KCL TEST: an open-source inspired asymptomatic SARS-CoV-2 surveillance programme in an academic institution.

Rapid and accessible testing was paramount in the management of the COVID-19 pandemic. Our university established KCL TEST: a SARS-CoV-2 asymptomatic testing programme that enabled sensitive and accessible PCR testing of SARS-CoV-2 RNA in saliva. Here, we describe our learnings and provide our blueprint for launching diagnostic laboratories, particularly in low-resource settings. Between December 2020 and July 2022, we performed 158277 PCRs for our staff, students, and their household contacts, free of charge. Our average turnaround time was 16 h and 37 min from user registration to result delivery. KCL TEST combined open-source automation and in-house non-commercial reagents, which allows for rapid implementation and repurposing. Importantly, our data parallel those of the UK Office for National Statistics, though we detected a lower positive rate and virtually no delta wave. Our observations strongly support regular asymptomatic community testing as an important measure for decreasing outbreaks and providing safe working spaces. Universities can therefore provide agile, resilient, and accurate testing that reflects the infection rate and trend of the general population. Our findings call for the early integration of academic institutions in pandemic preparedness, with capabilities to rapidly deploy highly skilled staff, as well as develop, test, and accommodate efficient low-cost pipelines.

Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted antiviral functions of APOBEC3G.

Following cell entry, the RNA genome of HIV-1 is reverse transcribed into double-stranded DNA that ultimately integrates into the host-cell genome to establish the provirus. These early phases of infection are notably vulnerable to suppression by a collection of cellular antiviral effectors, called restriction or resistance factors. The host antiviral protein APOBEC3G (A3G) antagonizes the early steps of HIV-1 infection through the combined effects of inhibiting viral cDNA production and cytidine-to-uridine-driven hypermutation of this cDNA. In seeking to address the underlying molecular mechanism for inhibited cDNA synthesis, we developed a deep sequencing strategy to characterize nascent reverse transcription products and their precise 3'-termini in HIV-1 infected T cells. Our results demonstrate site- and sequence-independent interference with reverse transcription, which requires the specific interaction of A3G with reverse transcriptase itself. This approach also established, contrary to current ideas, that cellular uracil base excision repair (UBER) enzymes target and cleave A3G-edited uridine-containing viral cDNA. Together, these findings yield further insights into the regulatory interplay between reverse transcriptase, A3G and cellular DNA repair machinery, and identify the suppression of HIV-1 reverse transcriptase by a directly interacting host protein as a new cell-mediated antiviral mechanism.

A triple-arginine motif in the amino-terminal domain and oligomerization are required for HIV-1 inhibition by human MX2.

We have employed molecular genetic approaches to understand the domain organization of the HIV-1 resistance factor myxovirus resistance 2 (MX2). First, we describe an essential triple-arginine motif in the amino-terminal domain. Second, we demonstrate that this 91-residue domain mediates antiviral activity when appended to heterologous proteins, and we provide genetic evidence that protein oligomerization is required for MX2 function. These insights will facilitate future work aiming to elucidate MX2's mechanism of action.

Matrix mediates the functional link between human immunodeficiency virus type 1 RNA nuclear export elements and the assembly competency of Gag in murine cells.

Human immunodeficiency virus type 1 (HIV-1) assembles poorly in murine cells, reflecting inefficient targeting of the Gag structural polyprotein to the plasma membrane. Virus particle production can be restored by replacing the cis-acting Rev response element (RRE) in Gag-Pol mRNAs with multiple copies of the CTE (4xCTE), suggesting a mechanistic link between HIV-1 RNA trafficking and productive Gag assembly. In this report, we demonstrate that Gag molecules generated from RRE-dependent transcripts are intrinsically defective for assembly in murine 3T3 cells. When controlled for the intracellular Gag level, modulations of the Gag matrix (MA) domain that enhance Gag membrane association (e.g., deletion of the MA globular head) substantially improve assembly for Gag derived from RRE- but not 4xCTE-dependent transcripts. Gag mutants carrying a leucine zipper replacement of the nucleocapsid (NC) domain remain largely assembly defective when derived from RRE-dependent transcripts, indicating that the defect does not reflect aberrant NC/RNA-driven Gag multimerization. We further demonstrate that single changes in uncharged amino acids implicated in Gag/MA myristoyl switch regulation, most notably replacing the leucine at position 21 with serine, improve assembly for Gag derived from RRE-dependent transcripts. In sum, we provide genetic evidence to suggest that HIV-1 RNA metabolism specifically modulates the activation of MA-dependent membrane targeting.

