Current diagnostic protocols of acute Zika virus (ZIKV) infection focus on detection of viral RNA in serum or urine using reverse transcription quantitative polymerase chain reaction (RT-qPCR); however, detecting infection can be a challenge, given that 80% of people with acute ZIKV infection are asymptomatic, and the window to detect viremia in serum is short. The ability to extend that window is needed to detect ZIKV at later time points after infection, particularly in high-risk individuals such as pregnant women. We evaluated RNA extraction methods to optimize detection of ZIKV in various body fluids using RT-qPCR as a means of improving the analytical sensitivity of detection. We optimized methods for ZIKV RNA recovery from a number of body fluids by spiking with three varying concentrations of virus, then comparing recovery with that of spiked buffer control. RNA extraction protocols were adjusted as necessary for maximum RNA recovery. Adjustment of the elution step was essential for improved ZIKV RNA recovery from whole blood, saliva, vaginal secretions, and breast milk. Optimal recovery from urine samples required the addition of Urine Conditioning Buffer, and the use of RLT Plus buffer and RNeasy Mini Spin Columns was necessary for RNA extractions from semen samples. Optimized QIAamp MinElute Virus Spin Kit  protocol followed by the singleplex ZIKV RT-qPCR assay provided a reliable method for detection of ZIKV RNA in a variety of biological samples. Improved diagnostics are crucial for timely detection and diagnosis, particularly during pregnancy when the consequences of ZIKV infection can greatly impact the developing fetus. Flavivirus genus, emerged in the Americas in 2015 and is a significant public health concern.3 The virus has been known to circulate with limited reported activity in the Old World since before the middle of the twentieth century.3 It first appeared in the continental New World in Brazil in March 2015, initiating an explosive outbreak that resulted in millions of infections throughout South, Central, and North America, including the U.S. territory of Puerto Rico, within 1 year.3 Most of the clinical cases reported in the U.S. states have been associated with travel to the affected areas.4 Since 2015, more than 5,000 such travel-associated cases have been documented, mostly in 2016. The number of locally acquired ZIKV cases in the United States is about 20 times lower than travel-associated cases, with limited autochthonous transmission confined to Florida and south Texas.4Zika virus (ZIKV), a member of Aedes spp. mosquitoes.3 Humans develop a level of viremia high enough to propagate the transmission cycle.3 Other routes of ZIKV transmission in humans have been documented, including sexual, congenital, breast milk, and blood transfusion.8 Transmission through organ transplantation is a possibility for ZIKV as it was reported for another flavivirus, West Nile virus (WNV).9Zika virus is mosquito-borne, transmitted to humans by a bite of 3 Clinical manifestations of ZIKV infection are typically mild with acute onset of fever with maculopapular rash, arthralgia, conjunctivitis, myalgia, and headache, lasting from several days to 2 weeks.3 In rare cases, ZIKV infection can result in serious complications, including Guillain– Barré syndrome.3 The most severe ZIKV infection complications occur during pregnancy, leading to severe birth defects, death, and poor clinical outcomes in the newborn, with the condition collectively known as congenital Zika syndrome.10 The U.S. Zika Pregnancy Registry reported ZIKV-associated birth defects, including congenital microcephaly, in up to 5% of neonates born to mothers with possible recent ZIKV infection during pregnancy.11About 80% of infections with ZIKV are asymptomatic.3 Therefore, nucleic acid tests on paired serum and urine samples are recommended for the laboratory diagnosis of acute ZIKV infection.2 Detection of viral RNA in sera or urine provides conclusive diagnosis of infection, but the timeframe for it is limited to 1–2 weeks post onset of symptoms because of fast decline of virus presence in serum and urine.3 Tests for ZIKV-specific IgM antibodies in serum are also used as a diagnostic evidence for ZIKV infection and might increase the window for diagnosis to several months, but ZIKV serology is complicated by considerable cross-reactivity with other related flaviviruses, particularly in dengue-endemic areas.2 Considering these diagnostic constraints, it would be ideal to optimize the ability to detect RNA for a longer window of time, particularly in high-risk individuals , or individuals with serious complications such as Guillain–Barré syndrome. We recently reported our laboratory’s experience in detecting ZIKV RNA in saliva and whole blood up to 14 and 81 days post onset of symptoms in one acute ZIKV case, respectively.12 Given these findings, the purpose of this study was to identify viral RNA extraction methods that maximized ZIKV RNA recovery from a given sample type to further strengthen diagnostic protocols for detection of ZIKV in various body fluids.Zika disease onset usually coincides with viremia and viruria.12 plasma was separated and discarded, and the remaining blood cells and a small portion of plasma were mixed. Analogous to the setup and description from a previous WNV study,13 this fraction is referred to as whole blood from this point forward, as it contains all major components of whole blood: erythrocytes, leukocytes, thrombocytes, and a portion of plasma. Urine specimens were spun at 1,000 × g and the supernatant was collected for testing. Saliva swabs and vaginal secretions swabs were obtained with BD BBL CultureSwab Sterile Single Swab (BD), which is free of media or preservatives. Semen specimens were collected in a sterile urine cup, allowed for liquefaction, and used without separation. Breast milk specimens were collected using the subject’s own breast pump, then transferred to a sterile specimen cup.Collection of clinical specimens was performed by standard procedures under a research protocol enrolling suspected or confirmed cases of ZIKV infection in U.S. residents. The studies were approved by Baylor College of Medicine Institutional Review Board, protocols H-30533 and H-39865. Serum specimens were obtained by collection and processing of venous blood in BD SST vacutainers . Whole blood specimens were obtained by collection and processing of venous blood in BD K2EDTA vacutainers (BD). As it is shown that ZIKV remains associated with red blood cell fraction of the whole blood,Zika virus–positive control, strain MEX I-7, was provided by University of Texas Medical Branch World Reference Center for Emerging Viruses and Arboviruses. The strain was isolated in 2016 from mosquito samples collected in Mexico