Codon Preference Optimization Increases Heterologous PEDF Expression

Pigment epithelium-derived factor (PEDF) is widely known for its neurotrophic and antiangiogenic functions. Efficacy studies of PEDF in animal models are limited because of poor heterologous protein yields. Here, we redesigned the human PEDF gene to preferentially match codon frequencies of E coli without altering the amino acid sequence. Following de novo synthesis, codon optimized PEDF (coPEDF) and the wtPEDF genes were cloned into pET32a containing a 5′ thioredoxin sequence (Trx) and the recombinant Trx-coPEDF or Trx-wtPEDF fusion constructs expressed in native and two tRNA augmented E coli hosts - BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP, carrying extra copies of tRNAarg,ile,leu and tRNAarg,pro genes , respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor Xα. Protein purity and identity were confirmed by western blot, MALDI-TOF, and UV/CD spectral analyses. Expression of the synthetic gene was ∼3.4 fold greater (212.7 mg/g; 62.1 mg/g wet cells) and purified yields ∼4 fold greater (41.1 mg/g; 11.3 mg/g wet cell) than wtPEDF in the native host. A small increase in expression of both genes was observed in hosts supplemented with rare tRNA genes compared to the native host but expression of coPEDF was ∼3 fold greater than wtPEDF in both native and codon-bias-adjusted E coli strains. ΔGs at −3 to +50 of the Trx site of both fusion genes were −3.9 kcal/mol. Functionally, coPEDF was equally as effective as wtPEDF in reducing oxidative stress, promoting neurite outgrowth, and blocking endothelial tube formation. These findings suggest that while rare tRNA augmentation and mRNA folding energies can significantly contribute to increased protein expression, preferred codon usage, in this case, is advantageous to translational efficiency of biologically active PEDF in E coli. This strategy will undoubtedly fast forward studies to validate therapeutic utility of PEDF in vivo

A 2-month field cohort study of SARS-CoV-2 in saliva of BNT162b2 vaccinated nursing home workers

peer reviewe

Loss of PIM2 enhances the anti-proliferative effect of the pan-PIM kinase inhibitor AZD1208 in non-Hodgkin lymphomas

Background A promising therapeutic approach for aggressive B-cell Non-Hodgkin lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma (BL) is to target kinases involved in signal transduction and gene regulation. PIM1/2 serine/threonine kinases are highly expressed in activated B-cell-like DLBCL (ABC-DLBCL) with poor prognosis. In addition, both PIM kinases have a reported synergistic effect with c-MYC in mediating tumour development in several cancers, c-MYC gene being translocated to one of the immunoglobulin loci in nearly all BLs. Methods For these reasons, we tested the efficiency of several PIM kinase inhibitors (AZD1208, SMI4a, PIM1/2 inhibitor VI and Quercetagetin) in preventing proliferation of aggressive NHL-derived cell lines and compared their efficiency with PIM1 and/or PIM2 knockdown. Results We observed that most of the anti-proliferative potential of these inhibitors in NHL was due to an off-target effect. Interestingly, we present evidence of a kinase-independent function of PIM2 in regulating cell cycle. Moreover, combining AZD1208 treatment and PIM2 knockdown additively repressed cell proliferation. Conclusion Taken together, this study suggests that at least a part of PIM1/2 oncogenic potential could be independent of their kinase activity, justifying the limited anti-tumorigenic outcome of PIM-kinase inhibitors in NHL

Epidemiology of neurodegenerative diseases in sub-Saharan Africa: a systematic review

BACKGROUND:Sub-Saharan African (SSA) countries are experiencing rapid transitions with increased life expectancy. As a result the burden of age-related conditions such as neurodegenerative diseases might be increasing. We conducted a systematic review of published studies on common neurodegenerative diseases, and HIV-related neurocognitive impairment in SSA, in order to identify research gaps and inform prevention and control solutions. METHODS: We searched MEDLINE via PubMed, 'Banque de Donnees de Sante Publique' and the database of the 'Institut d'Epidemiologie Neurologique et de Neurologie Tropicale' from inception to February 2013 for published original studies from SSA on neurodegenerative diseases and HIV-related neurocognitive impairment. Screening and data extraction were conducted by two investigators. Bibliographies and citations of eligible studies were investigated. RESULTS: In all 144 publications reporting on dementia (n=49 publications, mainly Alzheimer disease), Parkinsonism (PD, n=20), HIV-related neurocognitive impairment (n=47), Huntington disease (HD, n=19), amyotrophic lateral sclerosis (ALS, n=15), cerebellar degeneration (n=4) and Lewy body dementia (n=1). Of these studies, largely based on prevalent cases from retrospective data on urban populations, half originated from Nigeria and South Africa. The prevalence of dementia (Alzheimer disease) varied between <1% and 10.1% (0.7% and 5.6%) in population-based studies and from <1% to 47.8% in hospital-based studies. Incidence of dementia (Alzheimer disease) ranged from 8.7 to 21.8/1000/year (9.5 to 11.1), and major risk factors were advanced age and female sex. HIV-related neurocognitive impairment's prevalence (all from hospital-based studies) ranged from <1% to 80%. Population-based prevalence of PD and ALS varied from 10 to 235/100,000, and from 5 to 15/100,000 respectively while that for Huntington disease was 3.5/100,000. Equivalent figures for hospital based studies were the following: PD (0.41 to 7.2%), ALS (0.2 to 8.0/1000), and HD (0.2/100,000 to 46.0/100,000). CONCLUSIONS: The body of literature on neurodegenerative disorders in SSA is large with regard to dementia and HIV-related neurocognitive disorders but limited for other neurodegenerative disorders. Shortcomings include few population-based studies, heterogeneous diagnostic criteria and uneven representation of countries on the continent. There are important knowledge gaps that need urgent action, in order to prepare the sub-continent for the anticipated local surge in neurodegenerative diseases

A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and Euchromatin

© 2016 Janssen et al. Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains

Repetitive saliva‐based mass screening as a tool for controlling SARS‐CoV‐2 transmission in nursing homes

Nursing home (NH) residents and staff have been severely affected by the COVID-19 pandemic. The aim of this study was to examine the use of weekly saliva RT-qPCR testing for SARS-CoV-2 detection among NH workers as a strategy to control disease transmission within NHs in Belgium. From 16 November to 27 December 2020, a voluntary and anonymous weekly screening was implemented in a cohort of 50,000 workers across 572 NHs in the Walloon region of Belgium to detect asymptomatic cases of SARS-CoV-2 via saliva RT-qPCR testing and using the Diagenode saliva sample collection device. Positive workers were isolated to avoid subsequent infections in residents and other staff. RT-qPCR testing was based on pooled saliva sampling techniques from three workers, followed by individual testing of each positive or inconclusive pool. The majority of NHs (85%) and 55% of their workers participated. Pooling did not affect sensitivity as it only induced a very decrease in sensitivity estimated as 0.33%. Significant decreases in the prevalence (34.4–13.4%) and incidence of NHs with either single (13.8–2%) or multiple positive workers (3.7–0%) were observed over time. In addition, deaths among NH residents and NH worker absences decreased significantly over time. Weekly saliva RT-qPCR testing for SARS-CoV-2 demonstrated large-scale feasibility and efficacy in disrupting the chain of transmission. Implementation of this testing strategy in NHs could also be extended to other settings with the aim to control viral transmission for maintaining essential activities