Targeting alternative splicing as a new cancer immunotherapy-phosphorylation of serine arginine-rich splicing factor (SRSF1) by SR protein kinase 1 (SRPK1) regulates alternative splicing of PD1 to generate a soluble antagonistic isoform that prevents T cell exhaustion.

BACKGROUND: Regulation of alternative splicing is a new therapeutic approach in cancer. The programmed cell death receptor 1 (PD-1) is an immunoinhibitory receptor expressed on immune cells that binds to its ligands, PD-L1 and PD-L2 expressed by cancer cells forming a dominant immune checkpoint pathway in the tumour microenvironment. Targeting this pathway using blocking antibodies (nivolumab and pembrolizumab) is the mainstay of anti-cancer immunotherapies, restoring the function of exhausted T cells. PD-1 is alternatively spliced to form isoforms that are either transmembrane signalling receptors (flPD1) that mediate T cell death by binding to the ligand, PD-L1 or an alternatively spliced, soluble, variant that lacks the transmembrane domain. METHODS: We used PCR and western blotting on primary peripheral blood mononuclear cells (PBMCs) and Jurkat T cells, IL-2 ELISA, flow cytometry, co-culture of melanoma and cholangiocarcinoma cells, and bioinformatics analysis and molecular cloning to examine the mechanism of splicing of PD1 and its consequence. RESULTS: The soluble form of PD-1, generated by skipping exon 3 (∆Ex3PD1), was endogenously expressed in PBMCs and T cells and prevents cancer cell-mediated T cell repression. Multiple binding sites of SRSF1 are adjacent to PD-1 exon 3 splicing sites. Overexpression of phosphomimic SRSF1 resulted in preferential expression of flPD1. Inhibition of SRSF1 phosphorylation both by SRPK1 shRNA knockdown and by a selective inhibitor, SPHINX31, resulted in a switch in splicing to ∆Ex3PD1. Cholangiocarcinoma cell-mediated repression of T cell IL-2 expression was reversed by SPHINX31 (equivalent to pembrolizumab). CONCLUSIONS: These results indicate that switching of the splicing decision from flPD1 to ∆Ex3PD1 by targeting SRPK1 could represent a potential novel mechanism of immune checkpoint inhibition in cancer.

Clinical implications of the arterial supplies and their anastomotic territories in the nasolabial region for avoiding arterial complications during soft tissue filler injection.

INTRODUCTION: The nasolabial fold (NLF) causes particular concern during aging in the middle face region. However, arterial complications of filler injections at this site have been continually reported during recent years. The aim of this study was to investigate the arterial locations and their anastomotic pathways related to filler injection sites in the NLF. MATERIALS AND METHODS: Thirty hemi-faces of 15 embalmed Thai cadavers were dissected. Three anatomical landmarks of NLFs were assigned: the inferior margin level (NLF1), the mid-philtral horizontal line level (NLF2), and the inferior alar level (NLF3). Ten hemi-faces of five soft embalmed Thai cadavers underwent a modified Sihler's staining procedure to investigate the arterial anastomoses. RESULTS: The artery closest to all of the landmarks was the facial artery. It was located inferomedial to NLF1 in 28%, and the mean distances along the X- and Y-axes were 3.53 ± 2.11 mm and 3.53 ± 1.75 mm, respectively. It was also located medial to NLF2 in 52.1% with an X-axis distance of 4.93 ± 1.53 mm. Several arteries were located close to NLF3, including the facial (33.3%), lateral nasal (33.3%), and infraorbital (30.0%) arteries. Anastomoses of the nasolabial arteries served to connect both the external-external and internal-external carotid systems. CONCLUSIONS: Several arteries are located close to NLF1-NLF3. To prevent arterial injury, the locations and anastomotic pathways, as possible sources of severe complications, should be recognized prior to NLF filler injection.

Localization and Topography of the Arteries on the Middle Forehead Region for Eluding Complications Following Forehead Augmentation: Conventional Cadaveric Dissection and Ultrasonography Investigation.