and passaged a total of five times on Vero cells.Ten aliquots of each sample matrix were prepared, with 200 µL volume for liquid samples. AVE buffer, the elution buffer from the QIAamp MinElute Virus Spin Kit , was used as the spiking reference control buffer to generate expected cycle threshold (Ct) values. The aliquots were mixed with 200 µL of the lysis buffer from the kit, which is AL buffer containing 28 ng/µL of carrier RNA. The aliquots were spiked in triplicates with 10 µL of viral cell culture supernatant containing three levels of viral load, and a single aliquot was left unspiked to confirm ZIKV-negative status of the samples from the donors. To obtain high, medium, and low spiking loads, the original viral cell culture supernatant was diluted in culture media so that the reference buffer samples’ final Ct values were in the range of 19–21, 27–29, and 33–35, respectively. Spiked samples were stored at −80°C before proceeding to RNA extraction.To facilitate lysis of 200 µL of packed blood cells specimens, which approximate 400 µL of the original venous blood specimens, a total of 400 µL of AL along with 200 µL of phosphate buffered saline (PBS) were added to whole blood samples . AL for Saliva and vaginal secretions swabs were first incubated in 250 µL of AL/carrier RNA for 10 minutes at room temperature and were then compressed against the inside of the tube to expel the liquid before removing and discarding. Remaining volume of the lysis buffer was about 200 µL, and 200 µL of PBS was added to adjust the total sample volume according to the QIAGEN protocol.14 with elution in 30 µL of AVE buffer. All centrifugations were performed at maximum speed , except where indicated below. Each extraction batch included positive (ZIKV MEX I-7 diluted supernatant) and negative (AVE) extraction controls. Depending on the sample type, three sets of adjustments to the manufacturer’s protocol (Optimization A through C) were shown to be beneficial for ZIKV RNA recovery. The details of the three optimizations are described in the following text and The baseline method of RNA extraction from the study specimens was the QIAamp MinElute Virus Spin Kit (QIAGEN) according to the manufacturer’s protocolg , followed by 1 minute centrifugation at maximum speed. Elution incubation , followed by 1 minute centrifugation at maximum speed. The column was washed and dried per QIAamp MinElute Virus Spin Kit protocol, and the elution incubation step  was added, and the sample was incubated at room temperature for 5 minutes. After adding 1.4 mL of ethanol, the sample was loaded onto the RNeasy Mini Spin Column (QIAGEN) at 900 × Step 14,  was perfg , and pellet was resuspended in a mix of 200 µL AVE, 200 µL AL/carrier RNA, and 25 µL Protease. The sample was further processed starting with Step 4 of the manufacturer’s protocol  of spiking material was added directly to freshly collected urine. Seventy microliters of Urine Conditioning Buffer  was mixed in. Samples were centrifuged at 3,000 × protocol . Two intSupplemental Tables 1 and 2.Additional procedures and reagents/supplies were tested to improve viral RNA recovery from semen and urine samples, including buffers and columns of RNeasy Plus Mini Kit or QIAamp Viral RNA Mini Kit, QIAzol, QIAshredder columns, ATL buffer , Direct-zol RNA MiniPrep Kit (Zymo Research), RNasin Plus RNase Inhibitor , SUPERase In RNase Inhibitor , Protector RNase Inhibitor , ProtectRNA RNase Inhibitor , and Aptima Urine Specimen Transport Tubes . The summary of these experiments is presented in 15 The following controls were included in each RT-qPCR run: six points of ZIKV RNA standard curve (in vitro transcribed oligonucleotide), extraction negative control, extraction positive control, and no template control, all in duplicate.Five microliters of extracted RNA were used in a duplicate 20-µL reaction with TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) on ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). The previously published TaqMan ZIKV 1107 assay was used.Ct values and copies/µL values from a duplicate of RT-qPCR for each sample were used to calculate mean Ct values and mean copies/µL values for each extracted sample. Each triplicate of mean copies/µL values per viral load was used to calculate average concentration in spiked matrix for each level of viral load.Supplemental Table 3).To determine expected Ct values and average concentration in spiked reference control, three sets of 10 spiked reference controls samples  were independently extracted and run on RT-qPCR (one set was run twice). Expected Ct value was calculated as average across all mean Ct values from a duplicate of RT-qPCR for each reference sample of a given viral load. Average concentration in spiked reference control was calculated as average across all mean copies/µL values from a duplicate of RT-qPCR for each reference sample of a given viral load  × 100%.12 was subjected to five freeze/thaw cycles: an aliquot was taken after each thaw cycle and kept at 4°C in AL buffer/carrier RNA until extraction later on the same day. Each freeze cycle was for 20 minutes at −80°C.A known ZIKV-positive serum specimen aliquotSupplemental Table 4). Because most of the tested matrices contained high amounts of cellular DNA/RNA, the extraction protocol was optimized by addition of 5 minutes incubation at 56°C at the elution step. Slow loading on the extraction column  was also added to the protocol to potentially improve recovery of sample nucleic acids. These modifications restored recovery of ZIKV RNA in whole blood, vaginal secretions, and breast milk samples and maintained full recovery in saliva samples .Initial testing of the spiked matrices with RNA extracted by the QIAamp MinElute Virus Spin Kit manufacturer’s protocol yielded poor recovery values for all sample types, except for serum and saliva . A series of alternative extraction protocols were tested for semen specimens and eventually one was selected that yielded 89% of the replicates within two Ct units of the expected Ct values and an average of 29% ZIKV RNA recovery .The extraction protocol optimized as before did not improve the recovery of ZIKV RNA in urine and semen specimens . The UCB protocol was applied to spiked urine specimens from two additional negative control donors. In these urine samples, the protocol did not yield expected Ct values, but the results were still the most favorable compared with other available urine preservation methods tested . However, an abbreviated single spiking load test of two previously available urine specimens, which were stored frozen, showed complete recovery of ZIKV RNA even with the original protocol . These data can be explained by variation in urine properties from donor to donor.Similarly, a number of additional extraction