Current treatment and future directions in the management of anal cancer.

Although rare, the rate of squamous cell carcinoma of the anus (SCCA) is rising globally. Most patients present with nonmetastatic disease and are curable with appropriate treatment, which has evolved significantly over the last several decades. Before the 1970s, SCCA was managed with radical surgery, resulting in a permanent colostomy. Researchers found that preoperative treatment with chemotherapy and concurrent radiation could achieve a pathologic complete response. After this observation, definitive therapy shifted from radical surgery to sphincter-preserving chemoradiation. Investigations into the necessity of chemotherapy and the optimal regimen found that chemotherapy with mitomycin-C and 5-fluorouracil is required for cure. Further studies evaluating the addition of induction or maintenance chemotherapy, monoclonal antibody therapy, or higher radiation doses have demonstrated no significant benefit to disease control. Advanced radiation delivery with intensity-modulated radiotherapy techniques is now considered the standard of care because of its prospectively determined, favorable acute toxicity profile compared with 3-dimensional conformal radiation. It is important to note that chemoradiation treatment response may be slow (up to 26 weeks) and should be assessed through serial clinical examinations. Today, surgical management of SCCA is reserved only for the lowest risk, early stage tumors or for recurrent/persistent disease. Current studies are evaluating radiation dose de-escalation in early stage disease and radiation dose escalation and the addition of immune checkpoint inhibitors in locally advanced cancers. In reviewing how and why modern-day treatment of SCCA was established, the objective of this report is to reenforce adherence to current treatment paradigms to assure the best possible outcomes for patients.

Moving Immune Therapy Forward Targeting TME.

The host immune system shapes the fate of tumor progression. Hence, manipulating patients' immune system to activate host immune responses against cancer pathogenesis is a promising strategy to develop effective therapeutic interventions for metastatic and drug-resistant cancers. Understanding the dynamic mechanisms within the tumor microenvironment (TME) that contribute to heterogeneity and metabolic plasticity is essential to enhance the patients' responsiveness to immune targeted therapies. Riera-Domingo et al. (Riera-Domingo C, Audige A, Granja S, Cheng WC, Ho PC, Baltazar F, Stockmann C, Mazzone, M. Physiol Rev 100: 1-102, 2020) describe the immune landscape within the TME and highlight the significance of metabolic and hypoxic signatures that impact immune function and response to immunotherapy strategies. Current literature in this field confirms that targeting tumor metabolism and the acidic microenvironment commonly associated with tumors may present viable strategies to modulate the host immune system in favor of response to immune targeted therapies. However, development of better tools to understand tumor-immune interactions and identify mechanisms driving nonresponders, more innovative clinical trial design, and new therapies will need to be identified to move the field forward. Personalized immune therapies incorporating metabolic and microbiome-based gene signatures to influence the therapeutic response and novel methods to generate immunologically "hot" tumors are at the forefront of immunotherapy currently. The combination of these approaches with clinically approved immunotherapies will be valuable moving forward.

Structure of a B12-dependent radical SAM enzyme in carbapenem biosynthesis.

Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.

AAV-delivered suppressor tRNA overcomes a nonsense mutation in mice.

Gene therapy is a potentially curative medicine for many currently untreatable diseases, and recombinant adeno-associated virus (rAAV) is the most successful gene delivery vehicle for in vivo applications1-3. However, rAAV-based gene therapy suffers from several limitations, such as constrained DNA cargo size and toxicities caused by non-physiological expression of a transgene4-6. Here we show that rAAV delivery of a suppressor tRNA (rAAV.sup-tRNA) safely and efficiently rescued a genetic disease in a mouse model carrying a nonsense mutation, and effects lasted for more than 6 months after a single treatment. Mechanistically, this was achieved through a synergistic effect of premature stop codon readthrough and inhibition of nonsense-mediated mRNA decay. rAAV.sup-tRNA had a limited effect on global readthrough at normal stop codons and did not perturb endogenous tRNA homeostasis, as determined by ribosome profiling and tRNA sequencing, respectively. By optimizing the AAV capsid and the route of administration, therapeutic efficacy in various target tissues was achieved, including liver, heart, skeletal muscle and brain. This study demonstrates the feasibility of developing a toolbox of AAV-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) to rescue pathogenic nonsense mutations and restore gene function under endogenous regulation. As nonsense mutations account for 11% of pathogenic mutations, AAV-NoSTOP can benefit a large number of patients. AAV-NoSTOP obviates the need to deliver a full-length protein-coding gene that may exceed the rAAV packaging limit, elicit adverse immune responses or cause transgene-related toxicities. It therefore represents a valuable addition to gene therapeutics.

