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Localised squamous cell carcinoma of the anus is treated with radical chemoradiotherapy. Cure rates are high, but treatment can result in substantial acute and long-term morbidity. We aimed to assess whether lower dose chemoradiotherapy maintains high local control rates in patients with early-stage disease, with the secondary aim of reducing toxicity. ACT4 is a phase 2, prospective, multicentre, open-label, two-arm non-comparative, randomised, controlled trial, investigating reduced-dose intensity-modulated radiotherapy (rd-IMRT: 41·4 Gy in 23 fractions) in patients with early-stage anal cancer; T1–2 (≤4 cm) N0–NxM0. Eligible patients were at least 16 years of age, with an Eastern Cooperative Oncology Group performance status of 0–1. The primary outcome is 3-year loco-regional failure rates. Patients were randomly assigned 1:2 (with stratification by T stage, N stage, gender, HIV status, and randomising site) to standard-dose IMRT (sd-IMRT: 50·4 Gy in 28 fractions) or rd-IMRT with concurrent mitomycin and capecitabine chemotherapy. Here, we report the pre-planned, modified intention-to-treat analysis of secondary endpoints 6 months after treatment end—complete clinical response, compliance, patient-reported outcomes (EORTC QLQ-C30 and ANL27), and safety data. The trial is registered at the ISRCTN registry (ISRCTN88455282) and is ongoing but no longer recruiting. 163 patients were recruited from 28 UK tertiary centres between April 24, 2017, and Dec 1, 2020. 160 patients were included in the primary analysis (sd-IMRT n=55; dr-IMRT n=105). Data on ethnicity were not collected. The median patient age was 66 years (IQR 58–72 years); 117 (73%) were female and 43 (27%) male; and 129 (94%) of 138 evaluable samples were p16 positive. Complete clinical responses at 6 months were 87% (46 of 53) for sd-IMRT and 92% (89 of 97) for rd-IMRT. Radiotherapy interruptions of 3 days or more occurred in 14 (26%) of 55 patients in sd-IMRT and 16 (15%) of 105 patients in rd-IMRT. Chemotherapy modifications occurred in 27 (49%) of 55 patients in sd-IMRT and 39 (37%) of 105 patients in rd-IMRT. Grade 3 or worse acute toxicity was reported in 25 (46%) of 55 patients in sd-IMRT and 37 (35%) of 105 patients in rd-IMRT. The most common grade 3 or worse adverse events were radiation dermatitis (seven [13%] of 55 in sd-IMRT and ten [10%] of 105 in rd-IMRT), and diarrhoea (four [7%] of 55 in sd-IMRT and nine [9%] of 105 in rd-IMRT). Serious adverse events occurred in eight (15%) of 55 patients in sd-IMRT and ten (10%) of 105 patients in rd-IMRT. Patient-reported outcomes for most issues deteriorated at the end of treatment and resolved to baseline by 6 weeks in both groups. Poorer sexual function for men and women was observed at 6 months following sd-IMRT. Good 6-month complete clinical responses rates were seen in both groups. Early results suggest rd-IMRT is well tolerated with oncological outcomes maintained. 3-year locoregional failure rates are awaited. Cancer Research UK and Stand Up to Cancer.

Nanobodies (Nbs) are single domain antibody fragments derived from heavy-chain antibodies found in members of the Camelidae family. They have become a relevant class of biomolecules for many different applications because of several important advantages such as their small size, high solubility and stability, and low production costs. On the other hand, synthetic Nb libraries are emerging as an attractive alternative to animal immunization for the selection of antigen-specific Nbs. Here, we present the design and construction of a new synthetic nanobody library using the phage display technology, following a structure-based approach in which the three hypervariable loops were subjected to position-specific randomization schemes. The constructed library has a clonal diversity of 108 and an amino acid variability that matches the codon distribution set by design at each randomized position. We have explored the capabilities of the new library by selecting nanobodies specific for three antigens: vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF) and the glycoprotein complex (GnGc) of Andes virus. To test the potential of the library to yield a variety of antigen-specific Nbs, we introduced a biopanning strategy consisting of a single selection round using stringent conditions. Using this approach, we obtained several binders for each of the target antigens. The constructed library represents a promising nanobody source for different applications.

