A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease.

A major limitation of gene therapy for sickle cell disease (SCD) is the availability and access to a potentially curative one-time treatment, due to high treatment costs. We have developed a high-titer bifunctional lentiviral vector (LVV) in a vector backbone that has reduced size, high vector yields, and efficient gene transfer to human CD34+ hematopoietic stem and progenitor cells (HSPCs). This LVV contains locus control region cores expressing an anti-sickling ßAS3-globin gene and two microRNA-adapted short hairpin RNA simultaneously targeting BCL11A and ZNF410 transcripts to maximally induce fetal hemoglobin (HbF) expression. This LVV induces high levels of anti-sickling hemoglobins (HbAAS3 + HbF), while concurrently decreasing sickle hemoglobin (HbS). The decrease in HbS and increased anti-sickling hemoglobin impedes deoxygenated HbS polymerization and red blood cell sickling at low vector copy per cell in transduced SCD patient CD34+ cells differentiated into erythrocytes. The dual alterations in red cell hemoglobins ameliorated the SCD phenotype in the SCD Berkeley mouse model in vivo. With high titer and enhanced transduction of HSPC at a low multiplicity of infection, this LVV will increase the number of patient doses of vector from production lots to decrease costs and help improve accessibility to gene therapy for SCD.

Assessment of Urinary Dysfunction After Midurethral Sling Placement: A Comparison of Two Voiding Trial Methods.

STUDY OBJECTIVE: Temporary urinary retention after midurethral sling (MUS) surgery requiring indwelling catheter or self-catheterization usage is common. Different methods for assessment of immediate postoperative urinary retention have been described. This study aimed to compare postoperative voiding trial (VT) success after active vs passive VT in women undergoing MUS surgery. DESIGN: Comparative retrospective cohort study. SETTING: Female pelvic medicine and reconstructive surgery practice at a university-affiliated tertiary medical center. PATIENTS: Patients with stress urinary incontinence who underwent surgical treatment during the study period were eligible for inclusion. Excluded were patients younger than the age of 18 years, combined cases with other surgical services, planned laparotomy, and a history of urinary retention and patients for whom their VT was performed on postoperative day 1. The cohort was divided into 2 groups: (1) patients who underwent an active retrofill of their bladder using a Foley catheter and (2) patients who were allowed to have a spontaneous void. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 285 patients met the inclusion criteria for the study. Of these subjects, 94 underwent an active VT and 191 underwent a passive VT. There were no statistically significant differences in immediate postoperative urinary retention (30.8% vs 29.3%; p = .79) or time from surgery end to VT (233.0 ± 167.6 minutes vs 203.1 ± 147.8 minutes; p = .13) between groups. Urinary retention, as defined by a failed VT, increased from 10% to 29.3% when MUS placement was accompanied by concomitant prolapse repair procedure. Multivariate logistic regression analysis revealed that undergoing a combined anterior and posterior colporrhaphy (odds ratio [OR], 5.13; p <.001) and undergoing an apical prolapse procedure (OR, 2.75; p = .004) were independently associated with immediate postoperative urinary retention whereas increased body mass index (OR, 0.89; p <.001) lowered likelihood of retention. CONCLUSION: The method used to assess immediate postoperative urinary retention did not affect VT success. Concomitant combined anterior and posterior colporrhaphy and apical suspension were correlated with greater likelihood of VT failure whereas increased body mass index decreased odds of retention.

Postoperative complications and unanticipated healthcare encounters following mini-laparotomy vs. laparoscopic/robotic-assisted sacrocolpopexy: a comparative retrospective study.

