Optical Detection of CoV-SARS-2 Viral Proteins to Sub-Picomolar Concentrations

The emergence of a new strain of coronavirus in late 2019, SARS-CoV-2, led to a global pandemic in 2020. This may have been preventable if large scale, rapid diagnosis of active cases had been possible, and this has highlighted the need for more effective and efficient ways of detecting and managing viral infections. In this work, we investigate three different optical techniques for quantifying the binding of recombinant SARS-CoV-2 spike protein to surface-immobilized oligonucleotide aptamers. Biolayer interferometry is a relatively cheap, robust, and rapid method that only requires very small sample volumes. However, its detection limit of 250 nM means that it is not sensitive enough to detect antigen proteins at physiologically relevant levels (sub-pM). Surface plasmon resonance is a more sensitive technique but requires larger sample volumes, takes longer, requires more expensive instrumentation, and only reduces the detection limit to 5 nM. Surface-enhanced Raman spectroscopy is far more sensitive, enabling detection of spike protein to sub-picomolar concentrations. Control experiments performed using scrambled aptamers and using bovine serum albumin as an analyte show that this apta-sensing approach is both sensitive and selective, with no appreciable response observed for any controls. Overall, these proof-of-principle results demonstrate that SERS-based aptasensors hold great promise for development into rapid, point-of-use antigen detection systems, enabling mass testing without any need for reagents or laboratory expertise and equipment.fals

Identification of genes involved in exoprotein release using a high-throughput exoproteome screening assay in Yersinia entomophaga

Bacterial protein secretion is crucial to the maintenance of viability and pathogenicity. Although many bacterial secretion systems have been identified, the underlying mechanisms regulating their expression are less well explored. Yersinia entomophaga MH96, an entomopathogenic bacterium, releases an abundance of proteins including the Yen-Tc into the growth medium when cultured in Luria Bertani broth at ≤ 25˚C. Through the development of a high-throughput exoproteome screening assay (HESA), genes involved in MH96 exoprotein production were identified. Of 4,080 screened transposon mutants, 34 mutants exhibited a decreased exoprotein release, and one mutation located in the intergenic region of the Yen-Tc operon displayed an elevated exoprotein release relative to the wild-type strain MH96. DNA sequencing revealed several transposon insertions clustered in gene regions associated with lipopolysaccharide (LPSI and LPSII), and N-acyl-homoserine lactone synthesis (quorum sensing). Twelve transposon insertions were located within transcriptional regulators or intergenic regions. The HESA will have broad applicability for identifying genes associated with exoproteome production in a range of microorganisms

Optical Detection of CoV-SARS-2 Viral Proteins to Sub-Picomolar Concentrations

The emergence of a new strain of coronavirus in late 2019, SARS-CoV-2, led to a global pandemic in 2020. This may have been preventable if large scale, rapid diagnosis of active cases had been possible, and this has highlighted the need for more effective and efficient ways of detecting and managing viral infections. In this work, we investigate three different optical techniques for quantifying the binding of recombinant SARS-CoV-2 spike protein to surface-immobilized oligonucleotide aptamers. Biolayer interferometry is a relatively cheap, robust, and rapid method that only requires very small sample volumes. However, its detection limit of 250 nM means that it is not sensitive enough to detect antigen proteins at physiologically relevant levels (sub-pM). Surface plasmon resonance is a more sensitive technique but requires larger sample volumes, takes longer, requires more expensive instrumentation, and only reduces the detection limit to 5 nM. Surface-enhanced Raman spectroscopy is far more sensitive, enabling detection of spike protein to sub-picomolar concentrations. Control experiments performed using scrambled aptamers and using bovine serum albumin as an analyte show that this apta-sensing approach is both sensitive and selective, with no appreciable response observed for any controls. Overall, these proof-of-principle results demonstrate that SERS-based aptasensors hold great promise for development into rapid, point-of-use antigen detection systems, enabling mass testing without any need for reagents or laboratory expertise and equipment

Linocin M18 protein from the insect pathogenic bacterium Brevibacillus laterosporus isolates