protocols were tested for urine specimens. The most successful protocol for RNA extraction from urine specimens included addition of UCB and was shown to work with 1-mL aliquots of fresh urine, or frozen “as is.” Spiked urine specimens from a negative control donor were tested and showed 89% of the replicates within two Ct units of the expected Ct values, and an average of 51% ZIKV RNA recovery .Spiked urine specimens from negative control donors #1 and #3 were tested to compare addition of UCB to a urine specimen stored frozen as is with the addition of UCB to a fresh UR specimen and then freezing and storing the resulting pellet of nucleic acids. There was no difference between these two urine processing methods . The adjustments also included slow load of the sample onto the column, but the main effect of the RNA recovery restoration was achieved because of the modification of the elution step. The specimen types that benefited from this Optimization A protocol have significant cellular content, and it was assumed that more stringent elution conditions would be required to release high amount of cellular nucleic acids  from the column filter. Despite excellent performance of the original protocol for saliva specimens, optimized extraction protocol would be recommended for this matrix because of potential variation in the amount of cellular DNA/RNA content.Whole blood, vaginal secretions swabs, and breast milk specimens required extended incubation at elevated temperature at the elution step for optimal recovery of viral RNA . Of the several tested methods, these two simple modifications proved to be the best technique of managing extremely high DNA content in semen specimens. Although we could not reach full recovery of ZIKV RNA from this matrix, our result (29%) was very similar to that of a recently published study from the Centers for Disease Control and Prevention (CDC) group in Puerto Rico.16 The investigators showed an average of 31% ZIKV RNA recovery from semen specimens using Trioplex Real-time RT-PCR Assay testing platform, with magnetic beads–based automated sample RNA extraction.Semen specimens required the use of high volume of RLT Plus buffer and RNeasy Mini Spin Columns . However, because the extractions with this protocol are performed from 1 mL of urine sample, compared with 0.2 mL in the original protocol, expected Ct values are decreased by 2.3 units. In that regard, although average recovery was 51%, Ct values of all replicates were actually lower than expected Ct values when extracted from 0.2 mL of urine sample, suggesting that this extraction protocol offers a more sensitive test. We also showed that addition of UCB could be performed before or after freezing the specimen for storage, with the same effect on RNA recovery. However, in our experiment, specimens were kept as is at −80°C only for 1 day. Therefore, storing of urine specimens preserved with UCB right after the sample collection should be preferable because of potential decrease in recovery of low levels of ZIKV RNA when stored as is for prolonged time, as it was shown in a recent study.18 On the other hand, we used the UCB method with archived ZIKV-positive urine specimens (stored for more than 9 months at −80°C without any preservatives), and were able to achieve better Ct values when compared with extractions performed right after specimen collection by the standard protocol ,20 in saliva up to 60 DPO,26 in vaginal secretions up to 14 DPO,28 and in breast milk up to 8 days post delivery.29 A recent Puerto Rican prospective cohort study of 150 symptomatic participants who tested positive by RT-PCR at presentation showed the following medians and 95th percentiles for the time until loss of ZIKV RNA detection: 14 and 54 days, respectively, in serum; 8 and 39 days in urine, and 34 and 81 days in semen.16 In animal models, ZIKV RNA was shown to persist for several weeks after infection in saliva, lymph nodes, and colorectal tissue in cynomolgus and rhesus macaques.25Our long-term goal is to apply these methods for optimal RNA recovery to determine shedding time in different body fluids among a cohort of ZIKV-infected patients to identify opportunities for a longer window of time to diagnose infection. In addition to specimen type and timeframe recommendations for viral RNA tests in laboratory diagnosis of acute infections, cerebrospinal fluid (CSF) and amniotic fluid specimens are also acceptable for testing, but must have a paired serum specimen as recovery of RNA from these alternative specimens can be highly variable. Zika virus RNA has been detected in other body fluids and, for some matrices, far past the acute stage of the disease. This RNA persistence was shown in clinical studies and in animal models.16 However, even the same study showed only about 30% recovery of ZIKV RNA from semen specimens with their assay. Semen, as well as urine and whole blood, are known for their complex biochemical and cellular composition that present challenges for complete and PCR inhibitor-free extractions of RNA or DNA.35 Several studies have compared the effectiveness of different nucleic acid extraction methods from these body fluids,36 rarely showing actual viral RNA recovery compared with buffer control.37Studies that generated aforementioned results on prolonged detection of ZIKV RNA in tissues and body fluids mostly used the generalized RNA extraction protocols provided by the extraction kit manufacturers, without confirming (or at least without reporting) the level of viral RNA recovery for a given specimen type and a given RNA extraction method. One exception is the Puerto Rican cohort study where ZIKV RNA detection was performed by Trioplex Real-time RT-PCR Assay, which is validated by CDC for ZIKV for use with serum; and with urine, CSF, and amniotic fluid only alongside the patient-matched serum specimen.14 Another concern of adding a step of plasma separation in the whole blood testing procedure is an increased risk of contamination of the sample. This risk increase can be alleviated by strict adherence to high standards of laboratory practices.A main limitation to our study was that we have not tested stability of ZIKV in various body fluids as applied to time from collection to time of addition of lysis buffer and following processing of the sample. Our study was focused on effect of a matrix in terms of its composition on RNA extraction and coupled RT-qPCR. Hence, the specimens were first lysed, and then spiked, to differentiate form any effect of processes that might happen in a clinical specimen in its natural state. We were also limited in testing variations of RNA extraction as applied to a single commercially available kit. Although this QIAamp MinElute Virus Spin Kit is widely used in viral research, there are