Structurally modulated codelivery of siRNA and Argonaute 2 for enhanced RNA interference.

Small interfering RNA (siRNA) represents a promising class of inhibitors in both fundamental research and the clinic. Numerous delivery vehicles have been developed to facilitate siRNA delivery. Nevertheless, achieving highly potent RNA interference (RNAi) toward clinical translation requires efficient formation of RNA-induced gene-silencing complex (RISC) in the cytoplasm. Here we coencapsulate siRNA and the central RNAi effector protein Argonaute 2 (Ago2) via different delivery carriers as a platform to augment RNAi. The physical clustering between siRNA and Ago2 is found to be indispensable for enhanced RNAi. Moreover, by utilizing polyamines bearing the same backbone but distinct cationic side-group arrangements of ethylene diamine repeats as the delivery vehicles, we find that the molecular structure of these polyamines modulates the degree of siRNA/Ago2-mediated improvement of RNAi. We apply this strategy to silence the oncogene STAT3 and significantly prolong survival in mice challenged with melanoma. Our findings suggest a paradigm for RNAi via the synergistic coassembly of RNA with helper proteins.

Orbital and Ocular Adnexal Manifestations of Adult T-Cell Leukemia/Lymphoma: a Case Report and Systematic Review.

PURPOSE: To describe a patient with orbital adult T-cell leukemia/lymphoma (ATLL) and to review the literature on presentation, diagnostics, management, and clinical course of this rare disease. METHODS: A systematic literature review. PubMed/MEDLINE and Google Scholar databases were searched for all well-documented cases of orbital/ocular adnexal ATLL. RESULTS: Sixteen patients were included in the final analysis. The median age at diagnosis was 47 years (range, 20-85), 9/16 patients (56%) were male, and patients were of Japanese (10/16, 63%), Caribbean (5/16, 31%), or African (1/16, 6%) origin. Proptosis (6/15, 40%) and visual loss (5/15, 33%) were the most common presenting signs. Involvement of adjacent structures was documented in 8 of 16 (50%) patients. All patients had evidence of systemic ATLL, which was identified concurrently with orbital/ocular adnexal disease in 9 of 15 (60%) patients. Management included multi-agent chemotherapy with steroids (9/13, 69%), antivirals (2/13, 15%), biologic agents (4/13, 31%), and umbilical cord blood transplantation (1/13, 8%). Most patients (8/12, 67%) experienced at least partial remission with disease relapse occurring in 6 of 8 patients (75%). The median survival time was 28 months (95% CI, 5.5-50.5 months). CONCLUSIONS: Adult T-cell leukemia/lymphoma should be considered in the differential diagnosis of orbital and ocular adnexal space-occupying lesions, particularly in male patients from endemic regions. Orbital disease is frequently locally aggressive and presents concurrently with systemic ATLL, highlighting the importance of comprehensive multimodal work-up and multidisciplinary management. Emerging targeted therapies and hematopoietic stem cell transplant may prolong survival.

Ultrasensitive Detection of Attomolar Protein Concentrations by Dropcast Single Molecule Assays.

Measurements of very low levels of biomolecules, including proteins and nucleic acids, remain a critical challenge in many clinical diagnostic applications due to insufficient sensitivity. While digital measurement methods such as Single Molecule Arrays (Simoa), or digital ELISA, have made significant advances in sensitivity, there are still many potential disease biomarkers that exist in accessible biofluids at levels below the detection limits of these techniques. To overcome this barrier, we have developed a simple strategy for single molecule counting, dropcast single molecule assays (dSimoa), that enables more target molecules to be counted through increased sampling efficiency and with a simpler workflow. In this approach, beads are simply dropcast onto a microscope slide and dried into a monolayer film for digital signal readout. The dSimoa platform achieves attomolar limits of detection, with an up to 25-fold improvement in sensitivity over Simoa, the current state of the art for ultrasensitive protein detection. Furthermore, due to its simple readout process and improved cost-effectiveness compared to existing digital bioassays, dSimoa increases amenability to integration into point-of-care platforms. As an illustration of the potential utility of dSimoa, we demonstrate its ability to measure previously undetectable levels of Brachyury, a tissue biomarker for chordoma, in plasma samples. With its significantly enhanced sensitivity and simplicity, dSimoa can pave the way toward the discovery of new biomarkers for early disease diagnosis and improved health outcomes.