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium. Insect pathogenic strains have been characterised in New Zealand, and two isolates, Bl 1821L and Bl 1951, are under development for use in biopesticides. However, growth in culture is sometimes disrupted, affecting mass production. Based on previous work, it was hypothesised that Tectiviridae phages might be implicated. While investigating the cause of the disrupted growth, electron micrographs of crude lysates showed structural components of putative phages including capsid and tail-like structures. Sucrose density gradient purification yielded a putative self-killing protein of ∼30 kDa. N-terminal sequencing of the ∼30 kDa protein identified matches to a predicted 25 kDa hypothetical and a 31.4 kDa putative encapsulating protein homologs, with the genes encoding each protein adjacent in the genomes. BLASTp analysis of the homologs of 31.4 kDa amino acid sequences shared 98.6% amino acid identity to the Linocin M18 bacteriocin family protein of Brevibacterium sp. JNUCC-42. Bioinformatic tools including AMPA and CellPPD defined that the bactericidal potential originated from a putative encapsulating protein. Antagonistic activity of the ∼30 kDa encapsulating protein of Bl 1821L and Bl 1951 during growth in broth exhibited bacterial autolytic activity. LIVE/DEAD staining of Bl 1821L cells after treatment with the ∼30 kDa encapsulating protein of Bl 1821L substantiated the findings by showing 58.8% cells with the compromised cell membranes as compared to 37.5% cells in the control. Furthermore, antibacterial activity of the identified proteins of Bl 1821L was validated through gene expression in a Gram-positive bacterium Bacillus subtilis WB800N

Tokophobia and fear of birth: a workshop consensus statement on current issues and recommendations for future research

Objective: The workshop aimed to discuss and develop a statement on the current state of the evidence and opinion in the field of Fear of Childbirth (FoC) and Tokophobia (Tocophobia), and to provide some recommendations for research. Background: A group of international researchers, clinicians and a service user met in 2019 to discuss the state of clinical and academic knowledge relating to FoC/Tokophobia. Five key areas were identified and agreed as the focus of discussion at the meeting. Methods: 12 clinicians and researchers, invited for their known expertise in this or a closely related area (e.g. PTSD) met in Hull, UK to discuss their understanding of the evidence for FoC/ Tokophobia and current practice. The meeting focused on identifying areas of uncertainty, key areas of knowledge, emerging research and possible future research within the field. The consensus described in this paper constitutes the expression of the general opinion of the participants and does not necessarily imply unanimity Keys points for discussion: Whilst there is a body of work in the field of FoC, work specifically focussed on tokophobia is more recent. It was agreed that there remains a wide range of issues, for which we need further evidence, which were addressed in the workshop including complexity in defining prevalence, a theoretical lack of understanding of tokophobia, which gives rise to challenges for robust assessment and the identification of risk factors. An improved understanding of the aetiological and developmental aspects of tokophobia is required to underpin appropriate, effective and evidence-based interventions. The development and evaluation of pathways of care and the interventions these might incorporate, should be a focus of future research to explore the potential for positive outcomes. Conclusion: Significant gaps remain within the FoC/tokophobia knowledge base. Further research continues to be needed across all areas identified

The Transcription Factor NFAT5 Is Required for Cyclin Expression and Cell Cycle Progression in Cells Exposed to Hypertonic Stress

Background: Hypertonicity can perturb cellular functions, induce DNA damage-like responses and inhibit proliferation. The transcription factor NFAT5 induces osmoprotective gene products that allow cells to adapt to sustained hypertonic conditions. Although it is known that NFAT5-deficient lymphocytes and renal medullary cells have reduced proliferative capacity and viability under hypertonic stress, less is understood about the contribution of this factor to DNA damage responses and cell cycle regulation. Methodology/Principal Findings: We have generated conditional knockout mice to obtain NFAT5−/− T lymphocytes, which we used as a model of proliferating cells to study NFAT5-dependent responses. We show that hypertonicity triggered an early, NFAT5-independent, genotoxic stress-like response with induction of p53, p21 and GADD45, downregulation of cyclins, and cell cycle arrest. This was followed by an NFAT5-dependent adaptive phase in wild-type cells, which induced an osmoprotective gene expression program, downregulated stress markers, resumed cyclin expression and proliferation, and displayed enhanced NFAT5 transcriptional activity in S and G2/M. In contrast, NFAT5−/− cells failed to induce osmoprotective genes and exhibited poorer viability. Although surviving NFAT5−/− cells downregulated genotoxic stress markers, they underwent cell cycle arrest in G1/S and G2/M, which was associated with reduced expression of cyclins E1, A2 and B1. We also show that pathologic hypertonicity levels, as occurring in plasma of patients and animal models of osmoregulatory disorders, inhibited the induction of cyclins and aurora B kinase in response to T cell receptor stimulation in fresh NFAT5−/− lymphocytes. Conclusions/Significance: We conclude that NFAT5 facilitates cell proliferation under hypertonic conditions by inducing an osmoadaptive response that enables cells to express fundamental regulators needed for cell cycle progression.Molecular and Cellular Biolog