many other RNA extraction kits on the market, including automation equipment/kits used in clinical diagnostic laboratories. Our results might provide guidance in optimizing RNA recovery when using these alternative methods. Finally, our study design was limited in testing whole blood specimens that lack most of the plasma component. Different amounts of plasma in separated versus complete whole blood specimens might change the performance of the RNA extraction kit. However, we would expect the effect to be minimal as plasma is one of the specimen types for which the QIAamp MinElute Virus Spin Kit was developed.Optimal methods for ZIKV detection in various body fluids will allow accurate monitoring of viral RNA presence during the disease progression in the future studies aimed to improving our understanding of natural history of ZIKV infection and duration of viral persistence in various body fluids. Optimized methods can potentially be successfully used with other arboviruses, as improved diagnostics are crucial to timely detection and response to emerging infections such as ZIKV.Supplemental figure


If life can emerge at least once in such a large volume, it is not in contradiction with our observations of life on Earth, even if the expected number of abiogenesis events is negligibly small within the observable universe that contains only 1022 stars. Here, a quantitative relation is derived between the minimum RNA length lmin required to be the first biological polymer, and the universe size necessary to expect the formation of such a long and active RNA by randomly adding monomers. It is then shown that an active RNA can indeed be produced somewhere in an inflationary universe, giving a solution to the abiotic polymerization problem. On the other hand, lmin must be shorter than ~20 nucleotides for the abiogenesis probability close to unity on a terrestrial planet, but a self-replicating activity is not expected for such a short RNA. Therefore, if extraterrestrial organisms of a different origin from those on Earth are discovered in the future, it would imply an unknown mechanism at work to polymerize nucleotides much faster than random statistical processes.Abiotic emergence of ordered information stored in the form of RNA is an important unresolved problem concerning the origin of life. A polymer longer than 40–100 nucleotides is necessary to expect a self-replicating activity, but the formation of such a long polymer having a correct nucleotide sequence by random reactions seems statistically unlikely. However, our universe, created by a single inflation event, likely includes more than 10 A prominent feature of life is the ordered information stored in DNA/RNA, and how such information appeared from abiotic processes is a crucial issue. The RNA world hypothesis8 postulates an early era when RNA played both the genetic and catalytic roles before the DNA-protein world came into being. This is widely accepted due to strong supporting evidence including catalytic activities of RNA, especially its central role in a ribosome. However, a more fundamental and unsolved problem is how an RNA polymer long enough to have a self-replicating RNA polymerase activity  emerged from prebiotic conditions and then triggered Darwinian evolution.In spite of recent rapid development of biology, chemistry, Earth science and astronomy, the origin of life (abiogenesis) is still a great mystery in science9. RNA polymerase ribozymes produced by in vitro experiments so far have a length longer than 100 nt12. Furthermore, formation of just a single long strand may not be sufficient to initiate an abiogenesis event. Instead a pair of identical strands may be necessary if one serves as a replicase ribozyme and the other as a template.A key quantity is the minimum RNA length required to show a self-replicating ability. RNA molecules shorter than 25 nucleotides (nt) do not show a specified function, but there is a reasonable hope to find a functioning replicase ribozyme longer than 40–60 nt13. Reactions at inorganic catalytic sites  may be particularly efficient15. Some experiments yielded production of up to 40-mers of RNA17, which may be long enough to have some biological activities. However, these results have not been reproducible, and only short oligomers of up to 10-mers were produced conclusively in recent experiments, with the abundance rapidly decreasing with the oligomer length20. This trend is also consistent with the theoretical expectation for random adding of monomers (see below). An experimental difficulty is that aggregates may easily be mistaken for polymers, depending on detection methods21.Polymerization of RNA in water is a thermodynamically uphill process, and hence reacting monomers need to be activated. Non-enzymatic reactions of adding activated monomers  to an RNA oligomer have been experimentally studied22, but there is no experimental or quantitative demonstration of this starting from realistic prebiotic conditions. A report23 of experimental production of long polymers (>120 nt) by ligation has been subject to reproducibility and the aggregate/polymer discrimination problem24. A high concentration of oligomers is necessary for ligation to work efficiently, but this may be difficult because oligomer abundance rapidly decreases with oligomer length in polymerization by monomers, even if such a ligase activity exists.It is theoretically speculated that terminal ligation of oligomers may hierarchically produce further longer polymers27 because the chance probability tab∕t⊕ ~ 0.1 is not negligible assuming a constant abiogenesis rate, where tab is the time of abiogenesis elapsed from the birth of Earth28, and t⊕ the present age of Earth. It may well be possible that early Earth was a more favorable environment for abiogenesis than present30. There may also be an anthropic selection effect favoring earlier abiogenesis on Earth, because an intelligent life must emerge before the increasing solar luminosity causes an end to Earth’s habitable state (estimated to be ~1 Gyr in future)31.If we consider only the conservative abiotic polymerization, i.e., statistically adding monomers, the probability of abiogenesis may be extremely low on a terrestrial planet. This case is not in contradiction with our existence on Earth, because we would find ourselves on a planet where abiogenesis happened. The life on Earth is believed to have descended from the single last universal common ancestor (LUCA) with no evidence for multiple abiogenesis events, and we do not know any life of a different origin in the universe. The emergence of life early in the history of Earth is often used to argue for a high abiogenesis rate, but an arbitrarily low rate cannot be robustly excluded11 Sun-like stars32 or in the whole observable universe