Development and Pilot Screen of Novel High Content Assay for Down Regulators of Expression of Heterogenous Nuclear Ribonuclear Protein H2.

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late-stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al., Cell Physiol Biochem 2019;53:656-686). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H2 that can be used for melanoma therapy and research. METHODS: We established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. RESULTS: All plate-based QC parameters were acceptable: %CV = 6.7±0.3, S/B = 21±2.1, Z' = 0.75±0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis. CONCLUSION: We developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNPH1 and 2 in a large compound collection and, therefore, is amenable to a large-scale screening effort.

Septic Arthritis With Superimposed Acute Gouty Arthritis in a Rheumatoid Arthritis Patient.

Septic arthritis is a rare but serious complication of both rheumatoid and gouty arthritis and can lead to significant morbidity and even mortality. Here, we report a case of septic arthritis with bacteremia, monosodium urate crystals, and hyperuricemia in a 75-year-old male with long-standing rheumatoid arthritis. Arthrocentesis revealed gram-positive cocci representing group B streptococcus (Streptococcus agalactiae) infection and monosodium urate crystals. A diagnosis of septic arthritis with superimposed acute gouty arthritis was made and the patient was treated accordingly. Management included surgical irrigation and debridement, antibiotic therapy, and systemic glucocorticoids which resulted in a significant improvement in the patient's clinical status.

Assessing the quality of randomization methods in randomized control trials.

IMPORTANCE: The randomization process is considered among the most important components of a randomized control trial (RCTs) and a core advantage of RCTs. Proper randomization should eliminate most population biases, in which some populations, or members of a population are more likely to be selected or not selected than others, such that similar comparison groups are produced to evaluate treatments.4,5 OBJECTIVE: To assess the methodologic quality of the descriptions of randomization methods used to allocate participants to comparison groups in randomized controlled trials. EVIDENCE REVIEW: A cross-sectional review of phase 3 clinical trials reported in Clinicaltrials.gov. Beginning at all records available (n = 345,278) we included studies only listed for stage 3 RCTs in the U.S. National Library of Medicine database. A total of 1528 protocols were identified as of June 1, 2020. Exclusion criteria involved no protocol listed or non-randomized studies, of which 517 were excluded. There were 693 text articles excluded due to unclear methods of randomization. Inclusion criteria involved randomization methods based on "A review of randomization methods in clinical trials" by Berger and Antsygina.1 Each study protocol was extracted to identify the randomization methods described by three independent reviewers. Classification of randomization methods described in the study protocols for randomized clinical trials. FINDINGS: Only 20.8 % of the study protocols described a method for randomly assigning participants to groups. Of this subset that defined protocols, the Permuted-Block Design was used most often (85.9 %). More than three quarters of all study protocols (77.7 %) provided incomplete descriptions about the type of randomization method (i.e. no protocol, n/a, unclear). CONCLUSIONS: and Relevance:Proper randomization is required to generate unbiased comparison groups in controlled trials, yet the majority of study protocols for RCTs currently in Clinicaltrials.gov provide inadequate or unacceptable information regarding their randomization methods.