Bovine viral diarrhea virus (BVDV) affects cattle worldwide causing severe productive and economic loss. In this study, we investigated the subgenotypes of BVDV circulating in cattle samples from the Aysén region, an active cattle breeding area located in southern Chile. Partial amplification of the 5' untranslated region (UTR) was performed by polymerase chain reaction (PCR), and twelve samples were analyzed by Sanger sequencing and phylogenetic analysis. Eight samples were identified as belonging to Pestivirus bovis subgenotype 1e, three to 1-b, and one to 1-d. The phylogenetic analyses performed revealed a marked distance between these now-identified strains and those previously reported in the country. These findings support the need to continually expand the analysis of the variability of the viral phylogeny for the currently circulating BVDV strains and to update the vaccines recommended for this livestock area and surrounding areas.

Background In high grade serous ovarian cancer (HGSOC), there is a spectrum of sensitivity to first line platinum‐based chemotherapy. This study molecularly characterizes HGSOC patients from two distinct groups of chemotherapy responders (good vs. poor). Methods Following primary debulking surgery and intravenous carboplatin/paclitaxel, women with stage III–IV HGSOC were grouped by response. Patients in the good response (GR) and poor response (PR) groups respectively had a progression‐free intervals (PFI) of ≥12 and ≤6 months. Analysis of surgical specimens interrogated genomic and immunologic features using whole exome sequencing. RNA‐sequencing detected gene expression outliers and inference of immune infiltrate, with validation by targeted NanoString arrays. PD‐L1 expression was scored by immunohistochemistry (IHC). Results A total of 39 patient samples were analyzed (GR = 20; PR = 19). Median PFI for GR and PR patient cohorts was 32 and 3 months, respectively. GR tumors were enriched for loss‐of‐function BRCA2 mutations and had a significantly higher nonsynonymous mutation rate compared to PR tumors (p = 0.001). Samples from the PR cohort were characterized by mutations in MGA and RAD51B and trended towards a greater rate of amplification of PIK3CA, MECOM, and ATR in comparison to GR tumors. Gene expression analysis by NanoString correlated increased PARP4 with PR and increased PD‐L1 and EMSY with GR. There was greater tumor immune cell infiltration and higher immune cell PD‐L1 protein expression in the GR group. Conclusions Our research demonstrates that tumors from HGSOC patients responding poorly to first line chemotherapy have a distinct molecular profile characterized by actionable drug targets including PARP4. In patients with suboptimally‐debulked, advanced high grade serous ovarian cancer (HGSOC), the spectrum of responses to first line platinum‐based chemotherapy (PtC) vary from durable to non‐existent, highlighting an unmet medical need for those with poor responses. Our study molecularly characterizes a cohort of HGSOC patients with distinct responses, either good or poor, to PtC. The good and poor responders have median progression‐free intervals of 32 and 3 months, respectively, and molecular analysis of tumors identifies differences in DNA damage response pathways and immunity, namely, tumors from poor responders to first line PtC have a distinct molecular profile characterized by actionable drug targets including PARP4.

Human inborn errors of immunity mediated by the cytokines interleukin-17A and interleukin-17F (IL-17A/F) underlie mucocutaneous candidiasis, whereas inborn errors of interferon-γ (IFN-γ) immunity underlie mycobacterial disease. We report the discovery of bi-allelic RORC loss-of-function mutations in seven individuals from three kindreds of different ethnic origins with both candidiasis and mycobacteriosis. The lack of functional RORγ and RORγT isoforms resulted in the absence of IL-17A/F–producing T cells in these individuals, probably accounting for their chronic candidiasis. Unexpectedly, leukocytes from RORγ- and RORγT-deficient individuals also displayed an impaired IFN-γ response to Mycobacterium. This principally reflected profoundly defective IFN-γ production by circulating γδ T cells and CD4+CCR6+CXCR3+ αβ T cells. In humans, both mucocutaneous immunity to Candida and systemic immunity to Mycobacterium require RORγ, RORγT, or both.