containing 1022 stars33 inside a spherical volume with a comoving radius of 46.3 Gly (or 13.8 Gly as a light travel time distance)34. However, the size of the observable universe is not related at all to its whole physical size. According to the widely accepted view of the inflationary cosmology40, the physical size of the universe created by an inflation event should be much larger, likely including more than 10100 stars (see below). In that case, even if the expected number of abiogenesis events is much less than unity in a volume size of the observable universe, it may still be consistent with our observations provided that abiogenesis is expected to occur somewhere in an inflationary universe.For the case of a low abiogenesis rate, the number of abiogenesis events is often considered in the Milky Way Galaxy containing about 1041 considered implications of the eternal inflation theory for the origin of life. In this scenario, most part of the universe inflates forever, self-reproducing many subregions that undergo a conventional inflation followed by a hot big-bang universe. Then an infinite number of stars and galaxies would be formed, and we expect emergence of life even if the abiogenesis probability is infinitely small. Though eternal inflation is a theoretically likely scenario42, it is difficult to confirm observationally, and a quantitative discussion is impossible. It is then interesting to ask if life can emerge within the homogeneous region in which we exist, assuming its minimal size necessary to explain observations. This work tries to give a quantitative answer to this question.The aim of this work is to examine this possibility quantitatively, assuming that the first biological RNA polymer was produced by randomly adding monomers. Koonin43.The observable universe is highly homogeneous and spatially flat on scales many orders of magnitude larger than the causally connected scale (horizon) in the early universe, which are called the horizon and flatness problems, and cannot be explained by the standard big bang cosmology. Cosmic inflation is currently the only widely accepted solution to these problems, and furthermore, it naturally generates scale-invariant quantum density fluctuations that serve as the seed of galaxy formation and the large scale structure in the present universe. Its prediction is in quantitative agreement with the observations of the cosmic microwave background radiation anisotropy, already constraining some theoretical models42, but all of them consider an epoch of exponential expansion as a ∝ exp(Hi ti), where a is the scale factor of the universe, Hi the Hubble parameter at the time of inflation, and ti the duration of inflation. If the inflation occurred at the energy scale of the grand unified theory of particle physics (1016 GeV), Hi would be about 1037 s−1. To solve the horizon and flatness problems, the e-folding number of inflation (Ni ≡ Hi ti) must be larger than45Ni,min ~ 60. If Ni = Ni,min, a causal patch region expanded by the inflation has now the same size as the observable universe. It would be a fine tuning if the inflation duration is extremely close to the minimal value to solve the problems . Rather, we naturally expect Ni − Ni,min ≳ Ni,min. If the inflation duration is twice (three times) as much as that required to solve the problems, the homogeneous universe should extend e60 (e120) times as much as the currently observable universe, which is 1078 (10156) times as large in volume, thus including about 10100 (10178) stars.There are many models and scenarios about how inflation occurred in the early universe18 as a model case. Non-RNA nucleic acid analogues may have carried genetic information before the RNA world emerged1, but the formulations below can also be applied to such cases. Let xl be the abundance of l-nt long oligomers. After the injection of activated monomers at the time of initialization (t = 0), evolution of xl is described by the following differential equations: κ (probability of a reaction with a monomer per unit time) does not depend on the oligomer length, which is approximately consistent with the trend found in the experiment18. We consider initial conditions of xl = 0 for l ≥ 2, and x1 can be approximated to be constant in the early phase. The second term on the right hand side can be neglected when xl+1 ≪ xl. Solving the equations iteratively under these conditions, the abundance xl at a time t is obtained as pr ≡ κ t is the reaction probability with a monomer up to the time t. A similar result is obtained by considering the Poisson distribution with an expectation value of pr; the only difference is a factor of exp(−pr) that is not important at pr ≲ 1. We should consider only the regime of pr ≲ 1, because by the time t ~ κ−1, a significant fraction of activated monomers are lost by the reactions, and hence the approximation of constant x1 is no longer valid and efficient polymerization is not expected beyond this point. If activated monomers are lost earlier by some other processes (e.g. hydrolysis), pr would be smaller than unity.Here we consider a cycle of RNA polymerization by randomly adding activated monomers to an oligomer as a Poisson process, taking an experiment on clay surfacesκ should be proportional to the concentration of activated monomers adsorbed on the clay surface. This clay-phase monomer concentration increases with that in aqueous phase, but according to the Langmuir adsorption isotherm, it saturates when the adsorbed monomer abundance reaches that of the exchangeable cations on clay surface. In the experiment18, montmorillonite has 0.8 mmol exchangeable cations per gram, and it starts to saturate at an aqueous monomer concentration of ~ 0.01 M (=mol/L). At the saturated clay-phase monomer concentration, the reaction rate is κ ~ 1 h−1, and thus pr ~ 1 is reached within a few hours, which is much shorter than the hydrolysis time scale of activated monomers. Aqueous monomer concentration needs to be higher than a certain level to keep κ large enough for pr ~ 1, and this may be achieved at some points during a cycle, for example, by variable amount of water expected in dry-wet cycles around warm little ponds29.In RNA oligomerization on clay surfaces, the coefficient lmin be the minimum length of an RNA that needs to be abiotically formed for an emergence of life, and suppose that a lmin-nt long, randomly polymerized RNA molecule acquires the necessary activity with a probability Pac by a correct informational sequence of nucleotides. Once such an active polymer is produced, it proceeds to the stage of Darwinian evolution with a probability Pev, thus completing an abiogenesis process. Then we can calculate the number of abiogenesis events in a region of the universe containing N* stars as fpl is the number