Ultra-Sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression. METHODS: We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of patients with coronavirus disease (COVID-19). We studied plasma from 64 patients who were COVID-19 positive, 17 who were COVID-19 negative, and 34 prepandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 patients with COVID-19. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes. RESULTS: SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95% CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral-antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within 1 day) was statistically significant. CONCLUSIONS: The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of patients who were COVID-19 positive to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease.

Delayed Retinal Breaks and Detachments after Acute Posterior Vitreous Detachment.

PURPOSE: To determine the incidence and timing of delayed retinal breaks and retinal detachments (RDs) after acute posterior vitreous detachment (PVD) and associated risk factors. DESIGN: Retrospective case-control study. PARTICIPANTS: Acute PVD eyes treated between October 2015 and August 2018 at a single academic retina practice. METHODS: Eyes with a PVD diagnosis and history of extended ophthalmoscopic examination on presentation were identified using diagnostic billing codes. The number of eyes with a history of laser retinopexy, cryotherapy for retinal tear, or RD repair was determined using procedural billing codes, and the duration between initial and treatment visits was measured. Records of eyes with a delayed retinal break or RD and of a reference group comprising the first 100 presenting eyes with no initial or delayed retinal break or RD were reviewed to determine and compare the presence of select risk factors on initial examination. MAIN OUTCOME MEASURES: Development of a delayed retinal break or RD. RESULTS: Of 7999 eyes with acute PVD, 1280 (16.0%) showed a retinal break and 499 (6.2%) showed an RD on presentation. Delayed retinal breaks and RDs were found in 209 (2.6%) and 80 (1.0%) eyes, respectively. Of delayed breaks, 116 (55.5%) were found in 6 weeks or less and 93 (44.5%) were found more than 6 weeks after presentation. Of delayed RDs, 26 (32.5%) were found in 6 weeks or less and 54 (67.5%) were found more than 6 weeks after presentation. Compared with the reference group, vitreous hemorrhage (hazard ratio, 2.53 [P < 0.001] and 2.80 [P = 0.001]) and male gender (hazard ratio, 1.36 [P = 0.03] and 1.87 [P = 0.02]) were risk factors for delayed retinal breaks and RDs, respectively. Pseudophakia (hazard ratio, 2.10; P = 0.004) was also a risk factor for delayed RD; older age (odds ratio, 0.96; P = 0.01) was slightly protective. Vitreous hemorrhage was a risk factor for earlier retinal breaks (≤6 weeks vs. >6 weeks; odds ratio, 3.58; P < 0.001). CONCLUSIONS: Clinically significant rates of newly detected retinal breaks and RDs may occur after acute PVD, suggesting that repeat examination may be prudent in these patients.

Ocular trauma during COVID-19 stay-at-home orders: a comparative cohort study.

PURPOSE OF REVIEW: The aim of this study was to report characteristics of patients presenting with serious ocular injuries during the COVID-19 stay-at-home orders. RECENT FINDINGS: Of 1058 patients presenting for emergency evaluation during the stay-at-home order, 62 (5.9%) patients [mean (SD) age, 41.1 (19.2) years; 19 (31%) women; 31 (50%) white] presented with severe ocular trauma. The daily mean (SD) number of patients who presented for emergency evaluation decreased from 49.0 (9) to 36.4 (6) during the quarantine (P < 0.001). Patients presenting during the stay-at-home order were less likely to have health insurance [odds ratio (OR), 0.33; 95% confidence interval (95% CI), 0.13-0.90, P = 0.024], more likely to have a delayed presentation (difference, 22.7 h, 95% CI, 5.8-39.5, P < 0.001, more likely to travel farther to seek emergency care (difference, 10.4 miles, 95% CI, 2.6-18.2, P < 0.001) and more likely to have an injury occur at home (OR, 22.8; 95% CI, 9.6-54.2, P < 0.001). Of injuries occurring at home, there was a significant increase in injuries arising from home improvement projects during the stay-at-home order (28 vs. 0%, P = 0.02). SUMMARY: During the COVID-19 pandemic, patients with ocular trauma were more likely to have injuries sustained at home and have additional barriers to care. These changes underscore a need for targeted interventions to optimize emergent eye care during a pandemic.