Combination Targeted Therapy in Relapsed Diffuse Large B-Cell Lymphoma.

BACKGROUND: The identification of oncogenic mutations in diffuse large B-cell lymphoma (DLBCL) has led to the development of drugs that target essential survival pathways, but whether targeting multiple survival pathways may be curative in DLBCL is unknown. METHODS: We performed a single-center, phase 1b-2 study of a regimen of venetoclax, ibrutinib, prednisone, obinutuzumab, and lenalidomide (ViPOR) in relapsed or refractory DLBCL. In phase 1b, which included patients with DLBCL and indolent lymphomas, four dose levels of venetoclax were evaluated to identify the recommended phase 2 dose, with fixed doses of the other four drugs. A phase 2 expansion in patients with germinal-center B-cell (GCB) and non-GCB DLBCL was performed. ViPOR was administered every 21 days for six cycles. RESULTS: In phase 1b of the study, involving 20 patients (10 with DLBCL), a single dose-limiting toxic effect of grade 3 intracranial hemorrhage occurred, a result that established venetoclax at a dose of 800 mg as the recommended phase 2 dose. Phase 2 included 40 patients with DLBCL. Toxic effects that were observed among all the patients included grade 3 or 4 neutropenia (in 24% of the cycles), thrombocytopenia (in 23%), anemia (in 7%), and febrile neutropenia (in 1%). Objective responses occurred in 54% of 48 evaluable patients with DLBCL, and complete responses occurred in 38%; complete responses were exclusively in patients with non-GCB DLBCL and high-grade B-cell lymphoma with rearrangements of MYC and BCL2 or BCL6 (or both). Circulating tumor DNA was undetectable in 33% of the patients at the end of ViPOR therapy. With a median follow-up of 40 months, 2-year progression-free survival and overall survival were 34% (95% confidence interval [CI], 21 to 47) and 36% (95% CI, 23 to 49), respectively. CONCLUSIONS: Treatment with ViPOR was associated with durable remissions in patients with specific molecular DLBCL subtypes and was associated with mainly reversible adverse events. (Funded by the Intramural Research Program of the National Cancer Institute and the National Center for Advancing Translational Sciences of the National Institutes of Health and others; ClinicalTrials.gov number, NCT03223610.).

An Isoprene Lipid-Binding Protein Promotes Eukaryotic Coenzyme Q Biosynthesis.

The biosynthesis of coenzyme Q presents a paradigm for how cells surmount hydrophobic barriers in lipid biology. In eukaryotes, CoQ precursors-among nature's most hydrophobic molecules-must somehow be presented to a series of enzymes peripherally associated with the mitochondrial inner membrane. Here, we reveal that this process relies on custom lipid-binding properties of COQ9. We show that COQ9 repurposes the bacterial TetR fold to bind aromatic isoprenes with high specificity, including CoQ intermediates that likely reside entirely within the bilayer. We reveal a process by which COQ9 associates with cardiolipin-rich membranes and warps the membrane surface to access this cargo. Finally, we identify a molecular interface between COQ9 and the hydroxylase COQ7, motivating a model whereby COQ9 presents intermediates directly to CoQ enzymes. Overall, our results provide a mechanism for how a lipid-binding protein might access, select, and deliver specific cargo from a membrane to promote biosynthesis.

Variable carbon isotope fractionation of photosynthetic communities over depth in an open-ocean euphotic zone.

