PTB Domain-Directed Substrate Targeting in a Tyrosine Kinase from the Unicellular Choanoflagellate Monosiga brevicollis

Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition

Clinical utility of circulating tumor RNA (ctRNA) in a combined circulating tumor DNA (ctDNA) and ctRNA next-generation sequencing (NGS) pan-cancer liquid biopsy assay

3016 Background: Liquid biopsies are increasingly used in the real world for cancer therapy selection, disease monitoring, and early detection. Combining RNA with DNA sequencing for the detection of FDA-actionable gene fusions is already recommended as standard of care in guidelines as fusions are inherently challenging to detect by DNA-only methods. However, the clinical utility of profiling plasma ctRNA in addition to ctDNA has not yet been quantified in large studies. Here we report results from the first large real-world study establishing the clinical utility of combining ctRNA and ctDNA in a single liquid biopsy assay. Methods: A total of 1,007 consecutive plasma samples from 979 cancer patients across the USA and Asia underwent real-world liquid biopsy testing with a combined ctDNA and ctRNA assay (LiquidHALLMARK) in two CAP-accredited CLIA-certified laboratories from Jun 2021 to Dec 2024. The combined amplicon-based assay profiles genomic alterations in 80 genes on ctDNA and up to 37 genes on ctRNA. Only gene fusions (including MET exon 14 skipping) were included in this analysis. The limit of detection for gene fusions of the ctDNA and ctRNA panel were validated as 0.5% and 10 copies. Results: Plasma ctRNA was successfully analyzed in 99.6% (1003/1007) of samples across 30 cancer types. The top 5 cancer types (lung, prostate, breast, colorectal, and pancreas) comprised 84.2% of the clinical volume. Gene fusions were detected across 11 genes ( ALK , RET , ROS1 , MET , NRG1 , NTRK1 , FGFR2 , FGFR3 , ESR1 , ERG , ETV4 ) in 7.8% (78/1003) of cases, primarily in prostate and lung, but also in breast, bile duct, thyroid, liver, and bladder cancers. A total of 80 fusions were detected, of which 25 were detected by both ctDNA and ctRNA, while ctDNA and ctRNA each exclusively detected 27 and 28 fusions. Among the 28 ctRNA-only fusions, 5 were not covered by the ctDNA panel (1 ATP1B1 - NRG1 , 1 ESR1-CCDC170 , 1 ESR1 - AKAP12 , and 2 SLC45A3 - ERG fusions). Eleven (11/28) ctRNA-only fusions were actionable; all 11 were found in lung cancer. Two of these co-occurred with another lung driver mutation, highlighting potential resistance mechanisms to targeted therapy. Of the remaining 9, 3 were treated with fusion-matched targeted therapy. Two patients had real-world response rates available; both exhibited partial response to treatment. Overall, inclusion of ctRNA analysis increased the diagnostic yield of all fusions by 53.8% and actionable fusions by 36.7%. Conclusions: This is the first large study showing that adding ctRNA to ctDNA liquid biopsy increases total actionable diagnostic yield by 36.7%, highlights potential resistance mechanisms, and can broaden panel coverage to include gene fusions not amenable to detection by conventional DNA-based methods. These findings support recommendations for combined DNA/RNA testing of fusions in both tissue and liquid samples

3rd College of Physicians’ Lecture – Translational Research: From Bench to Bedside and From Bedside to Bench; Incorporating a Clinical Research Journey in IgA Nephritis (1976 to 2006)