of habitable planets per star, td the time during which abiotic RNA polymerization cycles continue, and rp the production rate of lmin-nt long RNA polymers on a planet. The production rate by the Poissonian process can be expressed using Eq. Nm is the number of activated monomers participating in a cycle of polymerization on a planet, tc the repeating time interval of polymerization cycles, and an approximation of lmin ~ lmin − 1 is used for simplicity. The baseline value of pr is set to unity in the following analysis.Let entclass1pt{minimaPac can be expressed as Nnb is the number of nucleobase types participating in polymerization, and Nac is the number of active sequences among all the possible sequences of a lmin-nt long RNA polymer. We adopt Nnb = 4 as the baseline from RNA/DNA of life as we know it, but probably this is an underestimate for abiotic polymerization, because regioselectivity, homochiral selectivity, or any other reacting molecules that stop further polymerization would effectively increase Nnb. The parameter Nac is highly uncertain. Here we convert this parameter into Δl (or leff ≡ lmin − Δl) defined by the relation lmin = 40, there are 440 ~ 1024 possible sequences of 40-mers, and perhaps Nac = 104 sequences out of them may have a replicase activity8, in this case Δl = 6.6. Here we take Δl = 0 as the baseline value, which is valid when Δl ≪ lmin.The probability Nlife = 1 and taking a logarithm of Eq. C. Obviously there are huge uncertainties, probably more than 10 orders of magnitude in total. However, these parameters appear only logarithmically, and we will find that these uncertainties hardly affect the main conclusions derived in this work.Requiring fpl = 0.1 as the baseline for the planet parameter46, which is the least uncertain among these, owing to the rapid development of exoplanet studies in recent years. The baseline for td is set to 0.5 Gyr as a plausible time scale from the birth of Earth to the abiogenesis28, and that for tc is set to 1 yr supposing a seasonal cycle (e.g. ref. 29), though 1 day may also be reasonable for a day-night cycle. The parameter Pev is highly uncertain, but Pev = 1 is set as the baseline, which is optimistic but may not be unreasonable because a long RNA polymer assembled by the Poisson process would be rare and there would be no competitor or predator around it. Any other essential factors involved in the origin of life, e.g., encapsulation by membrane vesicle formation, may significantly reduce this parameter. It has been known that both RNA polymerization and vesicle assembly are accelerated on clay surfaces14.We use Nm, is probably the most uncertain parameter among the five in C. An upper limit may be estimated by the number of nucleotides in the present life on Earth, Nm = 7 × 1037 (3.7 × 1016 g in mass), which is estimated by the total biomass of 550 Gt-C (3.7 × 1012 wet ⋅ t)47 assuming that nucleic acids constitute 1% of the wet biomass. A rough amount of nucleobases delivered from space by meteorites can be estimated as follows (see ref. 29 for a more detailed modeling). The mass delivery rate of meteoroids from 4.5 to 4.0 Ga is 1020−25 kg/Gyr, and 0.1% of this mass belongs to meteoroids of a diameter 40–80 m, which efficiently deliver nucleobases avoiding melting or vaporization. Carbonaceous meteorites contain nucleobases with a mass fraction of 10−7, and they are deposited into warm little ponds which cover a fraction of 10−6 on the Earth surface. These nucleobases survive for 1 yr, i.e., a seasonal cycle before they are destroyed by UV radiation or seepage. Then we expect 1020−25 nucleobases (0.01–103 g in mass) in the ponds. Instead, nucleobases may also be produced on Earth, and it would not be unreasonable to assume a similar nucleobase/carbon mass ratio to that found in carbonaceous meteorites (10−5). Assuming a carbon abundance similar to the present seawater, we expect 1027 nucleobases in the ponds assuming their depth to be 1 m. We then use Nm = 1025 as the baseline, though it could be wrong by many orders of magnitude, depending on various scenarios of nucleotide formation and their activation under prebiotic conditions. Using the baseline parameter values thus determined, we find lnC = 75.3.The amount of monomers, N* versus lmin for Nlife = 1 calculated by Eq. lmin = 21, 27 and 32 to expect one abiogenesis event for a survey of a single star (lgN* = 0), a galaxy (lgN* = 11), and the observable universe (lgN* = 22), respectively. These lmin values are not sufficiently large compared with that (~40–100) required to expect an RNA replicase activity from a biological viewpoint, implying that abiogenesis is not easy even if we consider the entire volume of the observable universe. For lmin = 40 we find lgN* = 39. If we try to reduce this to lgN* = 22 or 0 for the same lmin by the uncertainty in C, this parameter needs to be increased by a factor of 1017 or 1039, respectively.Figure N* = 100, 178, and 256), lmin becomes 66, 97, and 127, respectively. These lmin lengths now allow us to expect a self-replicating activity of an RNA molecule. If an identical pair of RNA strands is required for abiogenesis, lmin should be effectively twice as large as each of the identical strands. Then an inflationary universe can produce a pair with a length of ~33–64 nt for each, and we can still expect a replicase activity. It is also possible that the inflation duration is even longer than the examples considered here.However, if we request one abiogenesis event somewhere in the whole physical volume created by an inflation, the chance of abiogenesis greatly increases. In a volume created by a twice, three, and four times as long inflation as that required to create the observable universe . A sufficient number of abiogenesis events may not be expected when pr ≪ 1, even in the total volume of an inflationary universe.Figure ϵs of oligomers survive to the next cycle. If polymerization of an oligomer continues over m cycles, the most efficient path to form a lmin-nt polymer would be to repeat m times the process of adding lmin∕m monomers. Then the polymer production rate rp of Eq. m = 5 and ϵs = 0.2 is shown in Fig. lmin/m is not an integer. In this case lmin becomes 42 for lgN* = 22, implying a possibility that abiogenesis has occurred more than once inside the observable universe. Though m and ϵs are highly uncertain, this possibility should not be overlooked.A possibly important process is polymerization over multiple cycles. In polymerization on clay surfaces, inactive monomers and oligomers left from the previous cycle must be released from a clay surface for the next cycle to work, but a fraction of long oligomers may remain on the surface. Adding newly activated monomers to such oligomers over many cycles may be an efficient way to assemble