The constitution, physical properties and biocompatibility of modified accelerated cement.

OBJECTIVES: The aim of this study was to determine constitution and physical properties of a prototype material based on Portland cement and assess biocompatibility compared with glass-ionomer cement by evaluating cell morphology. MATERIALS AND METHODS: Analysis of the material was performed using energy dispersive analysis (EDAX) and X-ray diffraction (XRD) analysis. Compressive strength and the effect of changing the mixing and curing conditions on the compressive strength of the materials were evaluated. Dimensional stability was evaluated by measuring water uptake of the materials. Biocompatibility was assessed at 1 and 28 days using a cell-culture technique and semi-quantitative cell morphological evaluation was performed by SEM. RESULTS: Analysis of the material showed that it was primarily composed of tricalcium silicate and dicalcium silicate. The compressive strength of the prototype cement and variants was comparable to Ketac Molar (47.98 N mm(-2) after 1 day, P>0.05). Vacuum mixing did not improve the compressive strength of the prototype cements at any age. Wet curing was detrimental to the neat cement at 1 day (35.98 N mm(-2), P=0.011) and 7 days (44.08 N mm(-2), P=0.025). The filler-replaced cement prototypes were more stable and less susceptible to changes in compressive strength by varying the curing method (P>0.05). The prototype material took up more water (0.9%) than glass-ionomer cement (1.7%) with P=0 after 1 day. Curing at 100% humidity resulted in a net loss of weight for all the materials tested. The test materials were less biocompatible than glass-ionomer cement at 1 day but their biocompatibility improved as the material aged. CONCLUSIONS: The constitution of the prototype material was broadly similar to that of mineral trioxide aggregate. The prototype cement could be a potential dental restorative material as its compressive strength compared well to an established restorative material. However, the material did not support cell growth, with biocompatibility being similar to that of glass-ionomer cement.

Cytokine release by osteoblast-like cells cultured on implant discs of varying alloy compositions.

OBJECTIVE: The aims of this study were (i). to assess the morphological features of osteo-blast-like, osteosarcoma cells (cell line SaOS-2) cultured on implant surfaces of varying alloys and (ii). to evaluate the biological activity of these cells, following their adhesion onto these surfaces. MATERIALS AND METHODS: SaOS-2 cells (6 x 104) were grown on titanium discs (diameter 30 mm), each with a surface of differing composition and roughness (commercially pure titanium, titanium-aluminium-vanadium alloy, oxide-blasted titanium and Astra-Tech special treatment titanium; the alloys are directly comparable with those used to construct implants). The cells were grown for time periods of 1, 3, 5 and 7 days, the media were collected and the cells were fixed with 2.5% glutaraldehyde. The media were then assayed (using enzyme-linked immunosorbant assay) for the levels of interleukin (IL)-1, interleukin-6, interleukin-18 and osteoprotegerin (OPG) produced by the cells. The discs, with the cells fixed on them, were viewed under scanning electron microscopy (SEM, x 2.0 k) to evaluate cell morphology. RESULTS: Following attachment, the cells changed their morphology and released local factors known to activate osteoclasts. Commercially pure titanium stimulated the cells the most and titanium-aluminium-vanadium alloy the least. All implant materials stimulated production of IL-1, IL-6, IL-18 above that produced by cells grown on Petri dishes (polystyrene). The titanium-aluminium-vanadium alloy allowed cell attachment but levels of IL-1 in this medium were significantly lower (31.5 +/- 5.2 pg/ml on same day) than cultures with pure titanium (201.8 +/- 11.5 pg/ml at day 5). The same pattern was observed with the IL-6, IL-18, and OPG with polystyrene appearing to stimulate most production of OPG. Titanium-aluminium-vanadium produced the least biological response.