In recent years, it has become evident that a generational gap has developed in the community of arbovirus research. This apparent gap is due to the dis-investment of training for the next generation of arbovirologists, which threatens to derail the rich history of virus discovery, field epidemiology, and understanding of the richness of diversity that surrounds us. On the other hand, new technologies have resulted in an explosion of virus discovery that is constantly redefining the virosphere and the evolutionary relationships between viruses. This paradox presents new challenges that may have immediate and disastrous consequences for public health when yet to be discovered arboviruses emerge. In this review we endeavor to bridge this gap by providing a historical context for the work being conducted today and provide continuity between the generations. To this end, we will provide a narrative of the thrill of scientific discovery and excitement and the challenges lying ahead. Stegomyia fasciata (Aedes aegypti). Lazear, who like his colleagues, had been stationed by the US Army in Cuba to study the disease, died of yellow fever in September 1900 after being exposed experimentally to mosquitos that had fed on sick patients. At about the same time in South Africa, James Spreull and Sir Arnold Theiler demonstrated that bluetongue disease of sheep is caused by an “ultravisible” agent that could be transmitted by the injection of an infected serum. Epidemiological evidence suggested that the agent was vector-borne, and it was subsequently shown by R.M. du Toit that the disease occurred in sheep inoculated experimentally with suspensions of wild-caught biting midges (Culicoides imicola). These and other seminal discoveries precipitated a century of research into vector-borne and zoonotic viral diseases, resulting in the discovery and isolation of many hundreds of novel viruses from insects or vertebrate hosts. Some were identified as important human or veterinary pathogens. Many other viruses were archived in reference collections, with only basic characterization of their biological or molecular properties. In recent years, the advent of next generation sequencing (NGS) has transformed this situation. Complete genome sequences are now available for many of the archived isolates, allowing more accurate taxonomic assignments, analysis of their phylogenetic and evolutionary relationships with other viruses, and evaluation of the potential risks they may present to humans and wild or domestic animal populations. NGS has also opened the door to viral metagenomics, which has greatly increased the pace of new virus discovery from a wide range of hosts, usually with complete or near-complete viral coding sequences, but no virus isolate and minimal biological data. This has presented both opportunities and challenges for virologists and epidemiologists, as well as viral taxonomists, evolutionary biologists, and bioinfomaticians. Sadly, this technological revolution has been accompanied by a period of progressive disinvestment in training in classical virology. In this review, we recall the rich history of the discovery of arboviruses and other zoonotic viruses in various settings around the world and the many outstanding scientists who have contributed to the endeavour. We also consider the impacts of NGS and metagenomic analysis, and the implications of these new technologies for the future of this important field of research.Almost 120 years have passed since Walter Reed, James Carroll, Aristides Agramonte, and Jesse Lazear established that yellow fever is caused by a filterable infectious agent which is transmitted by the bite of a mosquito, then known as Aedes aegypti;Confirmation of earlier work by the Reed Commission in Cuba, demonstrating that yellow fever was caused by a filterable agent, yellow fever virus (YFV), that was transmitted by the bite of infected mosquitoes, Discovery that the rhesus monkey and the white mouse are susceptible to infection with YFV, providing models for subsequent studies on the pathogenesis, transmission, epidemiology, and control of the disease;Demonstration that convalescent sera of humans and animals infected with YFV neutralize the virus. This discovery led to the development of the mouse neutralization test, which allowed investigators to map the geographic distribution of the virus;Ae. aegypti in the transmission and maintenance of the virus;Discovery of the forest or sylvan cycle of YFV and the importance of mosquitoes other than Development of the 17D vaccine strain of YFV and its first human trials. Max Theiler, son of South African bluetongue researcher Sir Arnold Theiler, received a Nobel Prize in 1951 for this work.The Rockefeller Foundation (RF) was organized in 1913 for “the well-being of mankind throughout the world” . Much ofAs a by-product of the overseas yellow fever investigations, RF-funded researchers also isolated a number of other previously unknown arboviruses, including West Nile, Zika, Semliki Forest, Bunyamwera, Bwamba, Uganda S, Ilheus, and Anopheles A and B viruses .The threat and onset of World War II changed the priorities of the RF, and most of its overseas YF research activities ceased during this period. Many of the former American RF staff became involved in studies of diseases of military importance, such as typhus, malaria, sandfly fever, and dengue, some as civilians and others as members of the U.S. Armed Forces.In 1950, after the end of the war, the RF decided to develop a major new program to study arthropod-borne viruses and to discover “what might be out there”, as well as their disease associations, life cycles, and vectors. This led to the development of the Rockefeller Foundation Virus Program, a world-wide virus discovery program that was funded from 1951 to 1970 . In EgypThe RF Virus Program was extremely productive and many novel arboviruses, as well as non-arthropod-borne viruses , were discovered by RF-funded investigators during this period. The productivity of this search strategy in detecting novel viruses affecting humans was recently reviewed by Rosenberg et al. . The virIn 1960, the RF made a decision to phase-out its Virus Program and to devote more of its efforts and resources to other projects, such as population control and increasing food production (“the green revolution”). Over the next 10 years, the RF-funded investigators working in overseas laboratories were withdrawn and the respective laboratories were turned over to local institutions or governments. In 1964, the RF made arrangements with Yale University to transfer its Arbovirus Group to New Haven. Accordingly, the Virus Laboratory in New York City was closed, and most of the remaining investigators , their equipment, virus stocks, and reagents were moved to New Haven, with a small endowment to temporarily support them. The newly established group was designated the Yale Arbovirus Research Unit (YARU) and was housed in the newly constructed Laboratory of Epidemiology and Public Health building on the grounds of the Yale University School of Medicine. Upon arrival, the former RF staff members in turn became members of the Yale faculty . Wilbur The YARU was subsequently designated by WHO as an International Reference Center for Arboviruses, and for a number of years, it received virus samples from investigators worldwide for identification and confirmation . Under SEmerging Infections: Microbial Threats to Health in the United States, which highlighted the threat that emerging infections pose for the U.S. and global public health , international organizations , and foreign governments. In 1992, Shope was a co-editor of the Institute of Medicine’s seminal publication, c health . Shope wc health ,16, and Consultancies, service on government committees and free diagnostic laboratory support (the old RF model) could not provide sufficient funds to maintain and recruit new personnel, upgrade equipment, or support a major research program at a university in the U.S. In turn, leaders at Yale Medical School decided that arbovirology was passé and that other areas, such as HIV/AIDS, environmental health, and molecular biology, offered better funding and research opportunities for the 1980s. Thus, YARU gradually lost university support, faculty positions, and space. In 1995, after 30 years at Yale, Shope retired. Along with his colleague Robert Tesh, he moved to the newly established Center for Tropical Diseases at the University of Texas Medical Branch in Galveston . The extSince the establishment of the Institut Pasteur in Paris in 1888, Louis Pasteur sent collaborators to various countries, mainly in the French colonies of Indochina and Africa. In that early time, Pasteur wanted to set up rabies centers where the disease was highly and dramatically prevalent; naturally, the research potential of these centers was rapidly extended to tropical infectious diseases [Another French multidisciplinary worldwide institution, the French Institute of Research for Development  joined the RIIP’s efforts in the intertropical zone of Africa in its fight against infectious diseases, by bringing expertise on medical entomology and vector transmitted diseases. ORSTOM was created in 1946 from a previously existing Office of Colonial Research , established by Charles de Gaulle in 1943.Aedes africanus, Aedes opok, Aedes furcifer-taylori, and Aedes luteocephalus), thus elucidating the mechanism of YFV maintenance in nature. Epidemiological observations during this period allowed Max Germain to formulate the concept of a “yellow fever zone of emergence” in West and Central Africa [Ae. furcifer-taylori) allowed the documentation of vertical transmission, allowing for virus maintenance in the inter-epidemic periods [Yellow fever was to play a key role in the research at IPD. In the 1930s, IPD had developed a vaccine against yellow fever (strain FNV) and produced it commercially. The re-emergence of yellow fever in Africa in the 1960s occurred mainly in the savannah zone and revealed the lack of knowledge about the natural maintenance of the virus during the inter-epidemic period. A large study to understand the emergence and re-emergence of sylvatic yellow fever was established between the IPD, Institute Pasteur Abidjan (IPA) in Côte d’Ivoire, and Institute Pasteur Bangui (IPB) in the Central African Republic, in order to detect the circulation of the YFV and map its emergence in the different ecozones. Permanent research stations were developed in rain forests and savannas to detect virus circulation in vectors and hosts from the tree canopies to the savanna ground. Year-round mosquito and monkey sampling over a period of more than 10 years made possible the detection of YFV in various monkey and mosquito species , led by Paul Brés. CRORA laboratories were created at various IPs throughout Africa, including IPD, IPA, and IPB, as well as IP Yaoundé (Cameroon), and IP Tananarive (Madagascar). One of the major activities was to establish an inventory of arboviruses circulating in various ecosystems. Although virus isolations were made at various IPs, virus characterization and identification were carried out at the CRORA reference center at IPD, followed by confirmation at YARU, with final registration in the International Arbovirus Catalogue .At the end of the 1970s, following an outbreak of the Ebola epidemic in Zaire in 1976, IPB set up a research program on viral hemorrhagic fevers that lasted until 1983 . This reAedes (stegomiya) africanus and Ae. (st.) opok, inside the forest gallery, Aedes vittatus, in the savannah, and Anopheles gambiae and An. Funestus, in the houses of the village of Bozo. A total of 