Marine particulate organic carbon (POC) contributes to carbon export, food webs, and sediments, but uncertainties remain in its origins. Globally, variations in stable carbon isotope ratios (δ13C values) of POC between the upper and lower euphotic zones (LEZ) indicate either varying aspects of photosynthetic communities or degradative alteration of POC. During summertime in the subtropical north Atlantic Ocean, we find that δ13C values of the photosynthetic product phytol decreased by 6.3‰ and photosynthetic carbon isotope fractionation (εp) increased by 5.6‰ between the surface and the LEZ-variation as large as that found in the geologic record during major carbon cycle perturbations, but here reflecting vertical variation in δ13C values of photosynthetic communities. We find that simultaneous variations in light intensity and phytoplankton community composition over depth may be important factors not fully accounted for in common models of photosynthetic carbon isotope fractionation. Using additional isotopic and cell count data, we estimate that photosynthetic and non-photosynthetic material (heterotrophs or detritus) contribute relatively constant proportions of POC throughout the euphotic zone but are isotopically more distinct in the LEZ. As a result, the large vertical differences in εp result in significant, but smaller, differences in the δ13C values of total POC across the same depths (2.7‰). Vertical structuring of photosynthetic communities and export potential from the LEZ may vary across current and past ocean ecosystems; thus, LEZ photosynthesis may influence the exported and/or sedimentary δ13C values of both phytol and total organic carbon and affect interpretations of εp over geologic time.

Decoupling of bird migration from the changing phenology of spring green-up.

The green-up of vegetation in spring brings a pulse of food resources that many animals track during migration. However, green-up phenology is changing with climate change, posing an immense challenge for species that time their migrations to coincide with these resource pulses. We evaluated changes in green-up phenology from 2002 to 2021 in relation to the migrations of 150 Western-Hemisphere bird species using eBird citizen science data. We found that green-up phenology has changed within bird migration routes, and yet the migrations of most species align more closely with long-term averages of green-up than with current conditions. Changing green-up strongly influenced phenological mismatches, especially for longer-distance migrants. These results reveal that bird migration may have limited flexibility to adjust to changing vegetation phenology and emphasize the mounting challenge migratory animals face in following en route resources in a changing climate.

Prevention efficacy of the broadly neutralizing antibody VRC01 depends on HIV-1 envelope sequence features.

In the Antibody Mediated Prevention (AMP) trials (HVTN 704/HPTN 085 and HVTN 703/HPTN 081), prevention efficacy (PE) of the monoclonal broadly neutralizing antibody (bnAb) VRC01 (vs. placebo) against HIV-1 acquisition diagnosis varied according to the HIV-1 Envelope (Env) neutralization sensitivity to VRC01, as measured by 80% inhibitory concentration (IC80). Here, we performed a genotypic sieve analysis, a complementary approach to gaining insight into correlates of protection that assesses how PE varies with HIV-1 sequence features. We analyzed HIV-1 Env amino acid (AA) sequences from the earliest available HIV-1 RNA-positive plasma samples from AMP participants diagnosed with HIV-1 and identified Env sequence features that associated with PE. The strongest Env AA sequence correlate in both trials was VRC01 epitope distance that quantifies the divergence of the VRC01 epitope in an acquired HIV-1 isolate from the VRC01 epitope of reference HIV-1 strains that were most sensitive to VRC01-mediated neutralization. In HVTN 704/HPTN 085, the Env sequence-based predicted probability that VRC01 IC80 against the acquired isolate exceeded 1 µg/mL also significantly associated with PE. In HVTN 703/HPTN 081, a physicochemical-weighted Hamming distance across 50 VRC01 binding-associated Env AA positions of the acquired isolate from the most VRC01-sensitive HIV-1 strain significantly associated with PE. These results suggest that incorporating mutation scoring by BLOSUM62 and weighting by the strength of interactions at AA positions in the epitope:VRC01 interface can optimize performance of an Env sequence-based biomarker of VRC01 prevention efficacy. Future work could determine whether these results extend to other bnAbs and bnAb combinations.

DMRT1-mediated regulation of TOX3 modulates expansion of the gonadal steroidogenic cell lineage in the chicken embryo.