Single-molecule measurements in microwells for clinical applications.

The ability to detect and analyze proteins, nucleic acids, and other biomolecules is critical for clinical diagnostics and for understanding the underlying mechanisms of disease. Current detection methods in clinical and research laboratories rely upon bulk measurement techniques such as immunoassays, polymerase chain reaction, and mass spectrometry to detect these biomarkers. However, many potentially useful protein or nucleic acid biomarkers in blood, saliva, or other biofluids exist at concentrations well below the detection limits of current methods, necessitating the development of more sensitive technologies. Single-molecule measurements are poised to address this challenge, vastly improving sensitivity for detecting low abundance biomarkers and rare events within a population. Microwell arrays have emerged as a powerful tool for single-molecule measurements, enabling ultrasensitive detection of disease-relevant biomolecules in easily accessible biofluids. This review discusses the development, fundamentals, and clinical applications of microwell-based single-molecule methods, as well as challenges and future directions for translating these methods to the clinic.

Inadequate Boston Bowel Preparation Scale scores predict the risk of missed neoplasia on the next colonoscopy.

BACKGROUND AND AIMS: The risks of missed findings after inadequate bowel preparation are not fully characterized in a diverse cohort. We aimed to evaluate the likelihood of missed polyps after an inadequate preparation as assessed by using the Boston Bowel Preparation Scale (BBPS). METHODS: In this observational study of prospectively collected data within a large, national, endoscopic consortium, we identified patients aged 50 to 75 years who underwent average-risk screening colonoscopy (C1) followed by a second colonoscopy for any indication within 3 years (C2). We determined the polyp detection rates (PDRs) and advanced PDRs during C2 stratified by C1 BBPS scores. RESULTS: Among segment pairs without polyps at C1 (N = 601), those with inadequate C1 BBPS segment scores had a higher PDR at C2 (10%) compared with those with adequate bowel preparation at C1 (5%; P = .04). Among segment pairs with polyps at C1 (N = 154), segments with inadequate bowel preparation scores at C1 had higher advanced PDRs at C2 (20%) compared with those with adequate bowel preparation scores at C1 (4%; P = .03). In multivariable analysis, the presence of advanced polyps at C1 (adjusted odds ratio [OR] 3.5; 95% confidence intervals [CIs], 1.1-10.8) but not inadequate BBPS scores at C1 (adjusted OR 1.8; 95% CI, 0.6-5.1) was associated with a significantly increased risk of advanced polyps at C2. CONCLUSIONS: Inadequate BBPS segment scores generally are associated with higher rates of polyps and advanced polyps at subsequent colonoscopy within a short timeframe. The presence of advanced polyps as well as inadequate BBPS segment scores can inform the risk of missed polyps and help triage which patients warrant a timely repeat colonoscopy.

Promoting Careers in Neurology: Mentorship of Medical Students.

Research in neurological disorders is expanding at a phenomenal pace, and the need for neurologists is increasing as the population ages. This results in a critical requirement for medical students entering the neurology pipeline. Mentoring, whether formal or informal, ensures that students are inspired and supported to enter the field of neurology. Students should also receive structured mentoring throughout their longitudinal curriculum. Informal mentoring programs enable expanded opportunities for collaborations in education, research, and outreach. Faculty, residents, fellow medical students, and other health professionals all participate in medical student mentoring. Each relationship offers unique aspects to individual growth and development. Specific programmatic guidance and mentoring qualities are presented in this paper.