321 viruses were isolated and assigned to 24 different species. These included chikungunya, Bagaza, Bouboui, Bozo, Bwamba, Ilesha, Kamese, Kedougou, Middleburg, Mossuril, M’Poko, Nyando, Orungo, Pata, Pongola, Simbu, Sindbis, Tataguine, Wesselsbron, West Nile, yellow fever, and Zika viruses. Altogether, six arboviruses were found in the forest gallery, including Bouboui, Bozo, chikungunya, Orungo, yellow fever, and Zika viruses, vectored primarily by Ae. africanus.Research at IPB was also critical in elucidating the etiology of exanthematous fevers, coined “Congolese red fevers”, that have long been attributed to rickettsia. A total of 16 arboviruses  were associated with these syndromes. Symptoms, consisting of fever, diffuse pain, and exanthem, were present in more than 60% of the cases, with etiology dominated by four viruses—Chikungunya, Ilesha, Bwamba, and Tataguine ,32. BetwResearch at IPD and IPB was also instrumental in demonstrating the expanded range of the Crimean–Congo hemorrhagic fever virus (CCHV) in West and Central Africa ,34,35, fSupport for RIIP laboratories located in Africa was provided by the IPP and OSTROM teams, as well as through collaboration with various research centers in the U.S., such as Harvard University, supporting the initial study on yellow fever and FNV vaccine; YARU, as a partner for new arbovirus classification; the Centers for Diseases control (CDC) at Fort Collins Colorado, for the study of arboviruses; the CDC in Atlanta and the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), for the initiation of viral hemorrhagic fever research in Central and West Africa.The era of virus discovery in Australia can be traced to the summer of 1950–1951, when a major epidemic of encephalitis swept through southeastern Australia. There were clinical cases reported in Victoria, New South Wales, and South Australia, of which 19 42%) were fatal . A simil% were faAedes vigilax mosquitoes collected in Townsville [The subsequent development of arbovirology and the pathway to virus discovery in Australia were linked intimately with the 1947 establishment of the Queensland Institute of Medical Research (now the QIMR-Berghofer Institute) at Herston in Brisbane . Ralph Lwnsville . He subswnsville . He alsownsville ,47. In 1wnsville . RRV andwnsville .Centropus phasianinus) collected at Kowanyama from 1963 to 1966 yielded three novel viruses  [Cryptoblepharus virgatus) at Kowanyama in 1966 [Rattus leucopus) captured near Mossman in 1970 [Doherty’s program of virus discovery continued until 1977 with a team that included several other notable scientists, including Harry A. Standfast, Brian H. Kay, Edwin G. Westaway, Barry M. Gorman, and John G. Aaskov. During 1960 and 1961, Doherty isolated 60 strains of 11 viruses from 25,901 mosquitoes of 32 species from Kowanyama , Normanton, and Cairns in far north Queensland. These included four novel flaviviruses , three orthobunyaviruses  and one orbivirus (Corriparta) . The stuviruses) . In 1969viruses) ,52. Furtviruses) . Leanyerviruses) . Viruses in 1966  and the  in 1970 .Ornithodoros capensis) from sooty tern (Onychoprion fuscatus) colonies on the Great Barrier Reef near Cairns, from which two viruses (Upolu and Johnston Atoll viruses) were isolated [Ixodes uriae) associated with sea birds, resulting in the isolation of two novel viruses (Nugget and Taggert viruses) [Eudyptes chrysolophus schlegeli) rookeries [In collaboration with Doherty and his QIMR team, expeditions were also conducted to isolate viruses from ticks associated with sea birds. In 1966, Harald N. Johnson from YARU collected soft ticks (isolated . The Sauisolated . In 1972viruses) . A seconookeries .Pteropus poliocephalus) [Syconycteris crassa). In Western Australia, Stanley and colleagues surveyed for arbovirus activity in the Ord River Valley from 1972 to 1976 [Cx. annulirostris, suggesting the region may be an endemic focus in Australia [Other research groups in Australia also joined the hunt for arboviruses during the late 1960s and early 1970s, including Ian D. Marshall at the Australian National University in Canberra and Neville F. Stanley at the University of Western Australia. From 1965 to 1975, Marshall and his colleagues conducted surveys for arbovirus activity, particularly RRV and MVEV, in coastal regions of New South Wales and in the Murray River Valley. In addition to these and other known arboviruses, Marshall and colleagues isolated several novel arboviruses from mosquitoes, including Gan Gan, Yacaaba, Tilligerry and Termeil, Paroo River, Picola, and Barmah Forest viruses ,62 and aephalus) . Like thephalus) ,65, withephalus) . The Ganephalus) . Marshal to 1976 ,69. Fromustralia ,72,73,74In 1968, CSIRO established a new virology laboratory at Long Pocket in Brisbane, headed by Toby D. St. George, to investigate endemic diseases of livestock in northern Australia. In 1967, Doherty and colleagues had isolated bovine ephemeral fever virus (BEFV) from cattle during a major epizootic in Queensland  but, desArgas robertsi) (Vinegar Hill and Lake Clarendon viruses) [The isolation of BTV-20 and the consequences for international trade dramatically changed the landscape with respect to virus discovery and characterization in Australia. An immediate consequence was the approval of government expenditure for the establishment of the $230 million CSIRO Australian Animal Health Laboratory (AAHL) in Geelong, Victoria, providing high-level biosecure containment for laboratory work and live animal studies. The discovery also led to the establishment of a permanent veterinary virology capability at the Berrimah Veterinary Laboratory in Darwin under Geoff P. Gard, and a national program for serological monitoring of sentinel cattle herds and the collection of insect vectors. Efforts to isolate viruses from arthropods and livestock intensified. In the Northern Territory, a second novel serotype of bluetongue virus (BTV-21) was isolated from a healthy sentinel cow at Victoria River Station in 1979 , and fouviruses) ,85,86,87viruses) ,89.Pteropus alecto) was found at Ballina in New South Wales. Tissue homogenates from the euthanized bat were injected into mice, resulting in the isolation of a novel lyssavirus, subsequently named Australian bat lyssavirus (ABLV) [The 1990s also saw several significant disease emergence events in Australia, which drew particular attention to bats as reservoir hosts of highly pathogenic viruses. In September 1994, an outbreak of a severe respiratory disease occurred in horses at a stable in Brisbane. Of the 21 affected horses 14 were euthanized or died of the disease. One of two severely affected humans who had contact with the horses also died. Cooperation between the Queensland Government, the newly established CSIRO Australian Animal Health Laboratory, and others resulted in rapid isolation of the Hendra virus, a novel paramyxovirus ,92. The s (ABLV) . Three fs (ABLV) ,96. The s (ABLV) . In Apris (ABLV) . Two hums (ABLV) . The rolIn all, more than 80 novel RNA viruses representing 9 families and 16 genera have been isolated from humans, livestock, wildlife, and arthropods in Australia and Papua New Guinea, and reported in the literature . Many otPresbytis entellus). This followed reports of an epizootic of unknown etiology causing large numbers of deaths in non-human primates and a number of cases of severe febrile illness in villages close to forested areas where dead monkeys had been found. The virus was shown to be closely related to the Russian spring–summer encephalitis (RSSE) serocomplex of flaviviruses, now known as the tick-borne encephalitis (TBE) serocomplex of flaviviruses. The virus was also isolated from some larvae and nymphs of Hemaphysalis spinigera ticks [Ixodes granualtus, collected from forest rats caught near Kuala Lumpur, Malaysia, by C.E. Gordon Smith, then working at the Institute for Medical Research in Kuala Lumpur [South and Southeast Asia have also been a fertile area for virus discovery, particularly novel mosquito-borne and tick-borne flaviviruses and viruses with reservoirs in bat species. Early studies in India, partly funded by the Rockefeller Foundation, by Telford Work and his Indian colleagues from the Virus Research Centre in Pune, including D.P. Murthy, P.N. Bhatt, H. Trapido and K. Pavri, led to the discovery of the Kyasanur Forest disease (KFD) virus . The virra ticks . KFD in a Lumpur , but it Aedes aegypti and Culex tritaeniorhynchus mosquitoes collected during a major outbreak of epidemic hemorrhagic fever in Manila, Philippines, by W.M. Hammon, A. Rudnick, and colleagues at the University of Pittsburgh [Cx. tritaeniorhynchus mosquitoes collected in Chiang Mai, Thailand, in 1992 [Aedes spp. and Uranotaenaia spp. mosquitoes and from the blood of a Cynopterus brachyotis fruit bat [Mansonia septempunctata mosquitoes collected in the Sepik District of Papua New Guinea [Cx. pseudovishnui group mosquitoes [A number of mosquito-borne flaviviruses were first isolated in Southeast Asia. The most important with respect to human disease are three of the four dengue serotypes. Although dengue serotype 1 had been first isolated independently by Hotta in Japan in 1943 , and shottsburgh . Other nttsburgh . The Temttsburgh  and was  in 1992 ; the Sit in 1992 ; and the in 1992 ,112. Neu in 1992  but the ruit bat . The Sepw Guinea . It was w Guinea . A novelsquitoes . Initialsquitoes .Cy. brachyotis fruit bats: the Carey Island virus was isolated from a bat in the Jugra Forest, Malaysia, in 1970 by A. Rudnick and colleagues from the Institute of Medical Research, Kuala Lumpur and the International Center for Medical Research, University of California [Two flaviviruses with no known vector have been isolated in Southeast Asia, both from lifornia , and thelifornia . A closeCx. gelidus mosquitoes [Tadarida plicata bats in Thailand.A considerable number of novel bunyaviruses have been isolated from South and Southeast Asia, particularly by scientists from the National Institute of Virology (formerly the Virus Research Centre) in Pune, including P.N. Bhatt, K. Pavri, K.R. Singh, C.N. Dandawate, F.M. Rodrigues, D.T. Mourya, P.D. Yadev, A.C. Mishra, and many others. Recently reviewed in , these vsquitoes  and the squitoes , and theBrahminy myna) collected in Vellore, Tamil Nadu, India, and most closely related to the mosquito-borne orbiviruses [Novel orbiviruses from South and Southeast Asia include the Sathuvachari virus, isolated from starlings  spp. collected in 1956, but although neutralising antibodies have been found in human sera, it has not been associated with human disease [Cx. gelidus mosquitoes collected in 1955 near Kuala Lumpur [Two novel alphaviruses were reported in Kuala Lumpur—Bebaru and Getah viruses. Bebaru was first isolated from  disease . Getah va Lumpur . It causPaguna larvata), through wet markets in southern China.The important role of bats as reservoirs of a wide range of viruses was underlined by a number of the viruses described above, and particularly by the discovery of their role as the reservoir of the Nipah virus in Malaysia in 1999  and subsRubulavirus, which was isolated from a Rousettus leschenaultia bat collected near Pune in India [Mastadenovirus was also isolated from the same fruit bat species caught in Maharashtra State [Rubulavirusi, isolated from Pteropus hypermelanus and related to the Australian Menangle virus, but not known to cause human disease [Continued studies of the Nipah virus in Malaysia, and subsequently in Bangladesh and India, and investigations of SARS-CoV in bats have resulted in an enormous explosion of knowledge of viruses carried by bats, with many examples from most viral families, although