug Components in Intracellular levels of Subjects Receiving FTC-TDF Final Results Of the 2499 HIV-negative men or transgender women, 10 subjects were found to have been infected with HIV at enrollment 100 became infected during follow-up (36 in the FTC-TDF group and 64 in the placebo group) That is a 44% reduction in the incidence of HIV (95% confidence interval, 15 to 63; P=.005) FTC-TDF leads to a 92% risk reduction in HIV Once daily oral FTC-TDF provided 44% additional protection from HIV among men or transgender women who have sex with men along with comprehensive package of

Journal Club Presentation

Transcript: 10% and 15% of patients experience a major complication within 1 year and 5 years, respectively. injury to abdominal viscera - gastrocolocutaneous fistula rate has been reported to range from 1.7% to 12.5%. bumper of the PEG can damage or tear the esophagus “buried bumper” syndrome Disruption of the tract small bowel obstruction operative tube replacement jejunojejunal intussusception bowel stricture small bowel hematoma Over all complication rates as high as 38% Early complication rates: similar open vs lap Tube dislodgement: 65% of patients within 5 years Excess granulation tissue mild peristomal leakage skin irritation cellulitis Shenoy S, Karunakar BP. Factors influencing the peripheral venous catheter survival in critically ill children in a pediatric intensive care unit. Indian J Pediatr. 2014;81:1293–1296. Optimization: MDT Discussion INR <1.6/platelet count >50,000/mm3 Site: Higher incidence of VTE and infections with femoral lines Case reports of tunnelling into the developing breast bud Pneumothorax is a rare complication of subclavian lines Radiological lines into lumbar, gonadal, mammary, or portal veins. 10% central line-associated sepsis (prophylaxis no use bt 70% ethanol works) Often difficult to clear coagulase- ve staphylococcus Luminal thrombus: removal after 3–5 days of therapeutic anticoagulation, and anticoagulation should be given for 6 weeks to 3 months. In neonates, erosion of a central catheter tip can result in a TPN effusion; (??Chylothorax) 30% of children in the first year of life require more than one insertion attempt to secure IV access 1894 - Stamm Mortality: 30% by the mid-20th Now: as low as 0% The depth of the UVC can be determined by the formula: (1.5 body weight in kilograms) + 5.5 cm pharyngoesophageal perforation: 4% for neonates weighing less than 750 g VeinViewers , AccuVeins , and VascuLumina- tors devices “nasogastric tube syndrome” Cricopharyngeus - higher perfs in neonates Dr. John W. Broviac (b. 1942) Journal Club Presentation Meert KL, Daphtary KM, Metheny NA. Gastric vs small-bowel feeding in critically ill children receiving mechanical ventilation: a randomized controlled trial. Chest. 2004;126:872–878. Gastrostomy Tubes Luck RP, Haines C, Mull CC. Intraosseous access. J Emerg Med. 2010;39:468–475. 24% on neonates Complications PICC Peripheral Access the “push” gastrostomy technique Intraosseous Lines Synopsis Dr Robert O. Hickman (b. 1927) WARNING!! axillary, subclavian, and innominate - thrombosis High thoracic injury perforates into the posterior mediastinum Distal esophageal injury - right- sided pneumothorax Mortality as low as 4% in one series PEG Vascular Access 1973: Lap-Assisted Gastrostomy sudden, life-threatening bilateral vocal cord paralysis, Nasal Complictions Intragastric and Postpyloric feeding Central Venous Access similar risks of aspiration minor differences in feeding capacity BUT NG is often less challenging, allows for physiologically “normal” bolus feeding 1979: The overall complication rate is o1%, No documentation of bone deformity or growth arrest The aspirate can be used Group and Save Doses used through IO lines are the same Typically removed within 24 h of placement. Mean lifespan 39hrs Tunnelled Lines Norfolk and Norwich Paediatric Surgery 17th February 2017 William Gauderer, a pediatric surgeon, first described his experience with percutaneous endoscopic gastrostomy (PEG) in 1980 1–6 months. Misplaced Tubes Maintenance Enteral Access primary inability to swallow severe gastroesophageal reflux chronic aspiration difficult oral medication regimen general failure to thrive PICC should sit in the superior vena cava or just in the atrium Cheaper and avoids PN risks: - liver failure - sepsis - catheter rated Nasojejunal Tubes Open "STAMM" Gastrostomy 2 lumen: Salem Sump vs Replogle "In extremely rare cases with other complicating factors, NG tubes have been misplaced into the brain, pericardial space, liver, spleen, and the urinary bladder." Umbilical Venous Catheter Nasogastric Tubes total parenteral nutrition chemotherapy administration of caustic medications central venous monitoring blood draws cell harvest apheresis dialysis. Enteral feeding support for more than 4 weeks Faster recovery times, and quick advancement to goal enteral feeds. ?? primary button placement In emergencies - can be placed 3–4 cm from the umbilicus below the hepatic circulation No topical antibiotic ointment should be applied low-dose heparin (0.25–1.0 U/mL) should be added to the fluid last 14 days Feeding jejunostomy paresis of the posterior cricoarytenoid muscles due to ulceration and infection over the posterior lamina of the cricoid no true reduction in aspiration events, aspiration pneumonia, or other complications were appreciated BUT possible higher caloric intake overall in ICU and gastric dysmotility Multiple studies since the 1970s: cause perforation of the duodenum or jejunum resulting in small bowel fistulas or peritonitis. NB Easy to miss in