Periodic-shRNA molecules are capable of gene silencing, cytotoxicity and innate immune activation in cancer cells.

Large dsRNA molecules can cause potent cytotoxic and immunostimulatory effects through the activation of pattern recognition receptors; however, synthetic versions of these molecules are mostly limited to simple sequences like poly-I:C and poly-A:U. Here we show that large RNA molecules generated by rolling circle transcription fold into periodic-shRNA (p-shRNA) structures and cause potent cytotoxicity and gene silencing when delivered to cancer cells. We determined structural requirements for the dumbbell templates used to synthesize p-shRNA, and showed that these molecules likely adopt a co-transcriptionally folded structure. The cytotoxicity of p-shRNA was robustly observed across four different cancer cell lines using two different delivery systems. Despite having a considerably different folded structure than conventional dsRNA, the cytotoxicity of p-shRNA was either equal to or substantially greater than that of poly-I:C depending on the delivery vehicle. Furthermore, p-shRNA caused greater NF-κB activation in SKOV3 cells compared to poly-I:C, indicating that it is a powerful activator of innate immunity. The tuneable sequence and combined gene silencing, immunostimulatory and cytotoxic capacity of p-shRNA make it an attractive platform for cancer immunotherapy.

Engineering Periodic shRNA for Enhanced Silencing Efficacy.

RNA interference (RNAi) provides a versatile therapeutic approach via silencing of specific genes, particularly undruggable targets in cancer and other diseases. However, challenges in the delivery of small interfering RNA (siRNA) have hampered clinical translation. Polymeric or periodic short hairpin RNAs (p-shRNAs)-synthesized by enzymatic amplification of circular DNA-are a recent development that can potentially address these delivery barriers by showing improved stability and complexation to enable nanoparticle packaging. Here, we modify these biomacromolecules via structural and sequence engineering coupled with selective enzymatic digestion to generate an open-ended p-shRNA (op-shRNA) that is cleaved over ten times more efficiently to yield siRNA. The op-shRNA induces considerably greater gene silencing than p-shRNA in multiple cancer cell lines up to 9 days. Furthermore, its high valency and flexibility dramatically improve complexation with a low molecular weight polycation compared to monomeric siRNA. Thus, op-shRNA provides an RNAi platform that can potentially be packaged and efficiently delivered to disease sites with higher therapeutic efficacy.

Polyamine-Mediated Stoichiometric Assembly of Ribonucleoproteins for Enhanced mRNA Delivery.

Messenger RNA (mRNA) represents a promising class of nucleic acid drugs. Although numerous carriers have been developed for mRNA delivery, the inefficient mRNA expression inside cells remains a major challenge. Inspired by the dependence of mRNA on 3'-terminal polyadenosine nucleotides (poly A) and poly A binding proteins (PABPs) for optimal expression, we complexed synthetic mRNA containing a poly A tail with PABPs in a stoichiometric manner and stabilized the ribonucleoproteins (RNPs) with a family of polypeptides bearing different arrangements of cationic side groups. We found that the molecular structure of these polypeptides modulates the degree of PABP-mediated enhancement of mRNA expression. This strategy elicits an up to 20-fold increase in mRNA expression in vitro and an approximately fourfold increase in mice. These findings suggest a set of new design principles for gene delivery by the synergistic co-assembly of mRNA with helper proteins.

Deletion of mitochondrial anchoring protects dysmyelinating shiverer: implications for progressive MS.

The demyelinating disease multiple sclerosis (MS) has an early inflammatory phase followed by an incurable progressive phase with subdued inflammation and poorly understood neurodegenerative mechanism. In this study, we identified various parallelisms between progressive MS and the dysmyelinating mouse model Shiverer and then genetically deleted a major neuron-specific mitochondrial anchoring protein Syntaphilin (SNPH) from the mouse. Prevailing evidence suggests that deletion of SNPH is harmful in demyelination. Surprisingly, SNPH deletion produces striking benefits in the Shiverer by prolonging survival, reducing cerebellar damage, suppressing oxidative stress, and improving mitochondrial health. In contrast, SNPH deletion does not benefit clinical symptoms in experimental autoimmune encephalomyelitis (EAE), a model for early-phase MS. We propose that deleting mitochondrial anchoring is a novel, specific treatment for progressive MS.