in many cases the information is from genomic fragments . Virus iin India . A novelra State . A numbe disease ; Pulau v disease ; Melaka  disease ; and Kam disease . The imp disease .Ixodes persulcatus ticks in the taiga, Zilber assumed that SSE was transmitted by ticks, in contrast to “summer encephalitis”, which is transmitted by mosquitoes [Ix. persulcatus ticks, and their ability to transmit the virus by biting laboratory animals was shown experimentally [Ix. ricinus ticks. At the same time, it was found that TBEV is also an etiological agent of some seasonal encephalitis or febrile illnesses, such as Central European encephalitis or biphasic milk fever [The history of arbovirus research in the USSR began in 1937, when an expedition under the leadership of Lev A. Zilber  went to the Russian Far East to study seasonal epidemic encephalitis. This disease with high mortality rates affected forest workers and soldiers stationed in the taiga, mainly those who came from other regions of the USSR. The disease had a pronounced seasonality—the cases started being recorded at the beginning of May, reached the peak in early June, and declined by August. Local doctors designated the disease “spring–summer encephalitis” (SSE) and assumed that it had been caused by some kind of virus. It has also been suggested that there were some similarities between SSE and “summer encephalitis”  described at the time, it was also assumed to be a toxic form of influenza . Howeversquitoes . Severalmentally . One of mentally ,143. So,mentally ,145,146.lk fever ,148,149.lk fever . Geneticlk fever ,152,153.Tick-borne encephalitis virus of the genus Flavivirus (Flaviviridae) [Ix. persulcatus and Ix. ricinus ticks, including Russia, Eastern and Central Europe, Baltic and Scandinavian countries [TBE is the most important arboviral infection in Russia. Despite significant progress in the development of anti-TBEV vaccines, thousands of cases are recorded annually in Russia, mainly in Siberian and Far Eastern regions . In the viridae) . TBEV isountries ,157.Dermacentor reticulates ticks, which are highly prevalent in the region [De. reticulates ticks collected in the natural foci of OHF [De. reticulatus ticks and their host, a narrow-headed vole , are considered an original natural reservoir of OHFV. The European water vole (Arvicola amphibius), the tundra vole (Microtus oeconomus), and some species of shrews are also involved in the circulation of OHFV [Ondatra zibethicus) to this region in 1935–1936 [Omsk hemorrhagic fever virus of the genus Flavivirus (family Flaviviridae) [The discovery of TBEV as a causative agent of SSE gave an impetus to studies of similar diseases throughout the USSR. During subsequent years, the major virological centres were established as parts of the Academy of Medical Science of the USSR (AMS USSR), such as the department of neurovirology at the Institute of Neurology (1942), the Institute of Virology (1944), the Institute of Poliomyelitis and Viral Encephalitis (1950), as well as departments of virology at regional medical institutes in Siberia and the Far East. Scientists from these centres were actively involved in the study of various zoonotic viral infections distributed in the USSR. Many participants of the first expeditions subsequently became famous virologists. One of the most notable ones is Michael P. Chumakov, who later headed the Institute of Virology AMS USSR (1950–1954) and the Institute of Poliomyelitis and Viral Encephalitis AMS USSR (1955–1972) in Moscow. Chumakov organized numerous expeditions that aimed to study the etiology of zoonotic human infections. In the 1940s, outbreaks of the disease, designated by local doctors as “atypical tularemia”, “anicteric leptospirosis”, and “Omsk spring–summer fever”, were recorded in several rural regions of the Omsk district in Western Siberia. Clinicians from the Omsk Medical Institute, under the leadership of Ahrem-Akhremovich, described the disease in detail and named it Omsk hemorrhagic fever (OHF), as the patients often developed hemorrhagic diathesis. They also suggested that OHF was transmitted by e region ,159. In i of OHF ,161. In  of OHFV . However935–1936 . Muskrat935–1936 ,163. In 935–1936 ,165. Bas935–1936 , and latviridae) . Genome viridae) ,168,169.Hyalomma (plumbeum) marginatum ticks. Despite the absence of virus isolates from specimens from CHF patients or from ticks, the viral etiology of CHF and its zoonotic nature were proven experimentally by infecting volunteers with the blood of CHF patients or a filtered suspension of ticks collected from a hare caught in the focus of the disease [Hy. marginatum nymphs isolated in southern Russia [Crimean–Congo hemorrhagic fever virus, of the genus Orthonairovirus .In 1944, virologists led by Chumakov studied the etiology of an outbreak of a febrile illness which was accompanied with hemorrhagic manifestations (“acute infectious capillary toxicoses”) in a rural area in the northwest part of the Crimean Peninsula. They designated the disease as Crimean hemorrhagic fever (CHF) and suggested that is transmitted by  disease . Sporadin Russia ,172. Latn Russia . CCHFV iIx. persulcatus ticks collected in the region where the outbreak occurred [Ix. ricinus ticks in Czechoslovakia in 1963 [Orbivirus (family Reoviridae) [Ix. persulcatus, Ix. ricinus, Ix. Pavlovsky, and De. reticulatus ticks varies from zero to 10.1% in different regions of the country [During spring and summer 1962, Chumakov, together with Libíková from the Institute of Virology in Bratislava (former Cžechoslovakia), investigated an outbreak of fibrile illness in the Kemerovo district in western Siberia. Initially, it was assumed that the patients were affected by TBE, but the sera of the patients did not react with TBEV-specific antigen in serological tests. On the contrary, a new virus, named the Kemerovo virus (KEMV), was isolated from the blood of patients. Several strains of KEMV were also isolated from occurred ,175. Sim in 1963 ,177. Basviridae) . The eco country ,180.Hy. marginatum ticks for the first time in the USSR while studying the natural foci of CCHFV in the southern region of Russia in 1964 [From the above, it follows that in the period 1930–1960, arboviruses in the USSR were studied mostly as causative agents of human disease. Examinations of arthropods and vertebrates in the natural foci of important human disease often led to exploring some other arboviruses. For example, Butenko isolated the West Nile virus (WNV) and Dhori virus (DHOV) from  in 1964 . In the California encephalitis orthobunyavirus) and Batai and Batai-like viruses (species Bunyamwera orthobunyavirus) in the genus Orthobunyavirus, family Peribunyaviridae [Alphavirus, family Togaviridae) [Lvov and colleagues isolated more than 500 strains of different mosquito-borne viruses, including viruses of the California encephalitis antigenic group  ,187,188.Ix. uriae ticks, which parasitize on colony-nesting sea birds. In 1969–1974, more than 240 virus strains were isolated from Ix. uriae ticks collected in the nests of sea birds on the coasts and islands in the Sea of Okhotsk, the Bering Sea, and the Barents Sea [Sakhalin orthonairovirus  [Uukuniemi phlebovirus  [Flavivirus, family Flaviviridae) [Great Island virus , have been studied in detail [One of the important subjects of D. Lvov’s research was ents Sea . The isoviridae) . Severalviridae) . The Tyuviridae) . The pren detail ,189,193.Hyalomma spp. ticks collected from sheep or cows in pasture lands [Orthonairovirus (family Nairoviridae) [Many new viruses were discovered by Lvov and his colleagues while exploring the territories of Central Asia and Transcaucasia. They isolated and studied the Issyk-Kul virus (ISKV), which is associated with bats of the family Vespertionidae and their argasid ticks ,195. Newre lands . Some nore lands . Morpholviridae) .In total, during the ecological and virological surveys of the 1970s to 2000s, thousands of strains of different arboviruses were isolated, some of which remain to be classified. Based on the studies conducted by Soviet and Russian virologists, we now know that at least 80 zoonotic viruses, assigned to eight viral families, circulate on the territories of the former USSR . Does thThe concept of viruses developed from the observations of Ivanovsky and Beijerinck of “filterable agents”, with the discovery of the causative agent of tobacco mosaic in the 1890s. Yellow fever virus and dengue virus were the first two arboviruses to be isolated early in the 20th century ,200. TheHirst observed in 1941 that chicken erythrocytes agglutinated in the presence of the influenza A virus and that virus-specific antibodies inhibited agglutination, forming the foundation for the hemagglutination inhibition (HI) test . A decadHistorically, these methods  served as the basis of arbovirus diagnosis for many years, augmenting electron microscopy (see section below), which allowed the visualization of viruses in infected tissues and cell cultures. However, an inherent limitation of the serology-based assays has been their inability to determine whether antibodies in the examined serum were the result of a recent or past infection. This conundrum was solved by determining whether antibodies were IgM (recent infection) or IgG (past infection), using the enzyme-linked immunosorbent assay (ELISA) . The intOverall, although identification of pathologic agents through serologic assays is quite straightforward, there are instances where accurate identification may not be possible due to cross-reactivity. For example, cross-reactivity among flaviviruses poses a challenge in their identification, even when the “gold standard” of PRNT for arbovirus detection is applied, especially in hyper-endemic settings of flavivirus circulation. Several diagnostic labs faced this challenge during the recent emergence and explosive spread of the Zika virus in the Americas. A similar challenge is also common for the serologic diagnosis of bunyavirus infections, which is attributed to their ability to reassort. In this scenario, a novel bunyavirus may be misidentified as a known pathogen due to the presence of the M segment (contributed by the known pathogen), which encodes the immune-reactive envelope proteins (reviewed in ).Since the beginning of modern virology in the 1950s, transmission electron microscopy (TEM) has been one of the most important and widely used techniques for the identification and characterization of new viruses. Two TEM techniques are usually used for this purpose: negative staining on an electron microscopic grid coated with a support film and (ultra) thin section TEM of infected cells, fixed, pelleted, dehydrated, and embedded in epoxy plastic. Negative staining can be conducted on highly concentrated suspensions of purified virus or cell culture supernatants. For some viruses, TEM can be conducted on contents of skin lesions  or concentrated stool material (rotaviruses and noroviruses). For successful detection of viruses in ultrathin sections of infected cells, at least 70% of cells must be infected, and so either high multiplicity of infection (MOI) or rapid virus multiplication is required.Viruses can be differentiated by their specific morphology (ultrastructure): shape, size, intracellular location or, for some viruses, from the ultrastructural cytopathology and specific structures forming in the host cell during virus replication. Usually, ultrastructural characteristics are sufficient for the identification of a virus at the level of a family. In certain cases, confirmation can be obtained by immuno-EM performed either on virus suspension before negative staining or on ultrathin