BACKGROUND: Pelvic organ prolapse is a debilitating condition impacting lives of millions of women worldwide. Sacrocolpopexy (SCP) is considered an effective and durable surgical technique for treatment of apical prolapse. The aim of this study was to compare short-term outcomes including postoperative complications and unanticipated healthcare encounters between patients who underwent SCP with a mini-laparotomy approach compared to patients treated with laparoscopic and robotic-assisted laparoscopic SCP. METHODS: This was a retrospective cohort study including patients treated for apical prolapse at a university affiliated urogynecology practice. Patients over the age of 18 who underwent abdominal SCP between 2019 and 2023 were included. The cohort was formed into two groups: (1) Patients who underwent SCP through a mini-laparotomy incision (Mini-lap group); (2) Patients who underwent laparoscopic or robotic-assisted laparoscopic SCP (Lap/Robot group). RESULTS: A total of 116 patients were included in the final analysis. Ninety patients underwent either laparoscopic or robotic-assisted SCP, whereas 26 patients underwent SCP with a mini-laparotomy approach. Study participants exhibited a mean age of 63.1 ± 10.3 years, mean body mass index (BMI) of 25.8 ± 4.9 Kg/m2, and 77.6% of them identified as Caucasian. Upon comparison of demographic and past medical history between groups there were no statistically significant differences in age, BMI, menopausal status, race, parity or comorbid conditions. Patients in the Mini-lap group were less likely to have undergone previous abdominal surgery (11.5% vs. 50.6%, p < 0.001) and had more severe apical prolapse (stage 4 prolapse, 40% vs. 21.2%, p < 0.001) than their counterparts in the Lap/robot group. Regarding intraoperative parameters, length of surgery was significantly shorter in the Mini-lap group compared to the Lap/robot group (97.3 ± 35.0 min vs. 242.0 ± 52.6 min, p < 0.001). When focusing on the primary outcome, postoperative complications within the first 30 days after surgery, there were no differences noted between groups. Additionally, the number of unanticipated healthcare encounters, such as phone calls, clinic visits, emergency department visits, urgent care visits, readmissions and reoperations were similar between groups. CONCLUSIONS: Mini-laparotomy approach for SCP is safe with comparable intra- and postoperative complications, and unanticipated healthcare encounters compared to conventional minimally invasive methods.

A Delphi Method for Development of a Barrett's Esophagus Question Prompt List as a Communication Tool for Optimal Patient-physician Communication.

BACKGROUND METHODS: The question prompt list content was derived through a modified Delphi process consisting of 3 rounds. In round 1, experts provided 5 answers to the prompts "What general questions should patients ask when given a new diagnosis of Barrett's esophagus" and "What questions do I not hear patients asking, but given my expertise, I believe they should be asking?" Questions were reviewed and categorized into themes. In round 2, experts rated questions on a 5-point Likert scale. In round 3, experts rerated questions modified or reduced after the previous rounds. Only questions rated as "essential" or "important" were included in Barrett's esophagus question prompt list (BE-QPL). To improve usability, questions were reduced to minimize redundancy and simplified to use language at an eighth-grade level (Fig. 1). RESULTS: Twenty-one esophageal medical and surgical experts participated in both rounds (91% males; median age 52 years). The expert panel comprised of 33% esophagologists, 24% foregut surgeons, and 24% advanced endoscopists, with a median of 15 years in clinical practice. Most (81%), worked in an academic tertiary referral hospital. In this 3-round Delphi technique, 220 questions were proposed in round 1, 122 (55.5%) were accepted into the BE-QPL and reduced down to 76 questions (round 2), and 67 questions (round 3). These 67 questions reached a Flesch Reading Ease of 68.8, interpreted as easily understood by 13 to 15 years olds. CONCLUSIONS: With multidisciplinary input, we have developed a physician-derived BE-QPL to optimize patient-physician communication. Future directions will seek patient feedback to distill the questions further to a smaller number and then assess their usability.

A Retrospective Analysis to Assess the Reasons for the Failure of Dental Implants.

Background: To assess the reasons for dental implant failures. Materials and Methods: A total of 100 implants were studied in 80 subjects. The radiographic investigations were performed to assess the record length of implant and diameter of implant. Risk factors, such as habit of smoking, history of diabetes, and hypertension, were recorded. The data were collected and assessed using Statistical Package for the Social Sciences (SPSS) software. The one-way analysis of variance (ANOVA) test was used to assess the failure rate in dental implants. A P-value less than 0.05 was considered statistically significant. Results: A total of 100 dental implants were included. The maximum dental implant failure was seen with a diameter <3.75 mm (30%) followed by 3.75-4.5 mm (16%) and >4.5 mm (6.15%). The difference was found to be significant (P < 0.05). Conclusion: Dental implant failure was high in dental implants with <3.75 mm diameter, dental implants with a length <10.0 mm, and among smokers.

Esophageal Motility Patterns in Paraesophageal Hernia Patients Compared to Sliding Hiatal Hernia: Bigger Is Not Better.