During gonadal sex determination, the supporting cell lineage differentiates into Sertoli cells in males and pre-granulosa cells in females. Recently, single cell RNA-seq data have indicated that chicken steroidogenic cells are derived from differentiated supporting cells. This differentiation process is achieved by a sequential upregulation of steroidogenic genes and downregulation of supporting cell markers. The exact mechanism regulating this differentiation process remains unknown. We have identified TOX3 as a previously unreported transcription factor expressed in embryonic Sertoli cells of the chicken testis. TOX3 knockdown in males resulted in increased CYP17A1-positive Leydig cells. TOX3 overexpression in male and female gonads resulted in a significant decline in CYP17A1-positive steroidogenic cells. In ovo knockdown of the testis determinant DMRT1 in male gonads resulted in a downregulation of TOX3 expression. Conversely, DMRT1 overexpression caused an increase in TOX3 expression. Taken together, these data indicate that DMRT1-mediated regulation of TOX3 modulates expansion of the steroidogenic lineage, either directly, via cell lineage allocation, or indirectly, via signaling from the supporting to steroidogenic cell populations.

Regulation of the Drosophila transcriptome by Pumilio and the CCR4-NOT deadenylase complex.

The sequence-specific RNA-binding protein Pumilio (Pum) controls Drosophila development; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we use knockdown and knockout approaches coupled with RNA-seq to measure the impact of Pum on the transcriptome of Drosophila cells in culture. We also use an improved RNA coimmunoprecipitation method to identify Pum-bound mRNAs in Drosophila embryos. Integration of these data sets with the locations of Pum-binding motifs across the transcriptome reveals novel direct Pum target genes involved in neural, muscle, wing, and germ cell development and in cellular proliferation. These genes include components of Wnt, TGF-ß, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. We identify the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pum-mediated repression, and observe concordant regulation of Pum:CCR4-NOT target mRNAs. Computational modeling reveals that Pum binding, binding site number, clustering, and sequence context are important determinants of regulation. In contrast, we show that the responses of direct mRNA targets to Pum-mediated repression are not influenced by the content of optimal synonymous codons. Moreover, contrary to a prevailing model, we do not detect a role for CCR4-NOT in the degradation of mRNAs with low codon optimality. Together, the results of this work provide new insights into the Pum regulatory network and mechanisms and the parameters that influence the efficacy of Pum-mediated regulation.

Epigenetic switch reshapes epithelial progenitor cell signatures and drives inflammatory pathogenesis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a complex inflammatory skin disease with undefined mechanistic underpinnings. Here, we investigated HS epithelial cells and demonstrated that HS basal progenitors modulate their lineage restriction and give rise to pathogenic keratinocyte clones, resulting in epidermal hyperproliferation and dysregulated inflammation in HS. When comparing to healthy epithelial stem/progenitor cells, in HS, we identified changes in gene signatures that revolve around the mitotic cell cycle, DNA damage response and repair, as well as cell-cell adhesion and chromatin remodeling. By reconstructing cell differentiation trajectory and CellChat modeling, we identified a keratinocyte population specific to HS. This population is marked by S100A7/8/9 and KRT6 family members, triggering IL1, IL10, and complement inflammatory cascades. These signals, along with HS-specific proinflammatory cytokines and chemokines, contribute to the recruitment of certain immune cells during the disease progression. Furthermore, we revealed a previously uncharacterized role of S100A8 in regulating the local chromatin environment of target loci in HS keratinocytes. Through the integration of genomic and epigenomic datasets, we identified genome-wide chromatin rewiring alongside the switch of transcription factors (TFs), which mediated HS transcriptional profiles. Importantly, we identified numerous clinically relevant inflammatory enhancers and their coordinated TFs in HS basal CD49fhigh cells. The disruption of the S100A enhancer using the CRISPR/Cas9-mediated approach or the pharmacological inhibition of the interferon regulatory transcription factor 3 (IRF3) efficiently reduced the production of HS-associated inflammatory regulators. Our study not only uncovers the plasticity of epidermal progenitor cells in HS but also elucidates the epigenetic mechanisms underlying HS pathogenesis.

Linking vertical movements of large pelagic predators with distribution patterns of biomass in the open ocean.