Journal Club Presentation

Transcript: GKEITA KAMIJ and RYUJI ABE Aftereffects of Cognitively Demanding Acute Aerobic Exercise on Working Memory Background Background Number of studies have shown chronic regular exercise improves cognitive function across lifespan Cognitive Function - intellectual process where you become aware of, perceive, or comprehend an idea perception, thinking, reasoning, and remembering Executive function - umbrella term for the neurologically-based skills involving mental control and self-regulation Working memory - immediate conscious perceptual and linguistic processing allows people to maintain and update information in their brains about things they’re working on, such as assignments, goals and strategies. Cognitively-demanding exercise - exercise performed while the subject engages in a cognitive activity Research has suggested sustained cognitive effort during exercise may not enhance cognitive function walking in nature vs walking in urban areas studies have shown cognitively demanding exercise to be less effective than simpler exercise in enhancing cognitive function in children and young adults exergaming and classroom settings Cognitively Demanding Exercise Cognitively Demanding Exercise Small number of studies have used a within-participants design to examine the aftereffects of cognitively demanding acute exercise Most studies have used a between-participant design Within-Participant Design Within-Participant Design Majority of acute exercise studies have focused on children, young adults, or older adults little is known about the effects of acute exercise on cognitive function in middle-age individuals Unknown if middle-age individuals experience effects of aerobic exercise on working memory like those found in other age groups If there's an association between aerobic exercise and working memory, it could encourage middle-age individuals to exercise during working hours What's Missing? Study Purpose Subjects Methodology 36 middle-age men were recruited from (40-52 y/o) No neurological disorders No CV disease No medications that influenced CNS Nonsmokers Normal or corrected-to-normal vision 28 subject completed study Two-back task - cognitive demand continuous sequence of single-digit numbers from 0 to 9 press one of two buttons with their index fingers corresponding to whether the currently presented single digit matched (right button) or did not match (left) the digit presented two previously in the sequence Switch task - cognitive function continuous sequence of single-digit numbers from 1 to 9, excluding 5 digits enclosed in solid or dashed square solid square - left button if was lower than 5 and right button if digit higher than five dashed square - left button if odd, right button if even Cognitive Tasks Cognitive Tasks https://www.cogstate.com/clinical-trials/computerized-cognitive-assessment/ Two-back task Two-back task https://www.psytoolkit.org/experiment-library/taskswitching.html Switch task Switch task Duration = 25 min each 5-min warm-up; workload was gradually increased 20 min of cycling at 60%-70% of age-predicted HRmax Participants instructed to maintain a cadence between 60 and 70 rpm RPE assessed every 5 min using the 15-point Borg scale Exercise intensity and duration sufficient to detect cognitive benefits after acute aerobic exercise Exercise Intervention Exercise Interventions 1. Rest-cognition condition sit quietly on a cycle ergometer for 5 min four blocks of a 3-min switch task 2-min breaks after each block to refocus 2. Exercise-cognition condition 5-min warm-up; workload was gradually increased 20 min of cycling exercise at 60%-70% of age-predicted HRmax During exercise, simultaneously performed four blocks of a 3-min switch task 2-min breaks after each block 3. Exercise-only condition identical to the exercise-cognition condition, except no switch task during exercise Experimental Protocol Protocol Protocol Diagram Hypothesis Research Hypothesis Working memory performance, as reflected in response accuracy and reaction times (RT) on two-back task, would improve after the interventions in both exercise conditions, but not in the rest-cognition condition No firm prediction regarding beneficial effects of acute exercise on working memory in the exercise-cognition versus exercise-only conditions Analysis Results Two-back task - response accuracy, RT, and SDRT analyzed using a 3x3 repeated-measures ANOVA Condition: rest-cognition, exercise-cognition, exerciseonly) Time: pretest, post 1, post 2 Switch task - response accuracy, RT, and SDRT analyzed using a 2x2 repeated-measures ANOVA Condition: rest-cognition, exercise-cognition Trial: switch, nonswitch HR and RPE were also analyzed using repeated-measures ANOVAs Working memory performance improved after moderate aerobic exercise, as indicated by greater hit accuracy and shorter correct rejection RT This supports existing evidence that simple aerobic exercise has a beneficial impact on working memory Positive effects of exercise were

