Superfast muscles (SFMs) are extremely fast synchronous muscles capable of contraction rates up to 250 Hz, enabling precise motor execution at the millisecond time scale. SFM phenotypes have been discovered in most major vertebrate lineages, but it remains unknown whether all SFMs share excitation-contraction coupling pathway adaptations for speed, and if SFMs arose once, or from independent evolutionary events. Here, we demonstrate that to achieve rapid actomyosin crossbridge kinetics bat and songbird SFM express myosin heavy chain genes that are evolutionarily and ontologically distinct. Furthermore, we show that all known SFMs share multiple functional adaptations that minimize excitation-contraction coupling transduction times. Our results suggest that SFM evolved independently in sound-producing organs in ray-finned fish, birds, and mammals, and that SFM phenotypes operate at a maximum operational speed set by fundamental constraints in synchronous muscle. Consequentially, these constraints set a fundamental limit to the maximum speed of fine motor control. Across animals, different muscle types have evolved to perform vastly different tasks at different speeds. For example, tortoise leg muscles move slowly over several seconds, while the flight muscles of a hummingbird move quickly dozens of times per second. The speed record holders among vertebrates are the so-called superfast muscles, which can move up to 250 times per second. Superfast muscles power the alarming rattle of rattlesnakes, courtship calls in fish, rapid echolocation calls in bats and the elaborate vocal gymnastics of songbirds. Thus these extreme muscles are all around us and are always involved in sound production.Did superfast muscles evolve from a common ancestor? And how do different superfast muscles achieve their extreme behavior? To answer these questions, Mead et al. studied the systems known to limit contraction speed in all currently known superfast muscles found in rattlesnakes, toadfish, bats and songbirds. This revealed that all the muscles share certain specific adaptations that allow superfast contractions. Furthermore, the three fastest examples – toadfish, songbird and bat – have nearly identical maximum speeds. Although this appears to support the idea that the adaptations all evolved from a shared ancestor, Mead et al. found evidence that suggests otherwise. Each of the three superfast muscles are powered by a different motor protein, which argues strongly in favor of the muscles evolving independently. The existence of such similar mechanisms and performance in independently evolved muscles raises the possibility that the fastest contraction rates measured by Mead et al. represent a maximum speed limit for all vertebrate muscles.Any technical failure in a racecar most likely will slow it down, while the same failure in a robustly engineered family car may not be so noticeable. Similarly in superfast muscle many cellular and molecular systems need to perform maximally. Therefore by understanding how these extreme muscles work, we also gain a better understanding of how normal muscles contract. Superfast muscles (SFMs) are the fastest known synchronous muscle phenotypes in vertebrates . Their a2+) stored in the sarcoplasmic reticulum (SR). Subsequent binding of Ca2+ to troponin in sarcomeres triggers the exposure of binding sites for the motor protein myosin along actin filaments, allowing the cyclical binding and unbinding of myosin motor head domains to actin, forming actomyosin crossbridges that generate force. Finally, force decreases when SR Ca2+-ATPases (SERCA) pump Ca2+ back into the SR, consecutively lowering the cytoplasmic free Ca2+ concentration ([Ca2+]i) and returning thin filament inhibition thus preventing further crossbridge formation. Extensive study of SFMs in the oyster toadfish (Opsanus tao) swimbladder has demonstrated that no single step in the ECC pathway is rate-limiting, but that multiple hallmark traits have adapted to allow superfast cycling rates. In swimbladder SFMs, a far greater proportion of cellular volume is dedicated to SR compared to locomotory muscles, which increases the number and density of SERCA pumps  pathway that consists of several sequential steps whose individual kinetics affect the maximally attainable force modulation speed. In brief, firing of a motor neuron first triggers the release of intracellular calcium ions (CaCA pumps . FurtherCA pumps  greatly y muscle . Additioy muscle . WhetherMYH) genes each with unique kinetic properties  determine whether SFMs share a common evolutionary origin and to (ii) improve our understanding of muscle function, because sequenced genomes   . BecauseMYH gene expression in the SFM of zebra finch (Taeniopygia guttata) syrinx and Daubenton’s bat (Myotis daubentonii) larynx. The employment of evolutionarily and ontologically distinct MYH genes suggests separate evolutionary origins of SFM. Furthermore, by quantifying cellular morphometry and [Ca2+]i signal transduction in all known SFM, we show that all SFMs share hallmark adaptations in the ECC pathway and have identical intracellular calcium dynamics at their operating temperatures in vivo. Our data suggest that SFMs converge at a maximum speed allowed by fundamental constraints in vertebrate synchronous muscle architecture. This implies that motor control of complex acoustic communication is fundamentally limited by synchronous vertebral muscle architecture.Here, we test if SFMs share a common evolutionary origin by characterizing Myotis lucifigus), an echolocating bat in the same genus as Daubenton’s bat, to identify the dominant MYH genes expressed in their SFMs. Among known MYH genes, orthologs of human MYH13 are prime candidates for superfast motor performance  are linked head-to-tail in what is known as the fast/developmental cluster   (Python molurus)  (Takifugu rubripes)  that does not appear to have descended from the ancestral fast/developmental locus (MYH3 (LOC103063479) at the expected 5’ end of the cluster, but the presence of a 3’ flanking MYH13 ortholog is unknown due to the presence of a scaffold end.To test the hypothesis that SFMs in the bat larynx and songbird syrinx share a common evolutionary origin , we usedformance . In huma cluster , which a cluster . Here, w genomes . To placampyrus) , chicken gallus) , Burmesemolurus) , clawed picalis) , and torubripes) . Phyloge studies . We also studies , though  studies . Some ra cluster , supportal locus . The pytMYH13 orthologs are expressed in bat laryngeal and zebra finch syringeal SFMs, we first used a panel of MYH13-specific commercial and custom-made antibodies . Surprisingly, we did not find immunoreactivity against MYH13 in bat laryngeal SFM  was expressed and we could not distinguish whether this myosin was expressed in different cells, as suggested by observation of fast myosin antibody staining in starlings , and also four additional MYH genes from the fast/developmental cluster  and was significantly upregulated progressively  in juvenile male syringeal SFM . This technique yields comparable values for SFMs in zebra finch, toadfish and bat, which were significantly elevated  5–10 times compared to intraspecific skeletal muscle and also compared to the comparable rat fast twitch m. extensor digitorum longus , while Daubenton’s bat larynx SFMs express an ortholog of the mammalian locomotory MYH4 (MyHC-2b). Our analysis is consistent with earlier findings  adaptations that affect the function of ryanodine receptors or the SERCA pumps  the employment of calcium-sequestering proteins such as parvalbumin  fasvalbumin  that havvalbumin . The potvalbumin , 2011. Tkinetics , but if on state , also waBoth mechanistic as well as functional characteristics are convergent in SFMs. Previous observations show that SFMs from diverse taxa have remarkably similar maximum performance characteristics (force and cycling frequency) , echolocWhat mechanisms set the maximum speed to power motion in vertebrates? In toadfish swimbladder SFMs, a trade-off between force-generating capacity and speed is well-established and results from two constraints related to (i) crossbridge dynamics and (ii) increased ECC pathway requirements . The conInterestingly, only arthropods have pushed the envelope of force/speed tradeoffs further than vertebrate SFMs. In insect asynchronous flight muscle force cycling is uncoupled from calcium cycling, minimizing force loss and allowing for high power production at similar cycling speeds to SFM . HoweverMYH13 ortholog expression in avian syrinx SFM corroborates earlier developmental studies  and allowances , particular to motor systems associated with sound production. Motor-driven acoustic modulation rates in complex communication are thus fundamentally limited by muscle architecture.All procedures were carried out in accordance with the Danish Animal Experiments Inspectorate .Songbird: All bird data presented were collected in zebra finches . All adult birds (older than 100 days) were kept in group aviaries at the University of Southern Denmark, Odense, Denmark, and juvenile zebra finches used for muscle kinetics measurements were raised in separate cages together with both parents and siblings at 13 hr light:11 hr dark photoperiod and given water and food ad libitum. The juvenile male zebra finches used for MYH mRNA quantification came from the long-term breeding colony at the Freie Universität Berlin, Germany. These animals were kept on a 14 hr light:10 hr dark photoperiod and given water and food ad libitum. We dissected the syringeal muscle tracheobronchialis ventralis (VTB) on ice immediately after isoflurane euthanasia , flight  and leg  muscles were collected as reference tissues.thanasia . ExtraocToadfish: Adult male oyster toadfish  were obtained from the Marine Biological Laboratories  and housed individually in 80 × 40×40 cm seawater tanks on constant flow-through of fresh seawater at the Fjord and Bælt field station, Kerteminde, Denmark. Toadfish were euthanized by a blow on the head and double pithing. Superfast swimbladder muscle was isolated bilaterally from the midsection of the swimbladder.Bats: Efforts were focused on Daubenton’s bat (Myotis daubentoni) where superfast behavior of the anterior portion of the laryngeal muscle m. cricothyroideus anterior (ACTM) was previously established (Myotis daubentoni from the Skov- og Naturstyrelsen Inspectorate (Denmark). After isoflurane euthanasia, the ACTM muscle was isolated and extraocular and leg  muscles were collected as reference tissues.ablished . We obtaRats: Adult male Sprague Dawley rats (Rattus norvegicus) were purchased from the Institute of Biomedicine, Odense University Hospital. The rats were housed in cages with a 12 hr:12 hr light:dark cycle and provided unrestricted access to water and food. The rats were killed by a blow on the head followed by cervical dislocation. M. soleus (SOL) and m. extensor digitorum longus (EDL) were collected.Myotis lucifigus and Taeniopygia guttata by BLASTP using ENSEMBL genome resources  with the rod domain of human MYH3 (conserved motor domain/rod junction proline 839 to C terminus) as query sequence. Seven Ensembl genebuild annotated predicted proteins were clustered on forward strand of the zebra finch chromosome 18. Six predicted proteins in bat were similarly clustered on the reverse strand of scaffold GL430049  was used to partially reconstruct the full-length gene, including loop 1 and loop two subdomains, from flanking genomic sequence  as query sequence. Torafugu sequences were obtained directly from . Human MYH sequences were obtained from the NCBI protein database . To remolustal W  in MacveMYH genes from zebra finch, little brown bat, chicken, large flying fox, Burmese python, clawed frog, torafugu and human genomes (MYH3). Here full-length rods were not aligned due to a region of unknown sequence in bat MYH13. Maximum likelihood trees were generated and branch lengths calculated in Macvector using the Neigbor Joining method  from human MYH13, little brown bat and flying fox MYH4; chicken and zebra finch MYH13. The three-dimensional homology model in SCR_013032) server  server  as in (B) server .AB_784724), M4276 (RRID:AB_477190), Sigma-Aldrich, MO, USA), diluted 1:100; a rabbit polyclonal myosin 13/Extraocular myosin (Hinge region) (MP4571 (RRID:AB_2713977), ECM Biosciences, KY, USA), diluted 1:50; and a rat monoclonal anti-Laminin 3 alpha antibody , Abcam, MA, USA), diluted 1:500 in PBS with BSA for 60 min at 37°C. The skeletal fast antibody MY-32 binds to MYH1, 2 and 4. To our knowledge no existing antibody can reliably distinguish between these myosins. After incubation with antiserum and PBS wash, a new incubation in 10% normal goat serum followed, after which the sections were incubated in goat anti-mouse IgG1, Human ads-TRITC , donkey anti-rabbit IgG-TRITC  and goat anti-Rat IgG Alexa 488  diluted 1:300 for 30 min at 37°C. The sections were thereafter washed in PBS and then mounted in Vectashield Mounting Medium (H-1500) with DAPI . Images were taken using Leica DM6000 at University of Pennsylvania Microscopy Core Facility.Muscle tissue was rapidly dissected and frozen in liquid nitrogen after sacrifice. Immunofluorescence staining was carried out on 5 µm thick frozen sections. After initial washing with PBS three times for 5 min, sections were incubated for 20 min in a 1% solution of Triton X-100  in 0.01 M PBS  and rinsed in PBS three times. The sections were then incubated in 10% normal goat serum  for 15 min, and were thereafter incubated with a mouse monoclonal anti-myosin  antibody , and tested one antibody used successfully in rabbits . All theMuscle tissues were rapidly dissected and frozen in liquid nitrogen after sacrifice. Tissues were homogenized in Trizol using an Ultra-turrax homogenizer and total RNA was extracted according to manufacturer´s instructions (Invitrogen). Reverse transcription was performed by incubating 1 µg of total RNA with 0.25 µg of random hexamers (Amersham Pharmacia Biotech) and 0.9 mM dNTPs at 65°C for 5 min, followed by incubation with 1x First Strand Buffer (Invitrogen), 10 mM DTT and 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies) at 37°C for 1 hr. For more details see . Quantit4) and 1.5% potassium ferrocyanide (K4Fe(CN)6) in 0.1 M sodium cacodylate buffer for 90 min at 4°C. After post-fixation, muscles were rinsed twice in 0.1 M sodium cacodylate buffer at 4°C, dehydrated through a graded series of alcohol at 4–20oC, infiltrated with graded mixtures of propylene oxide and Epon at 20°C, and embedded in 100% Epon at 30°C. Ultra-thin (60 nm) sections were cut  at three depths (separated by 150 µm) and contrasted with uranyl acetate and lead citrate. Sections were examined and photographed at 40,000x magnification in a pre-calibrated transmission electron microscope  and a Quemesa camera ). We quantified volumetric percentage estimates (VPE) of sarcoplasmatic reticulum (VPESR), mitochondria (VPEmt) and myofibrils (VPEmf). The VPESR and VPEmito and VPEmyo were determined by point counting using grid sizes of 135, 135 and 300 nm, respectively . For zebra finch TA all the estimates were based on 61 images obtained from 5 fibres in 2 animals. For bat CT all the estimates were based on 80 images obtained from 7 fibres in 2 animals, and for bat TA all the estimates were based on 48 images from 4 fibres in 2 animals. The estimated coefficient of error for ratio estimators were 0.05, 0.03 and 0.08 for the estimates of SR, myofibrils and mitochondria, respectively. The rest volume consisted of myoplasm, lipids, nuclei and glycogen particles. SR per myofibril ratio was defined as Crotalus atrox tailshaker and dorsal mid-body muscle  for 24 hr and subsequently rinsed four times in 0.1 M sodium cacodylate buffer. Following rinsing, muscles were post-fixed with 1% osmium tetroxide . We performed these measurements in zebra finch syringeal VTB muscle (N = 5) and toadfish swimbladder muscle (N = 2). The bat laryngeal CT muscle’s force data presented in Force development curves of zebra finch syringeal VTB muscle were measured at 50 (n = 4), 75 (n = 4) and 100 DPH (n = 4) at 39°C at optimal stimulus amplitude and muscle length as described previously , 2011. Rin vitro at relevant physiological temperatures, we could not optimize the real-time calcium imaging technique for bats and used an alternative technique to measure free calcium uptake and release by isolated SR vesicles in muscle homogenate . The number of technical and biological replicates are listed above with each essay. No outliers or data were excluded.No sample sizes were computed before the experiments. A technical replicate is a replicate of the measurement on the same preparation, and a biological replicate is an individual. Data are presented as mean values ± 1 SE.  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor. One of the reviewers, Lawrence Rome, has agreed to share his identity.Thank you for submitting your article "Fundamental Constraints In Synchronous Muscle Limit Superfast Motor Control In Vertebrates" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Superfast muscles (SFMs), the fastest known synchronous muscle of vertebrates, are known in fish, reptiles, birds and mammals where they are mainly linked to sound production. Early studies of SFMs that vibrate the toadfish swimbladder and rattlesnake tailshaker showed how the excitation-contraction coupling pathway underlying force generation in skeletal muscle allows an especially high rate of contraction and rapid modulation of sound pulses; the tradeoff being low force generation. These SFMs also exhibit a hypertrophy of the sarcoplasmic reticulum that allows for a more rapid recycling of calcium ions essential to the formation of actomyosin bridges that generate force, and thin myofibrils that reduce the diffusion distance for calcium. This new study characterizes the molecular basis for SFM physiological capacity and its evolutionary history by combining comparative genomic and quantitative (q) PCR analyses with morphometric and calcium dynamic analyses. To complement the prior studies of toadfish and rattlesnakes, this study includes the sound-producing SFMs of avian and mammalian species – the of zebra finch (ZF) syrinx and Daubenton's bat larynx. The results represent a novel contribution to our understanding of the functional architecture and evolutionary history of SFMs that are a prime example of innovations in skeletal muscle adapted to specific behavioral tasks.Essential revisions:1) It is sometimes unclear for the ZF studies if the dorsal or ventral tracheobronchialis muscle  is being studied: The text describing 2) Discussion, first paragraph: The hypoglossal nerve is considered to be homologous to occipital nerve roots in fish  so they are not as distinct as one might be led to think here – recent molecular evidence supports the hypothesis that sonic motor neurons in fish and birds arose from a common progenitor domain distinct from the one that gave rise to laryngeal motor neurons . This does not mean that the SFMS in birds and fish do not depend upon distinct MYH genes for their function and "descend from different evolutionary events" but the authors might want to be more circumspect in saying that occipital and hypoglossal nerves are "different cranial nerves".3) The last paragraph of the Discussion should include a comment on the genomic analysis that supports independent origins for the MYH in each group studied.4) There are too many abbreviations.5) The authors should justify their selection of a 100Hz cycle frequency as the boundary for super-fast muscles. 1) It is sometimes unclear for the ZF studies if the dorsal or ventral tracheobronchialis muscle  is being studied: The text describing We apologize for these errors and thank the reviewers for catching them. In our experiments we measured MYH content using qPCR separately for all syringeal muscles in adult males and females. In this manuscript, we decided to only include data on the VTB, for which superfast muscle performance has been established earlier. Unfortunately, we erroneously presented the DTB qPCR data in tracheobronchialis ventralis (VTB) on ice immediately after isoflurane euthanasia .”“We dissected the syringeal muscle Consequently we replaced Furthermore, in 2) Discussion, first paragraph: The hypoglossal nerve is considered to be homologous to occipital nerve roots in fish  so they are not as distinct as one might be led to think here – recent molecular evidence supports the hypothesis that sonic motor neurons in fish and birds arose from a common progenitor domain distinct from the one that gave rise to laryngeal motor neurons . This does not mean that the SFMS in birds and fish do not depend upon distinct MYH genes for their function and "descend from different evolutionary events" but the authors might want to be more circumspect in saying that occipital and hypoglossal nerves are "different cranial nerves".We thank the reviewers for pointing out this very interesting paper. As this new evidence renders our previous statement incorrect, we decided to omit the sentence. We tried adding a brief statement regarding the origin and physiological properties of the vocal/sonic motorneurons to our Discussion both at the end of the first paragraph and also at the last paragraph before the concluding paragraph. However, in both cases this introduced such a significant break of the reading flow and detracted from the main point of the paragraph that we decided to not do so. In the revised manuscript, the first Discussion paragraph ends as follows:MYH gene expression in vertebrates , the parsimonious conclusion is that the SFM phenotype found in the sound producing organs of bats, songbirds, and toadfish descend from different evolutionary events.”“Since extremely fast crossbridge kinetics are necessary, though not sufficient, for superfast performance , and these are set largely by 3) The last paragraph of the Discussion should include a comment on the genomic analysis that supports independent origins for the MYH in each group studied.We have rephrased the concluding paragraph into:“To conclude, the non-orthology of the dominant myosin heavy chains expressed in SFMs of birds, mammals, and fish indicates that SFM likely evolved independently in each lineage. […] Motor-driven acoustic modulation rates in complex communication are thus fundamentally limited by muscle architecture.”4) There are too many abbreviations.2+, SERCA -this abbreviation would otherwise be replaced by SR Ca2+-ATPase which is not helping readability). In the Discussion we kept essential gene and protein names .We acknowledge that we used many abbreviations and in our revision aimed to reduce the number of abbreviations as much as possible, especially in the abstract and main text. As such, we omitted six abbreviations in the main text , but kept the five abbreviations relevant to this paper , and some that are widely used  The authors should justify their selection of a 100Hz cycle frequency as the boundary for super-fast muscles.In our revised manuscript we use the definition by Rome (2006) where SFM range from approximately 90 to 250 Hz.Introduction: “Their ability to repetitively contract and relax fast enough to produce work at cycling rates over around 90 Hz and up to 250 Hz sets them apart from other muscles by almost two orders of magnitude and allows the execution of central motor commands with millisecond temporal precision (Rome, 2006; Rome et al., 1988; Elemans et al., 2004; Elemans et al., 2008; Elemans et al., 2011.”