BACKGROUND: In patients with paraesophageal hernias (PEH), the course of the esophagus is often altered, which may affect esophageal motility. High-resolution manometry (HRM) is frequently used to evaluate esophageal motor function prior to PEH repair. This study was performed to characterize esophageal motility disorders in patients with PEH as compared to sliding hiatal hernia and to determine how these findings affect operative decision-making. METHODS: Patients referred for HRM to a single institution from 2015 to 2019 were included in a prospectively maintained database. HRM studies were analyzed for the appearance of any esophageal motility disorder using the Chicago classification. PEH patients had confirmation of their diagnosis at the time of surgery, and the type of fundoplication performed was recorded. They were case-matched based on sex, age, and BMI to patients with sliding hiatal hernia who were referred for HRM in the same period. RESULTS: There were 306 patients diagnosed with a PEH who underwent repair. When compared to case-matched sliding hiatal hernia patients, PEH patients had higher rates of ineffective esophageal motility (IEM) (p<.001) and lower rates of absent peristalsis (p=.048). Of those with ineffective motility (n=70), 41 (59%) had a partial or no fundoplication performed during PEH repair. CONCLUSION: PEH patients had higher rates of IEM compared to controls, possibly due to a chronically distorted esophageal lumen. Offering the appropriate operation hinges on understanding the involved anatomy and esophageal function of each individual. HRM is important to obtain preoperatively for optimizing patient and procedure selection in PEH repair.

Maternal perinatal depression and child brain structure at 2-3 years in a South African birth cohort study.

Maternal perinatal depression is associated with risk of adverse child developmental outcomes and differences in offspring brain structure. Evidence from low- and middle-income countries is lacking as is an investigation of antenatal, postnatal, and persistent depression in the same sample. In a South African birth cohort, we investigated the effect of antenatal and postpartum maternal depressive symptoms on offspring brain structure at 2-3 years of age. Magnetic resonance imaging was performed, extracting cortical thickness and surface areas in frontal cortex regions of interest and subcortical volumes using FreeSurfer software. Maternal depressive symptoms were measured using the Edinburgh Postpartum Depression Scale and the Beck Depression Inventory II antenatally and at 6-10 weeks, 6 months, 12 months, and 18 months postpartum and analyzed dichotomously and continuously. Linear regressions were used controlling for child age, sex, intracranial volume, maternal education, age, smoking, alcohol use and HIV. 146 children were included with 38 (37%) exposed to depressive symptoms antenatally and 44 (35%) exposed postnatally. Of these, 16 (13%) were exposed to both. Postpartum, but not antenatal, depressive symptoms were associated with smaller amygdala volumes in children (B = -74.73, p = 0.01). Persistent maternal depressive symptoms across pregnancy and postpartum were also independently associated with smaller amygdala volumes (B = -78.61, p = 0.047). Differences in amygdala volumes among children exposed to postnatal as well as persistent maternal depressive symptomatology underscore the importance of identifying women at-risk for depression during the entire perinatal period.

A prospective, randomized trial comparing intravesical dimethyl sulfoxide (DMSO) to bupivacaine, triamcinolone, and heparin (BTH), for newly diagnosed interstitial cystitis/painful bladder syndrome (IC/PBS).

INTRODUCTION AND HYPOTHESIS: The primary aim of this study was to compare the effect of bladder instillations using dimethyl sulfoxide (DMSO) with triamcinolone versus bupivacaine, triamcinolone, and heparin (BTH) in women with newly diagnosed interstitial cystitis/painful bladder syndrome. The primary outcome was improvement in symptoms measured using the O'Leary-Sant Interstitial Cystitis Symptoms Index (ICSI) score. Secondary comparisons included changes in urinary frequency, nocturia, and bladder capacity. MATERIALS AND METHODS: This was a prospective, randomized study. Patients with a recent diagnosis of interstitial cystitis/painful bladder syndrome (IC/PBS) were randomized 1:1 to treatment with either 6 weekly bladder instillations of DMSO with triamcinolone or BTH. During follow-up visits, patients completed the ICSI questionnaire, and bladder capacity was determined through the retrograde filling of the bladder. The χ2 test or Student's t test were used for data analysis. RESULTS: A total of 83 patients were randomized, and final analysis included 70 participants who completed the 6 weekly instillations (42 DMSO, 28 BTH). The groups were similar in baseline demographics and clinical characteristics, except for cystometric maximum capacity (DMSO 338.62± 139.44 mL, BTH 447.43 ± 180.38 mL, p = 0.01). In the DMSO group, 63% of patients had a greater than 29.5% reduction in total ICSI score versus 43% in the BTH group (p = 0.15). Nocturia and pain were significantly reduced in the DMSO group. There was a significant increase from baseline in bladder capacity for both groups. CONCLUSION: In women with newly diagnosed IC/PBS, bladder instillations with DMSO and triamcinolone provide greater improvement in pain and nocturia compared to BTH.