GABA receptors in brain development, function, and injury.

This review presents a brief overview of the γ-aminobutyric acid (GABA) system in the developing and mature central nervous system (CNS) and its potential connections to pathologies of the CNS. γ-aminobutyric acid (GABA) is a major neurotransmitter expressed from the embryonic stage and throughout life. At an early developmental stage, GABA acts in an excitatory manner and is implicated in many processes of neurogenesis, including neuronal proliferation, migration, differentiation, and preliminary circuit-building, as well as the development of critical periods. In the mature CNS, GABA acts in an inhibitory manner, a switch mediated by chloride/cation transporter expression and summarized in this review. GABA also plays a role in the development of interstitial neurons of the white matter, as well as in oligodendrocyte development. Although the underlying cellular mechanisms are not yet well understood, we present current findings for the role of GABA in neurological diseases with characteristic white matter abnormalities, including anoxic-ischemic injury, periventricular leukomalacia, and schizophrenia. Development abnormalities of the GABAergic system appear particularly relevant in the etiology of schizophrenia. This review also covers the potential role of GABA in mature brain injury, namely transient ischemia, stroke, and traumatic brain injury/post-traumatic epilepsy.

Management of orthostatic hypotension in patients with Parkinson's disease.

Orthostatic hypotension is common in Parkinson's disease. The current recommended management of orthostatic hypotension related to Parkinson's disease involves first general measures and then medications with little risk of severe adverse side effects.

Molecular basis for antagonism between PDGF and the TGFbeta family of signalling pathways by control of miR-24 expression.

Modulation of the vascular smooth-muscle-cell (vSMC) phenotype from a quiescent 'contractile' phenotype to a proliferative 'synthetic' phenotype has been implicated in vascular injury repair, as well as pathogenesis of vascular proliferative diseases. Both bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFbeta)-signalling pathways promote a contractile phenotype, while the platelet-derived growth factor-BB (PDGF-BB)-signalling pathway promotes a switch to the synthetic phenotype. Here we show that PDGF-BB induces microRNA-24 (miR-24), which in turn leads to downregulation of Tribbles-like protein-3 (Trb3). Repression of Trb3 coincides with reduced expression of Smad proteins and decrease in BMP and TGFbeta signalling, promoting a synthetic phenotype in vSMCs. Inhibition of miR-24 by antisense oligonuclotides abrogates the downregulation of Trb3 as well as pro-synthetic activity of the PDGF-signalling pathway. Thus, this study provides a molecular basis for the antagonism between the PDGF and TGFbeta pathways, and its effect on the control of the vSMC phenotype.

Designing inhaled small molecule drugs for severe respiratory diseases: an overview of the challenges and opportunities.

INTRODUCTION: Inhaled drugs offer advantages for the treatment of respiratory diseases over oral drugs by delivering the drug directly to the lung, thus improving the therapeutic index. There is an unmet medical need for novel therapies for lung diseases, exacerbated by a multitude of challenges for the design of inhaled small molecule drugs. AREAS COVERED: The authors review the challenges and opportunities for the design of inhaled drugs for respiratory diseases with a focus on new target discovery, medicinal chemistry, and pharmacokinetic, pharmacodynamic, and toxicological evaluation of drug candidates. EXPERT OPINION: Inhaled drug discovery is facing multiple unique challenges. Novel biological targets are scarce, as is the guidance for medicinal chemistry teams to design compounds with inhalation-compatible features. It is exceedingly difficult to establish a PK/PD relationship given the complexity of pulmonary PK and the impact of physical properties of the drug substance on PK. PK, PD and toxicology studies are technically challenging and require large amounts of drug substance. Despite the current challenges, the authors foresee that the design of inhaled drugs will be facilitated in the future by our increasing understanding of pathobiology, emerging medicinal chemistry guidelines, advances in drug formulation, PBPK models, and in vitro toxicology assays.