ADAM17 /TACE (TNFα converting enzyme) has emerged as a potential therapeutic target in colorectal cancer (CRC) and other cancers, due in part to its role in regulating various tumor cell surface proteins and growth factors and cytokines in the tumor microenvironment. The emergence of MEDI3622, a highly potent and specific antibody-based ADAM17 inhibitor, has allowed testing of the concept that targeting ADAM17 may be an important new therapeutic approach for CRC patients. We demonstrate that MEDI3622 is highly efficacious on tumor growth in multiple human CRC PDX models, resulting in improved survival of animals bearing tumor xenografts. MEDI3622 was further found to impact Notch pathway activity and tumor-initiating cells. The promising preclinical activity seen here supports further clinical investigation of this treatment approach to improve therapeutic outcome for patients diagnosed with metastatic CRC, including patients with KRAS-mutant tumors for whom other therapeutic options are currently limited. Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States and a major and growing health problem around the world . SurgicaMT) and KRAS-wild type (KRASWT) models. We report here that MEDI3622 exhibits significant single agent activity in multiple CRC models, including a KRASMT CRC model, and acts, in part, by targeting stem cell frequency in the tumors.A disintegrin and metalloprotease domain-containing protein 17 (ADAM17), also known as TNF-alpha converting enzyme (TACE) has emerged as a potential therapeutic target in CRC and other cancers. ADAM17 has potential roles in regulating various cell surface proteins on the cancer cell surface as well as growth factors and cytokines in the tumor microenvironment. Most notably, the EGFR ligands, transforming growth factor-α (TGF-α) and amphiregulin are frequently observed to be upregulated in colorectal cancer (CRC) –7. ADAM1in vivo testing of MEDI3622 was carried out in five human CRC PDX models, selected for two features: their genomic heterogeneity  showed similar pEGFR expression levels to the responsive CRC 13-1333 model  function as measured by effects on tumorigenicity. Three models were chosen for study: COLO 205, which we previously showed was responsive to MEDI3622 , CRC 13-Ex vivo evaluation of bioluminescence was carried out on organs excised from mice that were euthanized, when the mice began to exhibit signs of the effects of their tumor burden , generally occurring at five weeks following implantation of the primary tumor  were orthotopically implanted into the cecum, and bioluminescence was assessed over time to monitor relative tumor burden at the primary site and distant organs. A time-dependent increase in bioluminescent signal was observed non-invasively Figure . Ex vivor Figure . At thatr Figure .ex vivo BLI after the first and second cycle of treatment. Ex vivo evaluation of MEDI3622-treated mice showed a significant reduction in bioluminescence in the liver but not in the primary tumor . The molecular mechanisms regulating presumptive CSC phenotypes in CRC have not been fully elucidated. However, studies from Ellis and his colleagues have established a role for ADAM17 in promoting the CSC phenotype and chemoresistance of CRC cells . The indThe ultimate therapeutic promise of any new experimental agent being developed for the treatment of metastatic CRC will depend on its ability to impair progression of previously disseminated disease at the time of diagnosis. We report here the development of an imaging-enabled model of CRC spontaneous metastasis that allowed us to evaluate efficacy of MEDI3622 on metastatic CRC lesions arising in the liver after orthotopic implantation in the cecum. Treatment was not initiated until metastasis had been confirmed. It is encouraging that treatment with MEDI3622 conferred a 9-day increase in survival in mice implanted with UM-CRM 12-1159 tumors, an aggressive PDX model that resulted in death of all of the control mice within 20 days of surgery. Since secondary surgeries to remove the orthotopic tumors are problematic, the increase in survival arising as a consequence of MEDI3622 treatment could be a function of the activity of this agent not only on metastatic lesions present at the time treatment was initiated, but also on the formation of new metastases. A prometastatic role for Notch signaling in CRC was recently reported, revealing a previously unknown connection between NOTCH and RHO signaling to regulate CRC invasion and metastasis . GenomicIn summary, we have demonstrated that targeting of ADAM17 with the antibody MEDI3622 significantly impairs tumor growth in multiple CRC PDX models. CRC CSC function was inhibited by MEDI3622 treatment, as reflected by reduction in tumor initiating cell frequency in tumor re-implantation studies at the conclusion of treatment. MEDI3622 was further found to inhibit growth of metastatic lesions arising in the liver after orthotopic implantation leading to increased survival. The promising preclinical activity seen here supports further evaluation of this treatment approach to improve therapeutic outcome for patients diagnosed with metastatic colorectal cancer.50 in biochemical assays of 0.46 nmol/L against ADAM17. Cell-based shedding assays were then carried out to optimize cellular potency leading to the generation of MEDI3622.MEDI3622 evolved from screening of a Dyax phage display library for Fabs capable of inhibiting recombinant ADAM17 as previously described . One of Prkdcscid Il2rgtm1Wjl/SzJ) using an 11G Trocar needle. Tumor growth was monitored. Fragments were subsequently passaged into 6–8 week old NSG mice. PDX models were maintained in nude mice for a maximum of four passages before fresh material from the freezer was used to regenerate the line.For establishing models designated either CRC or CRM, tumor and matched normal specimens were obtained from patients undergoing liver metastectomies or colon resections of primary disease at the University of Michigan University Hospital . All patients provided informed written consent and samples were procured with approval of the University of Michigan Institutional Review Board (HUM00065489). Specimens were obtained within four hours of surgery and immediately transferred to DMEM/F12 media supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin-Glutamine at 4°C. A portion of normal colon specimens were fixed in 10% neutral buffered formalin (NBF) and the remainder snap frozen in liquid nitrogen. Portions of tumor specimens were either fixed in 10% NBF, snap frozen in liquid nitrogen, or divided into fragments approximately 3 × 3 mm for subcutaneous implantation into female 6–7 week old CIEA NOG mice (NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac from Taconic) using an 11G Trocar needle. Tumor-implanted mice were monitored for tumor growth for up to four months following implantation. Xenografted tumors from the NOG mice were passaged into female 6–7 week old NCR nude mice (CrTac:NCr-Foxn1nu from Taconic) for model expansion. For the models designated GB-CO, colorectal tumor samples were purchased from Asterand Biosciences and in accordance with MedImmune policies and procedures. Tumor fragments were implanted into 6–8 week old NSG mice (NOD.Cg-Foxn1nu from Taconic) were implanted subcutaneously with low passage PDX tumor fragments (∼30 mg) into the region of the right axilla. Mice were randomized into treatment groups and treatments initiated when tumors reached 100 to 200 mg. Human IgG1 control or MEDI3622 antibody was administered twice weekly via intraperitoneal injection as a solution in phosphate-buffered saline (0.2 ml/20g body weight). Tumor volume and body weights were measured two or three times a week. Tumor volumes were calculated by measuring two perpendicular diameters with calipers and using the formula: tumor volume = (length × width2)/2. When mean tumor burden in the IgG control group reached ∼1000 mg, mice were euthanized 72 hours following the last treatment. Following euthanasia, whole blood was collected via cardiac puncture into serum separator tubes. Whole blood was kept at room temperature for 30 minutes, centrifuged, and the serum collected and stored at -80°C until use. Tumors were harvested from mice at the time of euthanasia and divided into three pieces. The first piece was fixed in 10% neutral buffered formalin for subsequent immunohistochemical analysis. The second piece was snap frozen in liquid nitrogen and stored at –80°C until use. The third piece was minced, slowly frozen in 90% FBS/10% DMSO, and stored at −80°C until use. Percent treated/control (%T/C) was calculated by dividing the median treated tumor weight by the median control tumor weight and multiplying by 100 on the last day of treatment. All procedures related to the handling, care, and treatment of animals was conducted in accordance with University of Michigan’s Committee on the Use and Care of Animals guidelines.Female 6–7 week old NCR nude mice , into the MCS of the bicistronic pLVX-EF1α-IRES-mCherry lentiviral expression vector (Clontech). The resulting construct expresses firefly luciferase and mCherry as separate proteins due to the internal ribosome entry site (IRES), with very tight (> 99%) correlation between mCherry and luciferase expression as assessed by colony formation assays (data not shown). Lentiviruses were prepared from 293T cells by calcium chloride transfection with plasmids Delta and VSV  and used to tranduce dissagregated primary xenografts, which were re-implanted immediately. FACS sorting of resulting bulk tumors was carried out to purify the mCherry-positive population prior to subcutaneous re-implantation. Resulting tumors were dissociated and the luciferase- and mCherry-tagged tumorsphere line was propagated ® absorbable gelatin sponge (Pfizer Injectables) so that each Gelfoam® piece contained a final concentration of 5 × 105 cells. Laparotomies were performed to exteriorize the cecum and ascending colon. Using magnification and microsurgery techniques, the serosa of CIEA NOG mice were disrupted and a piece of the Gelfoam® was positioned and sutured to the submucosal layer of the cecum. The bowel was then returned to the peritoneal cavity and the abdomen closed with sutures. Bioluminescent imaging was performed using an IVIS 200 device  to monitor growth of the primary mass and metastatic spread to the liver and other organs.On the day of