Large ciliary body melanocytoma with pseudocysts: a case report.

BACKGROUND: To describe the correlation between standardized A-scan echography and histopathology in a ciliary body melanocytoma. CASE PRESENTATION: We present a case of a large ciliary body melanocytoma with significant growth, vision loss, and elevated intraocular pressure that was diagnosed clinically as a melanoma, but the standardized A-scan findings correlated to the histopathological description of a melanocytoma with multiple pseudocysts. CONCLUSIONS: The reflectivity of this melanocytoma by standardized A-scan was consistent with multiple pseudocysts on pathological evaluation. This echographic pattern guided the differential diagnosis. Standardized A-scan is an important diagnostic tool in the differentiation of ciliary body melanocytomas from melanomas.

Soluble components from mesenchymal stromal cell processing exert anti-inflammatory effects and facilitate ischemic muscle regeneration.

BACKGROUND AIMS: Skeletal muscle regeneration after severe damage is reliant on local stem cell proliferation and differentiation, processes that are tightly regulated by macrophages. Peripheral artery disease is a globally prevalent cardiovascular disease affecting millions of people. Progression of the disease leads to intermittent claudication, subsequent critical limb ischemia and muscle injury. Tissue-derived and ex vivo-expanded mesenchymal stromal cells (MSCs) for skeletal muscle regeneration have been studied, but pre-clinical and clinical results have not been consistent. As a result, the potential therapeutic efficacy and associated repair mechanisms of MSCs remain unclear. Numerous studies have demonstrated the vulnerability of delivered MSCs, with a precipitous drop in cell viability upon transplantation. This has prompted investigation into the therapeutic benefit of apoptotic cells, microvesicles, exosomes and soluble signals that are released upon cell death. METHODS: In this study, we characterized various components produced by MSCs after cell death induction under different conditions. We discovered anti-inflammatory and pro-regenerative effects produced by cell components following a freeze and thaw (F&T) process on macrophage polarization in vitro. We further investigated the underlying mechanisms of macrophage polarization by those components resulting from severe cell death induction. RESULTS: We found potent therapeutic effects from F&T-induced cell debris are dependent on the externalization of phosphatidylserine on the plasma membrane. In contrast, effects from the supernatant of F&T-induced cell death primarily depends on the released protein content. We then applied the F&T-induced cell supernatant to an animal model of peripheral artery disease to treat muscle injury caused by severe ischemia. Treatment with the F&T supernatant but not the vulnerable MSCs resulted in significantly improved recovery of muscle function, blood flow and morphology and inflammation resolution in the affected muscles 2 weeks after injury. CONCLUSIONS: This study validates the therapeutic potential of F&T-induced supernatant obviating the need for a viable population from vulnerable MSCs to treat injury, thus providing a roadmap for cell-free therapeutic approaches for tissue regeneration.

Long term surgical outcomes of vaginal colposuspension using the Uphold Lite™ mesh system vs. vaginal vault uterosacral ligament suspension for treatment of apical prolapse.

OBJECTIVE: The aim of this study is to compare long-term outcomes (7-10 years) between vaginal hysterectomy with uterosacral ligament suspension (VHUSLS) and sacrospinous hysteropexy with the Uphold™ Lite mesh System (SHU) for management of apical prolapse. METHODS: Patients undergoing VHUSLS or SHU from 2008 to 2012 at a single tertiary referral center were included. Patients were contacted, asked to return for physical examination, and to complete the Pelvic Floor Distress Inventory (PFDI-20) questionnaire. Our primary outcome was anatomic failure defined as Stage 2 POP or higher of any compartment. The secondary outcome was subjective changes in symptoms based upon PFDI-20 responses. RESULTS: Two-hundred and two women were identified to have undergone the index surgeries from 2008 to 2012. Sixty-three agreed to return for follow up symptom evaluation and examination (30 VHUSLS and 33 SHU). Baseline characteristics were similar between groups. Clinical cure was high for both groups reaching 93.4 % and 94.0 % for the VHUSLS and SHU groups, respectively (p = 0.721). Anatomical success was lower with 44.7 % and 66.7 % of patients in the VHUSLS and SHU groups, respectively, meeting criteria for success (p = 0.138). There were no mesh complications among patients returning for exams. However, two patients who were contacted and were not interested in this study reported mesh complications and need for additional surgeries. Anterior vaginal wall support was noted to be significantly better supported for SHU (Ba -2.03 ± 0.75 vs -1.42 ± 0.92, p = 0.008). There were no differences between groups for overall PFDI-20 scores postoperatively. However, SHU patients reported higher rates of stress urinary incontinence compared to VHUSLS patients. CONCLUSION: In women with apical prolapse, VHUSLS and SHU afford similar long-term outcomes. SHU patients reported higher rates of stress urinary incontinence.

