Selective enrichment of founding reproductive microbiomes allows extensive vertical transmission in a fungus-farming termite.

Mutualistic coevolution can be mediated by vertical transmission of symbionts between host generations. Termites host complex gut bacterial communities with evolutionary histories indicative of mixed-mode transmission. Here, we document that vertical transmission of gut bacterial strains is congruent across parent to offspring colonies in four pedigrees of the fungus-farming termite Macrotermes natalensis. We show that 44% of the offspring colony microbiome, including more than 80 bacterial genera and pedigree-specific strains, are consistently inherited. We go on to demonstrate that this is achieved because colony-founding reproductives are selectively enriched with a set of non-random, environmentally sensitive and termite-specific gut microbes from their colonies of origin. These symbionts transfer to offspring colony workers with high fidelity, after which priority effects appear to influence the composition of the establishing microbiome. Termite reproductives thus secure transmission of complex communities of specific, co-evolved microbes that are critical to their offspring colonies. Extensive yet imperfect inheritance implies that the maturing colony benefits from acquiring environmental microbes to complement combinations of termite, fungus and vertically transmitted microbes; a mode of transmission that is emerging as a prevailing strategy for hosts to assemble complex adaptive microbiomes.

Indirect maternal effects via nest microbiome composition drive gut colonization in altricial chicks.

Gut microbial communities are complex and heterogeneous and play critical roles for animal hosts. Early-life disruptions to microbiome establishment can negatively impact host fitness and development. However, the consequences of such early-life disruptions remain unknown in wild birds. To help fill this gap, we investigated the effect of continuous early-life gut microbiome disruptions on the establishment and development of gut communities in wild Great tit (Parus major) and Blue tit (Cyanistes caeruleus) nestlings by applying antibiotics and probiotics. Treatment neither affected nestling growth nor their gut microbiome composition. Independent of treatment, nestling gut microbiomes of both species grouped by brood, which shared the highest numbers of bacterial taxa with both nest environment and their mother. Although fathers showed different gut communities than their nestlings and nests, they still contributed to structuring chick microbiomes. Lastly, we observed that the distance between nests increased inter-brood microbiome dissimilarity, but only in Great tits, indicating that species-specific foraging behaviour and/or microhabitat influence gut microbiomes. Overall, the strong maternal effect, driven by continuous recolonization from the nest environment and vertical transfer of microbes during feeding, appears to provide resilience towards early-life disruptions in nestling gut microbiomes.

Multiple mutations in the Nav1.4 sodium channel of New Guinean toxic birds provide autoresistance to deadly batrachotoxin.

Toxicity has evolved multiple times across the tree of life and serves important functions related to hunting, defence and parasite deterrence. Toxins are produced either in situ by the toxic organism itself or associated symbionts, or acquired through diet. The ability to exploit toxins from external sources requires adaptations that prevent toxic effects on the consumer (autoresistance). Here, we examine genomic adaptations that could facilitate autoresistance to the diet-acquired potent neurotoxic alkaloid batrachotoxin (BTX) in New Guinean toxic birds. Our work documents two new toxic bird species and shows that toxic birds carry multiple mutations in the SCN4A gene that are under positive selection. This gene encodes the most common vertebrate muscle Nav channel (Nav1.4). Molecular docking results indicate that some of the mutations that are present in the pore-forming segment of the Nav channel, where BTX binds, could reduce its binding affinity. These mutations should therefore prevent the continuous opening of the sodium channels that BTX binding elicits, thereby preventing muscle paralysis and ultimately death. Although these mutations are different from those present in Neotropical Phyllobates poison dart frogs, they occur in the same segments of the Nav1.4 channel. Consequently, in addition to uncovering a greater diversity of toxic bird species than previously known, our work provides an intriguing example of molecular-level convergent adaptations allowing frogs and birds to ingest and use the same neurotoxin. This suggests that genetically modified Nav1.4 channels represent a key adaptation to BTX tolerance and exploitation across vertebrates.

The chemical ecology of the fungus-farming termite symbiosis.

