Collagen fibril formation at the plasma membrane occurs independently from collagen secretion.

Collagen fibrils are the primary supporting scaffold of vertebrate tissues but how they are assembled is unclear. Here, using CRISPR-tagging of type I collagen and SILAC labelling, we elucidate the cellular mechanism for the spatiotemporal assembly of collagen fibrils, in cultured fibroblasts. Our findings reveal multifaceted trafficking of collagen, including constitutive secretion, intracellular pooling, and plasma membrane-directed fibrillogenesis.

Collagen IV assembly is influenced by fluid flow in kidney cell-derived matrices.

Kidney podocytes and endothelial cells assemble a complex and dynamic basement membrane that is essential for kidney filtration. Whilst many components of this specialised matrix are known, the influence of fluid flow on its assembly and organisation remains poorly understood. Using the coculture of podocytes and glomerular endothelial cells in a low-shear stress, high-flow bioreactor, we investigated the effect of laminar fluid flow on the composition and assembly of cell-derived matrix.

Mmp14 is required for matrisome homeostasis and circadian rhythm in fibroblasts.

The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon.

UK-wide major trauma center tertiary trauma survey pro forma review and aggregation and consolidation into a redesigned document.

Objectives: The trauma tertiary survey (TTS) is an essential part of the continued care for major trauma patients which is performed to ensure that all injuries have been identified and none have been overlooked during the patient's stay. Although the Advanced Trauma Life Support Course states a need for a tertiary survey, there is currently no standard for what this survey comprises.

Methods: Using local consultant expert opinion and a literature search we identified a set of 32 TTS potential features that may be included within a TTS pro forma.

Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells.

COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, therapeutics that can help manage the disease are still required until immunity has been achieved globally. The identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease.

Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells.

COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, vaccine escape variants might arise leading to a re-emergence of COVID. In anticipation of such a scenario, the identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease.

Investigating Radiotherapy Response in a Novel Syngeneic Model of Prostate Cancer.

The prostate cancer (PCa) field lacks clinically relevant, syngeneic mouse models which retain the tumour microenvironment observed in PCa patients. This study establishes a cell line from prostate tumour tissue derived from the mouse, termed DVL3 which when subcutaneously implanted in immunocompetent C57BL/6 mice, forms tumours with distinct glandular morphology, strong cytokeratin 8 and androgen receptor expression, recapitulating high-risk localised human PCa. Compared to the commonly used TRAMP C1 model, generated with SV40 large T-antigen, DVL3 tumours are immunologically cold, with a lower proportion of CD8+ T-cells, and high proportion of immunosuppressive myeloid derived suppressor cells (MDSCs), thus resembling high-risk PCa.

Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging.

The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery, but remains a major technical challenge. Here, we describe dynamic, high-sensitivity protein quantitation technique using NanoLuciferase (NLuc) tagging, which is effective across microscopy and multiwell platforms. Using collagen as a test protein, the CRISPR-Cas9-mediated introduction of nluc (encoding NLuc) into the Col1a2 locus enabled the simplification and miniaturisation of procollagen-I (PC-I) quantitation.

Preservation of circadian rhythms by the protein folding chaperone, BiP.

Dysregulation of collagen synthesis is associated with disease progression in cancer and fibrosis. Collagen synthesis is coordinated with the circadian clock, which in cancer cells is, curiously, deregulated by endoplasmic reticulum (ER) stress. We hypothesized interplay between circadian rhythm, collagen synthesis, and ER stress in normal cells.

The anti-psychotic drug pimozide is a novel chemotherapeutic for breast cancer.

Pimozide, an antipsychotic drug of the diphenylbutylpiperidine class, has been shown to suppress cell growth of breast cancer cells . In this study we further explore the inhibitory effects of this molecule in cancer cells. We found that Pimozide inhibited cell proliferation in a dose- and time-dependent manner in MDA-MB-231 breast cancer cells and A549 lung cancer cells.

ΔNp63γ/SRC/Slug Signaling Axis Promotes Epithelial-to-Mesenchymal Transition in Squamous Cancers.

To investigate the regulation of epithelial-to-mesenchymal transition (EMT) in head and neck squamous cell carcinoma (HNSCC) and its importance in tumor invasion. We use a three-dimensional invasive organotypic raft culture model of human foreskin keratinocytes expressing the E6/E7 genes of the human papilloma virus-16, coupled with bioinformatic and IHC analysis of patient samples to investigate the role played by EMT in invasion and identify effectors and upstream regulatory pathways. We identify SNAI2 (Slug) as a critical effector of EMT-activated downstream of TP63 overexpression in HNSCC.

The IGF axis in HPV associated cancers.

Human papillomaviruses (HPV) infect and replicate in stratified epithelium at cutaneous and mucosal surfaces. The proliferation and maintenance of keratinocytes, the cells which make up this epithelium, are controlled by a number of growth factor receptors such as the keratinocyte growth factor receptor (KGFR, also called fibroblast growth factor receptor 2b (FGFR2b)), the epithelial growth factor receptor (EGFR) and the insulin-like growth factor receptors 1 and 2 (IGF1R and IGF2R). In this review, we will delineate the mutation, gene transcription, translation and processing of the IGF axis within HPV associated cancers.