orthotopic implantation, tumorspheres were dissociated and diluted in a 1:1 mixture of DMEM/F12 and Matrigel. Cell mixtures were absorbed into 3 × 3 × 1 mm pieces of Gelfoam3 in calculated size using the formula: tumor volume = (length × widthViable and lineage depleted cells were re-suspended in DMEM/F12 and Matrigel (BD Biosciences). Cells were injected subcutaneously in female 6–7 week old NCR mice at the indicated number of cells per injection. Animals were monitored by palpation twice weekly for at least 10 weeks following injection to detect tumor formation. Tumors were counted as detectable at 50 mmPharmacodynamic activity of MEDI3622 in excised tumors was measured by testing lysates in a fluorometric assay to measure hydrolysis of a FRET-tagged substrate by TACE (Sigma-Aldrich MAK218).t = Ct notch-related gene – CtGAPDH. Results of tumors treated with MEDI3622 were normalized to IgG control-treated tumors.Total RNA was extracted from tumors using RNeasy Plus Mini Kit (Qiagen). Each RNA sample was reverse-transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The resulting cDNAs were used as starting material for singleplex real-time PCR using Taqman gene expression assays JAG1 (Hs01070032_m1), JAG2 (Hs00171432_m1), HES1 (Hs00172878_m1), HES2 (Hs01021800_g1), HEY1 (Hs01114113_m1), HEY2 (Hs00232622_m1) and GAPDH (Hs02758991_g1) according to the manufacturer’s protocol (Applied Biosystems). Quantitative gene expression analyses were performed using the ViiA7 Real-time PCR System (Applied Biosystems). Data were calculated as DChttp://bioinf.wehi.edu.au/software/elda/). For in vivo efficacy and life span studies, analysis was carried out using log-rank test and t-test using Prism Software (GraphPad).For limiting dilution analysis, the frequency of tumorigenic cells and the 95% confidence interval were calculated using extreme limiting dilution software (


To the Editor: Previous studies have reported avian influenza virus (AIV)–positive serum samples obtained from Adélie (Pygoscelis adeliae), chinstrap (Pygoscelis antarcticus), and gentoo (Pygoscelis papua) penguins , Arctowski Base , and Bernardo O’Higgins Base  and identified 5 serum samples positive for influenza. We also collected 513 cloacal swabs from Adélie, chinstrap . This virus was subtyped as an H5N5 and named A/chinstrap_penguin/Antarctica/B04/2015 (H5N5). Analysis of its cleavage site confirmed this was a typical low pathogenicity AIV (LPAIV) containing cleavage motif PQRETRGLF  in Chile (Stercorarius maccormicki), might represent natural reservoirs that play a role in the introduction and maintenance of AIVs into Antarctica.The timing of arrival of migratory birds that breed in Antarctica  overlaps with that of the penguins as they return to colonies for breeding and nesting during the summer in the Southern Hemisphere. These birds share a habitat, enabling close contact  and an unidentified shore bird at Delaware Bay . This finding suggests a plausible entryway of this gene into Antarctica from South America through the Atlantic or Pacific-American flyway, which are common routes used by shore birds, such as the ruddy turnstone, white-rumped sandpiper , and red knot  (The chinstrap penguin neuraminidase segment clustered within a Eurasian N5 clade that includes sequences from 2001–2010 Figure 3.As previously suggested for H11N2 viruses from Antarctica, our data supports the idea that these AIVs are evolutionarily distinct from other AIVs (The positive animals we identified originated from a single location on the Antarctic Peninsula, which suggests recent introduction of this AIV H5N5 in the colonies sampled. Antarctica is refuge for most penguin colonies, including the near-threatened emperor penguins. Previous reports suggested that AIV could have caused Adélie penguin chick death (These data provide novel insights on the ecology of AIV in Antarctica. Our findings also highlight the need for increased surveillance to understand virus diversity on this continent and its potential contribution to the genetic constellation of AIV in the Americas.Phylogenetic analyses of hemagglutinin and neuraminidase gene segments of avian influenza virus H5N5 obtained from chinstrap penguin in Antarctica in 2015, showing North American and Eurasian origins.


EGFR) mutation-positive lung adenocarcinoma patients, we evaluated feasibility of EGFR mutation detection using circulating cell-free DNA (cfDNA) and prognostic and predictive utility of cfDNA positivity (cfDNA+).In the Phase III LUX-Lung 3/6 (LL3/LL6) trials in epidermal growth factor receptor (Paired tumour and blood samples were prospectively collected from randomised patients. Mutations were detected using cfDNA from serum (LL3) or plasma (LL6) by a validated allele-specific quantitative real-time PCR kit.EGFR mutation detection rates in cfDNA were 28.6% (serum) and 60.5% (plasma). Mutation detection in blood was associated with advanced disease characteristics, including higher performance score, number of metastatic sites and bone/liver metastases, and poorer prognosis. In patients with common EGFR mutations, afatinib improved progression-free survival vs chemotherapy in cfDNA+  and cfDNA−  cohorts. A trend towards overall survival benefit with afatinib was observed in cfDNA+ patients.EGFR testing. Detectable mutation in blood was associated with more advanced disease and poorer prognosis. Afatinib improved outcomes in EGFR mutation-positive patients regardless of blood mutation status.Plasma cfDNA is a promising alternative to biopsy for  EGFR mutation-positive NSCLC. In this era of targeted therapies, identification of patients  who may derive benefit from these agents is a key factor for successful treatment  has consisted of platinum-based chemotherapy. However, over the past decade, the discovery of frequent molecular alterations in NSCLC, particularly epidermal growth factor receptor (EGFR) mutations, has led to a new treatment paradigm that includes targeted agents   . HoweverEGFR mutation detection using cfDNA from serum and/or plasma samples, as well as the prognostic value and potential utility of EGFR mutation positivity by cfDNA to predict clinical outcomes to EGFR-targeted therapies  isolated from blood . Clinical studies in patients with NSCLC have explored the feasibility of EGFR mutations in their tumours , have been previously reported  or plasma (LL6) and explores the association of clinical characteristics and outcomes with cfDNA-positive (cfDNA+) or -negative (cfDNA−) status in EGFR mutation-positive patients.This article describes the findings of two large, randomised Phase III trials (LUX-Lung 3 (LL3) and LUX-Lung 6 (LL6)), which compared the ErbB family blocker afatinib with standard platinum-doublet chemotherapy  in treatment-naive patients with advanced NSCLC harbouring n=345; recruited globally) and LL6  had pathologically confirmed and previously untreated stage IIIB/IV lung adenocarcinoma, an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, measurable disease according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 , as described in detail below.Details of the LL3 and LL6 study designs and patient eligibility criteria have been previously published (−1) or up to six cycles of intravenous pemetrexed (500 mg m−2) plus cisplatin (75 mg m−2) once every 21 days in LL3 or gemcitabine  plus cisplatin  every 21 days in LL6 (EGFR mutation type (Del19/L858R/other) and race . Patients were treated until disease progression, death, unacceptable adverse events or withdrawal of consent for any reason.In each study, patients were randomised (2 : 1) to receive oral afatinib   and NCT01121393 (LL6).EGFR mutation detection consisted of at least five 10 μm unstained sections mounted on a non-charged microscopic slide and containing at least 20% tumour tissue. In addition to the tumour tissue sample, a mandatory 9-ml blood sample was collected from each patient at the start of treatment  in both trials; however, different DNA extraction methodologies were used (see below for further details). The selection of the DNA extraction kit was based on the recommendation from the manufacturer at the time of each study conduct.Tumour tissue from each patient was obtained at an initial diagnostic procedure for NSCLC and was paraffin embedded. Tumour samples for EGFR mutations was conducted at a central laboratory using a validated allele-specific quantitative real-time PCR kit  designed to detect 29 EGFR mutations (19 deletions in exon 19 (collectively termed Del19), L858R, three insertions in exon 20 (collectively termed Ins20), L861Q, G719S, G719A, G719C (or G719X), T790M, and S768I) against a background of wild-type genomic DNA. Although a formal assessment of the amount of serum/plasma used for each sample was not conducted, it is important to note that the Therascreen EGFR 29 assay includes DNA-loading controls, which indicate when a DNA sample is too dilute or is non-amplifiable, and provides a means of performing quality control of the DNA during the assay.In both tumour tissue and paired serum/plasma samples, genotyping of Following phlebotomy (9 ml of venous blood collected over ethylenediaminetetraacetic acid (EDTA)), serum was prepared and frozen prior to shipment to the central laboratory. DNA preparation from frozen serum samples (3 ml) was performed using the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer's instructions.g in a fixed-angle rotor at+4 °C. Proteinase K solution (400 μl) was added to a 50-ml tube. The clarified plasma supernatant was carefully separated from the sediment layer, and a maximum of 4 ml was added to the Proteinase K solution. The sample volume was then adjusted to 4 ml as required using phosphate-buffered saline. Buffer ACL  was added to the 50-ml tube and the solution was incubated at 60 °C for 30 min. The Proteinase K-digested sample was then further processed as per the QIAamp Circulating Nucleic Acid Kit manufacturer's instructions (Qiagen), and the final DNA samples were eluted in 100 ml of buffer AVE.Following phlebotomy (9 ml of venous blood collected over EDTA), plasma was prepared and frozen prior to shipment to the central laboratory. The EDTA plasma sample was thawed at room temperature and then transferred to a 15-ml tube and centrifuged for 10 min at 16 000 × Tumour assessments were performed by computed tomography or magnetic resonance imaging every 6 weeks for the first 48 weeks and then