sections. This requires virus-specific primary antibodies, which might be not available in the case of a novel virus. For on-section immuno-EM, OsO4 post-fixation must be omitted and the partially dehydrated sample must be embedded in a water-miscible acrylic plastic . The ultrastructure of most common viruses is well documented in good atlases and book chapters ,234,235 The advent of next generation sequencing (NGS) has expanded the tool kit of the virus hunter. For many years, Sanger-based sequence analysis has been employed in the identification and characterization of viruses ,241,242.The application of NGS to the unbiased mass sequencing and bioinformatic analysis of total nucleic acids extracted from biological samples obtained from a wide range of sources has led to an explosive increase in the number of complete or near-complete viral genomes. For example, a recent study using NGS to sequence the transcriptomes of arthropods representing 70 species from four classes  identified 112 novel viruses, many of which are represented by complete or near-complete genomes . The novBas Congo tibrovirus) [Ekpoma 1 tibrovirus) and Ekpoma virus 2 , and a serological survey indicated that antibodies to these or similar viruses occur commonly in healthy humans in Nigeria [Culicoides spp.) in Australia and Florida [This mass sequencing approach, which has been called viral metagenomics, can also be applied in a more targeted way to identify viruses in clinical cases of diseases of unknown etiology or to survey for potentially novel zoonotic viruses that may represent a significant risk of transmission to humans. Indeed, NGS is being used increasingly in conjunction with real-time PCR as a front-line tool in medical and veterinary settings for rapid detection and identification of exotic or unknown emerging viruses. For example, in 2009, an outbreak of acute hemorrhagic fever occurred in Mangala, Democratic Republic of Congo (DRC), involving three human cases, two of which were fatal. As no positive diagnosis was obtained using real-time PCR for known viral hemorrhagic fevers in Africa, NGS was conducted on acute phase serum collected from the surviving patient, revealing the near-complete sequence of a novel rhabdovirus, Bas-Congo virus  . Althoug Nigeria . However Florida ,262,263. Florida ,264. TheThe availability of a rapidly expanding number of novel viral genomes identified by metagenomic studies has also presented challenges for virus taxonomy—a system for classification of viruses that is administered by the International Committee on Taxonomy of Viruses (ICTV). Should viruses detected only by their nucleotide sequence be classified and assigned to species and other higher taxa  alongside viruses for which we have a viable isolate? The ICTV  was established in 1966 at the ninth International Congress for Microbiology in Moscow, publishing its first report in 1971 . It operWhile the classic tools of virus discovery and characterization  are still widely used, NGS allowed for the rapid identification of an enormous repertoire of viruses, thus exponentially expanding the boundaries of the known virosphere. The resultant genomic sequences allowed for their accurate taxonomic assignments, analysis of their phylogenetic and evolutionary relationships with other viruses, and evaluation of the potential risks they may present to humans and wild or domestic animal populations. Below are a few representative examples of how NGS transformed the known relationships of arboviruses within their respective families.Rhabdoviridae currently comprises 20 genera containing 143 species and one unassigned species [Vesiculovirus, Ephemerovirus, Tibrovirus, Hapavirus, Ledantevirus, Curiovirus, and Sripuvirus) are considered to be arboviruses. Viruses assigned to the newly characterized genus Almendravirus appear to be insect-specific [Tupavirus have been isolated only from vertebrates but may possibly have arthropod vectors. Complete coding sequences are now available for more than 100 other rhabdoviruses, many of which have been isolated from or detected in arthropods, but they have not yet been formally classified. While the classic tools of viral discovery  are still widely used, NGS has played a central role in recent genome sequencing efforts, revealing diversity, not only in the ecology of rhabdoviruses but also in the structural diversity of genome architecture [Rhabdoviruses contain negative-sense (-) single-stranded RNA (ssRNA) genomes. They are amongst the most numerous and diverse of RNA viruses, naturally infecting mammals, birds, fish, reptiles, and amphibians, as well as insects, arachnids, crustaceans, nematodes, and a wide range of plants ,279. Mos species . Of thesspecific . Virusesitecture ,286,287.itecture  and reveitecture .Bunyavirales encompasses 10 families of segmented (-) ssRNA viruses—Arenaviridae, Hantaviridae, Nairoviridae, Peribunyaviridae and Cruliviridae, Mypoviridae, Fimoviridae, Wupedeviridae, Phasmaviridae, and Phenuiviridae—a classification based on structural, genetic, and antigenic characteristics [Bunyavirales is a diverse order with a large number of viruses associated with human, veterinary, and plant disease, as well as being vectored by arthropods  and infecting a wide range of other invertebrates. The recent discovery of Gouléako (GOLV) [Phlebovirus, familyPhenuiviridae, suggesting they are to be assigned to the new genus Goukovirus. In the past five years, NGS has revolutionized understanding of the vast diversity of this order, by allowing genetic identification of previously uncharacterized and unassigned bunyaviruses, which in turn provided a complimentary approach to the gold standard of classification  for a more refined taxonomic classification. Combined, these approaches will be instrumental for enhancing our understanding of their ecologic and geographic distribution, as well as public health impact.According to the current International Committee on Taxonomy of Viruses (ICTV), classification of the order eristics . Bunyavio (GOLV)  and Cumuo (GOLV)  viruses Flaviviridae comprises positive-sense (+) ssRNA viruses. The family contains several serious human pathogens, including dengue, yellow fever, Zika, Japanese encephalitis, West Nile, and tick-borne encephalitis viruses  and the hepatitis C virus (a member of the genus Hepacivirus). Members of the genus Flavivirus, like the alphaviruses , from Anopheles tesselatus in Borneo [Aedes albopictus laboratory colony that originated in Thailand [Aedes albopictus in China [Mansonia ssp. in Mozambique [The family uses see , are a dn Panama , Sabethen Brazil , the La  in Peru , the Lonn Borneo , Aedes fThailand , Xishuanin China , and thezambique .Flaviviridae and were more closely related to flaviviruses than members of other established genera. The Jingmen tick virus (JMTV) is unique in the family Flaviviridae with a four segment positive-sense genome [Rhipicephalus and Haemaphysalis spp.) [Culex ssp. mosquitoes [Toxocara canis, a larva roundworm, and tentatively named T. canis larva agent virus (TCLAV), are distantly related to JMTV and appear to be the first evidence of a flavivirus-line organism in a member of the phylum Nematoda [One unexpected finding was the discovery of segmented viruses that grouped in the family e genome . Traditiis spp.) . This prsquitoes . Additiosquitoes . InteresNematoda . The disThe alphaviruses are a diverse group of (+) ssRNA arthropod-borne viruses that are found on every continent except Antarctica . There wOrbivirus. Interestingly, because of the structure of the genomes of reoviruses and the conserved sequences at the 5’ and 3’ ends of the segments [Seadornavirus, Orthoreovirus,Dinovernavirus, and Cypovirus), all of which are shown in Reoviruses are a diverse family of double-stranded RNA (dsRNA) viruses that infect a wide range of hosts and have a wide range of characteristics. Of the 30 genera, there are only three for which novel viruses have been described using NGS, and the majority of these are in the genus segments , it is rNegevirus, related to genera of mite-infecting plant viruses  in the new family Kitaviridae [Cilevirus, Higrevirus, and Blunervirus, coupled with their heterogenous genome organization and architecture, provides support for the possibility that negeviruses are plant-like viruses that could eventually anchor a new virus family [Newly recognized viruses containing a (+) ssRNA genome have been proposed to form a new genus aviridae ,253. Oriaviridae . Their waviridae , Trinidaaviridae , Côte d’aviridae , Brasil s family .Mesoniviridae, a newly discovered family of (+) ssRNA viruses assigned to the order Nidovirales [Mesoniviridae comprise of a single genus Alphamesonivirus with nine recognized species: Alphamesonivirus 1, including Nam Dinh (NDiV) [Alphamesonivirus 2, including the single isolate of the Karang Sari (KSaV) virus, and the four Bontag Baru (BBaV) isolates sampled in the early 1980s in Indonesia, but only recently characterized [Alphamesonivirus 3, including the Dak Nong virus (DKNV), the three isolates of Kamphaeng Phet (KPhV), sampled in Indonesia and Thailand in the mid-1980s [Alphamesonivirus 4, including the Casuarina virus (CASV), isolated in Australia from Coquillettidia xanthogaster mosquitoes [Alphamesonivirus 5, the African Hana virus (HanaV) [Alphamesonivirus 6 and 7, including Ofaie (OFAV) and Kadiweu (KADV) viruses, isolated in the Pantanal of Brasil from Mansonia sp. mosquitoes, respectively [Alphamesonivirus-8 and 9, including the African Nse (NseV), and Meno (MenoV) viruses [Culex sp. mosquitoes in China.Members of the family ovirales ,319, appovirales ,324,325.h (NDiV) , Cavallyh (NDiV)  viruses;cterized ; Alphameid-1980s ,326; Alpsquitoes ; Alphame (HanaV) ; Alphameectively ; and Alp viruses ; and theAcross several generations, virus hunters have left a profound legacy, both to science and to the broader global community. In the field, their work has often been conducted in difficult, demanding, and sometimes dangerous circumstances. In the laboratory, they have developed and applied tools and methodologies that led to improved diagnosis, prevention, and treatment of viral disease, and still lie at the center of virology today. Others working at more fundamental scientific levels have used their virus isolates as scientific models, opening the door to a revolution in molecular and cellular biology. Yet, their work continues. Viral disease emergence remains one of the most serious threats to humanity, with potentially devastating social and economic consequences. Recent examples, such as SARS, MERS, henipa-, Ebola, and Zika viruses, illustrate the threat that unidentified or poorly characterized viruses can, and almost certainly will, continue to present. Equally devastating emerging diseases of livestock, fisheries, and crops threaten food production and livelihoods. As history has repeatedly shown us, technological revolutions have been often accompanied by periods of progressive disinvestment in training for the eclipsed technologies of the past, and in our case, classical virology. The recent emergence of the Zika virus in the Americas has reinforced the notion that traditional methods of virus discovery and new technologies offer a complimentary toolkit, especially in resource-poor settings that can be seamlessly integrated in the service of public health. It is our hope and expectation that new generations of virus hunters will appreciate the legacy of their predecessors and complement the power of NGS, metagenomics, and bioinformatics, not only to advance viral evolutionary biology, but to understand and limit the potential impacts of emerging viral disease.