Covering: September 1972 to December 2020Explorations of complex symbioses have often elucidated a plethora of previously undescribed chemical compounds that may serve ecological functions in signalling, communication or defence. A case in point is the subfamily of termites that cultivate a fungus as their primary food source and maintain complex bacterial communities, from which a series of novel compound discoveries have been made. Here, we summarise the origins and types of 375 compounds that have been discovered from the symbiosis over the past four decades and discuss the potential for synergistic actions between compounds within the complex chemical mixtures in which they exist. We go on to highlight how vastly underexplored the diversity and geographic distribution of the symbiosis is, which leaves ample potential for natural product discovery of compounds of both ecological and medical importance.

Insights into the Metabolomic Capacity of Podaxis and Isolation of Podaxisterols A-D, Ergosterol Derivatives Carrying Nitrosyl Cyanide-Derived Modifications.

Cultures of a termite-associated and a free-living member of the fungal genus Podaxis, revived from spores maintained in century-old herbarium collections, were analyzed for their insecticidal and antimicrobial effects. Their secondary metabolomes were explored to uncover possible adaptive mechanisms of termite association, and dereplication of LC-HRMS/MS data sets led to the isolation of podaxisterols A-D (1-4), modified ergosterol derivatives that result from a Diels-Alder reaction with endogenous nitrosyl cyanide. Chemical structures were determined based on HRMS/MS and NMR analyses as well as X-ray crystallography. The putative origin of the endogenous fungal nitrosyl cyanide and ergosterol derivatives is discussed based on results obtained from stable isotope experiments and in silico analysis. Our "omics"-driven analysis of this underexplored yet worldwide distributed fungal genus builds a foundation for studies on a potential metabolic adaptations to diverse lifestyles.

Symbiont-Mediated Digestion of Plant Biomass in Fungus-Farming Insects.

Feeding on living or dead plant material is widespread in insects. Seminal work on termites and aphids has provided profound insights into the critical nutritional role that microbes play in plant-feeding insects. Some ants, beetles, and termites, among others, have evolved the ability to use microbes to gain indirect access to plant substrate through the farming of a fungus on which they feed. Recent genomic studies, including studies of insect hosts and fungal and bacterial symbionts, as well as metagenomics and proteomics, have provided important insights into plant biomass digestion across insect-fungal mutualisms. Not only do advances in understanding of the divergent and complementary functions of complex symbionts reveal the mechanism of how these herbivorous insects catabolize plant biomass, but these symbionts also represent a promising reservoir for novel carbohydrate-active enzyme discovery, which is of considerable biotechnological interest.

Chemotherapy and radiotherapy in locally advanced head and neck cancer: an individual patient data network meta-analysis.

BACKGROUND: Randomised, controlled trials and meta-analyses have shown the survival benefit of concomitant chemoradiotherapy or hyperfractionated radiotherapy in the treatment of locally advanced head and neck cancer. However, the relative efficacy of these treatments is unknown. We aimed to determine whether one treatment was superior to the other. METHODS: We did a frequentist network meta-analysis based on individual patient data of meta-analyses evaluating the role of chemotherapy (Meta-Analysis of Chemotherapy in Head and Neck Cancer [MACH-NC]) and of altered fractionation radiotherapy (Meta-Analysis of Radiotherapy in Carcinomas of Head and Neck [MARCH]). Randomised, controlled trials that enrolled patients with non-metastatic head and neck squamous cell cancer between Jan 1, 1980, and Dec 31, 2016, were included. We used a two-step random-effects approach, and the log-rank test, stratified by trial to compare treatments, with locoregional therapy as the reference. Overall survival was the primary endpoint. The global Cochran Q statistic was used to assess homogeneity and consistency and P score to rank treatments (higher scores indicate more effective therapies). FINDINGS: 115 randomised, controlled trials, which enrolled patients between Jan 1, 1980, and April 30, 2012, yielded 154 comparisons (28 978 patients with 19 253 deaths and 20 579 progression events). Treatments were grouped into 16 modalities, for which 35 types of direct comparisons were available. Median follow-up based on all trials was 6·6 years (IQR 5·0-9·4). Hyperfractionated radiotherapy with concomitant chemotherapy (HFCRT) was ranked as the best treatment for overall survival (P score 97%; hazard ratio 0·63 [95% CI 0·51-0·77] compared with locoregional therapy). The hazard ratio of HFCRT compared with locoregional therapy with concomitant chemoradiotherapy with platinum-based chemotherapy (CLRTP) was 0·82 (95% CI 0·66-1·01) for overall survival. The superiority of HFCRT was robust to sensitivity analyses. Three other modalities of treatment had a better P score, but not a significantly better HR, for overall survival than CLRTP (P score 78%): induction chemotherapy with taxane, cisplatin, and fluorouracil followed by locoregional therapy (ICTaxPF-LRT; 89%), accelerated radiotherapy with concomitant chemotherapy (82%), and ICTaxPF followed by CLRT (80%). INTERPRETATION: The results of this network meta-analysis suggest that further intensifying chemoradiotherapy, using HFCRT or ICTaxPF-CLRT, could improve outcomes over chemoradiotherapy for the treatment of locally advanced head and neck cancer. FUNDINGS: French Institut National du Cancer, French Ligue Nationale Contre le Cancer, and Fondation ARC.