every 12 weeks thereafter until disease progression or start of new anticancer therapy , and positive predictive value (PPV) of the assay. Per eligibility criteria in LL3 and LL6, only patients with EGFR mutation-positive tumours were included . As such, calculations for sensitivity, specificity, PPV, and NPV in this analysis make use of test results for all seven mutation types within each individual patient.To formally define the performance of the EGFR Therascreen 29 assay, test results from a mixture of EGFR mutation test result from blood (cfDNA+ or cfDNA−). Separate efficacy analyses were performed on patients who were cfDNA+ or cfDNA− in each study. Comparisons of PFS and OS between treatment arms were made via a log-rank test. Cox proportional-hazard models were used to derive hazard ratios (HRs) and 95% confidence intervals (CIs) comparing the two treatment arms. Kaplan–Meier estimates were used to construct survival curves and calculate median PFS and OS. Logistic regression models were used to compare the rates of objective response and disease control between treatment groups. Additional Cox proportional-hazard models were used to assess whether the blood test result (cfDNA+ or cfDNA−) was prognostic for PFS or OS after adjusting for any effects of treatment. Further Cox proportional-hazard models were used, including the treatment-by-blood test result interaction term, in order to evaluate any evidence of heterogeneity. Efficacy analyses were restricted to those patients with an EGFR mutation blood test result whose tumour harboured a common EGFR mutation (Del19/L858R). Analyses were performed on all such patients and also for patients split by the type of mutation (Del19 or L858R) in the tumour. Efficacy data presented here are from the time point of the main OS analysis, when it was estimated that OS would be sufficiently mature  in LL3 and 60.5% (202 out of 334) in LL6. Detection rates for individual EGFR mutations, including the common mutations Del19 and L858R, as well as various uncommon mutations, are shown in In LL3 and LL6, 287 (serum) and 334 (plasma) blood samples were analysed for the presence of utations . The detEGFR exhibited characteristics associated with more advanced disease compared with cfDNA− patients. cfDNA+ patients more frequently had a higher ECOG performance status , greater tumour burden , more metastatic sites , and metastases to the bone (LL3 and LL6: P<0.0001) and liver  confirmed by tissue biopsy. Within this group of patients, comparisons between treatment arms (afatinib vs chemotherapy) were performed in those who were cfDNA+ or cfDNA− for any EGFR mutation based on their blood test result.Efficacy analyses were performed in NSCLC patients whose tumours harboured common vs chemotherapy in patients with common EGFR mutations in both the cfDNA+  and the cfDNA−group  or cfDNA−  and in cfDNA− patients in both trials . No difference in OS with afatinib vs chemotherapy was observed in the L858R group for those with or without a detectable EGFR mutation in the blood  was different for cfDNA+ or cfDNA− patients, as observed by the non-significant interaction term P values . Higher ORRs with afatinib vs chemotherapy in cfDNA+ patients (LL3: 78.0% vs 40.0% LL6: 76.3% vs 34.7%) and cfDNA− patients (LL3: 76.9% vs 45.2% LL6: 75.0% vs 33.3%) were also observed based on investigator assessment of tumour response.In both cfDNA+ and cfDNA− patients, higher objective response rates (ORRs) based on independent review were observed in those treated with afatinib EGFR-targeted agents. In the current analysis of NSCLC patients enrolled in the Phase III LL3 and LL6 trials, the EGFR mutation detection rates between paired tumour and blood samples suggest that the isolation of cfDNA from plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen) could be a potential alternative to tumour tissue for EGFR mutation testing in the absence of available tumour samples. Whether the difference in EGFR mutation detection rates between LL3 (28.6%) and LL6 (60.5%) in this analysis was attributable to the different DNA extraction kits used, the difference in DNA source (serum or plasma), or both is currently unclear. However, previous studies suggest that plasma may be a more reliable source than serum for tumour DNA owing to higher background levels of non-tumour wild-type DNA found in serum (BRAF mutation testing using amplification refractory mutation system (ARMS) methodology on cfDNA isolated from either serum or plasma collected from advanced melanoma patients, a greater amount of total cfDNA was isolated from serum vs plasma (using the QIAamp Circulating Nucleic Acid Kit (Qiagen) for each blood sample type); however, the proportion of tumour-derived mutant cfDNA was significantly greater in plasma than in serum (approximately 2 × higher)  cfDNA compared with paired tumour tissue samples have ranged from 31% to 97% (vs serum), differences in DNA extraction and mutation detection methodologies , disease stage of patients , which are associated with metastatic spread to the bone/liver. This association may be a result of increased escape of tumour cells and DNA into the blood in patients with greater tumour burden and more metastatic sites. Furthermore, the more advanced baseline disease characteristics of EGFR cfDNA+ patients are likely to contribute to the poorer prognosis of these patients, irrespective of treatment with afatinib or chemotherapy. The correlation between the presence of mutant EGFR in the blood and the increased number of metastatic sites has been reported in a previous analysis  ; The therapeutic sensitivity of ablished . In thisin serum  and plasEGFR mutation in the blood and the correlation of clinical characteristics and outcomes with cfDNA+ status. In this context, there are some limitations of the analyses that should be considered when interpreting the findings. First, blood samples were not collected from all screened patients in these studies, and the proportions of missing samples, while generally low, were different between LL3 and LL6, which may be a confounding factor in these analyses. Second, quantitative measurement of the extracted DNA was not performed; thus we were unable to determine whether there was a relationship between DNA quantity and test positivity. In addition, because all patients included in LL3 and LL6 had tumour tissue that was positive for one or more EGFR mutation, we were unable to formally determine the sensitivity and specificity of the methods to detect these mutations, as compared with wild-type EGFR, in serum or plasma. Further, the different sample media used in each study (plasma vs serum) prevented combining the study data. Although combination of the LL3 and LL6 data would have potentially improved the overall statistical power of the outcomes analyses, the separate analysis of plasma and serum did provide a preliminary assessment of which media may be better for the detection of EGFR mutations. As described in the Methods section, all analyses were post hoc and therefore considered exploratory. No adjustment for multiplicity was made to the P values presented, resulting in an increased chance of false-positive results; however, the results were generally consistent and of sufficient magnitude to suggest a true effect rather than a chance finding. Finally, serial postbaseline blood sampling was not conducted in LL3 and LL6; thus potential changes in blood mutation status during treatment and at the time of progression could not be assessed.To our knowledge, the current analysis of the LL3 and LL6 trials provides the largest cohort of data to date assessing both the feasibility of detecting EGFR mutation in cfDNA from plasma appears to be a promising and minimally invasive alternative to tumour biopsy for patients without available tissue samples. Patients with EGFR mutations detected in the blood (irrespective of mutation type) displayed more advanced disease characteristics and had worse prognosis compared with blood EGFR mutation-negative patients. Importantly, clinical benefit with afatinib over platinum-based chemotherapy was consistently observed in both cfDNA+ and cfDNA− populations. Improvements in the methodology of cfDNA testing for EGFR mutations are needed to increase sensitivity and to enable this to be offered to all patients considered for EGFR-targeted therapy, with the goal of reducing the overall burden of performing invasive biopsy procedures. Future prospective studies in patients with paired tumour tissue and blood samples will be important to further develop the utility of this methodology with regard to patient prognosis and predicting clinical outcomes.In summary, our findings suggest that detection of


Inflammatory infiltrates are sometimes present in solid tumors and may be coupled to clinical behavior or etiology. Infectious viruses contribute to tumorigenesis in a significant fraction of human neoplasias.Characterize inflammatory infiltrates and possible viral transcription in primary hyperparathyroidism.From the period 2007 to 2016, a total of 55 parathyroid tumors (51 adenomas and 4 hyperplasias) with prominent inflammatory infiltrates were identified from more than 2000 parathyroid tumors in the pathology archives, and investigated by immunohistochemistry for CD4, CD8, CD20 and CD45 and scored as +0, +1 or +2. Clinicopathological data were compared to 142 parathyroid adenomas without histological evidence of inflammation. Transcriptome sequencing was performed for 13 parathyroid tumors  to identify potential viral transcripts.P = 0.007) and oxyphilic differentiation (P = 0.002). Co-existent autoimmune disease was observed in 27% of patients with inflammatory infiltrates, which in turn was associated with oxyphilic differentiation (P = 0.041). Additionally, prescription of anti-inflammatory drugs was associated with lower serum ionized calcium (P = 0.037).Tumors had prominent germinal center-like nodular (+2) lymphocytic infiltrates consisting of T and B lymphocytes (31%) and/or diffuse (+1–2) infiltrates of predominantly CD8+ T lymphocytes (84%). In the majority of cases with adjacent normal parathyroid tissue, the normal rim was unaffected by the inflammatory infiltrates (96%). Presence of inflammatory infiltrates was associated with higher levels of serum-PTH (No evidence of virus-like sequences in the parathyroid tumors could be found by transcriptome sequencing, suggesting