SLC37A4 mutation, often require prolonged granulocyte colony stimulating factor (G‐CSF) therapy to prevent recurrent infections and hospital admission. There has been emerging evidence that prolonged exposure to G‐CSF in cases with congenital neutropenia other than GSD is associated with transformation to myelodysplastic syndrome/acute myeloid leukemia.Congenital neutropenias due to mutations in ELANE, SBDS or HAX1 or in the setting of glycogen storage disease (GSD) which is caused by  Deficiency in the translocase leads to intracellular accumulation of glycogen and disturbed glycogenolysis and gluconeogenesis.Glycogen storage disease (GSD) is an autosomal recessive disorder caused by mutations in the Clinical features of type Ib GSD include hypoglycemia, lactic acidosis, hyperuricemia, hyperlipidemia, hepatomegaly, inflammatory bowel disease, and growth retardation.We present our experience in the management of a young female with AML on a background of type Ib GSD and chronic neutropenia treated with G‐CSF therapy for almost 25 years. We highlight the plausible association between G‐CSF and leukemia risk, in a condition that may inherently be associated with an increased risk of AML, and to describe the supportive care required during induction chemotherapy for a patient with this rare metabolic condition.29/L, which was felt to be from hypersplenism. Two years later, she developed recurrent oral ulcers, skin infections, weight loss, and low‐grade fevers. A CBC at that time showed Hb 112 g/L, WBC 2.4 × 109/L, neutrophils 1.40 × 109/L, and platelets 107 × 109/L. Three months later, she developed pancytopenia with circulating blasts. A subsequent bone marrow aspiration demonstrated dysmegakaryopiesis, dyserythropiesis and 5% blasts with auer rods  of the thorax to investigate unilateral pleuritic chest pain showed a pulmonary macro‐nodule with ground glass opacity and cavitation, suspicious for invasive aspergillosis infection. Fiberoptic bronchoscopy and bronchoalveolar lavage was planned, requiring the patient to fast overnight prior to the procedure. Considering her meticulous metabolic requirements, our team consulted the metabolic genetics team. Upon fasting, she was started on 10% dextrose in water with 0.45% saline (D10W‐0.45%NaCl) by intravenous infusion with serum glucose monitoring every 3 hours. The IV fluids were titrated to maintain glucose level between 4 and 6 mmol/L (70‐110 mg/dL). UCCS was reintroduced 2 hours following the procedure, and she was weaned off IV fluids without incident. Results of the BAL came back negative. Due to subsequent progressive worsening of the pulmonary cavity, a CT‐guided biopsy was performed and the pathology report revealed infiltration with myeloblasts: MPO+, CD117+ (30%), CD68+ less than 20%, CD34−, with no fungal elements. The bone marrow, however, did not show evidence of progression. She was diagnosed with a granulocytic sarcoma and the decision was made to proceed with induction chemotherapy using daunorubicin and cytarabine (3 + 7) prior to allogeneic SCT.To address the unique metabolic considerations, the metabolic genetics team provided us with a protocol Table . IV mediRadiologic progression Figure A indicat3ELANE, SBDS or HAX1.GSD type Ib is a rare congenital metabolic disorder that manifests with neutropenia and multiorgan dysfunction. The Severe Chronic Neutropenic International Registry (SCNIR) and The French National Severe Chronic Neutropenia Registry have both conducted individual prospective cohort studies where transformation to MDS/AML was documented in congenital neutropenia syndromes including GSD..A literature search identified four published case reports, with ours being the fifth Table . Two werWhile G‐CSF has been the standard therapy for the neutropenia in the GSD Ib population, there is increasing evidence that the bone marrow is not the primary problem. Several studies have demonstrated that bone marrow production of cells is normal, but apoptosis is occurring in the neutrophils resulting in premature death and neutropenia.G‐CSF use in the GSD Ib population has been life sustaining, but it is now apparent that it is not treating the underlying disorder. Until new therapies are available, we recommend vigilant use of G‐CSF in this population. Patients with GSD appear to have a higher rate of complications from G‐CSF compared with other populations. Hypersplenism is almost universal even with low dosing, and the incidence of hematologic malignancies may be higher.In addition to contributing to the growing literature on malignancies in GSD, this case report demonstrates the meticulous supportive care required for these patients. The stress of chemotherapy can induce a metabolic crisis, and strict attention to the metabolic requirements of patients with GSD is crucial to avoid hypoglycaemia and lactic acidosis.Dina Khalaf: first author, wrote up the manuscript, performed literature review and took the lead in circulating the draft among the co‐authors, obtained an informed consent form the patient, as well as submission for publication.Heather Bell is a clinical dietician who collaborated with Drs. Faghfoury and Morel in drafting of the metabolic protocol Table .Drs. Vikas Gupta, David Dale, and David Weinstein reviewed and edited the manuscript. Drs. Tierens and Jiong from the hematopathology department provided the slides Figure A,B, and All of the above mentioned authors had substantial input in draft review, provided good follow up and critical edits to the manuscript, have approved the final draft and they agree to be accountable for this work.There are no competing interests to declare.Patient consent has been obtained. There are no observations on their behalf.Approval from the Institutional Committee for Care and Use of Laboratory Animals.


Synthetic oligonucleotides (oligos) are important tools in the fields of molecular biology and genetic engineering. For applications requiring a large number of oligos with high concentration, it is critical to perform high throughput oligo synthesis and achieve high yield of each oligo. This study reports a microreactor chip for oligo synthesis. By incorporating silica beads in the microreactors, the surface area of the solid substrate for oligo synthesis increases significantly in each microreactor. These beads are fixed in the microreactors to withstand the flushing step in oligo synthesis. Compared to conventional synthesis methods, this design is able to avoid protocols to hold the beads and integrate more microreactors on a chip. An inkjet printer is utilized to deliver chemical reagents in the microreactors. To evaluate the feasibility of oligo synthesis using this proof-of-concept synthesizer, an oligo with six nucleotide units is successfully synthesized. The four-step, solid-phase phosphoramidite cycle is the standard method for current oligo synthesis5. With the initial phosphoramidite nucleotide fixed on a solid substrate, the target oligo is synthesized by coupling new nucleotides. To achieve high product yield, controlled pore glass (CPG) is widely employed due to the significant enhancement of surface area. As the oligo synthesis is a cyclic process, the CPG is maintained in physical separated columns while the chemical waste between adjacent reactions is flushed away. The nature of this process minimizes the cross contamination between columns and leads to high quality products. This column-based process can typically automate 96–1536 oligos synthesis in parallel6.Synthetic oligonucleotide (oligo) serves as a unique tool to explore molecular biology, protein engineering, and genetic diagnosis9. Microarray-based oligo synthesis is well known for the extremely high throughput that can be as high as tens of thousands of oligos per chip10. In particular, oligo synthesis is performed at specific locations on the surface using light-activated reactions, inkjet printing technology, or electrochemical reactions16. The concentration of individual oligos is relatively low  and an amplification step ) is required for further applications21. To enhance the synthetic amount of each oligo, engineering designs, including paper-based devices, engineering filters, microfluidic valves, and optical tweezers, have been developed to generate in situ micro-columns to increase the surface area for the oligo synthesis27. However, these techniques do not fully address the challenge for simply and practically oligo synthesis.Microfluidics provides opportunities to achieve high throughput oligo synthesis at low costIn this study, we develop a microreactor chip for oligo synthesis that integrates fixed silica beads in the microreactors. The microreactors are fabricated on a silicon wafer using an anisotropic etching process. Silica beads are self-assembled in the microreactors to increase the surface area. The surface area of each microreactor with beads is 71 times more than an empty microreactor. As oligo synthesis occurs on the surface of the beads, the surface area is critical to the product yield. These beads are sintered and fixed in the microreactors, creating high mechanical strength bonds among the beads and between the beads and the microreactor walls. This design is able to retain the beads during flushing processes between reactions in oligo synthesis. An inkjet printer is utilized to deliver chemical reagents in the microreactors. As a proof-of-concept, an oligo with six phosphoramidite monomers is successfully synthesized using the proposed platform.The oligo synthesizer has been developed using a microreactor chip and an inkjet printer Fig. . The chi30. The dry film was lift-off from the substrate by applying acetone for 1 min to curve up the film. The extra beads between adjacent microreactors were removed due to lift-off of the bottom dry film  was obtained from Dow Corning.The silicon wafer is in (100) crystal direction and 400 μm in thickness. The dry film (Dupont Riston FX530) was obtained from Dupont. The thickness of the dry film is 30 μm. The silica beads were purchased from Unisize Technology (Changzhou) Co., Ltd. The diameter of the beads is 6.3 μm. To avoid cracks during the following sintering process, the beads were pretreated at 600 °C for 4 hours to remove surface physisorbed water before assembly in the microreactorThe inkjet printer  was purchased from Epson. This printer has six sets of identical piezoelectric printer heads that are able to independently deliver reagents through corresponding cartridges. The compatibility of the chemical reagents and the printer was examined by successfully printing acetonitrile (Sigma) stored in the black cartridge.To achieve alignment between the printed reagents and the substrate , the substrate was immobilized on a Teflon frame. By tuning the height of the Teflon frame, the gap between the substrate and the printer head was reduced to approximately 2 mm. This reduces lateral movement of the printing droplet and facilitates accurate printing. To mimic the washing step in oligo synthesis, the reagents in the microreactor can be collected from the backside of the chip that was placed on a Teflon frame and connected with a vacuum pump.2SO4) and hydrogen peroxide (H2O2) at a ratio of 3:1, at 100 °C for 1 hr. Then, the beads were subsequently aminated in a solution of (3-Aminopropyl)triethoxysilane (ATPES) and toluene at a ratio of 1:10 at 50 °C for 16 hr. Next, a spacer was bonded on the amino group. The spacer