Journal Club presentation

Transcript: Comparison of the accumulation and effectiveness of different sizes of long-circulating, drug-loaded (DACHPt) polymeric micelles in both highly and poorly permeable tumours BALB/c nude mice inoculated subcutaneously with C26 or BxPC3 cells to prepare tumour model Mice injected on days 0, 2 and 4 with oxaliplatin (8 mg/kg) or micelles (3 mg/kg on a platinum basis) to evaluate antitumour activity Mice injected with 100 µg micelles on a platinum basis for tumour accumulation experiments Haemotoxylin and eosin staining for histological investigations Fluorescent labelling of micelles to investigate size-dependent extravasation and penetration in tumours Immunofluorescent localization of platelet endothelial cell adhesion molecule-1 (PECAM-1) to show distribution of blood vessels Assessment of drug microdistribution in tumours by detecting element disposition (µ-SR-XRF) Observation of platinum and iron in tumour tissue to evaluate the distribution of haemoproteins and the location of the drug Use of DACHPt/m with diameters of 30 and 70 nm (critical differences in antitumour activity, tumour accumulation and microdistribution in BxPC3 model for diameters below and above 50 nm) Labelling of 30 nm micelles with Alexa 488 and 70 nm micelles with Alexa 594 Injection of fluorescently labelled micelles in mice bearing C26 or BxPC3 tumours (10 mg/kg) Microdistribution of micelles assessed 1h and 24h after injection using in vivo confocal laser scanning microscopy (fluorescent measurements relative to the fluorescence intensity in the vasculature immediately after injection (Vmax)) Low doses of a transforming growth factor ß (TGF-ß) inhibitor transiently decreases the pericyte coverage of the endothelium in the neovasculature of pancreatic tumours, resulting in enhanced accumulation and antitumour activity of 65 nm micelles and 90 nm Doxil What is the effect of TGF-ß inhibitors on the delivery of sub-100 nm DACHPt/m in BxPC3 tumours??? BxPC3-bearing mice received intraperitoneal injections of TGF-ß inhibitor (LY364947) at 1 mg/kg every second day (0, 2, 4, 6 & 8) for the antitumour acitivity experiment and 1h before the micelles injection for tumour accumulation studies 30 and 70 nm fluorescently labelled micelles (3mg/kg) were injected on days 0, 4 & 8 The tumoricidal efficiency of long-circulating polymeric micelles depends on the size of the micelles and the permeability of the tumour Sub-100 nm DACHPt/m show no size-dependent restrictions on extravasation and penetration in hypervascular tumours Only DACHPt/m < 50 nm can penetrate poorly permeable hypovascular tumours Increasing the permeability of hypovascular tumours using TGF-ß inhibitor improves the accumulation and distribution of larger (70 nm) micelles Clarification of tumour accumulation and intratumoural distribution of nanomedicines in the range between 5 and 30 nm Optimizing the size of nanomedicines by looking at the balance between antitumour efficacy and potential toxicity Accumulation of sub-100 nm polymeric micelles in poorly permeable tumours depends on size Method Nature reviews. Drug discovery [1474-1776] Davis, Mark yr:2008 vol:7 iss:9 pg:771 -782 Preliminary data C26 Similar extravasation and penetration of 30 and 70 nm micelles BxPC3 30 nm micelles crossed vascular wall (20% Vmax at 40µm from blood vessel), 70 nm micelles failed to reach interstitial space Extravasation points of 70 nm micelles surrounded the blood vessels Method (1) 70 nm micelles reduced the tumour growth rate as effectively as 30 nm micelles Accumulation of 70 nm micelles was reaching a level comparable to 30 nm micelles Results Prolonged circulation time acquired by size