Translational research (TR) can be defined as research where a discovery made in the laboratory (bench) can be applied in the diagnosis, treatment or prevention of a disease. Examples of medical discoveries contributing to translational medicine (TM) include the isolation of insulin by Banting (Nobel Laureate, 1923), the discovery of penicillin by Alexander Fleming (Nobel Laureate, 1945) and recently the discovery of the role of bacterium Helicobacter pylori in the causation of gastritis and peptic ulcer by Marshall and Warren (Nobel Laureates, 2005). Clinical research (CR) would be a more appropriate term for the bulk of research work undertaken by doctors. CR embraces both clinical based and laboratory-based research. The terminology “bedside to bench” applies more to CR as opposed to “bench to bedside” in the case of TR. But regardless of who does it, as long as the discovery can be translated to the bedside and results in improvement in patient care it can be considered a contribution to TM. Our work spans a 30-year period, involving laboratory-based research, clinical trials and genomics of IgA nephritis (Nx). This is a series of work to elucidate the pathogensis and therapy of IgANx. Plasma beta-thromboglobulin (BTG) an in-vivo index of platelet aggregation and anti-thrombin III increase due to a constant thrombogenecity resulting from platelet degranulation formed the basis for anti-platelet and low-dose warfarin therapy. A study of the natural history of IgANx revealed 2 courses, a slowly progressive course with end-stage renal failure (ESRF) at 7.7 years and a more rapid course at 3.3 years. Triple therapy (cyclophosphamide, persantin and low-dose warfarin) delayed progression to ESRF by about 8 years and for some patients up to 20 years. Documentation of abnormal suppressor T cell function provided the basis for immune therapy. Four patterns of proteinuria were present in IgANx and it is the quality and not so much the quantity of proteinuria which determined the prognosis. Low molecular weight proteinuria was a bad prognostic marker. A controlled therapeutic trial using ACEI/ATRA showed that therapy decreases proteinuria, improves renal function and converts non-selective to selective proteinuria. Subsequent work confirmed that it was the ATRA, not the ACEI which contributed to improved renal function. Individual anti proteinuria response to ATRA varies depending on ACE gene polymorphism. We found that the II genotype of the ACE gene was renoprotective and patients with this genotype had significantly reduced incidence of ESRF compared to those with the DD genotype. Patients responsive to ATRA therapy can retard progression to ESRF by up to 32 years. Mild renal failure can be reversed with possible regression of glomerulosclerosis because of glomerular remodelling by ATRA. Key words: Genomics, Glomerulonephritis, History, Therapy, Translational medicine</jats:p

Predição da força de reação do solo durante a corrida na água

Este estudo visou desenvolver um modelo para a predição da força de reação do solo na corrida subaquática. Participaram 20 sujeitos (9 homens e 11 mulheres), que realizaram corrida subaquática em dois níveis de imersão e três velocidades. Para cada sujeito foram coletadas seis passagens válidas em cada condição, com a utilização de uma plataforma subaquática de força. O modelo para predição da força foi construído por regressão linear múltipla. Foram consideradas variáveis dependentes a componente vertical e a componente ântero-posterior da força de reação do solo. As variáveis imersão, sexo, velocidade, massa corporal, densidade corporal e percentual de gordura foram consideradas independentes. Permaneceu no modelo final de regressão para a componente vertical a velocidade (pThis study aimed at developing a model to predict ground reaction force during deep-water running. A total of 20 subjects ((9 men, 11 women) ran in water at two immersion levels and three different speeds. Each subject performed six valid trials in each condition, data being captured by an underwater force plate. The force prediction model was build by multiple linear regression. Dependent variables were the vertical and anteroposterior components of the ground reaction force; independent variables were runners' immersion, sex, speed, body mass, body density, and percentage of fat. At the final regression model for the vertical component, only speed remained (p<0.001), while for the anteroposterior component, speed, immersion, and body mass were maintained (all at p<0.001). The obtained model for the anteroposterior component of ground reaction force may be found satisfactory, as adjusted determination coefficient was 0.79. However, the prediction model for the vertical component cannot be recommended for prediction during deep-water running, since that coefficient was 0.18. It must be noted that the proposed prediction model applies to subjects provided that they have similar characteristics to those who took part in this study

Tailoring of recommendations to reduce serious cutaneous adverse drug reactions: a pharmacogenomics approach

The Health Sciences Authority launched a pharmacogenetics initiative in 2008 to facilitate evaluation of pharmacogenetics associations pertinent for Chinese, Malays and Indians in Singapore. The aim was to reduce the incidence and unpredictability of serious adverse drug reactions, with a focus on serious skin adverse drug reactions. This paper describes the gathering of evidence and weighing of factors that led to different genotyping recommendations for HLA-B*15:02 with carbamazepine and HLA-B*58:01 with allopurinol, despite both having strong genetic associations. Translation of pharmacogenomics at a national level requires careful deliberation of the prevalence of at-risk allele, strength of genetic associations, positive predictive value, cost–effectiveness and availability of alternative therapies. Our experience provides a perspective on translating genomic discoveries in advancing drug safety. </jats:p