Risk factors for unplanned admission following surgical repair of apical prolapse.

INTRODUCTION AND HYPOTHESIS: Same-day discharge (SDD) is increasing in popularity following surgical repair of pelvic organ prolapse. The aim of this study was to evaluate factors associated with unplanned admission (UA) in women undergoing apical prolapse repair. METHODS: This retrospective, observational cohort study included patients who underwent apical prolapse repair and planned same-day discharge (SDD) between March 2019 and December 2021. The cohort was divided into two groups: patients who were discharged on the same day as surgery (SDD group) and patients who had an unplanned admission (UA group). Demographic, pre-, intra-, and post-operative data were collected. Risk factors associated with unplanned admission were evaluated using univariate and multivariate analyses. RESULTS: One-hundred and eighty-four cases of apical prolapse repair met the criteria for inclusion in the final analysis; this included 142 in the SDD group and 42 in the UA group. Patients in the UA group had significantly increased estimated blood loss, longer total operative time, later time arriving to the Post-Anesthesia Care unit (PACU) and longer overall stay in the PACU. No differences were observed in the 30-day complication rate, or 30-day unanticipated healthcare encounters, between groups. Multivariate analysis revealed that receiving ketorolac post-operatively was associated with a higher likelihood of SDD (OR=2.6, 95% CI 1.032-6.580, p=0.043). CONCLUSIONS: Among women undergoing apical prolapse repair, same-day discharge was associated with comparable immediate and 30-day complication rates. Within our cohort, post-operative treatment with ketorolac was associated with greater likelihood of SDD.

Lentiviral gene therapy for X-linked chronic granulomatous disease recapitulates endogenous CYBB regulation and expression.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene, resulting in the inability of phagocytic cells to eliminate infections. To design a lentiviral vector (LV) capable of recapitulating the endogenous regulation and expression of CYBB, a bioinformatics-guided approach was used to elucidate the cognate enhancer elements regulating the native CYBB gene. Using this approach, we analyzed a 600-kilobase topologically associated domain of the CYBB gene and identified endogenous enhancer elements to supplement the CYBB promoter to develop MyeloVec, a physiologically regulated LV for the treatment of X-CGD. When compared with an LV currently in clinical trials for X-CGD, MyeloVec showed improved expression, superior gene transfer to hematopoietic stem and progenitor cells (HSPCs), corrected an X-CGD mouse model leading to complete protection against Burkholderia cepacia infection, and restored healthy donor levels of antimicrobial oxidase activity in neutrophils derived from HSPCs from patients with X-CGD. Our findings validate the bioinformatics-guided design approach and have yielded a novel LV with clinical promise for the treatment of X-CGD.

Same-day Discharge vs Planned Admission after Surgical Treatment for Apical Prolapse.

STUDY OBJECTIVE: Same-day discharge (SDD) after surgery is becoming more common, with studies supporting the safety of this practice in gynecologic surgeries. The aim of this study was to compare short-term outcomes of SDD with planned admission in patients undergoing apical pelvic organ prolapse repair, through 30-day complications and 30-day unanticipated healthcare encounters. SETTING: Female pelvic medicine and reconstructive surgery practice at a university-affiliated tertiary medical center. PATIENTS: Patients with apical prolapse who underwent surgical treatment during the study period were eligible for inclusion. Excluded were patients under the age of 18, cases planned for SDD with an unexpected admission, cases with a planned open procedure, and those performed in combination with another surgical service. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 296 patients were included in the final analysis. A total of 154 patients had a planned admission, and 142 were discharged on the day of surgery (SDD group). There were no differences in reoperations, development of venous thromboembolism, and blood transfusions between the 2 groups. Patients in the SDD group were more likely to have no postoperative complications (95% vs 88.3%, p = .037). Number of unanticipated urogynecology office visits, urgent/immediate care visits, readmissions, or unplanned phone calls were also similar between the 2 groups. There was a statistically significant difference observed in mean emergency department (ED) visits (0.16 ± 0.40 in the planned admission group vs 0.06 ± 0.27 in the SDD group, p = .02); however, this number was low in both groups. CONCLUSION: This comparative study suggests that SDD after apical prolapse repair is safe and may be considered for patients interested in this option.