was prepared with 1-hydroxybenzotriazole (HOBT), 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), N,N-Diisopropylethylamine (DIPEA) in N,N-Dimethylformamide (DMF). The final concentration of the above four chemicals were 30 mM. This reagent was activated at 28 °C for 5 min before use. The beads were treated in this reagent at 28 °C for 3 hr. Finally, a linker was bonded to facilitate the product collection after the synthesis process. The linker was thoroughly mixed with 4-dimethylaminopyridine (DMAP), N,N-Dimethylformamide (DMF), and dichloromethane (DCM). The amount was 0.15 mmol, 0.03 mmol, 2.25 mL, and 0.75 mL, respectively. This reagent was mixed with dicyclohexylcarbodiimide  at a ratio of 10:1 (v/v). The beads were treated in this reagent at 37 °C for 24 hr.A chemical surface treatment was applied to initiate chemical bonding on the sintered beads for oligo synthesis. The detailed surface treatment is illustrated in the following reactions Fig. . First, The chemicals for oligo synthesis involve deblocking, capping, and oxidation reagents, and phosphoramidite monomers. The deblocking reagent was trichloroacetic acid (TCA) in dichloromethane (DCM) , capping reagents contained capping A and capping B , phosphoramidite monomers were phosphoramidite monomers with tetrazole , and oxidation reagent was iodine (0.015 M) in water/pyridine/THF . The capping A was acetic anhydride/pyridine/THF ) and capping B was N-methyl imidazole in acetonitrile . The washing reagent was acetonitrile. The reagents to cleave product from the silica beads was aqueous ammonium hydroxide and aqueous methylamine .Oligo synthesis was performed with this microreactor chip. After the surface treatment, 10 μL deprotecting reagent was pipetted to the single microreactor to remove the dimethyl terephthalate (DMT) and initiate active hydroxyl bonds on the silica beads. This reaction was set to 1 min. Target phosphoramidite monomer was printed into the microreactor to couple on the active hydroxyl bonds. In this case, the dG and dT were delivered from the black and yellow cartridges. The target was printed 40 times. This process was performed by printing 5 times of a Microsoft PowerPoint slide that consisted of 8 same patterns . This process took <15 s. To address the potential nozzle clogging issue, the printer heads were cleaned using the printer control software at the beginning of the reagent delivery process. The reagent occupied the distribution microchannels among the beads and the chemical reaction was accomplished on the beads surface. The reaction was 2 min after the printing. There was no significant evaporation observed in this process. It might be attributed to the short time and the capability of reagent preservation in the microchannels. The capping process was subsequently performed by pipetting 10 μL capping reagents in the microreactor to esterify failure sequences and prevent them for the following reactions. This step was set to 1 min. The phosphite internucleotide linkage was oxidized to the phosphotriester by pipetting 10 μL oxidation reagent to the microreactor. This step was set to 1 min. Flushing process was repeated 3 times between adjacent reactions using 100 μL acetonitrile. Repetitions of this four-step process with different phosphoramidite monomers produced the oligonucleotide with specific sequence. All the synthesis processes were performed in a nitrogen dry glove box. The oligo product was cleaved from the beads by immersing the chip in cleaving reagents at 60 °C for 2 hr. The product was purified with desalting and characterized with MALDI-TOF MS.Supplementary information- An oligonucleotide synthesizer based on a microreactor chip and an inkjet printer


Lime was applied at the rate of 250 kg (Ca(OH)2) ha−1, while SSP was applied at 30 kg P ha−1. The trial was carried out in a screen house with the treatments arranged in randomized complete block design. Results obtained show that the performance of SAMFIX 286 inoculated plants did not significantly (p < 0.05) differ from that of the un-inoculated treatment. Application of lime significantly increased root dry weight, shoot dry weight, nodule number and dry weight by 42.5%, 35.3%, 65.6% and 50%, respectively. Nodulation was significantly (p < 0.05) increased by SSP. The complimentary effect of lime with SSP significantly increased total shoot N concentration. Similarly, combined inoculation of SAMFIX 286 with lime and SSP increased N concentration by 31.9% and N derived from atmosphere (Ndfa) by 16.3% compared to the un-inoculated treatment. Inoculation of SAMFIX 286 with SSP was also effective on Ndfa by cowpea. It was concluded that lime and SSP were good combination with native rhizobia strain in improving cowpea nodulation and biological N2 fixation.The complimentary effects of a native rhizobia strain (SAMFIX 286), lime and single superphosphate (SSP) as components of ISFM were evaluated on the biomass, nodulation and N Low soil fertility is increasingly recognized as a fundamental biophysical cause for declining food security among small-farm households in sub-Saharan Africa  is one of the most important edible grain legumes in Africa contributing to food security and maintenance of environment for millions of small-scale farmers in sub-Saharan Africa (SSA)  of IAR. Field S13 is located on longitudes 007036.865′ E and latitudes 11011.018′N with an altitude of 701 m above sea level. On the basis of cropping history, it has been put into continuous cultivation with different mixtures of cowpea, groundnut and soybean over 20 years.The trial was carried out in 2014 in a screen house at the Institute for Agricultural Research (IAR), Ahmadu Bello University (ABU), Samaru, Zaria located in the northern Guinea savanna (NGS) zone of Nigeria within longitudes 0072 within an area of 100 m2. The sampling depths were 0–5 cm for microbial analysis, 0–20 cm for analysis of soil physical and chemical properties and the screen house experiment. The soil samples were bulked, air dried and sieved through 2 mm mesh for routine soil analysis and 4 mm mesh for screen house experiments. Organic carbon (C) and total nitrogen (N) were determined from soils sieved through 0.5 mm mesh. For sowing in the screen house, experimental pots (12 cm in diameter) were filled with 10 kg of soil each.Soil samples were randomly collected using an auger at every 10 m−1 for lime, 24 to 30 kg P ha−1, and 17 to 25 kg K ha−1) of current agronomic practices in the region. Briefly, lime (Ca (OH)2) was applied at 250 kg ha−1prior to sowing, single superphosphate (SSP) at 30 kg P ha−1, and muriate of potash (MOP) at 17 kg K ha−1 at sowing. Factorial combination was used to allot each treatment and treatment combinations to their respective pots  for 10 s, and 5% sodium hypochlorite (NaOCl) for 3 min and rinsed six times with sterile water. Distilled water was used to irrigate the pots throughout the experiment in order to reduce the nutrient load especially nitrates that may be supplied to the plants if tap water is used. The tap water could also be a source of high chlorine which is toxic to rhizobia. Five seeds per pot were sowed and later thinned to two seedlings per pot at 2 weeks after sowing.The application of lime was done prior to sowing. Seeds were surface sterilized prior to sowing by using 70% ethanol (C−1 to 5−6)  was used to estimate the number of viable native rhizobia present in the experimental soil using the plant infection method. Hydroponic culture using Broughton and Dilworth N-free plant nutrient solution  serial dilutions of the native Rhizobium inoculant was made by adding 1 ml of yeast mannitol broth (YMB) culture to 9 ml of sterile water. 0.01 ml of the aliquots were transferred aseptically to dried yeast mannitol agar (YMA) plate having eight equal sectors being radially marked off on the outside bottom of the Petri dishes. The aliquots were allowed to dry by absorption into the agar. Thereafter, the plates were inverted and incubated for five days at 26–28° C to allow for the growth of rhizobium colonies. At the end of the procedures, the CFU count was calculated as shown in Eq. The MPN, expressed as colony forming units (CFU) per ml of inoculants was determined using the drop plate method as described by Woomer et al. . Ten-folPlants  were harvested at 8 weeks after sowing, early period of the reproductive growth stage, for tissue analysis. Total N was determined by using the macro Kjeldahl digestion method as described by Bremner and Mulvaney .2-fixing reference crop in order to estimate the amount of N fixed and percent N derived from BNF. Both the leguminous and maize crops were planted with same soil and under identical conditions in the screen house. After harvesting, the shoots were oven dried and analysed for total N to quantify N fixation. The amount of N fixed and percent N derived from BNF are estimated with the Eqs. Alongside the leguminous crop, maize was planted as a non-NP < 0.05).Analysis of variance (ANOVA) was done using General Linear Model (GLM) procedure of SAS 9.3 Software SAS . Means wRhizobia inoculant used in this study. The particle size distribution (PSD) showed that the textural class of the soil was Sandy Loam according to the United State Department of Agriculture Research Services  in Nigeria was used in grading the inoculants as high or low in quality. Thus, SAMFIX 286 was graded as high quality inoculant.The results obtained from chemical analysis of the experimental soil Table  showed tces SPAW . The mosg−1Table . Analysip < 0.05) improved the nodule number in the presence of lime compared to SAMFIX 286 without lime and the un-inoculated control with and without lime. This could be related to the pH sensitivity of the native rhizobia strain (SAMFIX 286). The native rhizobia strain was collected in northern Nigeria but not on Field S13 where the present study soil was collected. However, the inoculation of SAMFIX 286 alone or with SSP and/or lime had no significant effect on biomass and nodulation whereas, application of lime had significant (p < 0.05) effect on both parameters (Table p < 0.05) increased nodulation, i.e. nodule number and dry weight.The only significant interaction was found between inoculants and lime, and for only nodule number Fig. . Inoculap < 0.05) improved % Ndfa compared to SAMFIX 286 without SSP  was less tolerant to low P conditions compared to other indigenous strains and thus, did not improve plant growth. Importantly, over the single effect of the treatments, the synergistic relationship between the treatments was found to be very important in improving the growth and yield of cowpea. It was concluded that lime and SSP were good combination with the native