Many predator species make regular excursions from near-surface waters to the twilight (200 to 1,000 m) and midnight (1,000 to 3,000 m) zones of the deep pelagic ocean. While the occurrence of significant vertical movements into the deep ocean has evolved independently across taxonomic groups, the functional role(s) and ecological significance of these movements remain poorly understood. Here, we integrate results from satellite tagging efforts with model predictions of deep prey layers in the North Atlantic Ocean to determine whether prey distributions are correlated with vertical habitat use across 12 species of predators. Using 3D movement data for 344 individuals who traversed nearly 1.5 million km of pelagic ocean in [Formula: see text]42,000 d, we found that nearly every tagged predator frequented the twilight zone and many made regular trips to the midnight zone. Using a predictive model, we found clear alignment of predator depth use with the expected location of deep pelagic prey for at least half of the predator species. We compared high-resolution predator data with shipboard acoustics and selected representative matches that highlight the opportunities and challenges in the analysis and synthesis of these data. While not all observed behavior was consistent with estimated prey availability at depth, our results suggest that deep pelagic biomass likely has high ecological value for a suite of commercially important predators in the open ocean. Careful consideration of the disruption to ecosystem services provided by pelagic food webs is needed before the potential costs and benefits of proceeding with extractive activities in the deep ocean can be evaluated.

The structure of the monobactam-producing thioesterase domain of SulM forms a unique complex with the upstream carrier protein domain.

Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation, and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain of SulM, which catalyzes an unusual ß-lactam-forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the monobactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstream holo peptidyl-carrier domain. The position of variant lid helices results in an active site pocket that is quite constrained, a feature that is likely necessary to orient the substrate properly for ß-lactam formation. Modeling of a sulfazecin tripeptide into the active site identifies a plausible binding mode identifying potential interactions for the sulfamate and the peptide backbone with Arg2849 and Asn2819, respectively. The overall structure is similar to the ß-lactone-forming thioesterase domain that is responsible for similar ring closure in the production of obafluorin. We further use these insights to enable bioinformatic analysis to identify additional, uncharacterized ß-lactam-forming biosynthetic gene clusters by genome mining.

Overexpression of AMPKγ2 increases AMPK signaling to augment human T cell metabolism and function.

Cellular therapies are currently employed to treat a variety of disease processes. For T cell-based therapies, success often relies on the metabolic fitness of the T cell product, where cells with enhanced metabolic capacity demonstrate improved in vivo efficacy. AMP-activated protein kinase (AMPK) is a cellular energy sensor which combines environmental signals with cellular energy status to enforce efficient and flexible metabolic programming. We hypothesized that increasing AMPK activity in human T cells would augment their oxidative capacity, creating an ideal product for adoptive cellular therapies. Lentiviral transduction of the regulatory AMPKγ2 subunit stably enhanced intrinsic AMPK signaling and promoted mitochondrial respiration with increased basal oxygen consumption rates, higher maximal oxygen consumption rate, and augmented spare respiratory capacity. These changes were accompanied by increased proliferation and inflammatory cytokine production, particularly within restricted glucose environments. Introduction of AMPKγ2 into bulk CD4 T cells decreased RNA expression of canonical Th2 genes, including the cytokines interleukin (IL)-4 and IL-5, while introduction of AMPKγ2 into individual Th subsets universally favored proinflammatory cytokine production and a downregulation of IL-4 production in Th2 cells. When AMPKγ2 was overexpressed in regulatory T cells, both in vitro proliferation and suppressive capacity increased. Together, these data suggest that augmenting intrinsic AMPK signaling via overexpression of AMPKγ2 can improve the expansion and functional potential of human T cells for use in a variety of adoptive cellular therapies.

Striated muscle: an inadequate soil for cancers.