control (ca. 5 - 100 nm), too small = leakage, too big = uptake by macrophages from the reticuloendothelial system (RES) hydrophylic surface (mostly PEG) prevents opsonization by macrophages and uptake by RES Goals of drug delivery systems: Minimize premature drug degradation Prevent undesirable side effects Increase drug bioavailability in pathological area Accumulation of nanomedicines in the pathological area by the enhanced permeability and retention (EPR) effect Results (24h post injection) Results (1) Higher P(Glu) ratio from homopolymer to copolymer results in increased micelle size Micelles of different sizes show similar drug release rates Differently sized DACHPt/m maintained their diameters for over 48h ( in cell culture media + 10% serum at 37 ⁰C) Differently sized DACHPt/m showed similar plasma clearance rates and plasma half lives Microdistribution of fluorescently labelled DACHPt/m of varying sizes in tumours Method (2) Nature reviews. Drug discovery [1474-1776] Duncan, R yr:2003 vol:2 iss:5 pg:347 -360 So far, several nanomedicines have shown significant antitumour activity in highly vascularized tumours (e.g. Doxil (90 nm) and Abraxane (130 nm)) However, most of these nanomedicines with large diameters (> 100 nm) have shown limited penetration and accumulation in hypovascular/hypopermeable

Journal club presentation

Transcript: Identification of Hemopexin as an Anti-Inflammatory Factor That Inhibits Synergy of Hemoglobin with HMGB1 in Sterile and Infectious Inflammation Hemopexin: Heme binding protein HMGB1: cytokine mediator of inflammation inflammation: complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants Introduction Tissue injury/necrosis ruptures red blood cells and releases hemoglobin into the extracellular space Cell-free hemoglobin degradation of protein aggregates mechanisms are only partially understood this paper reveals importance of 'vacuolar' pathway in MHCI presentation illustrates importance of autophagy and lysosomes for this pathway shows how modulations of autophagy effect MHCI presentation of viral antigen gB finds junctions where vacuolar and classical pathway probably intersect Isolation of bone marrow derived macrophages (BMDMs) from mice 1. treatment of BMDMs from C57/BL6 WT mice with HMGB1 alone or in combination with Hb- measure TNF and IL-6 2. treatment of BMDMs from TLR2, TLR4 and RAGE KO mice with HMGB1 alone or in combination with Hb-measure TNF 3. pre-treatment of BMDMs from C57/BL6 WT mice with inhibitors of ROS, Syk kinase-then stimulate with HMGB1 alone or HMGB1+Hb- measure TNF 4. treatment of BMDMs from C57/BL6 WT mice with HMGB1 alone or HMGB1+Hb in the presence or absence of Hemopexin-measure TNF and IL-6 5. treatment of BMDMs from C57/BL6 WT mice with HMGB1 alone or HMGB1+Hb in the presence or absence of LPS or Pam3Cys-measure TNF and IL-6 Results and Discussion (big) Conclusions MHCI response relies on different pathways to compensate for viral inhibition vacoular pathway is described to rely on autophagy and lysosomal antigen degradation classical pathway is the bottleneck for antigen presentation pre-autophago-somal structure coil from nucleus ? experimental procedure somehow: injection of antigen fragments into classic MHCI pathway export of degradation products to cytoplasm "xenophagy" in case intracellular parasites being targeted autophagy marker viral antigen nucleus autophagosomes are formed around large portions of cytoplasm fusion of autophagosomes and lysosomes initiates degradation conventional autophagosomes hydrolytic degradation probably ER and/or nucleus Tian Lin, Fatima Sammy, Huan Yang, Sujatha Thundivalappil, Judith Hellman, Kevin J. Tracey and H. Shaw Warren