Species-specific but not phylosymbiotic gut microbiomes of New Guinean passerine birds are shaped by diet and flight-associated gut modifications.

Animal hosts have evolved intricate associations with microbial symbionts, where both depend on each other for particular functions. In many cases, these associations lead to phylosymbiosis, where phylogenetically related species harbour compositionally more similar microbiomes than distantly related species. However, evidence for phylosymbiosis is either weak or lacking in gut microbiomes of flying vertebrates, particularly in birds. To shed more light on this phenomenon, we compared cloacal microbiomes of 37 tropical passerine bird species from New Guinea using 16S rRNA bacterial gene sequencing. We show a lack of phylosymbiosis and document highly variable microbiomes. Furthermore, we find that gut bacterial community compositions are species-specific and tend to be shaped by host diet but not sampling locality, potentially driven by the similarities in habitats used by individual species. We further show that flight-associated gut modifications, coupled with individual dietary differences, shape gut microbiome structure and variation, contributing to the lack of phylosymbiosis. These patterns indicate that the stability of symbiosis may depend on microbial functional diversity rather than taxonomic composition. Furthermore, the more variable and fluid host-microbe associations suggest probable disparities in the potential for coevolution between bird host species and microbial symbionts.

The Concept, Practice, Application, and Results of Locally Based Monitoring of the Environment.

Locally based monitoring is typically undertaken in areas in which communities have a close attachment to their natural resource base. We present a summary of work to develop a theoretical and practical understanding of locally based monitoring and we outline tests of this approach in research and practice over the past 20 years. Our tests show that locally based monitoring delivers credible data at local scale independent of external experts and can be used to inform local and national decision making within a short timeframe. We believe that monitoring conducted by and anchored in communities will gain in importance where scientist-led monitoring is sparse or too expensive to sustain and for ecosystem attributes in cases in which remote sensing cannot provide credible data. The spread of smartphone technology and online portals will further enhance the importance and usefulness of this discipline.

Connecting Top-Down and Bottom-Up Approaches in Environmental Observing.

Effective responses to rapid environmental change rely on observations to inform planning and decision-making. Reviewing literature from 124 programs across the globe and analyzing survey data for 30 Arctic community-based monitoring programs, we compare top-down, large-scale program driven approaches with bottom-up approaches initiated and steered at the community level. Connecting these two approaches and linking to Indigenous and local knowledge yields benefits including improved information products and enhanced observing program efficiency and sustainability. We identify core principles central to such improved links: matching observing program aims, scales, and ability to act on information; matching observing program and community priorities; fostering compatibility in observing methodology and data management; respect of Indigenous intellectual property rights and the implementation of free, prior, and informed consent; creating sufficient organizational support structures; and ensuring sustained community members' commitment. Interventions to overcome challenges in adhering to these principles are discussed.

You don't have the guts: a diverse set of fungi survive passage through Macrotermes bellicosus termite guts.