that other factors may contribute to attract the immune system to the parathyroid tumor tissue. MEN1) gene and the cell division cycle 73 (CDC73) gene as causative agents, but other genetic events seem to occur at very low frequencies (Primary hyperparathyroidism (pHPT) is a common endocrine disorder presenting as a single parathyroid adenoma, multiglandular disease or rarely as a parathyroid carcinoma. Parathyroid adenomas with histological features suggestive of malignancy without fulfilling the criteria for carcinoma are named atypical adenomas. Genetic studies have revealed recurrent inactivating mutations of the multiple endocrine neoplasia 1  ruled out contamination from adjacent vascular structures, which had previously been suggested as an underlying cause  presence of tumor inflammatory infiltrates would be associated with a different clinical presentation as compared to tumors without inflammation, (ii) nodal- and diffuse inflammation would be associated with different clinical presentations of parathyroid tumors respectively and (iii) that active transcription of viral sequences could be an etiological factor in pHPT. To investigate the composition of inflammatory infiltrates in parathyroid tumors we screened 10 years of pathology archives at the Karolinska University Hospital (containing more than 2000 parathyroid tumors). We identified and retrieved 55 parathyroid tumors from 53 patients with histological evidence of inflammation. These cases were reviewed and evaluated using immunohistochemistry for cluster of differentiation (CD) markers. Furthermore, a series of tumors was investigated using massive parallel transcriptome sequencing (RNA-seq) and screened for viral-like sequences without prior assumption of viral pathogens .Supplementary Table 1, see section on We searched the pathology archives of Karolinska University Hospital-Solna for the period 2007–2016 for parathyroid tumors presenting with inflammatory infiltrates visible at histological examination. From 2347 parathyroid tumor records, we identified a total of 56 parathyroid tumors with inflammatory infiltrates. After exclusion of a single parathyroid adenoma that presented with manifestation of lymphoma, the remaining 55 tumors were entered into the study on immunohistochemical and clinical characterization. These included 51 adenomas (from 51 patients with a single parathyroid adenoma and pHPT), 2 tumors from a patient with secondary HPT and 2 from a patient with tertiary HPT. Patient files were reviewed for clinical details, biochemical findings, presence of autoimmune diseases and medication at the time of surgery .All histological slides were reviewed and formalin-fixated paraffin-embedded (FFPE) materials were sectioned for new immunohistochemical analyses using a clinical routine laboratory. Slides were incubated with antibodies for CD4 , CD8 , CD20  and CD45  all supplied as ready-to-use. After review by two pathologists, two distinct patterns of inflammation were identified; nodular and diffuse. Both patterns were quantified for all CD markers by a pathologist using the following criteria: Diffuse infiltrates were scored as +0 (less than 2% of cells), +1 (2–10% of cells) or +2 (more than 10% of cells). Nodular inflammation was scored as +0 (not present), +1  or +2 . We observed that both parathyroid normal and tumor tissue sometimes stained positive for CD4 and abstained from scoring the CD4+ lymphocytes in these cases  have previously been published as a part of a larger cohort . HistoloTwo rounds of RNA-seq were carried out at SciLifeLab, Karolinska Institutet, Stockholm, Sweden using previously described methodology . Total Rhttp://sourceforge.net/projects/smalt/), and the number of reads mapping to each viral sequence was recorded. RNA-Seq data from 108 samples of healthy tissues from 31 different organs/tissues  were firstly mapped to the human reference genome (GRCh38) using STAR 2.4.0f  in order/tissues  sequence/tissues . This me  and Kruskal–Wallis (for comparison between more than two groups). Relationships between variables were assessed with Spearman’s ranked order correlation. Fisher’s exact test was used for comparison of distribution of categorical variables. All tests were considered two-tailed and a P value of <0.05 was taken as statistically significant.All statistical analyses were performed on IBM SPSS, version 21. Based on distribution of data, non-parametric statistical tests were used, including Mann–Whitney Supplementary Table 2).Immunophenotyping of the tumor-infiltrating lymphocytes in 55 parathyroid tumors with inflammatory infiltrates observed at regular histopathology, revealed two distinct patterns. Diffuse inflammations were characterized by lymphocyte infiltrates with CD8+ cytotoxic T-cells and varying levels of CD4+ T-helper cells. For nodular inflammations, germinal center like nodules were observed with CD20+ B lymphocytes, CD4+ T-helper cells and CD8+ T-cytotoxic cells. Examples of diffuse and nodular inflammation are shown in Among the 51 adenomas a total of 17 cases (33%) had prominent nodular inflammation (+2) and 42 cases (82%) with diffuse inflammation (+1 or +2) as determined by CD45. In 28/51 adenoma cases, we found an adjacent normal rim, which was unaffected by the inflammation in the vast majority of cases (96%). In several adenomas with a mixed cell composition of oxyphilic and chief cells, we observed that the inflammation was predominantly located to the oxyphilic areas .Figure Additionally, two patients presented with multiglandular disease in the context of renal failure. The first patient (sHPT) showed diffuse inflammation in two out of four excised hyperplastic glands. The second patient (tHPT) presented with nodular and diffuse inflammation in two excised adenomatous glands.None of our 55 tumors exhibited inflammatory mediated degeneration of parathyroid tissue. In our experience, tumor associated stromal fibrosis is observed without relation to inflammatory infiltrates, and the absence of fibrosis in our cohort would argue against a direct link between the two phenomenon.P = 0.002, U test, P = 0.007; n = 14) . No statistically significant differences were observed between inflammatory adenomas and non-inflammatory controls concerning gender, age, tumor weight or patient’s level of serum ionized calcium (including both pre- and postoperative measurements) or plasma phosphate . Patients with an underlying autoimmune disease more often presented with oxyphilic adenomas . We also observed that anti-inflammatory medication was significantly associated with lower preoperative (but not postoperative) serum ionized calcium .Twelve of 45 patients (27%) with inflammatory parathyroid adenoma had a known autoimmune disease, and a similar proportion  was prescribed anti-inflammatory medication at the time of surgery  and the ribosomal RNA-depleted cDNA libraries (12 374 reads) of the Merkel-cell carcinoma, thus validating the method to detect viral transcript in this material. No virus transcripts were detected in the cDNA libraries of the 13 parathyroid tumors investigated.In rare cases of parathyroid tumors, histological analysis reveals infiltrates of inflammatory cells. Inflammatory disorders in other endocrine organs are often associated with functional disorders, but little is known about the pathophysiological effects of inflammation in the parathyroids. To date, this is the largest study of parathyroid tumors presenting with prominent lymphocytic infiltrates. Consistent with previous case reports and studies , 5, we oWe identified 17 tumors with prominent, nodular germinal center-like inflammation based on CD45+ (scored as 2+). In the literature, only 15 such cases are reported, four of which have been obtained from autopsy series . Previous studies have investigated a putative autoimmune etiology for pHPT, including identification of antibodies targeting CASR in analogy with Graves’ disease . There het al. studied 20 parathyroid adenomas and five biopsies from normal parathyroid glands, identifying increased levels of inflammatory cells in the tumor as compared to the normal glands. They describe varying levels of CD8+ tumor-infiltrating lymphocytes (ranging from ~2% to 20%) suggesting that such infiltrates are not that uncommon in parathyroid adenomas (This study was based on a retrospective analysis of parathyroid tumors with histologically evident inflammatory infiltrates at the time of diagnosis, which could explain the different observations made by earlier reports of inflammatory infiltrates in parathyroid tissue. Shi adenomas . While tAs part of the clinical diagnostics of parathyroid tumors, tumor cell proliferation is sometimes determined by Ki-67 index, and the WHO classification of endocrine tumors from 2004 demand vigilant follow-up for tumors with proliferation counts exceeding 5%. Since lymphocytes are histologically similar to parathyroid cells, proliferating leukocytes could potentially give raise to falsely high Ki-67 counting in cases with diffuse inflammation. Review of the pathology reports revealed that no cases were reported to have a Ki-67 index above 2%, indicating that this problem is limited. Even so, a dual staining technique with CD45 would exclude the possibility of falsely high Ki-67 counting.In conclusion, histologically prominent inflammation in parathyroid tumors is rare. At least one fourth of patients with inflammatory parathyroid tumors had a concomitant autoimmune disease, indicative of a common denominator. Oxyphilic adenomas were significantly associated with an autoimmune disease and presence of inflammatory infiltrates. Nodular inflammation is histologically distinct, but showed little adverse clinical presentation. Lymphocyte infiltrates significantly correlated to higher levels of patient’s levels of PTH and could potentially indicate a cytokine mediated change in parathyroid tumor cell endocrine activity. For patients with inflamed tumors, prescription of anti-inflammatory drugs at the time of surgery was significantly associated with lower calcium levels. Finally, we were unable to find evidence supporting active viral transcription in the investigated cases.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this study.This study was supported by the Swedish Cancer Foundation, the Swedish Research Council, the Cancer Society in Stockholm, Karolinska Institutet and Stockholm County Council.