Forehead augmentation with filler injection is one of the most dangerous procedures associated with iatrogenic intravascular injection resulting in the severe complications. Nonetheless, few studies have determined the explicit arterial localization and topography related to the facial soft tissues and landmarks. Therefore, this study aimed to determine an arterial distribution and topography on the middle forehead region correlated with facial landmarks to grant an appropriate guideline for enhancing the safety of injection. Nineteen Thai embalmed cadavers were discovered with conventional dissection and 14 Thai healthy volunteers were investigated with ultrasonographic examination on the middle forehead. This study found that at the level of mid-frontal depression point, the transverse distance from the medial canthal vertical line to the superficial and deep branches of supraorbital artery were 9.1 mm and 15.1 mm, respectively. Whereas the depths from the skin of these arteries were 4.1 mm and 4.3 mm, respectively. Furthermore, the frontal branch of superficial temporal artery was detectable in 42.1% as an artery entering the forehead area. At the level of lateral canthal vertical line, the vertical distance of frontal branch was 31.6 mm, and the depth from skin of the artery was 2.7 mm. In conclusion, a proper injection technique could be performed based on an intensive arterial distribution and topography, and ultrasonographic examination before the injection is also suggested in order to restrict the opportunity of severe complications.

Knockdown of a novel G-protein pathway suppressor 2 (GPS2) leads to shrimp mortality by exuvial entrapment during ecdysis.

A novel G-protein pathway suppressor 2 (GPS2) has been identified from hemocytes of the whiteleg shrimp Penaeus vannamei (Pv) and appears to play a role in ecdysis. The full-length of PvGPS2 cDNA consisted of a 1230-bp open reading frame, encoding 409 deduced amino acids with significant sequence homology to GPS2 sequences of crustaceans and insects. RT-PCR revealed that PvGPS2 was expressed in all P. vannamei tissues examined, but that expression was molt stage specific in eyestalk tissue. Relative expression was higher in the period before molting (i.e., intermolt and pre-molt stages) than in the post-molt stage. When double-stranded RNA (dsRNA)-mediated RNA interference was employed to inhibit PvGPS2 formation in shrimp, it led to significant mortality due to unsuccessful separation of new cuticle from old cuticle (exuvial entrapment) during ecdysis.

Discovery and partial characterization of a non-LTR retrotransposon that may be associated with abdominal segment deformity disease (ASDD) in the whiteleg shrimp Penaeus (Litopenaeus) vannamei.

BACKGROUND: Abdominal segment deformity disease (ASDD) of cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei causes economic loss of approximately 10% in affected specimens because of the unsightliness of distorted abdominal muscles. It is associated with the presence of viral-like particles seen by electron microscopy in the ventral nerve cords of affected shrimp. Thus, shotgun cloning was carried out to seek viral-like sequences in affected shrimp. RESULTS: A new retrovirus-like element of 5052 bp (named abdominal segment deformity element or ASDE) was compiled by shotgun cloning and 3' and 5' RACE using RNA and DNA extracted from ventral nerve cords of ASDD shrimp. ASDE contained 7 putative open reading frames (ORF). One ORF (called the PENS sub-domain), had a deduced amino acid (aa) sequence homologous to the GIY-YIG endonuclease domain of penelope-like retrotransposons while two others were homologous to the reverse transcriptase (RT) and RNaseH domains of the pol gene of non-long terminal repeat (non-LTR) retrotransposons (called the NLRS sub-domain). No single amplicon of 5 kb containing both these elements was obtained by PCR or RT-PCR from ASDD shrimp. Subsequent analysis indicated that PENS and NLRS were not contiguous and that NLRS was a host genetic element. In situ hybridization using a dioxygenin-labeled NLRS probe revealed that NLRS gave positive reactions in abdominal-ganglion neurons of ASDD shrimp but not normal shrimp. Preliminary analysis indicated that long-term use of female broodstock after eyestalk ablation in the hatchery increased the intensity of RT-PCR amplicons for NLRS and also the prevalence of ASDD in mysis 3 offspring of the broodstock. The deformities persist upon further cultivation until shrimp harvest but do not increase in prevalence and do not affect growth or survival. CONCLUSIONS: Our results suggested that NLRS is a shrimp genetic element associated with ASDD and that immediate preventative measures could include shorter-term use of broodstock after eyestalk ablation and/or discard of broodstock that give strong RT-PCR reactions for NLRS. In the longer term, it is recommended, if possible, that currently used, domesticated shrimp lines be selected for freedom from NLRS. The molecular tools developed in this work will facilitate the management and further study of ASDD.