Polycyclic aromatic hydrocarbons (PAHs) are toxic, mutagenic and among the most damaging chemical compounds with regard to living organisms. Because of their persistence and wide distribution removal from the environment is an important challenge. Here we report a new Nano container matrix based on the deep sea archaea-derived RHCC-Nanotube (RHCC-NT), which rapidly and preferentially binds low molecular weight PAHs. Under controlled-laboratory conditions and using fluorescence spectroscopy in combination with X-ray crystallography and MD simulations, we quantified the real-time binding of low molecular weight PAHs (2–4 rings) to our substrate. Binding coefficients ranged from 5.4 ± 1.6 (fluorene) to 32 ± 7.0 μM (acenaphthylene) and a binding capacity of 85 pmoles PAH per mg RHCC-NT, or 2.12 μmoles in a standard 25 mg sampler. The uptake rate of pyrene was calculated to be 1.59 nmol/hr∙mol RHCC-NT (at 10  C). Our results clearly show that RHCC-NT is uniquely suited as a monitoring matrix for low molecular weight PAHs. They are extremely restive molecules that can persist in the environment due their hydrophobicity and low water solubility4. As a result, they are ubiquitous in air, water and soil at varying concentrations depending on proximity to point and non-point sources1. While they are generally lipophilic, some PAHs are more soluble in water and are highly toxic to aquatic biota, including fish and invertebrates5. Specifically, PAHs with 3–5 aromatic rings are the most important source of chronic toxicity to aquatic organisms exposed at e.g. oil spill sites7. Because of the wide distribution of PAHs, it is important to understand natural and anthropogenic sources of these compounds and to monitor their baseline concentrations in the aquatic environment8.PAHs are a class of organic compounds that consist of two ore more fused benzene rings forming ring-shaped super molecules. PAHs are important drivers of chronic toxicity to aquatic organismsStaphylothermus marinus is a heterotrophic hyperthermophilic archaea that requires elemental sulfur for growth. The ability of S. marinus to sequester elemental sulfur from its environment relies in part on the Right Hand Coiled-Coil Nanotube (RHCC-NT), a protein fragment in the surface layer of the microorganism. The RHCC-NT encapsulates elemental cyclo-octasulfur in a hydrophobic cavity with a diameter of ~8.4 Å, where it is then available for metabolism. These cavities, adapted to capture the small, hydrophobic cyclo-octasulfur are also of a suitable size to encapsulate low molecular weight PAHs9. Due to the extreme conditions of its origin the RHCC-NT can function with a wide range of temperature and chemical stability, and would easily maintain its fold and binding capacity in aquatic environments surrounding hydrocarbon extraction and transportation sites.Discovered growing on the periphery of deep-sea hydrothermal vents, Here we present an in-depth characterization of RHCC-NT and its unique uptake properties for PAHs Fig. . SpecifiKd = 17 ± 1.4 µM  anthracene were also tested Figure , they dier Kd = 3 ± 7.0 µM12. In comparison to the emission intensity of Band I at 373 nm, the intensity of Band III at 384 nm is significantly enhanced in hydrophobic environments. In contrast, the intensity of Band I is significantly higher than that of Band III in polar environments. The ratio of the fluorescence emission intensities of Band I/III is defined as Py-value14 and can be employed to detect polarity in the vicinity of the probed microenvironment. Our findings clearly demonstrate a Py shift from 1.7288 (RHCC-NT:Pyrene time = 0: (II/IIII = 1067230/617334) to Py = 0.6395 (RHCC-NT:Pyrene time = 75 hrs: (II/IIII = 834882/1305448). Figure ca. 1 min (data not shown).To probe the uptake kinetics of low MW PAHs, we examined the fluorescence characteristics of pyrene in solvent in comparison to RHCC-NT bound pyrene Fig. . As the vs liganded structures, there is no major conformational rearrangement at the protein backbone nor of individual side chains detectable. The large-sized cavities act as fixed size container, which are able to uptake large hydrophobic ring-like structures. Remarkably, in the unliganded RHCC-NT structures, the cavities are filled with water clusters (pdb:1FE6)15. As these cavities are hydrophobic in nature, it is expected that the presence of polar waters in the cavities is unfavorable relative to a less hydrophilic moiety. Indeed, both naphthalene and pyrene completely displace the water molecules within the binding pocket  = −10.0 ± 2.5,ΔGsolv(phenanthrene) = −16.2 ± 2.5, ΔGsolv(pyrene) = −18.91 ± 2.5 and show the same systematic trend through the series naphthalene → phenanthrene → pyrene. The calculated transfer free energies absolute binding energies in strongly hydrophobic systems. In particular, the absolute binding free energy of isobutyl-methoxy-pyrazine (IMBP) bound to porcine odorant binding proteins (OBP) is computed to be ~−67 kJ/mol17 compared to the experimental value of −38.5 kJ/mol (similar to the discrepancy in the current investigation) and the absolute binding free energy of FKBP, a protein from the immunophilin group, bound to several ligands has been shown to be systematically more negative than the experimental values by ~13.4 kJ/mol18. By contrast, the application of MCTI to more hydrophilic systems yields much better agreement. This behaviour appears to be independent of the choice of force field, sampling time or implicit versus explicit solvent models and has been attributed to the higher mobility of ligands in hydrophobic cavities although, thus far, no successful protocols have emerged for improving the discrepancy. Nevertheless, the calculated binding free energies do correctly reproduce the experimental systematics with The computed values of the transfer free energy for the three PAH ligands are listed in Table entclass1pt{minimaThe MD simulations predict that phenanthrene is also stable in both cavities, in spite of its lower degree of symmetry, with a transfer free energy intermediate between that for naphthalene and pyrene in cavity 2 and equal to that for pyrene in cavity 3. The discrepancy between MD simulations and experiment in the case of phenanthrene is most likely the result of a limitation on the fluorescence technique. Other techniques (GC-MS/MS) clearly show an uptake of all 2, 3 and 4 ring PAHs by RHCC-tetrabrachion, including phenanthrene, fluoranthrene and benz(a)anthracene.FBHW − restrained, λ = 0 trajectories frame-by-frame showed that, on average, the plane of all three PAH ligands maintained an orientation transverse to the central cavity of the RHCC-NT throughout the entire trajectory, as observed experimentally, with angular fluctuations Δθ ~± 10  relative to the initial orientation. However, all three PAH ligands exhibited rapid transitions between discrete orientations within the transverse plane, with an angular separation of Δϕ~30 .A visual examination of the fully interacting, 3 and allow for the uptake of PAHs up to 4-rings in size, while binding was not detected for any 5 or higher ring PAH systems. This suggests that such molecules may be too large to fit inside the cavities.Using fluorescence probe techniques, X-ray crystallography, and Molecular Dynamics Simulations we have demonstrated that the RHCC-NT can bind low MW PAHs with low equilibration times and high selectivity. To our knowledge, this is the first study showing that a proteinaceous Nano container can encapsulate PAHs. The cavities of RHCC-NT are ellipsoidal in shape with a volume of ~380 Å20. However, most recent studies have shown that RHCC-NT and COMPcc can uptake a number of different cargos, including vitamin D321, fatty acids22, S8 crowns9 and metallic Hg cluster23. Whereas COMPcc shows a very dynamic behavior and extensive “breathing effect” during uptake of the ligand via the open N-terminal chamber, the RHCC-NT is a highly rigid homo-tetramer, which seems to allow uptake of hydrophobic cargos just via the inter-helical space. The closed storage cavities of RHCC-NT are very rigid and do not show any sign of significant structural re-arrangements upon binding. A common feature for both systems is that strictly hydrophobic interior channels are releasing water clusters upon hydrophobic ligand binding.Coiled coils are mostly known as strict oligomerisation motifs which function as entropic clamps to oligomerise target systems and to increase the local copy number of protein domains24. The crystal structure of human P450 1A2 reveals a compact, highly adaptive active site pocket for positioning and oxidation of the relatively large planar disk-shaped structures25. In general, these metabolites are considerably more toxic than the PAH precursor molecules. There is a wealth of studies published describing the bioremediation of PAHs by pollutant-degrading microorganisms26. However, many environmental factors  play a crucial role in these processes and are difficult to control.The genotoxoicity of PAHs is linked to metabolic activation via cytochrome P450 family enzymes into highly reactive electrophiles interacting with DNA sites9, however the Nano container can also reduce and store metallic metal clusters23. Moreover, RHCC-NT can be seen as a new protein-based matrix for removing PAHs and heavy metals from water. Using fluorescence probe techniques and X-ray crystallography we have demonstrated that RHCC-NT provides many advantages over traditional Passive Sample Devices for low MW PAHs. With low equilibration times and selective binding, the biodegradable RHCC-NT is ideally suited for determining changes in PAH concentrations at an oil spill site. This is critical as proportions of the toxic 3–5 ring PAHs changes rapidly at a spill site due to weathering and traditional PAH PSD devices take too long to reach equilibrium for any meaningful short-term measurements. In addition, our RHCC-NT matrix is extremely robust and because it is not susceptible to chemical changes because of temperature and/or pH it has the potential to be used in a wide range of aquatic environments. Further work is ongoing in our group to evaluate the performance of the RHCC-NT in the field.In conclusion, we have successfully studied the unique encapsulation of low MW PAHs in Nano containers of the archaea RHCC-NT. As shown in a most recent study, the large-sized cavities of RHCC-NT are not only able to store elemental sulfur S8 crownsIndividual PAHs  were purchased from AccuStandard  as 1000 µg/µL solutions in methanol. Ethanol (99.8% HPLC grade), sodium chloride (CAS No. 7647-14-5), sodium dihydrogen phosphate monohydrate (CAS No. 10049-21-5), disodium hydrogen phosphate (CAS No. 7558-79-4), ammonium sulfate (CAS No. 7783-20-2) and Lysogeny broth (LB) (CAS No. 69-52-3) were purchased from Thermo Fisher Scientific. Antifoam 204 (A6426), IPTG (CAS No. 367-93-1), glucose (CAS No. 50-99-7), guanidine hydrochloride (CAS No. 50-01-1), and imidazole (CAS No. 288-32-4) were obtained from Sigma-Aldrich.E. coli BL21 (DE3) freshly transformed with pET-15b.his6-TCS-RHCC plasmid and incubated at 37 °C, 220 rpm. Once the OD600 value of inoculum was 30 (after 10 hrs) the bioreactor was inoculated with 500 mL of inoculum . High density E. coli growth in the 15 L New Brunswick BioFlo 115 (Eppendorf) was achieved using a fed-batch fermentation method. Ten liters (working volume) of LB containing 100 mg/L ampicillin was used as the initial fermentation medium. Temperature, pH, and dissolved oxygen were kept constant at 37 °C, 7.5, and 30ppm during the experiment, respectively. Antifoam 204 was added only when needed (between 12–18 hrs of fermentation). The inducer IPTG was added to the media after 9 hrs of fermentation (OD600 = 30) at a final concentration of 1 mM. Samples were taken periodically to monitor the cell growth and glucose concentration. Glucose concentrations were measured using a glucose meter . Feeding fermentation medium with 20% glucose solution was initiated when the glucose concentration dropped below 2 g/L, which occurred at 6 h of cultivation. Twenty hours from the start of fermentation, 3 L of medium containing cells removed from the