KLRC locus flanked by KLRA and KLRJ, and a novel KLRH-like gene has acquired an activating tail. This novel gene has duplicated several times within cattle, while other activating receptor genes have been selectively disrupted. Targeted genome enrichment in cattle identified varying levels of allelic polymorphism between the NKC genes concentrated in the predicted extracellular ligand-binding domains. This novel recombination and allelic polymorphism is consistent with NKC evolution under balancing selection, suggesting that this diversity influences individual immune responses and may impact on differential outcomes of pathogen infection and vaccination.Natural killer (NK) cells are a diverse population of lymphocytes with a range of biological roles including essential immune functions. NK cell diversity is in part created by the differential expression of cell surface receptors which modulate activation and function, including multiple subfamilies of C-type lectin receptors encoded within the NK complex (NKC). Little is known about the gene content of the NKC beyond rodent and primate lineages, other than it appears to be extremely variable between mammalian groups. We compared the NKC structure between mammalian species using new high-quality draft genome assemblies for cattle and goat; re-annotated sheep, pig, and horse genome assemblies; and the published human, rat, and mouse lemur NKC. The major NKC genes are largely in the equivalent positions in all eight species, with significant independent expansions and deletions between species, allowing us to propose a model for NKC evolution during mammalian radiation. The ruminant species, cattle and goats, have independently evolved a second The online version of this article (doi:10.1007/s00251-017-0973-y) contains supplementary material, which is available to authorized users. KIR) and the natural killer complex (NKC) containing multiple members of killer cell lectin-like receptor genes (KLR). Both gene complexes evolve rapidly, vary in gene content within and between species, and can encode both activating and inhibitory polymorphic receptors. Thus, a highly diverse NK cell repertoire containing multiple highly similar receptors allows for a finely-tuned ability to discriminate MHC class I expression between healthy and damaged cells.Natural killer (NK) cells are a diverse population of circulating lymphoid cells with cytotoxic and cytokine-secreting functions, particularly in response to intracellular pathogen infections and neoplasms. Although rare, primary NK cell immunodeficiency leads to complications and/or death from severe herpesviral infections, virus-associated tumor growth, leukemia, and mycobacterial infections Orange . DysreguKIR and KLR genes is highly variable between mammalian species, with primate and rodent species the best studied to date  and a single KLRA (Ly49) gene or pseudogene  possess a single KIR in their LRC, whereas mice (Mus musculus) possess two KIR genes on chromosome X and which are thought to have alternative functions , for example, possess an expanded KLRA repertoire of five polymorphic genes and a single putatively functional KIR3DL-like gene (Futas and Horin Microcebus murinus) has expanded a different NKC gene family, possessing five functional KLRC and three functional KLRD (CD94) , possess a single functional KIR and single functional KLRA, while their terrestrial relatives, cats (Felis catus) and dogs (Canis lupus), only appear to have a functional KLRA, with the KIR gene being disrupted or deleted, respectively  also possess a single KIR and a single KLRA, yet it is uncertain if either of these genes are functional  are unique in having expanded and diversified NK cell receptor genes within both the NKC and LRC  NKC using bacterial artificial chromosomes (BACs) and recent long-read genome assemblies    Act 1986 and approved by either The Pirbright Institute Ethics Committee or The Roslin Institute’s Animal Welfare and Ethics Committee. The Chillingham samples were from animals culled for welfare reasons. Blood sampling of Kuchinoshima-Ushi cattle was carried out in accordance with the Regulations for Animal Experiments in Nagoya University and the Guidelines for the Care and Use of Laboratory Animals by the Tokyo University of Agriculture.Peripheral blood samples from KLRA to immediately downstream of KLRE was extracted from the current genome builds within Ensembl  and serine/threonine/tyrosine kinase 1 (STYK1)  and two clones from the Hereford bull L1 Domino 99375 (CH240-60G5 and CH240-239G9). The GenBank accessions for the BAC clones used in the present study are TPI4222-343E2 (KX611578), TPI4222-309A12 (KX611577), TPI4222–102F7 (KX611576), RP42–162P15 (KX698608), RP42–154D6 (KX698607), CH240-239G9 (AC170009), and CH240-60G5 (AC156849).A BAC library was previously developed from a Friesian dairy bull Di Palma  and was 1) Table . A PCR-bBAC clones were expanded overnight and BAC DNA was purified using the Qiagen Large Construct Kit . Purified DNA from three clones  was sequenced using Illumina MiSeq with 250 × 250-bp paired-end reads  and de novo assembled using Velvet (Zerbino and Birney http://www.pacb.com/devnet/). The resulting contigs were circularized by comparing the contig ends against the whole contigs to identify overlap, then the cloning vector was identified and removed to produce contigs with the first base being the first beyond the 3′ end of the cloning vector accession sequence (AY487252). Potential errors remaining in the contig sequence were removed by re-mapping all of the subreads to the edited contigs, producing high-quality (<0.01% error) genomic sequences.The remaining clones were sequenced at the USDA-ARS Meat Animal Research Center  using the PacBio RSII platform . To further confirm the Illumina assemblies, TPI4222-309A12 and TPI4222–102F7 were re-sequenced in this manner, and these assemblies matched those generated using the Illumina data. Read filtering and assembly were conducted using the Pacific Biosciences SMRT Analysis software  sequences in cattle  Gene Nomenclature Committee (HGNC)-approved gene nomenclature is used. For ease of reference, common gene synonyms are also provided upon first usage for many of the NKC genes. B. taurus) belonging to an MHC defined herd at The Pirbright Institute , and a single Nelore (B. indicus). The gDNA sourced from each of the B. taurus and B. indicus animals was whole genome amplified using the REPLI-g Mini Kit  following manufacturers’ instructions.Heparinized peripheral blood was acquired from 15 Friesian cattle (Mononuclear cells (PBMCs) were separated from the heparinized peripheral blood using Histopaque-1083 (Sigma-Aldrich Corporation), and gDNA was isolated using the QIAamp DNA Mini Kit  following the manufacturers’ instructions. The quantity of purified gDNA was assessed for each animal with the Quant-iT PicoGreen dsDNA assay (Thermo Fisher Scientific) using the low-range standard curve. An aliquot of gDNA from every animal was sheared using a Covaris S220 Focused-ultrasonicator . Instrument parameters provided by the manufacturer were used to fragment the DNA to insert sizes between 500 and 650 bp. Indexed paired-end gDNA libraries were constructed using a low-throughput, low-sample number TruSeq DNA sample preparation kit . Four multiplexed sequencing libraries were prepared, one for each of four independent genome enrichment experiments. Each multiplexed library was constructed using 1-μg input DNA per animal, and ligation products were size selected (>500 bp) on an agarose gel and purified as described in the TruSeq protocol. An aliquot was removed from each and used as PCR template to assess the quality of the constructed sample library. PCR amplification was carried out as described in the TruSeq DNA Sample Preparation Guide. Sample library quality assessment was carried out by running PCR products on a DNA1000 chip using a 2100 Bioanalyzer instrument (Agilent Technologies). Amplification of each multiplexed sample library was performed as described in the NimbleGen SeqCap EZ Library SR User’s Guide (version 4.1). To enrich cattle gDNA from the NKC, custom oligonucleotide probes were designed and synthesized as a SeqCap EZ Developer Library (Roche Sequencing) and enrichment was performed as described in the manufacturers’ protocol. Human Cot-1 DNA was used to block repetitive regions of the cattle genome. DNA was purified at each stage of the Roche protocol using Agencourt AMPure XP DNA purification beads . The captured multiplex DNA sample was washed and recovered as outlined in the Roche protocol. Amplification of the enriched multiplex DNA sample libraries used LM-PCR as described in the Roche protocol. To determine how successful the enrichment was, DNA was analyzed on a DNA1000 chip using a 2100 Bioanalyzer instrument (Agilent Technologies) and using qPCR. The degree of enrichment measured across the four independent captures indicated that each library was successfully generated.The four enriched multiplex DNA sample libraries were independently sequenced using a MiSeq desktop sequencer  at The Pirbright Institute. The MiSeq Reagent Kit version 2  was used to produce either 2 × 230- or 2 × 250-bp paired-end reads per run. Multiplexed DNA sample libraries were diluted and sequenced at a final concentration of 8 pM. Each sequencing run used a PhiX control spike which was denatured and diluted to 12.5 pM. The final pool of sequenced DNA was comprised of 99% sample library and 1% PhiX. Library preparation, sample loading, and MiSeq preparation steps were carried out as described in the manufacturers’ protocol. Resultant reads were mapped to the cattle genome build UMD_3.1 using