a long polymer. Such a polymerization process may be limited by a time scale of RNA oligomer destruction, e.g., by hydrolysis or UV radiation during the dry phase. As a toy model to consider this, suppose that a fraction N* on an astronomical scale and lmin on a biologically microscopic scale, and uncertainties of other parameters are not important because most of them appear logarithmically. This reminds us of an ouroboros.It has been shown that the first RNA polymer with a replicase activity can be abiotically assembled by the most conservative polymerization process, i.e., random Poissonian adding of monomers, if we require that it occurs more than once somewhere in the physical volume of a universe created by an inflation, rather than inside the observable universe for us. This gives a simple solution to the abiotic polymerization problem to initiate the RNA world. Equation Nnb twice as large as when ignoring chirality. As shown in Fig. Nnb by a factor of two does not seriously affect the expected number of abiogenesis events in an inflationary universe.The result of this work may also give an explanation for the homochirality of life. Even if activated monomers supplied to the polymerization cycle are a racemic mixture, life emerging from them would be homochiral, if homochirality is a necessary requirement for an RNA polymer to show biological activities. Simply it needs more time or volume for a homochiral polymer to be assembled by random polymerization, with 78 of the whole universe created by an inflation, and there is no strong reason to expect more than one abiogenesis event in such a small region. Even if Earth is the only planet that harbors life inside the observable universe, it does not contradict the Copernican principle, because life would have emerged on countless planets in the whole inflationary universe in which we exist.On the other hand, the expected number of abiogenesis events is much smaller than unity when we observe a star, a galaxy, or even the whole observable universe. This gives an explanation to the Fermi’s paradox. The observable universe is just a tiny part, whose volume is likely smaller than 1∕1049. It should be noted, however, that the case of a high abiogenesis rate (Nlife ≳ 1 for N* = 1) cannot be excluded by this work, because we assumed that abiotic RNA polymerization occurs only by the random Poisson process of adding monomers. Potential roles of much more efficient processes on the origin of life, such as non-linear auto- or cross-catalytic reactions, have been studied theoretically50, though it is highly uncertain whether such processes really worked in realistic prebiotic conditions. If organisms having a different origin from those on Earth are found in future, it would suggest that such a mechanism is working at abiogenesis to reduce lmin. Although this possibility should not be excluded, what is shown by this work is that such a hypothetical process is not necessary if we request abiogenesis events to occur somewhere in an inflationary universe.In the picture presented here, the probability of finding biosignatures from planets or satellites in the Solar System or from exoplanets is negligibly small, unless we consider interplanetary or interstellar traveling of microorganismsN*-lmin relation for Nlife = 1, lgN* rapidly increases from 0 (a star) to 22 (the observable universe) in a short range of lmin = 21–32. Even if a non-linear process is working at some stages, the initial polymerization is likely statistical and random as considered here. Then it would be an extreme fine tuning if a biological parameter lmin is just close to the value corresponding to Nlife ~ 1 for a star (N* = 1). Rather, Nlife ≫ 1 or Nlife ≪ 1 is much more likely when we observe just one planetary system. As we have argued, the case of Nlife ≪ 1 is not in contradiction with observations, but the opposite case may be in tension with the lack of evidence for multiple abiogenesis events in the history of Earth or in laboratories.It is also worth pointing out that, in the lgin vitro experiments. This work considered only a single homogeneous region in the universe created by an inflation event, obeying the same physical laws that we observe. However, the multiverse hypothesis51 implies existence of other universes created by different inflation events, in which physical laws may be different from ours. A theoretically intriguing question is whether a chemical RNA-like long polymer is easily formed to contain information and show biological activities eventually leading to higher organisms, when physical laws are arbitrarily made, e.g., by random choices of fundamental physical constants. Perhaps this may be the ultimate mystery regarding the origin of life, which is, of course, far beyond the scope of this work.A fundamental assumption in this work is that an abiotically assembled RNA polymer acquires a self-replicating ability if it is sufficiently long and has a correct nucleotide sequence. This may be rather trivial under the physical laws ruling this universe, because we know that ribozymes are actually working in life and can also be produced by


Although metastatic tumor is the most common intraocular malignancy, choroidal metastasis from gastric cancer is relatively rare. We present the exact course of a spontaneous rupture of the eyeball with choroidal metastasis from gastric carcinoma (GC) and the applied surgical interventions.A 59-year-old male presented with pain and vision loss on his left eye for 6 months. He was diagnosed with GC, for which he received systemic chemotherapy for a year.Preoperative B-scan, color fundus photography, computed tomography, and magnetic resonance imaging showed a choroidal tumor in his left eye. The spontaneous rupture of the corneoscleral limbus from 2 to 5 o’clock, combined secondary glaucoma, exudative retinal detachment and choroidal detachment were found.The ruptured corneoscleral limbus was sutured and the orbit was lavaged with 0.4% cisplatin during the enucleation.Histopathology confirmed high homology of the choroidal metastasis and GC. He survived for 2 months after surgery, without pain or orbital neoplasms.Choroidal metastasis from GC rapidly progressed to spontaneous rupture of the eyeball. Careful eyeball enucleation followed by orbital lavage with chemotherapeutics may reduce metastasis risk beyond the eyeball. Additional therapeutic interventions should be considered in patients resistant to single systemic chemotherapy. Here, we have presented the case of a patient with choroidal metastasis of GC, in whom the corneoscleral limbus spontaneously ruptured, despite systemic chemotherapy, due to uncontrolled disease course.Metastatic tumor is the most common type of intraocular malignancy. The uveal tissue, particularly of the