Sociodemographic Factors Associated with Emergent Eye-Related Emergency Department Visits: A Multicenter Analysis.

OBJECTIVE: To compare sociodemographic factors in patients presenting to the emergency department (ED) with emergent and non-emergent eye-related concerns. DESIGN: Cross-sectional multicenter study. SUBJECTS: 60,677 patients with eye-related concerns who visited EDs at Bascom Palmer Eye Institute, Wills Eye Hospital, Massachusetts Eye and Ear, and Johns Hopkins Hospital/Wilmer Eye Institute from January 1st, 2019 until December 31st, 2019. METHODS: Descriptive statistics were performed using STATA 17. MAIN OUTCOME MEASURES: 1) Sociodemographic factors associated with emergent diagnoses, 2) Visit patterns across ED settings (i.e. standard ED vs eye ED), and 3) the most common emergent and non-emergent diagnoses. RESULTS: A total of 60,677 eye-related ED encounters were included in the study, including 22,434 at Bascom Palmer Eye Institute, 16,124 at Wills Eye Hospital, 15,487 at Massachusetts Eye and Ear, and 6,632 at Johns Hopkins Hospital/Wilmer Eye Institute. Most patients had non-emergent diagnoses (56.7%). Males (OR 1.85, 95% CI 1.79-1.92) were more likely to have an emergent diagnosis than females. Patients with private/employer-based insurance (OR 0.88, 95% CI 0.81-0.96), Medicare (OR 0.80, 95% CI 0.72-0.87), and Medicaid (OR 0.81, 95% CI 0.74-0.89) were all less likely to have an emergent diagnosis than uninsured patients. Those with veteran/military insurance (OR 1.08, 95% CI 0.87-1.34) were equally likely to have an emergent diagnosis compared to uninsured patients. Non-White Hispanic patients (OR 1.26, 95% CI 1.12-1.42) were more likely to present with an emergent condition than White patients. Patient seen in the standard ED setting were more likely to have emergent diagnoses than those who visited standalone eye EDs (P < 0.001). The most common emergent diagnoses were corneal abrasion (12.97%), extraocular foreign body (7.61%), and corneal ulcer (7.06%). The most common non-emergent diagnoses were dry eye (7.90%), posterior vitreous detachment (7.76%), and chalazion (6.57%). CONCLUSIONS: ED setting was associated with the acuity of patient diagnoses. Lack of insurance coverage and non-White Hispanic race/ethnicity were associated with emergent eye-related ED visits. Improving access to ophthalmic care in these populations may reduce the incidence of preventable eye emergencies related to untreated chronic conditions. This combined with measures to redirect non-emergent issues to outpatient clinics may alleviate ED overload.

Squamous cell carcinoma of the anal sac in a spotted hyena (Crocuta crocuta).

A 25-yr-old spayed female spotted hyena (Crocuta crocuta) developed intermittent right pelvic limb lameness that persisted following conservative medical therapy. No obvious musculoskeletal lesions were noted on initial physical exam; however, spinal radiography was suspicious for possible intervertebral degenerative joint disease or discospondylitis. Despite prolonged medical therapy, the lameness progressed to minimal weight bearing and marked muscle atrophy of the right pelvic limb. Electromyography showed spontaneous activity in the muscles of right sciatic nerve distribution. Sensory and motor nerve conduction velocities in the right tibial and peroneal nerves were undetectable and markedly reduced, respectively. A magnetic resonance imaging (MRI) scan revealed a large, space-occupying mass on the right side of the sacrum and pelvis. Antemortem fine-needle aspiration of the mass and postmortem histopathology resulted in diagnosis of a high-grade squamous cell carcinoma of the anal sac. Squamous cell carcinoma of the anal sac is very rare in domestic dogs and previously unreported in spotted hyenas.