BACKGROUND: Monoculture farming poses significant disease challenges, but fungus-farming termites are able to successfully keep their monoculture crop free from contamination by other fungi. It has been hypothesised that obligate gut passage of all plant substrate used to manure the fungal symbiont is key to accomplish this. Here we refute this hypothesis in the fungus-farming termite species Macrotermes bellicosus. RESULTS: We first used ITS amplicon sequencing to show that plant substrate foraged on by termite workers harbour diverse fungal communities, which potentially could challenge the farming symbiosis. Subsequently, we cultivated fungi from dissected sections of termite guts to show that fungal diversity does not decrease during gut passage. Therefore, we investigated if healthy combs harboured these undesirable fungal genera, and whether the presence of workers affected fungal diversity within combs. Removal of workers led to a surge in fungal diversity in combs, implying that termite defences must be responsible for the near-complete absence of other fungi in functioning termite gardens. CONCLUSIONS: The rapid proliferation of some of these fungi when colonies are compromised indicates that some antagonists successfully employ a sit-and-wait strategy that allows them to remain dormant until conditions are favourable. Although this strategy requires potentially many years of waiting, it prevents these fungi from engaging in an evolutionary arms race with the termite host, which employs a series of complementary behavioural and chemical defences that may prove insurmountable.

Spatiotemporal patterns of avian host-parasite interactions in the face of biogeographical range expansions.

Exploration of interactions between hosts and parasitic symbionts is important for our understanding of the temporal and spatial distribution of organisms. For example, host colonization of new geographical regions may alter levels of infections and parasite specificity, and even allow hosts to escape from co-evolved parasites, consequently shaping spatial distributions and community structure of both host and parasite. Here we investigate the effect of host colonization of new regions and the elevational distribution of host-parasite associations between birds and their vector-transmitted haemosporidian blood parasites in two geological and geographical settings: mountains of New Guinea and the Canary Islands. Our results demonstrate that bird communities in younger regions have significantly lower levels of parasitism compared to those of older regions. Furthermore, host-parasite network analyses demonstrate that blood parasites may respond differently after arriving to a new region, through adaptations that allow for either expanding (Canary Islands) or retaining (New Guinea) their host niches. The spatial prevalence patterns along elevational gradients differed in the two regions, suggesting that region-specific biotic (e.g., host community) and abiotic factors (e.g., temperature) govern prevalence patterns. Our findings suggest that the spatiotemporal range dynamics in host-parasite systems are driven by multiple factors, but that host and parasite community compositions and colonization histories are of particular importance.

Nocardia macrotermitis sp. nov. and Nocardia aurantia sp. nov., isolated from the gut of the fungus-growing termite Macrotermes natalensis.

The taxonomic positions of two novel aerobic, Gram-stain-positive Actinobacteria, designated RB20T and RB56T, were determined using a polyphasic approach. Both were isolated from the fungus-farming termite Macrotermes natalensis. Results of 16S rRNA gene sequence analysis revealed that both strains are members of the genus Nocardia with the closest phylogenetic neighbours Nocardia miyunensis JCM12860T (98.9 %) and Nocardia nova DSM44481T (98.5 %) for RB20T and Nocardia takedensis DSM 44801T (98.3 %), Nocardia pseudobrasiliensis DSM 44290T (98.3 %) and Nocardia rayongensis JCM 19832T (98.2 %) for RB56T. Digital DNA-DNA hybridization (DDH) between RB20T and N. miyunensis JCM12860T and N. nova DSM 44481T resulted in similarity values of 33.9 and 22.0 %, respectively. DDH between RB56T and N. takedensis DSM44801T and N. pseudobrasiliensis DSM44290T showed similarity values of 20.7 and 22.3 %, respectively. In addition, wet-lab DDH between RB56T and N. rayongensis JCM19832T resulted in 10.2 % (14.5 %) similarity. Both strains showed morphological and chemotaxonomic features typical for the genus Nocardia, such as the presence of meso-diaminopimelic acid (A2pm) within the cell wall, arabinose and galactose as major sugar components within whole cell-wall hydrolysates, the presence of mycolic acids and major phospholipids (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol), and the predominant menaquinone MK-8 (H4, ω-cyclo). The main fatty acids for both strains were hexadecanoic acid (C16 : 0), 10-methyloctadecanoic acid (10-methyl C18 : 0) and cis-9-octadecenoic acid (C18 : 1 ω9c). We propose two novel species within the genus Nocardia: Nocardia macrotermitis sp. nov. with the type strain RB20T (=VKM Ac-2841T=NRRL B65541T) and Nocardia aurantia sp. nov. with the type strain RB56T (=VKM Ac-2842T=NRRL B65542T).