Exploring the Influence of Gender on Surgical Clerkship Grades and Test Scores: A Single Institution, Multisite Comparison.

OBJECTIVE: General surgery remains a male-dominated specialty. Women constitute 54% of medical students at the University of Washington, but only 3.4% of full professors within the Department of Surgery. Many believe surgical attrition and "the leaky pipeline" starts during medical school clerkships, but the exact deterrents remain undefined. This study examined the impact of gender on grading during the third-year surgical clerkship. DESIGN: Retrospective analysis of confidential final clerkship grades, examination scores and subjective clerkship grades was conducted. These were compared by gender, time period, and type of clerkship site. Chi-square analyses were performed. SETTING: Clerkship sites across multiple academic (n = 6) and nonacademic (n = 14) locations. PARTICIPANTS: All third-year medical students undergoing a core surgical clerkship over 2 time periods-2007 to 2010 (period 1) and 2016 to 2019 (period 2)-were included. RESULTS: There were 539 medical students in period 1 and 792 in period 2. The percentage of women was stable over time (52.0% vs 54.2%, p = 0.43). Final clerkship grades of Honors increased significantly from period 1 to 2 (22.3% vs 44.3%, p < 0.0001) and was similarly distributed by gender (women: 21.4% vs 48.0%, p < 0.0001; men 23.2% vs 39.9%, p < 0.0001). Honors on examinations remained stable over time and did not differ by gender. Women earned more final clerkship honors than men at academic sites in period 2 (48.4% vs 30.9%, p < 0.001). This finding was not identified in period 1, nor at nonacademic sites. CONCLUSION: There was a significant increase in surgical clerkship honors over the past decade, independent of gender. Women attained more clinical and final clerkship honors than men and similar exam grades as time progressed, suggesting that gender bias in the subjective grading of women at this institution does not directly contribute to the loss of talented women as they progress from medical student to faculty within the department, with said gender imbalance not related to clerkship evaluations.

The impact of the COVID-19 pandemic on medical student education: Implementation and outcome of a virtual general surgery curriculum.

BACKGROUND: Due to the COVID-19 pandemic, medical schools were forced to adapt clinical curricula. The University of Washington School of Medicine created a hybrid in person and virtual general surgery clerkship. METHODS: The third year general surgery clerkship was modified to a 4-week in person and 2-week virtual clerkship to accommodate the same number of learners in less time. All students completed a survey to assess the impact of the virtual clerkship. RESULTS: The students preferred faculty lectures over national modules in the virtual clerkship. 58.6% indicated they would prefer the virtual component before the in-person experience. There was no change from previous years in final grades or clerkship exam scores after this hybrid curriculum. CONCLUSIONS: If the need for a virtual general surgery curriculum arises again in the future, learners value this experience at the beginning of the clerkship and prefer faculty lectures over national modules.

A Critical Review of Zebrafish Models of Parkinson's Disease.

A wide variety of human diseases have been modelled in zebrafish, including various types of cancer, cardiovascular diseases and neurodegenerative diseases like Alzheimer's and Parkinson's. Recent reviews have summarized the currently available zebrafish models of Parkinson's Disease, which include gene-based, chemically induced and chemogenetic ablation models. The present review updates the literature, critically evaluates each of the available models of Parkinson's Disease in zebrafish and compares them with similar models in invertebrates and mammals to determine their advantages and disadvantages. We examine gene-based models, including ones linked to Early-Onset Parkinson's Disease: PARKIN, PINK1, DJ-1, and SNCA; but we also examine LRRK2, which is linked to Late-Onset Parkinson's Disease. We evaluate chemically induced models like MPTP, 6-OHDA, rotenone and paraquat, as well as chemogenetic ablation models like metronidazole-nitroreductase. The article also reviews the unique advantages of zebrafish, including the abundance of behavioural assays available to researchers and the efficiency of high-throughput screens. This offers a rare opportunity for assessing the potential therapeutic efficacy of pharmacological interventions. Zebrafish also are very amenable to genetic manipulation using a wide variety of techniques, which can be combined with an array of advanced microscopic imaging methods to enable in vivo visualization of cells and tissue. Taken together, these factors place zebrafish on the forefront of research as a versatile model for investigating disease states. The end goal of this review is to determine the benefits of using zebrafish in comparison to utilising other animals and to consider the limitations of zebrafish for investigating human disease.