JOURNAL CLUB PRESENTATION

Transcript: Instagram and Facebook influence on young women’s’ image: how it affects their self-esteem. Instagram and Facebook influence on young women’s’ image: how it affects their self-esteem. Vitória Magalhães Machado Research tools SURREY SEARCH athenas data base EBSCO DATA BASE GOOGLE SCHOLAR Terms definition Terms Self-esteem Not only the physical aspect but also the emotional evaluation of a person’s self-worth will have an impact on self-esteem (Duschene et al., 2017). Therefore, the self-esteem is also considered as an informer of psychological well-being. (Clay, Vignoles, & Dittmar, 2005). "'Dude, I am really pretty today!' 'Girl, you really are!'" (Ilustraclementine, 2020) Social media platforms imply the presence of others, connecting to others despite the distance. Added to which, social media such as Instagram and Facebook are considered as social facilitators, whose impact will be examined further on the literature review (Wolf, Sims, & Yang, 2018) social media The perception the individual has of themselves physically and how they perceive the reaction of others to their body (Spurr, Berry and Walker 2013). BODY IMAGE "What can not miss in a 'beach body'? Sunscream!" (Ilustra Gabs, 2020) main article ARTICLE Body Dissatisfaction and Psychological distress in adolescents: is self-esteem a mediator? Duchesne, A., Dion, J., Lalande, D., Bégin, C., Èmonde, C., Lalande, G. & McDuff, P. (2016) Introduction INTRO Aim of the article test whether if self-esteem is mediator between body dissatisfaction and psychological distress During adolescence self-esteem is being shaped. Besides that, considering their body changes due puberty it reflects on how they perceive their bodies (Duchesne, A., Dion, J., Lalande, D., Bégin, C., Èmonde, C., Lalande, G. & McDuff, P., 2016). why adolescents "Just like the moon, enjoy your phases." (Ilustragabs, 2019) LITERATURE BACKUP There is a wild range of research relating body dissatisfation with anxiety and depression (Duchesne, 2016); Other authors have shown self-esteem as an influence in body dissatisfaction and depression, however only one research test it (Duchesne, 2016). METHODS OF RESEARCH (Prezi Figures, 2020) pARTICIPANTS Ethics Tests used rESULTS and discussion Results results 59,7% were dissatisfied with their bodies datas 409 participants Girls were more dissatisfied with their bodies (63,5%) and lower self-esteem; also more syntoms of psychological distress DISCUSSION The research verified that the hypothesis is true: body dissatisfaction and psychological distress are mediated by self- esteem BODY DISSATISFATION SELF-ESTEEM PSYCHOLOGICAL DISTRESS analysis