bioreactor and replaced with the same volume (3 L) of LB containing 100 mg/L ampicillin. After 28 hrs of cultivation, cells were pelleted for protein purification. RHCC-NT was purified as described previously. Briefly, pelleted cells are suspended in buffer containing 6 M guanidine hydrochloride and lysed via sonication. After centrifugation  to remove cellular debris, the lysate is passed through a cobalt affinity column and washed with resuspension buffer. The RHCC-NT is eluted with buffer containing 200 mM imidazole. The polyhistidine tag is then removed by thrombin cleavage. A cartoon showing the amino acid sequence is presented in Figure The inoculum was grown in Lysogeny broth (LB) containing 100 mg/L ampicillin as selective pressure. Two 2.5 L shake flasks each containing 500 mL of LB medium were inoculated from 2 quartz cuvette. The obtained fluorescence data was used to calculate the binding constant Kd assuming that each PAH binds to one of two equal bindings sites in the RHCC-NT, cavity 2 or 3. The fraction of PAH bound and the concentration of unbound RHCC-NT was determined based on the following equation:F is the experimental fluorescence signal of each sample, F∞ is the fluorescence of the PAH at infinite protein concentration, F0 is the fluorescence of the free PAH in solution, Bmax is a constant and equal to 1. The y values thus obtained were plotted as a function of free RHCC concentrations. The plotted data were then fit to Equation . Excitation wavelength was set to 275 nm for naphthalene, 300 nm for acenaphthene, 350 nm for anthracene, 334 nm for chrysene and pyrene, 290 nm for acenaphthylene and fluorene. Excitation and emission slits were set to 1 nm band pass resolution. All samples were measured at 20 °C in a 10 × 3 mmThe time-scale assay was set-up by addition of pyrene to a 35-fold molar excess of RHCC in 20 mM sodium phosphate solution buffered at pH 7 and adjusted to 150 mM ionic strength. Both solutions were equilibrated to 10 °C prior to the assay. Data was collected on a Fluorolog-3 Horiba Jobin Yvon Spectrofluorometer . Excitation wavelength was set to 334 nm; emission was monitored at 384 nm; excitation and emission slits were set to 1 nm band pass resolution. Temperature was kept at 10 °C and controlled with a water bath throughout the experiment. Data was acquired every 1 minute for the first 25 minutes, following by 30 minute intervals until fluorescence signal plateaued and equilibrium achieved.15. For RHCC-NT:Pyrene the search model was truncated to two of the four chains of the RHCC tetramer. The structures were built manually and refined refined crystallographically using the Coot suite and the Phenix software package. Coordinates and chemical restraints for pyrene were generated using JLigand and eLBOW.RHCC-NT:Naphthalene was formed by adding a 5-fold molar excess of naphthalene from a 50% (w/v) naphthalene solution in 95% ethanol to 11.2 mg/mL RHCC in 10 mM Tris pH 7.5 I = 154 mM (NaCl). Due to the lower solubility of pyrene in water, RHCC-NT:Pyrene was formed by heating 225 mg pyrene with 1.6 mL of 5.0 mg/mL RHCC in 20 mM Tris pH 8.0 I = 154 mM (NaCl) to 70 °C and incubating for 1 week. After cooling to 20 °C, excess insoluble pyrene was removed by centrifugation and the complex concentrated to 11.0 mg/mL for crystallization. RHCC-NT:Naph crystal setups were performed by mixing 2 μL protein and 2 μL reservoir containing 1.5 M ammonium sulfate and 0.1 M Tris pH 8.5 at 277 K with a 1 mL reservoir. Crystals appeared after 1 week. RHCC-NT:Pyrene crystals were grown in a 0.7 μL protein and 0.7 μL reservoir solution drop over 50 μL reservoir composed of 25% PEG 3350 m 0.1 M Bis-Tris pH 5.5, 200 mM NaCl . Crystals were soaked with 15% glycerol in reservoir solution for 1 min prior to flash freezing in LN2. Data were collected on a Rigaku rotating anode MM-007HF diffractometer with a wavelength of 1.54178 Å in 1° wedges at 100 K. Data were processed with XDS and the CCP4-package. Phases were calculated in Phaser using a polyserine RHCC model based on a previously solved RHCC structure (pdb code 1FE6)28 with the Gromos 43a2 force field and an SPC water model. Each complex is representative of a particular ring structure, one ring , two rings (phenanthrene) and three rings (pyrene). In each case, the simulation data were used to compute the standard free energies for transferring the ligand to cavity 2 and cavity 3 of the RHCC-NT from the solvent. For the RHCC-naphthalene complex and the RHCC-pyrene complex, the starting structures for the simulations were the measured X-ray crystal structures 5VKF and 5VH0, respectively. As no structure of phenanthrene bound to the RHCC-NT was available, it was necessary to construct the RHCC-phenanthrene complex by inserting phenanthrene manually into the RHCC-NT cavities in the same position and orientation as the naphthalene and pyrene ligands. The topologies of all three ligands were generated by PRODRG29.Molecular dynamics simulations were performed on three RHCC-PAH complexes  using the GROMACS molecular dynamics simulation package9 of RHCC−NT and only the principle features are reproduced here. Each RHCC-PAH complex was solvated in a rectangular simulation box with dimensions 5.2 nm × 5.2 nm × 9.8 nm containing 7900 SPC water molecules and a set of charge neutralizing Na+ ions . A minimum distance of 1.2 nm was maintained between the surface of the RHCC-PAH complex and the walls of the simulation box. Particle mesh Ewald was employed to treat electrostatic interactions and the cut-off radius for non-bonded interactions was taken to be 1.0 nm. The initial X-ray diffraction structure was energy minimized using the method of steepest descent until convergence was achieved, typically around 40–50 kJ/mol. Solvation free energies were calculated by performing an independent series of simulations on a single energy-minimized PAH ligand in a periodic box with dimensions identical to those listed above.The MD simulation protocols and free energy analysis were based closely on those employed by the authors in a previous investigationT = 300K and P = 1atm with thermostats and barostats of the Berendsen type and time constants τ = 0.1 ps.The system was subjected to three position-restrained equilibrations at 50 K, 150 K and 300 K each for a period of 20 ps. During the 2 ns production simulation, the position restraints were removed and the temperature and pressure of the system (RHCC-NT/cavity ligand/solvent bath) were held at 32 using the thermodynamic paths in Figure 33 with functional form UFBHW(r < r0) = 0; UFBHW(r > r0) = k(r − r0)2 where k = 1000 kJmol−1nm−2, r0 = 0.55 nm.  ΔG1 is the free energy for this conversion. ΔG2 is the free energy cost of removing the FBHW restraint, which includes a correction for the standard concentration. The lower path in Figure G3 = −ΔGsolv is the associated free energy (ΔGsolv is the solvation free energy). The point symmetry group of each PAH ligand would, in principle, require the inclusion of another term in the free energy ΔGsymm = −RT lnσ, where σ is the rotational symmetry number. For phenanthrene with point group symmetry Cv2, ΔGsymm = −RT ln4 = −3.5 kJ mol−1, while for naphthalene and pyrene with symmetry group Dh2, ΔGsymm = −RT ln8 = −5.2 kJ mol−1. However, given the absence of body restraints that would artificially restrict the orientation of the PAH ligands, as mentioned above, this term was omitted from the current analysis. The absolute free energy for transferring a PAH ligand to the cavity from the solvent bath, corrected for the standard state, isThe absolute standard state free energy for transferring each PAH ligand from the solvent into one of the cavities of RHCC-NT was computed with the method of double-decouplingG1 and ΔG3 were calculated with the method of Multi-Configurational Thermodynamic Integration (MCTI)37. Linear scaling of the nonbonded parameters was accomplished using a coupling parameter λ which was varied from λ = 0 (ligand interacting) to λ = 1 (ligand non-interacting) while holding the bond lengths and bond angles fixed. The restraint free energy ΔG2 was calculated analytically9 using:V0 is the standard volume V0 = 1.660 nm3 corresponding to a concentration of 1M = 1molecule/1.660 nm3.The terms ΔdG/dλ for each λ. The mean value dG/dλ was computed using the Gromacs g_analyse routine and the errors were calculated by block averaging. Soft-core interactions with a soft-core parameter α = 0.1 were included to obtain smooth dG/dλ curves and to eliminate discontinuities in the non-bonded parameters and ensure convergence in the limit λ → 1.The minimization and equilibration protocols were repeated for each λ before generating the 2 ns production runs. Structures were saved every 0.5 ps and the accumulated set of 4000 structures was used to compute Supplement Information


Lactobacillus curvatus NFH-Km12, isolated from this unique food, is reported.Kabura-zushi is a traditional Japanese fermented food made from yellowtail, rice, salt, and kōji. In this study, the complete genomic sequence of  Lactobacillus curvatus NFH-Km12, isolated from this unique food, is reported. NFH-Km12 has a 1.9-Mbp chromosome and contains 5 plasmids.Kabura-zushi is a traditional Japanese fermented food made from yellowtail, rice, salt, and kōji. In this study, the complete genomic sequence of  Lactobacillus curvatus is a lactic acid bacterium widely associated with food fermentation   . BecauseLactobacillus de Man-Rogosa-Sharpe (MRS) broth  under 30°C overnight and provided for DNA extraction by the phenol-chloroform method. Whole-genome sequencing of NFH-Km12 was carried out using Illumina MiSeq and Oxford Nanopore minION platforms. For sequencing, from almost 1 µg of extracted DNA, 2 × 300-bp paired-end libraries and around 10-kbp sheared libraries prepared using g-TUBE  were used for MiSeq and minION sequencing, respectively. Hybrid de novo assembly of the 1.41 million reads from MiSeq sequencing and 82,421 reads from minION sequencing that were expected to have ∼540× coverage was performed using Unicycler (L. curvatus strains.NFH-Km12 was cultured by nicycler . The strPRJDB6985, and the assembly has been deposited in GenBank under the accession numbers AP018699 to AP018704.This whole-genome shotgun project has been deposited in ENA under BioProject number


The pneumomediastinum and subcutaneous emphysema can repetitively occur in human H5N6 virus pneumonia. Prompt treatment of this uncommon complication is important for successful rescue of patients with H5N6 virus pneumonia. The pneumomediastinum and subcutaneous emphysema can repetitively occur in human H5N6 virus pneumonia. Prompt treatment of this uncommon complication is important for successful rescue of patients with H5N6 virus pneumonia. After one day, mediastinal and chest wall subcutaneous emphysema occurred as revealed by CT scan  Figure A, which n Figure C, which n Figure D after tn Figure E. The pan Figure F demonstThe spontaneous pneumomediastinum and subcutaneous emphysema is uncommon in avian influenza A human pneumonia. It occurred in a patient with H1N1 virus pneumonia.None declared.ZX and XW: captured the images, coconducted the literature review, and cowrote the paper. JW and QZ: treated the patient, coconducted the literature review, and cowrote the paper. SPJ: analyzed the case, coconducted the literature review, and cowrote the paper. JS: cowrote the paper and coordinated and corrected the paper.