Streptomyces smaragdinus sp. nov., isolated from the gut of the fungus growing-termite Macrotermes natalensis.

The taxonomic position of a novel aerobic, Gram-positive actinobacteria, designated strain RB5T, was determined using a polyphasic approach. The strain, isolated from the gut of the fungus-farming termite Macrotermes natalensis, showed morphological, physiological and chemotaxonomic properties typical of the genus Streptomyces. Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbour of RB5T was Streptomyces polyrhachis DSM 42102T (98.87 %). DNA-DNA hybridization experiments between strain RB5T and S. polyrhachis DSM 42102T resulted in a value of 27.4 % (26.8 %). The cell wall of strain RB5T contained ll-diaminopimelic acid as the diagnostic amino acid. Mycolic acids and diagnostic sugars in whole-cell hydrolysates were not detected. The strain produced the following major phospholipids: diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol-mannoside and phosphatidylserine. The menaquinone profile showed hexa- and octahydrogenated menaquinones containing nine isoprene units [MK-9(H6) and MK-9(H8)]. The strain exhibited a fatty acid profile containing the following major fatty acids: 12-methyltridecanoic acid (iso-C14 : 0) 12-methyltetradecanoic acid (anteiso-C15 : 0), 13-methyltetradecanoic acid (iso-C15 : 0) and 14-methylpentadecanoic acid (iso-C16 : 0). Here, we propose a novel species of the genus Streptomyces - Streptomyces smaragdinus with the type strain RB5T (=VKM Ac-2839T=NRRL B65539T).

Asexual and sexual reproduction are two separate developmental pathways in a Termitomyces species.

Although mutualistic symbioses per definition are beneficial for interacting species, conflict may arise if partners reproduce independently. We address how this reproductive conflict is regulated in the obligate mutualistic symbiosis between fungus-growing termites and Termitomyces fungi. Even though the termites and their fungal symbiont disperse independently to establish new colonies, dispersal is correlated in time. The fungal symbiont typically forms mushrooms a few weeks after the colony has produced dispersing alates. It is thought that this timing is due to a trade-off between alate and worker production; alate production reduces resources available for worker production. As workers consume the fungus, reduced numbers of workers will allow mushrooms to 'escape' from the host colony. Here, we test a specific version of this hypothesis: the typical asexual structures found in all species of Termitomyces-nodules-are immature stages of mushrooms that are normally harvested by the termites at a primordial stage. We refute this hypothesis by showing that nodules and mushroom primordia are macro- and microscopically different structures and by showing that in the absence of workers, primordia do, and nodules do not grow out into mushrooms. It remains to be tested whether termite control of primordia formation or of primordia outgrowth mitigates the reproductive conflict.

18F-FDG Metabolic Tumor Volume: Association with Short- and Long-Term Feeding Tube Use in Head and Neck IMRT.