Journal Club Presentation

Transcript: (A) In human cells, DSBs may be repaired through the double-strand break repair (DSBR) pathway or the non-crossover synthesis-dependent strand annealing (SDSA) pathway. Both crossover and non-crossover DSBR can occur. (B) The HBD locus from the 7 recombined 3PN embryos were similarly examined as above. * Indicates that the HBD locus failed to be amplified in two of the embryos. (C) In human embryos, repair of DSBs generated by CRISPR/Cas9 occurs mainly through NHEJ. If HDR is utilized, the non-crossover pathway is preferred. Six Cas9-cleaved embryos were randomly selected (three each from groups 2 and 3) for whole-exome sequencing. Concentrations of the Cas9/gRNAs used for injections are indicated. Candidate off-target sites were also confirmed by T7E1 assay. A representative sequencing chromatogram of the region spanning the target site in Cas9-cleaved 3PN embryos. Double peaks near the PAM sequence (green) are indicated. Five embryos with double peaks near the PAM sequence were randomly selected for the T7E1 assay. Blue arrowhead indicates the expected size for uncut PCR products. Control, amplified products from target regions with no double peaks near the PAM sequence. 293T cells were transfected with increasing concentrations (1 μg, 2 μg, 3 μg, 4 μg) of the G1 gRNA-Cas9 vector. A GFP expression vector was used as transfection control. Regions spanning the top 7 predicted off-target sites for each gRNA were PCR amplified for the T7E1 assay. OT, off-target. HBB, on-target editing in the HBB gene locus. Embryo No.16 from group 3 was used to PCR amplify sequences spanning the gRNA target regions of the HBB gene. The PCR products were then subcloned and sequenced. A total of 50 clones were examined, and the number of clones for each pattern indicated. PAM, green. G1 gRNA sequence, blue. Point mutations, red. The ssDNA was co-transfected with the G1 gRNA-Cas9 construct (pX330-G1) into 293T cells. At 48 h after transfection, genomic DNA was extracted to PCR amplify the region spanning the G1 target site. The PCR products were then subcloned into TA cloning vectors for sequencing analysis. Representative sequencing chromatographs for wild- type and edited alleles are shown with the mutated target region underlined in red. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes Four groups of 3PN zygotes were injected intra-cytoplasmically with GFP mRNA (50 ng/L) and Cas9 gRNA/ssDNA in different concentration combinations. The genomes of GFP + embryos were first amplified by multiplex displacement amplification. The region spanning the target site was then PCR amplified, subcloned into TA vectors, and sequenced. * Indicates that target fragments in 5 GFP + embryos failed to be PCR amplified. 293T cells were individually transfected with the three gRNA-Cas9 expression vectors and harvested for genomic DNA isolation 48 h after transfection. A GFP expression vector was used as a control. The regions spanning the gRNA target sites were then PCR amplified for the T7E1 assay. Blue arrowhead indicates the expected size for uncut PCR products. Off-target cleavage in human embryos was summarized here. PAM sequence are labeled in green. HBB, on-target cleavage of the HBB locus. OT1–7, the top 7 predicted off-target sites. HBD, the predicted off-target site in the HBD locus. Mismatched nucleotides compared to the HBB locus are labeled in red. Some of the off-target sites failed to be amplified by PCR in this experiment. Sequencing chromatographs of the wild-type allele and recombined allele generated by homologous recombination between HBB and HBD are shown here. The region with base substitution is underlined with red line. A ssDNA oligo (Oligo donor) encoding 6 silent mutations (indicated in red) was synthesized By: Puping Liang, Yanwen Xu, Xiya Zhang, Chenhui Ding, Rui Huang, Zhen Zhang, Jie Lv, Xiaowei Xie, Yuxi Chen, Yujing Li, Ying Sun, Yaofu Bai, Zhou Songyang, Wenbin Ma, Canquan Zhou&, Junjiu Huang The region within the HBD locus that is highly similar to the G1 gRNA-Cas9 target sequence was analyzed by a T7E1 assay. It shows that although the HBD locus is similar to the HBB locus, no cleavage occured at that site. Three gRNA targeting sites were selected for the HBB locus, and the sequence for each gRNA (G1, G2, and G3) is shown with the PAM sequence in green. The three common HBB mutations found in βB-thalassemia are indicated in red. Exons are represented by deep blue boxes with yellow arrows indicating transcriptional direction.