the Burrows-Wheeler Aligner  , and two clones were from a Hereford bull . The Hereford BAC library was the source for the minimum BAC tiling path sequenced as the primary basis for the existing UMD_3.1 assembly, supplemented by whole-genome shotgun sequence from a daughter (L1 Dominette 01449) of the same bull , the NKC region spanning , the NKCLRA Fig.  and two KLRA to KLRE spans approximately 584 kb and contains a total of 53 sequence gaps  and a new long-read de novo goat assembly, ARS1, that has also used PacBio sequencing to improve assembly contiguity . Recurrence plot sequence identity analysis revealed the presence of a highly repetitive region approximately 280 kb in size midway between KLRA and KLRJ  possessed a cytoplasmic tail containing two immunoreceptor tyrosine-based inhibition motifs (ITIMs) and a predicted TM region containing an arginine residue creating the potential for both inhibitory and activating functions, respectively , of which three are putatively functional , although one of these contains two non-canonical splice sites (bota_KLRH6). Furthermore, one of two KLRH genes in the goat has likewise aquired a KLRC-like tail (cahi_KLRH2). However, exon 2 of this tail appears to have been subsequently deleted from cahi_KLRH2. Moreover, two non-canonical splice sites in exons 3 and 4 and a frameshift in exon 5 have likely destroyed its functionality, despite maintenance of an open reading frame across exon 1 to exon 3. Two additional genes in cattle (bota_KLRH1 and bota_KLRH7) and one in goats (cahi_KLRH1) do not appear to be associated with exons encoding the N-terminal intracellular and TM domains. An 80-bp fragment most similar to a KLRA-like N-terminal region was identified approximately 4 kb upstream of cahi_KLRH1 using the NCBI conserved domain database  encode potentially activating receptors  possesses an intact open reading frame with canonical splice sites. We conclude from this diversity of disabling mutations that both the recombinant KLRH genes with activating KLRC2-like tails and those apparently lacking tails were independently disrupted after their expansion.All four of the cattle pseudogenes and both goat pseudogenes that contain the full complement of exons (bota_KLRI2 is mutated (ATG− > AAG) in both the UMD_3.1 and ARS-UCDv0.1 assemblies. Although a potential alternative start site exists several codons downstream, a non-canonical splice site at the end of exon 1 may additionally disrupt the gene. We observed an additional 2-bp frameshift that results in multiple downstream stop codons in both of the overlapping RPCI-42 BAC clones, arguing that this gene has become non-functional in the cattle genome. In contrast, no disabling mutations were observed within the genes encoding inhibitory receptors. This type of selective disruption of activating genes is also a feature of the cattle KIR  were identified within the coding regions of KLRA, KLRJ, KLRI2, KLRI1, KLRK, KLRD1, KLRD2, and KLRE , pig (Sscrofa10.2), and horse (Equ Cab 2) that are all based on short sequence reads. As expected based on our findings with the UMD_3.1 and CHIR_1.0 assemblies, the NKCs of these reference genomes contain numerous sequence gaps, making conclusions about detailed genomic structure and allele content provisional , and three KLRC1 pseudogenes were identified in total. Thus, despite expansion, the horse KLRC gene cluster has retained only two putatively functional activating members.The horse shared a common ancestor with cattle approximately 80 Ma  or the rat (Rnor_6.0) genomes  in the opposite orientation as KLRA and KLRJ, as seen in the other species  genes, for which the gene content is not substantially different between these species apart from rodents, which have expanded their CLEC2D and KLRB loci. However, within the KLR gene cluster, our analyses revealed extensive species-specific expansions and contractions within a generally conserved framework of genes. In particular, the KLRA locus has undergone extensive expansion and contraction during mammalian evolution, as evidenced by its highly variable gene content between species and the duplication and subsequent divergence of the closely related KLRH and KLRJ genes. All three of these genes originated prior to the divergence of the Laurasiatheria  and the Euarchontoglires  approximately 92 Ma . Although different in rodents, KLRH is structurally similar in the mouse lemur and the Laurasiatherians, indicating that its sequence inverted and the intracellular and TM domains were deleted prior to the divergence of these two major clades. KLRH in rodents, on the other hand, is encoded in the same orientation as KLRA and possesses an inhibitory intracellular tail. This suggests either a duplication and subsequent contraction or that a recombination occurred between the lectin domains of KLRH and a KLRA gene during rodent evolution. In either case, the inverted KLRH-like sequences were deleted in the rodent lineage. It seems likely that in early ruminant evolution, this inverted region became a template for non-allelic homologous recombination (NAHR), in which sequence similarity between the regions containing KLRC1 and KLRH established a conversion tract with KLRC1 in the new location. The locus was then further expanded by duplication events and additional conversions.This study examined the structure and gene content of the KLRC genes was unable to resolve the sequence of duplications, although it is likely to have initially occurred with an ancestor homologous to bota_KLRC1–7. Shortly after the initial expansion, one of the now duplicated KLRH genes acquired the intracellular tail from an activating KLRC2 gene. This new KLRH/KLRC2 hybrid then subsequently duplicated along with the rest of the locus, giving rise to five such hybrids in cattle  and one in goats (cahi_KLRH2). Three of these in cattle  are putatively functional. As the knockout mutations differ between the remaining three full-length pseudogenes, it is apparent that following the initial recombination event and subsequent expansion, these genes were independently disabled. This suggests that the KLRH/KLRC2 hybrids were functional following the recombination event and prior to their expansion. Intriguingly, the open reading frame for the C-type lectin domain of KLRH remains functionally preserved in cattle, sheep, goats, pigs, horses, and mouse lemurs, despite the lack of recognizable intracellular and TM domains. As it seems unlikely for this preservation to be coincidence, this gene may be functional, perhaps expressed as a soluble receptor.Phylogenetic analysis of the C-type lectin domains of the ruminant KLRC1 subgroup pseudogenes appear to have undergone block duplication along with a functional KLRC1, thus expanding a net inhibitory receptor reservoir. The disruption of activating KLRC2-1 in both cattle and goats appears to predate species divergence, as they share the same loss of the last two lectin domain exons. In cattle, the loss of activating KLRI2 function is either relatively recent or ongoing due to the less obvious nature of the knockout mutations. That this gene is missing in pigs and horses suggests that it has either been lost or its expansion has never occurred in their ancestry. The most parsimonius explanation, however, is that they evolved once and subsequently homogenized within their respective species. Thus, as a result of gene conversion, the origin of many paired NK cell receptors is ambiguous.The apparent conversion of activating receptors to pseudogenes in ruminants fits with a paradigm that activating genes are quickly expanded in response to strong selection pressure, then quickly lost once beneficial function is lost  representations of repetitive regions. But even with relatively short stretches of haploid DNA, short-read technologies have difficulty creating unbroken assemblies when the repeat length greatly exceeds the read length. Recently, long-read technologies have overcome many of the constraints of assembling these types of genomic regions  sequencing was insufficient for mapping and assessing the diversity across this region. Furthermore, the potential for genes containing domains that have recombined from other genes, such as the case for the cattle KLRH/KLRC2 hybrids, may complicate efforts to transcriptomically assess gene expression when using standard short-read sequencing. To resolve these problems, we recommend that such repetitive immune gene clusters be sequenced and transcriptomically analyzed using long-read technology.Our results also indicate the utility of using a probe-based capture method to enrich and sequence genomic regions to elucidate allelic variation. However, given the highly repetitive nature of the recently expanded KLRC expansions in cattle and horses, the discovery of KLRE and KLRI outside of rodents, the presence of KLRJ in the described species, and the identification of novel KLRH genes bearing KLRC2-like activating tails in cattle and goats. Finally, polymorphisms across the cattle NKC, and likely other species, further expands the available NK cell receptor repertoire, particularly in the KLRC/D and KLRA systems. These results fill an important evolutionary link in our understanding of the NKC and will inform future investigations of NK cell receptor diversity, assist in identifying their potential ligands, and aid in the identification of genotypes associated with differential disease outcome.Our annotations of the cattle, goat, sheep, pig, and horse NKC regions have identified a large proportion of NK cell receptor gene family members that may have been subjected to expansion and contraction due to NAHR. We described the extensive ESM 1(DOCX 359 kb)