Most humans are exposed to Tropheryma whipplei (Tw). Whipple’s disease (WD) strikes only a small minority of individuals infected with Tw (<0.01%), whereas asymptomatic chronic carriage is more common (<25%). We studied a multiplex kindred, containing four WD patients and five healthy Tw chronic carriers. We hypothesized that WD displays autosomal dominant (AD) inheritance, with age-dependent incomplete penetrance. We identified a single very rare non-synonymous mutation in the four patients: the private R98W variant of IRF4, a transcription factor involved in immunity. The five Tw carriers were younger, and also heterozygous for R98W. We found that R98W was loss-of-function, modified the transcriptome of heterozygous leukocytes following Tw stimulation, and was not dominant-negative. We also found that only six of the other 153 known non-synonymous IRF4 variants were loss-of-function. Finally, we found that IRF4 had evolved under purifying selection. AD IRF4 deficiency can underlie WD by haploinsufficiency, with age-dependent incomplete penetrance. In 1907, George Hoyt Whipple – an American pathologist working at Johns Hopkins University in Baltimore – described a new inflammatory disease that affects the intestine. Patients with this condition, now known as Whipple’s disease, experience diarrhea, weight loss, and abdominal and joint pain. The disease is rare; it affects about one in a million people, mostly those over the age of 50 who are of European descent. Later it was discovered that bacteria called Tropheryma whipplei cause Whipple’s disease and that antibiotics can cure it. These bacteria are widespread and yet only a small minority of individuals infected with T. whipplei goes on to develop Whipple’s disease. In some populations, over 50% of individuals have been infected with the bacteria at some point in their lives, but most will get rid of the infection. This raised the question: when exposed to the same microbe, why do some individuals develop a severe disease, while others remain unharmed? From the 1950s onwards, scientists identified a few families with multiple members who have developed Whipple’s disease. These observations suggested that human genes may play a role in determining whether a person infected with T. whipplei becomes ill. Rare genetic mutations that affect the immune system have also been linked to the development of life-threatening cases of influenza or tuberculosis. Now, Guérin et al. report that, in one French family, an extremely rare mutation in the gene that codes for a protein called IRF4 may contribute to the development of Whipple’s disease. This family had four members with Whipple’s disease, all of whom had one copy of the gene with this rare mutation and one normal copy of the gene. The IRF4 protein acts like a switch that turns on and off some genes involved in the body’s response to infection. In patients with this mutation, the IRF4 protein does not work as it should. Guérin et al. suggest that Whipple’s disease may be caused by specific genetic mutations affecting the immune system in subjects infected by T. whipplei. More studies are needed to see if other genetic mutations also contribute to other cases of Whipple’s disease. Such studies may help physicians to better understand why some people become sick after T. whipplei infections while others do not. They may also help physicians to diagnose the disease, and even lead to better treatments.

Deep single-cell multi-omic profiling offers a promising approach to understand and overcome drug resistance in relapsed or refractory (rr) acute myeloid leukemia (AML). Here, we combine single-cell ex vivo drug profiling (pharmacoscopy) with single-cell and bulk DNA, RNA, and protein analyses, alongside clinical data from 21 rrAML patients. Unsupervised data integration reveals reduced ex vivo response to the Bcl-2 inhibitor venetoclax (VEN) in patients treated with both a hypomethylating agent (HMA) and VEN, compared to those pre-exposed to chemotherapy or HMA alone. Integrative analysis identifies both known and unreported mechanisms of innate and treatment-related VEN resistance and suggests alternative treatments, like targeting increased proliferation with the PLK inhibitor volasertib. Additionally, high CD36 expression in VEN-resistant blasts associates with sensitivity to CD36-targeted antibody treatment ex vivo. This study demonstrates how single-cell multi-omic profiling can uncover drug resistance mechanisms and treatment vulnerabilities, providing a valuable resource for future AML research. The molecular mechanisms underlying drug resistance in relapsed or refractory (rr) acute myeloid leukemia (AML) remain to be explored. Here, the use of bulk and single cell multi-omics and ex vivo drug profiling for 21 rrAML patients reveals mechanisms of resistance to the Bcl-2 inhibitor venetoclax and treatment vulnerabilities.

The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans. Here, the authors describe biallelic loss-of-function variants in human SHARPIN in individuals with autoinflammation and immunodeficiency, termed sharpenia. They also successfully treat one of these individuals with TNF inhibitors.

Direct and accurate radiocarbon dating of lipid residues preserved in ceramics is a recently established method that allows direct dating of specific food products and their inception in human subsistence strategies. The method targets individual fatty acids originating from animal fats such as ruminant dairy, ruminant adipose, non-ruminant adipose and aquatic fats. Horse lipid residues found in Central Asian pottery vessels are also directly dateable using this new method. Here we present the identification of equine lipid residues preserved in two pottery assemblages from the Neolithic and Eneolithic in Kazakhstan and their direct 14 C dating. The site of Botai, previously radiocarbon-dated to the 4th millennium BC, was used as a reference to evaluate the dates obtained directly on horse lipids. The direct dating of equine products extracted from Botai potsherds are shown to be compatible with previous 14 C dates at the site. The site of Bestamak, lacking previous 14 C measurements, had been relatively dated to the Neolithic based on pottery typologies. The direct dating of equine residues made it possible to anchor the pottery assemblage of Bestamak in the 6th millennium BC confirming their Neolithic attribution. These findings demonstrate the potential for dating horse products through a compound-specific approach, while highlighting challenges in 14 C dating individual fatty acids from lipid extracts in which their abundances differ substantially.