The purpose of this study was to investigate whether the metabolic tumor volume (MTV) of head and neck primary tumors may be a significant prognostic factor for feeding tube (FT) use and FT dependence. Seventy-nine patients with evaluable primary tumors, pre-therapy FDG-PET scans, treated with definitive intensity-modulated radiotherapy (IMRT) (± concurrent chemotherapy) for head and neck mucosal cancers were included. MTV was quantified and recorded for the primary lesion using a minimum standardized uptake value (SUV) threshold of 2.0. Patients were recommended prophylactic FT and followed up by a dietician for at least eight weeks of post-radiotherapy. Associations between MTV, dose to swallowing organs at risk, FT use, and FT dependence were analyzed. MTV was positively correlated with gross tumor volume (GTV) (r = 0.7357; p < 0.0001). MTVs larger than 17 cc were associated with higher rates of FT use (87.8% vs. 69.5%, p = 0.0067) and FT dependence at six weeks (76.7% vs. 41.7%, p = 0.0024) and six months (25.0% vs. 8.7%, p = 0.0088). Increasing MTV was associated with increasing mean dose to the oral cavity (p = < 0.0001), tongue base (p = 0.0009), and superior (SPCM) (p = 0.0001) and middle pharyngeal constrictor muscles (MPCM) (p = 0.0005). Increasing MTV was associated with increasing maximum dose to oral cavity (p = 0.0028), tongue base (p = 0.0056), SPCM (p = 0.0037), and MPCM (p = 0.0085). Pre-treatment MTV is a reproducible parameter that can be generated at or prior to a pre-treatment Multidisciplinary Tumor Board and may expedite decisions regarding placement of prophylactic FTs. Prospective evaluation in larger series is required to determine whether MTV is a more useful prognostic variable for FT use than clinical T-classification.

Enzyme Activities at Different Stages of Plant Biomass Decomposition in Three Species of Fungus-Growing Termites.

Fungus-growing termites rely on mutualistic fungi of the genus Termitomyces and gut microbes for plant biomass degradation. Due to a certain degree of symbiont complementarity, this tripartite symbiosis has evolved as a complex bioreactor, enabling decomposition of nearly any plant polymer, likely contributing to the success of the termites as one of the main plant decomposers in the Old World. In this study, we evaluated which plant polymers are decomposed and which enzymes are active during the decomposition process in two major genera of fungus-growing termites. We found a diversity of active enzymes at different stages of decomposition and a consistent decrease in plant components during the decomposition process. Furthermore, our findings are consistent with the hypothesis that termites transport enzymes from the older mature parts of the fungus comb through young worker guts to freshly inoculated plant substrate. However, preliminary fungal RNA sequencing (RNA-seq) analyses suggest that this likely transport is supplemented with enzymes produced in situ Our findings support that the maintenance of an external fungus comb, inoculated with an optimal mixture of plant material, fungal spores, and enzymes, is likely the key to the extraordinarily efficient plant decomposition in fungus-growing termites.IMPORTANCE Fungus-growing termites have a substantial ecological footprint in the Old World (sub)tropics due to their ability to decompose dead plant material. Through the establishment of an elaborate plant biomass inoculation strategy and through fungal and bacterial enzyme contributions, this farming symbiosis has become an efficient and versatile aerobic bioreactor for plant substrate conversion. Since little is known about what enzymes are expressed and where they are active at different stages of the decomposition process, we used enzyme assays, transcriptomics, and plant content measurements to shed light on how this decomposition of plant substrate is so effectively accomplished.

Expanding the Rubterolone Family: Intrinsic Reactivity and Directed Diversification of PKS-derived Pyrans.

We characterized two key biosynthetic intermediates of the intriguing rubterolone family (tropolone alkaloids) that contain a highly reactive pyran moiety (in equilibrium with the hydrolyzed 1,5-dione form) and undergo spontaneous pyridine formation in the presence of primary amines. We exploited the intrinsic reactivity of the pyran moiety and isolated several new rubterolone derivatives, two of which contain a unique thiazolidine moiety. Three rubterolone derivatives were chemically modified with fluorescence and biotin tags using peptide coupling and click reaction. Overall, eight derivatives were fully characterized by HRMS/MS and 1D and 2D NMR spectroscopy and their antimicrobial, cytotoxic, anti-inflammatory and antiparasitic activities evaluated.

Nursing Music Protocol and Postoperative Pain.