Many organs of the body are susceptible to cancer development. However, striated muscles-which include skeletal and cardiac muscles-are rarely the sites of primary cancers. Most deaths from cancer arise due to complications associated with the development of secondary metastatic tumours, for which there are few effective therapies. However, as with primary cancers, the establishment of metastatic tumours in striated muscle accounts for a disproportionately small fraction of secondary tumours, relative to the proportion of body composition. Examining why primary and metastatic cancers are comparatively rare in striated muscle presents an opportunity to better understand mechanisms that can influence cancer cell biology. To gain insights into the incidence and distribution of muscle metastases, this review presents a definitive summary of the 210 case studies of metastasis in muscle published since 2010. To examine why metastases rarely form in muscles, this review considers the mechanisms currently proposed to render muscle an inhospitable environment for cancers. The "seed and soil" hypothesis proposes that tissues' differences in susceptibility to metastatic colonization are due to differing host microenvironments that promote or suppress metastatic growth to varying degrees. As such, the "soil" within muscle may not be conducive to cancer growth. Gaining a greater understanding of the mechanisms that underpin the resistance of muscles to cancer may provide new insights into mechanisms of tumour growth and progression, and offer opportunities to leverage insights into the development of interventions with the potential to inhibit metastasis in susceptible tissues.

Open-Source Automated Insulin Delivery in Type 1 Diabetes.

BACKGROUND: Open-source automated insulin delivery (AID) systems are used by many patients with type 1 diabetes. Data are needed on the efficacy and safety of an open-source AID system. METHODS: In this multicenter, open-label, randomized, controlled trial, we assigned patients with type 1 diabetes in a 1:1 ratio to use an open-source AID system or a sensor-augmented insulin pump (control). The patients included both children (defined as 7 to 15 years of age) and adults (defined as 16 to 70 years of age). The AID system was a modified version of AndroidAPS 2.8 (with a standard OpenAPS 0.7.0 algorithm) paired with a preproduction DANA-i insulin pump and Dexcom G6 CGM, which has an Android smartphone application as the user interface. The primary outcome was the percentage of time in the target glucose range of 70 to 180 mg per deciliter (3.9 to 10.0 mmol per liter) between days 155 and 168 (the final 2 weeks of the trial). RESULTS: A total of 97 patients (48 children and 49 adults) underwent randomization (44 to open-source AID and 53 to the control group). At 24 weeks, the mean (±SD) time in the target range increased from 61.2±12.3% to 71.2±12.1% in the AID group and decreased from 57.7±14.3% to 54.5±16.0% in the control group (adjusted difference, 14 percentage points; 95% confidence interval, 9.2 to 18.8; P<0.001), with no treatment effect according to age (P = 0.56). Patients in the AID group spent 3 hours 21 minutes more in the target range per day than those in the control group. No severe hypoglycemia or diabetic ketoacidosis occurred in either group. Two patients in the AID group withdrew from the trial owing to connectivity issues. CONCLUSIONS: In children and adults with type 1 diabetes, the use of an open-source AID system resulted in a significantly higher percentage of time in the target glucose range than the use of a sensor-augmented insulin pump at 24 weeks. (Supported by the Health Research Council of New Zealand; Australian New Zealand Clinical Trials Registry number, ACTRN12620000034932.).

Secondary Antibiotic Prophylaxis for Latent Rheumatic Heart Disease.