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Transcript: Author : Anto P. Rajkumar, MD, PhD Background Results Antipsychotic-related risks Dealing with co-morbidity : Diabetes Mellitus Background Incidence of DM Analysis lightbulb Created by Scott Lewis from the Noun Project People with schizophrenia are at increased risk for diabetes regardless of their family history of diabetes and their antipsychotic drug exposure Endogenous risk for DM Exposure to antipsychotics Max. period of follow-up : 35.96yr No statistically significant difference (Chi square=0.13, df=1, p=0.72) 1. Risks for early onset DM in young people with schizophrenia 2. Antipsychotics related risk for diabetes : first-line therapeutic decision – 1st generation vs 2nd generation 3. Adjustment for the effects of potential confounders People with Diabetes - Diabetogenic psychotropic medications : tricyclic, tetracyclic antidepressants, valproate - Gender, urbanicity - Family history of diabetes Covariates of interest Aim of the study Study sample Background - The largest sample size to date, the longest follow-up - The focus on early-onset diabetes in young people - Could not reliably distinguish between type 1 and type 2 diabetes - Limited generalizability : focusing on early onset type 2 DM predominantly Caucasian population from a single country - People from the Danish Civil Registration System - Each Danish citizen is assigned a unique 10-digit personal ID number - Cohort : all people who were born in Denmark Jan.1, 1977. ~ Jan.1, 2013. Journal Club Critique Discussions Background Antipsychotics – related metabolic syndrome “제 2의 tardive dyskinesia” 발표 : R1 김재성 지도 : 김민아 교수님 2017. 05. 16 1. Endogenous risk for diabetes Antipsychotic naive people with SPR vs. Antipsychotic naive people without SPR Follow-up : began at date of birth ended on the date of DM diagnosis, first prescription of antipsychotics, emigration, death, or on Jan. 1, 2013. 2. Antipsychotic-related risk for diabetes People with schizophrenia who were antipsychotic naive at the time of diagnosis Follow-up : began on the date of first diagnosis of SPR ended on the date of DM diagnosis, emigration, death or on Jan. 1, 2013. “Bringing them back into the community, as fully accredited human beings” Position : Clinical lecturer, King's college, London Research interest : Schizophrenia, geriatric psychiatry, mood disorders Sensitivity analyses The Danish National Patient Register, since 1977 - ICD-8 : codes 249, 250 (until the end of 1993) - ICD-10 : codes E10-14, H36.0, O24, excluding O24.4 The Danish National Prescription Registry, since 1995 - Antidiabetic drugs available only by prescription Women age 20 or more treated only with metformin : excluded Study population Methods Background - Excluding 19 people who developed diabetes more than 12 months after the last prescription for any antipsychotics - Including women ages 20 or more years receiving treatment only with metformin (N=8,551) People with schizophrenia Conclusion A total of 14,118 (0.52%) Incidence rate - People without SPR (N=2,727,565) : 0.28 (95% CI 0.28-0.29) - People with SPR (N=8,945) : 4.29 (95% CI 3.74-4.92) The Danish Psychiatric Central Research Register, since 1969 - ICD-8 : code 295, excluding 295.79 (until the end of 1993) - ICD-10 : code F20 The Danish National Prescription Registry, since 1995 Included all incident schizophrenia cases since Jan. 1, 1996 - Impression : 데이터의 위력 - 우리에게는 심평원이 있다 - More to think about Potential confounders : obesity, smoking, diet, substance abuse … 어느 쪽의 surveillance가 더 활발할까 Risk associated with selected antipsychotics The Danish National Prescription Registry, since 1995 - All oral and depot antipsychotic prescriptions - Antipsychotics subdivided into 1st generation, 2nd generation, olanzapine, aripiprazole, clozapine Blood sugar studies in dementia praecox and manic-depressive insanity (Raphael, T. & Parsons, J. P., 1921) TCF7L2 & IGF2BP2 감사합니다

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