HPV16 Down-Regulates the Insulin-Like Growth Factor Binding Protein 2 to Promote Epithelial Invasion in Organotypic Cultures.

Cervical cancer is a multi-stage disease caused by human papillomaviruses (HPV) infection of cervical epithelial cells, but the mechanisms regulating disease progression are not clearly defined. Using 3-dimensional organotypic cultures, we demonstrate that HPV16 E6 and E7 proteins alter the secretome of primary human keratinocytes resulting in local epithelial invasion. Mechanistically, absence of the IGF-binding protein 2 (IGFBP2) caused increases in IGFI/II signalling and through crosstalk with KGF/FGFR2b/AKT, cell invasion.

p63 drives invasion in keratinocytes expressing HPV16 E6/E7 genes through regulation of Src-FAK signalling.

Using microarray information from oro-pharyngeal data sets and results from primary human foreskin keratinocytes (HFK) expressing Human Papilloma Virus (HPV)-16 E6/E7 proteins, we show that p63 expression regulates signalling molecules which initiate cell migration such as Src and focal adhesion kinase (FAK) and induce invasion in 3D-organotypic rafts; a phenotype that can be reversed by depletion of p63. Knockdown of Src or FAK in the invasive cells restored focal adhesion protein paxillin at cell periphery and impaired the cell migration. In addition, specific inhibition of FAK (PF573228) or Src (dasatinib) activities mitigated invasion and attenuated the expression/activity of matrix metalloproteinase 14 (MMP14), a pivotal MMP in the MMP activation cascade.

IGF-Binding Protein 2 - Oncogene or Tumor Suppressor?

The role of insulin-like growth factor binding protein 2 (IGFBP2) in cancer is unclear. In general, IGFBP2 is considered to be oncogenic and its expression is often observed to be elevated in cancer. However, there are a number of conflicting reports in vitro and in vivo where IGFBP2 acts in a tumor suppressor manner.

AKT in stromal fibroblasts controls invasion of epithelial cells.

The tumour microenvironment has an important role in cancer progression and recent reports have proposed that stromal AKT is activated and regulates tumourigenesis and invasion. We have shown, by immuno-fluorescent analysis of oro-pharyngeal cancer biopsies, an increase in AKT activity in tumour associated stromal fibroblasts compared to normal stromal fibroblasts. Using organotypic raft co-cultures, we show that activation of stromal AKT can induce the invasion of keratinocytes expressing the HPV type 16 E6 and E7 proteins, in a Keratinocyte Growth Factor (KGF) dependent manner.

Regulation of epithelial differentiation and proliferation by the stroma: a role for the retinoblastoma protein.

Signaling between the epithelium and stromal cells is crucial for growth, differentiation, and repair of the epithelium. Although the retinoblastoma protein (Rb) is known to regulate the growth of keratinocytes in a cell-autonomous manner, here we describe a function of Rb in the stromal compartment. We find that Rb depletion in fibroblasts leads to inhibition of differentiation and enhanced proliferation of the epithelium.

Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion.

Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described.

Role of DeltaNp63gamma in epithelial to mesenchymal transition.

Although members of the p63 family of transcription factors are known for their role in the development and differentiation of epithelial surfaces, their function in cancer is less clear. Here, we show that depletion of the ΔNp63α and β isoforms, leaving only ΔNp63γ, results in epithelial to mesenchymal transition (EMT) in the normal breast cell line MCF10A. EMT can be rescued by the expression of the ΔNp63α isoform.

Acetylation of Rb by PCAF is required for nuclear localization and keratinocyte differentiation.

Although the retinoblastoma protein (Rb) functions as a checkpoint in the cell cycle, it also regulates differentiation. It has recently been shown that Rb is acetylated during differentiation; however, the role of this modification has not been identified. Depletion of Rb levels with short hairpin RNA resulted in inhibition of human keratinocyte differentiation, delayed cell cycle exit and allowed cell cycle re-entry.

p300 alters keratinocyte cell growth and differentiation through regulation of p21(Waf1/CIP1).

Background: p300 functions as a transcriptional co-activator to regulate many cellular responses such as cell growth, transformation, development and differentiation. It has been shown to affect the transcriptional activity of p53 which regulates p21(Waf1/CIP1) expression, however, the role of p300 in differentiation remains unclear.

Methodology And Principal Findings: Knockdown of p300 protein with short hairpin RNA (shRNA) molecules delays human neonatal foreskin keratinocyte (HFKs) differentiation.

Tumor suppressor Ras-association domain family 1 isoform A is a novel regulator of cardiac hypertrophy.

Background: Ras signaling regulates a number of important processes in the heart, including cell growth and hypertrophy. Although it is known that defective Ras signaling is associated with Noonan, Costello, and other syndromes that are characterized by tumor formation and cardiac hypertrophy, little is known about factors that may control it. Here we investigate the role of Ras effector Ras-association domain family 1 isoform A (RASSF1A) in regulating myocardial hypertrophy.

Specific role of neuronal nitric-oxide synthase when tethered to the plasma membrane calcium pump in regulating the beta-adrenergic signal in the myocardium.

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates beta-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart.