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The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings.

Objectives To conduct a case–control study of abnormalities in the semen of genitourinary (GU) medicine clinic attendees compared with general practice (GP) controls and in patients with asymptomatic and symptomatic non-specific urethritis (NSU) before and after the urethritis resolves. Methods Rates of semen abnormalities were compared between the different groups (19 with symptomatic and 27 with asymptomatic NSU, seven with symptomatic non-NSU and 64 clinic controls) and between clinic attendees and 417 patients attending GP for the first investigation of possible infertility. Those with symptomatic or asymptomatic NSU gave repeat semen samples on resolution of the NSU. Results The study included 117 clinic volunteers. They were shown to have statistically significantly worse total sperm counts (p=0.002), volume of semen (p<0.001) and percentage of abnormal forms (p<0.04) compared with 417 GP controls. Compared with the rest of the clinic volunteers, asymptomatic NSU patients had statistically significantly lower total sperm counts (p<0.02). Asymptomatic NSU patients had statistically significantly lower total sperm counts compared with symptomatic NSU patients (p<0.02). Compared with GP controls, clinic controls had statistically significantly inferior total sperm counts (p=0.009) and semen volume (p<0.001). Conclusions GU clinic attendees are more likely to have abnormalities of semen than patients attending GP for the first check for possible infertility. A high rate of abnormal semen findings are found in patients with and without NSU but the highest rate occurred in those with asymptomatic NSU. Is asymptomatic NSU therefore pathogenic and does it require treatment like symptomatic NSU?

Over the past three years many genitourinary medicine (GUM) clinics have anecdotally reported large numbers of persons with insecure immigration or seeking asylum (PIISA) attending their facilities. We conducted a national survey to assess the prevalence and demographic background of PIISA who were attending GUM clinics in the UK during 2001 and 2002 and the effect on service provision. A questionnaire was circulated in April 2003 to 182 consultants in the UK of whom 128 (70%) responded. Amongst those centres that responded, 89 (69%) had provided GUM/HIV services for PIISA in 2002. One-third of clinics had accurate data collection systems and less than a quarter used computerized databases in order to identify the associated workload. Of the HIV-positive patients attending these clinics during 2002, 1140 (42%) were identified as PIISA. Eighty-two (95.3%) and 62 (48.8%) clinics had cared for PIISA from Africa and Europe respectively. Co-infection with HIV and tuberculosis was higher in patients from the PIISA group compared with the non-PIISA group (85% vs 15%, P = 0.001) for both 2001 and 2002. Clinics reported many problems associated with the service for PIISA. Forty-five percent of the clinics reported difficulties with funding for the increased workload associated with PIISA. The survey shows that GUM services have an important role in the management of PIISA and that the programme of dispersal is having a significant impact on the workload of clinics outside London. Services report that they are significantly overstretched and underfunded. An immediate investment in GUM services is necessary to improve the health of this client group. Any delay in diagnosis of sexually transmitted infections and HIV will have implications for public health and acute services.

Recent evidence suggests that asymptomatic nonspecific urethritis (NSU), which is not routinely tested for, is a clinically significant pathology.The aim of this pilot study was to determine if testing for urinary threads, leucocyte esterase (LE) or both in asymptomatic men is a good screening tool for NSU. Of the126 asymptomatic men, 8% met microscopic criteria for the diagnosis of NSU. The positive predictive value for NSU was 71% (95% confidence interval (CI): 29.3–95.5%) and the negative predictive value was 96% (95% CI: 92.8–99.5%). The absence of threads and negative LE makes urethritis highly unlikely, making urinary chlamydia (Chlamydia trachomatis) and gonorrhoea (Neisseria gonorrhoeae) testing sufficient. Incidental findings of further pathology occurred in 7%.

Inclusion at academic events is facing increased scrutiny as the communities these events serve raise their expectations for who can practically attend. Active efforts in recent years to bring more diversity to academic events have brought progress and created momentum. However, we must reflect on these efforts and determine which underrepresented groups are being disadvantaged. Inclusion at academic events is important to ensure diversity of discourse and opinion, to help build networks, and to avoid academic siloing. All of these contribute to the development of a robust and resilient academic field. We have developed these Ten Simple Rules both to amplify the voices that have been speaking out and to celebrate the progress of many Equity, Diversity, and Inclusivity practices that continue to drive the organisation of academic events. The Rules aim to raise awareness as well as provide actionable suggestions and tools to support these initiatives further. This aims to support academic organisations such as the Deep Learning Indaba, Neuromatch Academy, the IBRO-Simons Computational Neuroscience Imbizo, Biodiversity Information Standards (TDWG), Arabs in Neuroscience, FAIRPoints, and OLS (formerly Open Life Science). This article is a call to action for organisers to reevaluate the impact and reach of their inclusive practices.

Mutant selective drugs targeting the inactive, GDP-bound form of KRAS G12C have been approved for use in lung cancer, but resistance develops rapidly. Here we use an inhibitor, (RMC-4998) that targets RAS G12C in its active, GTP-bound form, to treat KRAS mutant lung cancer in various immune competent mouse models. RAS pathway reactivation after RMC-4998 treatment could be delayed using combined treatment with a SHP2 inhibitor, which not only impacts tumour cell RAS signalling but also remodels the tumour microenvironment to be less immunosuppressive. In an immune inflamed model, RAS and SHP2 inhibitors in combination drive durable responses by suppressing tumour relapse and inducing development of immune memory. In an immune excluded model, combined RAS and SHP2 inhibition sensitises tumours to immune checkpoint blockade, leading to efficient tumour immune rejection. These preclinical results demonstrate the potential of the combination of RAS(ON) G12C-selective inhibitors with SHP2 inhibitors to sensitize tumours to immune checkpoint blockade. GDP-bound form of KRASG12C inhibitors have been approved for the treatment of patients with advanced KRASG12C mutant non-small cell lung cancer (NSCLC), however resistance to these drugs is often observed. Here the authors report that combining a GTP-bound RAS G12C-selective inhibitor with SHP2 inhibition can sensitize lung tumours to immune checkpoint blockade.

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS) 1 , 2 . Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive 1 , 2 . Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma 3 . We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response. In lung adenocarcinoma, antibodies against endogenous retroviruses promote anti-tumour activity, and expression of endogenous retroviruses can predict outcomes of immunotherapy.

Mutations in oncogenes such as KRAS and EGFR cause a high proportion of lung cancers. Drugs targeting these proteins cause tumour regression but ultimately fail to cure these cancers, leading to intense interest in how best to combine them with other treatments, such as immunotherapies. However, preclinical systems for studying the interaction of lung tumours with the host immune system are inadequate, in part due to the low tumour mutational burden in genetically engineered mouse models. Here we set out to develop mouse models of mutant KRAS-driven lung cancer with an elevated tumour mutational burden by expressing the human DNA cytosine deaminase, APOBEC3B, to mimic the mutational signature seen in human lung cancer. This failed to substantially increase clonal tumour mutational burden and autochthonous tumours remained refractory to immunotherapy. However, by establishing clonal cell lines from these tumours we generated an immunogenic syngeneic transplantation model of KRAS mutant lung adenocarcinoma that was sensitive to immunotherapy. Unexpectedly, we found that anti-tumour immune responses were not directed against neoantigens but instead targeted derepressed endogenous retroviral antigens. The ability of KRASG12C inhibitors to cause regression of KRASG12C-expressing versions of these tumours was markedly potentiated by the adaptive immune system, providing a unique opportunity for the study of combinations of targeted and immunotherapies in immune-hot lung cancer. Competing Interest Statement J.D. has acted as a consultant for AstraZeneca, Bayer, Jubilant, Theras, Vividion and Novartis. R.S.H is a co founder, shareholder, and consultant of ApoGen Biotechnologies Inc. S.R. is an employee of AstraZeneca. C.S. receives grant support from Archer Dx, AstraZeneca, BoehringerIngelheim and Ono Pharmaceutical; has consulted for AstraZeneca, Bicycle Therapeutics, Celgene, Genentech, GRAIL, GSK, Illumina, Medicxi, MSD, Novartis and the Sarah Cannon Research Institute; receives grant support and has consulted for Bristol Myers Squibb, Pfizer and RocheVentana; is an advisory board member and is involved in trials sponsored by AstraZeneca; has stock options in Apogen Biotechnologies, Epic Sciences, GRAIL; and has stock options and is a cofounder of Achilles Therapeutics. The other authors declare that they have no competing interests. Footnotes * This version of the manuscript has been revised to reflect our finding that endogenous CD8+ T cell responses are directed against derepressed endogenous retrovirus antigens expressed by the KPAR1.3 tumour cell line, but evidence of T cell responses against APOBEC3B induced mutant neoantigens was not found.

Mutant selective drugs targeting the inactive, GDP-bound form of KRASG12C have been approved for use in lung cancer, but responses are short-lived due to rapid development of resistance. In this study we use a novel covalent tri-complex inhibitor, RMC-4998, that targets RASG12C in its active, GTP-bound form to investigate treatment of KRAS mutant lung cancer in various immune competent mouse models. While this RASG12C(ON) inhibitor was more potent than the KRASG12C(OFF) inhibitor adagrasib, rapid pathway reactivation was still observed. This could be delayed using combined treatment with a SHP2 inhibitor, RMC-4550, which not only impacted RAS pathway signalling within the tumour cells but also remodelled the tumour microenvironment (TME) to be less immunosuppressive and promoted interferon responses. In an inflamed, hot, mouse model of lung cancer, RASG12C(ON) and SHP2 inhibitors in combination drive durable responses by suppressing tumour relapse and inducing development of immune memory, which can also be induced by combination of RASG12C(ON) and PD-1 inhibitors. In contrast, in an immune excluded, cold, mouse model of lung cancer, combined RASG12C(ON) and SHP2 inhibition does not cause durable responses, but does sensitise tumours to immune checkpoint blockade, enabling efficient tumour rejection, accompanied by significant TME reorganization, including depletion of immunosuppressive innate immune cells and recruitment and activation of T and NK cells. These preclinical results demonstrate the potential of the combination of RASG12C(ON) inhibitors with SHP2 inhibitors to sensitize anti-PD-1 refractory tumours to immune checkpoint blockade by stimulating anti-tumour immunity as well as by targeting KRAS-driven proliferation in tumour cells.Competing Interest StatementJ.D. has acted as a consultant for AstraZeneca, Jubilant, Theras, Roche and Vividion and has funded research agreements with Bristol Myers Squibb, Revolution Medicines and AstraZeneca. S.C.T has acted as a consultant for Revolution Medicines. C.B., E.Q. and J.A.M.S. are employees of Revolution Medicines. The other authors declare that they have no competing interests.Footnotes* In this revision, corrections have been made to the bibliography which contained errors in referencing. In addition, minor changes have been made to the methods and acknowledgement sections. The rest of the paper is unchanged.