The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system

Transforming growth factor-beta (TGF-beta) has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stages. In contrast to the mechanisms by which TGF-beta induces growth arrest, the pathways that mediate tumor invasion are not well understood. Here, we describe a TGF-beta-dependent invasion assay system consisting of spheroids of MCF10A1 normal breast epithelial cells (M1) and RAS-transformed (pre-)malignant derivatives (M2 and M4) embedded in collagen gels. Both basal and TGF-beta-induced invasion of these cell lines was found to correlate with their tumorigenic potential; M4 showing the most aggressive behavior and M1 showing the least. Basal invasion was strongly inhibited by the TGF-beta receptor kinase inhibitor SB-431542, indicating the involvement of autocrine TGF-beta or TGF-beta-like activity. TGF-beta-induced invasion in premalignant M2 and highly malignant M4 cells was also inhibited upon specific knockdown of Smad3 or Smad4. Interestingly, both a broad spectrum matrix metalloproteinase (MMP) inhibitor and a selective MMP2 and MMP9 inhibitor mitigated TGF-beta-induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-beta was found to strongly induce MMP2 and MMP9 expression in a Smad3- and Smad4-dependent manner. This collagen-embedded spheroid system therefore offers a valuable screening model for TGF-beta/Smad- and MMP2- and MMP9-dependent breast cancer invasion.Urolog

Segmental Duplications Arise from Pol32-Dependent Repair of Broken Forks through Two Alternative Replication-Based Mechanisms

The propensity of segmental duplications (SDs) to promote genomic instability is of increasing interest since their involvement in numerous human genomic diseases and cancers was revealed. However, the mechanism(s) responsible for their appearance remain mostly speculative. Here, we show that in budding yeast, replication accidents, which are most likely transformed into broken forks, play a causal role in the formation of SDs. The Pol32 subunit of the major replicative polymerase Polδ is required for all SD formation, demonstrating that SDs result from untimely DNA synthesis rather than from unequal crossing-over. Although Pol32 is known to be required for classical (Rad52-dependant) break-induced replication, only half of the SDs can be attributed to this mechanism. The remaining SDs are generated through a Rad52-independent mechanism of template switching between microsatellites or microhomologous sequences. This new mechanism, named microhomology/microsatellite-induced replication (MMIR), differs from all known DNA double-strand break repair pathways, as MMIR-mediated duplications still occur in the combined absence of homologous recombination, microhomology-mediated, and nonhomologous end joining machineries. The interplay between these two replication-based pathways explains important features of higher eukaryotic genomes, such as the strong, but not strict, association between SDs and transposable elements, as well as the frequent formation of oncogenic fusion genes generating protein innovations at SD junctions

A Microhomology-Mediated Break-Induced Replication Model for the Origin of Human Copy Number Variation

Chromosome structural changes with nonrecurrent endpoints associated with genomic disorders offer windows into the mechanism of origin of copy number variation (CNV). A recent report of nonrecurrent duplications associated with Pelizaeus-Merzbacher disease identified three distinctive characteristics. First, the majority of events can be seen to be complex, showing discontinuous duplications mixed with deletions, inverted duplications, and triplications. Second, junctions at endpoints show microhomology of 2–5 base pairs (bp). Third, endpoints occur near pre-existing low copy repeats (LCRs). Using these observations and evidence from DNA repair in other organisms, we derive a model of microhomology-mediated break-induced replication (MMBIR) for the origin of CNV and, ultimately, of LCRs. We propose that breakage of replication forks in stressed cells that are deficient in homologous recombination induces an aberrant repair process with features of break-induced replication (BIR). Under these circumstances, single-strand 3′ tails from broken replication forks will anneal with microhomology on any single-stranded DNA nearby, priming low-processivity polymerization with multiple template switches generating complex rearrangements, and eventual re-establishment of processive replication