Pain has always been a major concern for patients and nurses during the postoperative period. Therapies, medicines, and protocols have been developed to improve pain and anxiety but have undesirable risks to the patient. Complementary and alternative medicine therapies have been studied but have not been applied as regular protocols in the hospital setting. Music is one type of complementary and alternative medicine therapy that has been reported to have favorable results on reducing postoperative pain, anxiety, and opioid usage. However, music lacks a protocol that nurses can implement during the perioperative process. This paper is an in-depth literature review assessing a best practice recommendation and protocol that establishes a consensus in the use of music therapy. The results suggest that music therapy may consist of calming, soft tones of 60-80 beats per minute for at least 15-30 minutes at least twice daily during the pre- and postoperative periods. It is suggested that music only be used in conjunction with standards of care and not as the primary intervention of pain or anxiety. This evidence suggests that proper use of music therapy can significantly reduce surgical pain. Implementing these protocols and allowing the freedom of nursing staff to use them may lead to greater reductions in surgical pain and anxiety and a reduction in opioid use.

Role of radiotherapy fractionation in head and neck cancers (MARCH): an updated meta-analysis.

BACKGROUND: The Meta-Analysis of Radiotherapy in squamous cell Carcinomas of Head and neck (MARCH) showed that altered fractionation radiotherapy is associated with improved overall and progression-free survival compared with conventional radiotherapy, with hyperfractionated radiotherapy showing the greatest benefit. This update aims to confirm and explain the superiority of hyperfractionated radiotherapy over other altered fractionation radiotherapy regimens and to assess the benefit of altered fractionation within the context of concomitant chemotherapy with the inclusion of new trials. METHODS: For this updated meta-analysis, we searched bibliography databases, trials registries, and meeting proceedings for published or unpublished randomised trials done between Jan 1, 2009, and July 15, 2015, comparing primary or postoperative conventional fractionation radiotherapy versus altered fractionation radiotherapy (comparison 1) or conventional fractionation radiotherapy plus concomitant chemotherapy versus altered fractionation radiotherapy alone (comparison 2). Eligible trials had to start randomisation on or after Jan 1, 1970, and completed accrual before Dec 31, 2010; had to have been randomised in a way that precluded prior knowledge of treatment assignment; and had to include patients with non-metastatic squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx, or larynx undergoing first-line curative treatment. Trials including a non-conventional radiotherapy control group, investigating hypofractionated radiotherapy, or including mostly nasopharyngeal carcinomas were excluded. Trials were grouped in three types of altered fractionation: hyperfractionated, moderately accelerated, and very accelerated. Individual patient data were collected and combined with a fixed-effects model based on the intention-to-treat principle. The primary endpoint was overall survival. FINDINGS: Comparison 1 (conventional fractionation radiotherapy vs altered fractionation radiotherapy) included 33 trials and 11 423 patients. Altered fractionation radiotherapy was associated with a significant benefit on overall survival (hazard ratio [HR] 0·94, 95% CI 0·90-0·98; p=0·0033), with an absolute difference at 5 years of 3·1% (95% CI 1·3-4·9) and at 10 years of 1·2% (-0·8 to 3·2). We found a significant interaction (p=0·051) between type of fractionation and treatment effect, the overall survival benefit being restricted to the hyperfractionated group (HR 0·83, 0·74-0·92), with absolute differences at 5 years of 8·1% (3·4 to 12·8) and at 10 years of 3·9% (-0·6 to 8·4). Comparison 2 (conventional fractionation radiotherapy plus concomitant chemotherapy versus altered fractionation radiotherapy alone) included five trials and 986 patients. Overall survival was significantly worse with altered fractionation radiotherapy compared with concomitant chemoradiotherapy (HR 1·22, 1·05-1·42; p=0·0098), with absolute differences at 5 years of -5·8% (-11·9 to 0·3) and at 10 years of -5·1% (-13·0 to 2·8). INTERPRETATION: This update confirms, with more patients and a longer follow-up than the first version of MARCH, that hyperfractionated radiotherapy is, along with concomitant chemoradiotherapy, a standard of care for the treatment of locally advanced head and neck squamous cell cancers. The comparison between hyperfractionated radiotherapy and concomitant chemoradiotherapy remains to be specifically tested. FUNDING: Institut National du Cancer; and Ligue Nationale Contre le Cancer.