BACKGROUND: Rheumatic heart disease affects more than 40.5 million people worldwide and results in 306,000 deaths annually. Echocardiographic screening detects rheumatic heart disease at an early, latent stage. Whether secondary antibiotic prophylaxis is effective in preventing progression of latent rheumatic heart disease is unknown. METHODS: We conducted a randomized, controlled trial of secondary antibiotic prophylaxis in Ugandan children and adolescents 5 to 17 years of age with latent rheumatic heart disease. Participants were randomly assigned to receive either injections of penicillin G benzathine (also known as benzathine benzylpenicillin) every 4 weeks for 2 years or no prophylaxis. All the participants underwent echocardiography at baseline and at 2 years after randomization. Changes from baseline were adjudicated by a panel whose members were unaware of the trial-group assignments. The primary outcome was echocardiographic progression of latent rheumatic heart disease at 2 years. RESULTS: Among 102,200 children and adolescents who had screening echocardiograms, 3327 were initially assessed as having latent rheumatic heart disease, and 926 of the 3327 subsequently received a definitive diagnosis on the basis of confirmatory echocardiography and were determined to be eligible for the trial. Consent or assent for participation was provided for 916 persons, and all underwent randomization; 818 participants were included in the modified intention-to-treat analysis, and 799 (97.7%) completed the trial. A total of 3 participants (0.8%) in the prophylaxis group had echocardiographic progression at 2 years, as compared with 33 (8.2%) in the control group (risk difference, -7.5 percentage points; 95% confidence interval, -10.2 to -4.7; P<0.001). Two participants in the prophylaxis group had serious adverse events that were attributable to receipt of prophylaxis, including one episode of a mild anaphylactic reaction (representing <0.1% of all administered doses of prophylaxis). CONCLUSIONS: Among children and adolescents 5 to 17 years of age with latent rheumatic heart disease, secondary antibiotic prophylaxis reduced the risk of disease progression at 2 years. Further research is needed before the implementation of population-level screening can be recommended. (Funded by the Thrasher Research Fund and others; GOAL ClinicalTrials.gov number, NCT03346525.).

Cell lineage specification and signalling pathway use during development of the lateral plate mesoderm and forelimb mesenchyme.

The lateral plate mesoderm (LPM) is a transient tissue that produces a diverse range of differentiated structures, including the limbs. However, the molecular mechanisms that drive early LPM specification and development are poorly understood. In this study, we use single-cell transcriptomics to define the cell-fate decisions directing LPM specification, subdivision and early initiation of the forelimb mesenchyme in chicken embryos. We establish a transcriptional atlas and global cell-cell signalling interactions in progenitor, transitional and mature cell types throughout the developing forelimb field. During LPM subdivision, somatic and splanchnic LPM fate is achieved through activation of lineage-specific gene modules. During the earliest stages of limb initiation, we identify activation of TWIST1 in the somatic LPM as a putative driver of limb bud epithelial-to-mesenchymal transition. Furthermore, we define a new role for BMP signalling during early limb development, revealing that it is necessary for inducing a somatic LPM fate and initiation of limb outgrowth, potentially through activation of TBX5. Together, these findings provide new insights into the mechanisms underlying LPM development, somatic LPM fate choice and early initiation of the vertebrate limb.

Woolly mutation with the Get02 locus overcomes the polygenic nature of trichome-based pest resistance in tomato.

Type-IV glandular trichomes, which only occur in the juvenile developmental phase of the cultivated tomato (Solanum lycopersicum), produce acylsugars that broadly protect against arthropod herbivory. Previously, we introgressed the capacity to retain type-IV trichomes in the adult phase from the wild tomato, Solanum galapagense, into the cultivated species cv. Micro-Tom (MT). The resulting MT-Galapagos enhanced trichome (MT-Get) introgression line contained 5 loci associated with enhancing the density of type-IV trichomes in adult plants. We genetically dissected MT-Get and obtained a subline containing only the locus on Chromosome 2 (MT-Get02). This genotype displayed about half the density of type-IV trichomes compared to the wild progenitor. However, when we stacked the gain-of-function allele of WOOLLY, which encodes a homeodomain leucine zipper IV transcription factor, Get02/Wo exhibited double the number of type-IV trichomes compared to S. galapagense. This discovery corroborates previous reports positioning WOOLLY as a master regulator of trichome development. Acylsugar levels in Get02/Wo were comparable to the wild progenitor, although the composition of acylsugar types differed, especially regarding fewer types with medium-length acyl chains. Agronomical parameters of Get02/Wo, including yield, were comparable to MT. Pest resistance assays showed enhanced protection against silverleaf whitefly (Bemisia tabaci), tobacco hornworm (Manduca sexta), and the fungus Septoria lycopersici. However, resistance levels did not reach those of the wild progenitor, suggesting the specificity of acylsugar types in the pest resistance mechanism. Our findings in trichome-mediated resistance advance the development of robust, naturally resistant tomato varieties, harnessing the potential of natural genetic variation. Moreover, by manipulating only 2 loci, we achieved exceptional results for a highly complex, polygenic trait, such as herbivory resistance in tomato.