pH and proton-sensitive receptors in brain ischemia.

Extracellular proton concentration is at 40 nM when pH is 7.4. In disease conditions such as brain ischemia, proton concentration can reach µM range. To respond to this increase in extracellular proton concentration, the mammalian brain expresses at least three classes of proton receptors. Acid-sensing ion channels (ASICs) are the main neuronal cationic proton receptor. The proton-activated chloride channel (PAC), which is also known as (aka) acid-sensitive outwardly rectifying anion channel (ASOR; TMEM206), mediates acid-induced chloride currents. Besides proton-activated channels, GPR4, GPR65 (aka TDAG8, T-cell death-associated gene 8), and GPR68 (aka OGR1, ovarian cancer G protein-coupled receptor 1) function as proton-sensitive G protein-coupled receptors (GPCRs). Though earlier studies on these GPCRs mainly focus on peripheral cells, we and others have recently provided evidence for their functional importance in brain injury. Specifically, GPR4 shows strong expression in brain endothelium, GPR65 is present in a fraction of microglia, while GPR68 exhibits predominant expression in brain neurons. Here, to get a better view of brain acid signaling and its contribution to ischemic injury, we will review the recent findings regarding the differential contribution of proton-sensitive GPCRs to cerebrovascular function, neuroinflammation, and neuronal injury following acidosis and brain ischemia.

Alagille Syndrome and Repeat Oxygenator Failure during Cardiopulmonary Bypass: A Word of Caution.

Alagille syndrome is an autosomal dominant disorder that is caused by heterozygous mutation of JAG1 or NOTCH2 gene that impacts several multisystem organs including but may not be limited to the liver, heart, musculoskeletal, skin, and the eyes. The most common congenital heart defect associated with Alagille syndrome is multilevel right ventricular outflow tract obstruction with multiple central and peripheral branch pulmonary arterial stenoses occurring in up to two-thirds of these patients. We report two cases of Alagille syndrome who underwent extensive pulmonary arterial branch rehabilitation and experienced unusual oxygenator failure during cardiopulmonary bypass (CPB). We present lessons learned from these two cases and the changes that we implemented in our practice that facilitated smooth conduct of CPB in other cases that we performed subsequently.

Improved lentiviral vector titers from a multi-gene knockout packaging line.

Lentiviral vectors (LVs) are robust delivery vehicles for gene therapy as they can efficiently integrate transgenes into host cell genomes. However, LVs with lengthy or complex expression cassettes typically are produced at low titers and have reduced gene transfer capacity, creating barriers for clinical and commercial applications. Modifications of the packaging cell line and methods may be able to produce complex vectors at higher titer and infectivity and may improve production of many different LVs. In this study, we identified two host restriction factors in HEK293T packaging cells that impeded LV production, 2'-5'-oligoadenylate synthetase 1 (OAS1) and low-density lipoprotein receptor (LDLR). Knocking out these two genes separately led to ∼2-fold increases in viral titer. We created a monoclonal cell line, CRISPRed HEK293T to Disrupt Antiviral Response (CHEDAR), by successively knocking out OAS1, LDLR, and PKR, a previously identified factor impeding LV titers. Packaging in CHEDAR yielded ∼7-fold increases in physical particles, full-length vector RNA, and vector titers. In addition, overexpressing transcription elongation factors, SPT4 and SPT5, during packaging improved the production of full-length vector RNA, thereby increasing titers by ∼2-fold. Packaging in CHEDAR with over-expression of SPT4 and SPT5 led to ∼11-fold increases of titers. These approaches improved the production of a variety of LVs, especially vectors with low titers or with internal promoters in the reverse orientation, and may be beneficial for multiple gene therapy applications.