choroid, on account of its high blood flow, is the most favored site of metastasis.22 once and oral tegafur 100 mg/d for 14 consecutive days per 3 weeks and erbitux 250 mg/m2 intravenously per 3 weeks for targeted therapy. Oxycontin was used to alleviate pain. He denied any history of either ocular disease or intraocular surgery.A retrospective chart review of the patient was conducted at the Hangzhou Red-Cross Hospital, after obtaining approval by the Institutional Review Board. A 59-year-old male with GC was referred to our institution with pain and decreased vision in his left eye, over a 6-month period after being diagnosed in another hospital with an intraocular tumor and secondary retinal detachment according to B-scan and color fundus photographs Fig. . A year The patient presented with total loss of vision in his left eye. The anterior segment revealed mixed-congestion and edema of conjunctiva, rupture of corneoscleral limbus from 2 to 5 o’clock, edema of cornea, very shallow and squeezed central anterior chamber with complete loss of peripheral anterior chamber detected by ultrasound biomicroscopy, and hyphemia  scan of the orbits showed an enhanced mass at the nasal and posterior side of the choroid of the left eyeball Fig. . MagnetiSubsequently, the patient underwent enucleation. To reduce the risk of orbital metastasis, we sutured the ruptured corneoscleral limbus in advance and used 0.4% cisplatin solution to lavage the orbit after enucleation during surgery. Nasal-posterior solid neoplasm was found in the enucleated eyeball, accompanied by massive hematocele Fig. . The cho3 followed by the iris,,11 eyelid and conjunctiva, optic nerve, and skin, as shown in a few case studies.Gastric cancer is one of the most common causes of cancer-related mortality in the world. The ocular metastasis of gastric cancer is most often located on the choroid,,3,9 Patients with choroid metastases from GC most often complain of visual disorder, due to early onset of macular edema, which was associated with accumulation of subretinal fluid and involvement of the macular region.,14–17 At the initial visit, the main complaint of our case was eye pain, which was probably related with secondary glaucoma, iridocyclitis, and scleral invasion of the tumor. Gradual decrease in visual acuity occurred earlier than eye pain in our patient, and his negative attitude to treatment delayed his first visit to the ophthalmology clinic. Eventually, massive hemorrhage and increased intraocular pressure caused spontaneous rupture of the corneoscleral limbus in the eyeball.Choroid metastasis manifests as blurred vision (70%–81%), flashes and floaters (5%–12%), eye pain (5%–14%), and 9% to 11% of patients do not exhibit any symptoms. and choroid metastasis of GC is similar to that of the lung and breast,,9 as presented by our patient at his first ophthalmologic visit. Choroidal metastasis from GC has been reported previously, with macular edema being a common feature, besides exudative retinal detachment and subretinal hemorrhage.,14–17 In fact, choroid metastasis usually exhibits a higher incidence of exudative retinal detachment compared to uveal melanomas. Ultrasonography revealed a choroidal mass with moderate-to-high internal reflectivity. A well-circumscribed subretinal metastatic carcinoma, appearing as an isointense signal on the T1-weighted image and as a hypointense signal on the T2-weighted image was revealed by MRI. In addition, optical coherence tomography examination shows irregular elevation of choroidal tissue and aids in early diagnosis. Based on the history of GC, manifestations of fundoscopy, and the results of ultrasonography, CT, and MRI, our patient was diagnosed with choroidal metastasis from GC, which was confirmed by histopathology after surgery.Choroid metastasis usually manifests as a yellow subretinal mass (94%) accompanied with subretinal fluid (73%)–24 Radiotherapy and photocoagulation were the most common treatments due to photosensitivity of the choroid.,9 However, systemic chemotherapy is commonly used for choroidal metastasis from GC as it is insensitive to radiotherapy. A combination of systemic paclitaxel and local intensity-modulated radiotherapy provided remission in a case of choroidal metastasis from GC, while chemotherapy with oral S-1 and intravenous cisplatin reduced choroidal lesions in another case. However, choroidal metastasis from GC was found to be resistant to systemic imatinib.Possible treatment options for choroidal metastasis include systemic chemotherapy, intravitreal injection of anti-vascular endothelial growth factor injections, localized radiation, transpupillary thermotherapy, photodynamic therapy, and surgical resection. enucleated the affected eye when choroidal tumor from GC infiltrated the macular area and extended around the optic nerve head, orbital recurrence still occurred. Indeed, no orbital metastasis-related symptoms were reported by our patient during the 2-month postoperative follow-up. The patients with choroidal metastasis have a poor prognosis.,27 The reported cases with choroidal metastasis from GC had survival durations of 9 months and 3 years, and our patient died 8 months after the first diagnosis of choroidal metastasis. Poor response to chemotherapy and long duration of choroidal metastasis from GC may have led to spontaneous rupture of the eyeball.Choroidal metastasis of our patient occurred 6 months after GC was diagnosed. Although the patient received systemic chemotherapy with oxaliplatin, tegafur, and erbitux, choroidal metastasis in the patient progressed, inducing spontaneous rupture of the eyeball. While our enucleation procedure reduced the risk of orbital recurrence of the tumor, Melo et alThe case of a patient with choroidal metastasis from GC was presented. The rapid progression led to spontaneous rupture of the eyeball. Careful enucleation of the eyeball followed by lavage of the orbit with chemotherapy drugs may reduce the risk of metastasis beyond the eyeball structure.Conceptualization: Qiu-Shi Li.Data curation: Shuang-Qing Wu, Qiu-Shi Li, Yu Zhang.Funding acquisition: Shuang-Qing Wu.Investigation: Qiu-Shi Li, Liwei Zhu.Methodology: Liwei Zhu.Resources: Shuang-Qing Wu, Yu Zhang, Liwei Zhu.Supervision: Liwei Zhu.Writing – original draft: Shuang-Qing Wu, Qiu-Shi Li.Writing – review & editing: Shuang-Qing Wu, Liwei Zhu.


Lepeophtheirus salmonis was determined. The viral genome contains five open reading frames encoding three unknown proteins , a putative glycoprotein (G), and a large (L) protein. Phylogenetic analysis placed LsNSRV-1 in the recently established mononegaviral family Artoviridae. LsNSRV-1 showed a prevalence of around 97% and was detected in all L. salmonis developmental stages. Viral genomic and antigenomic RNA was localized to nerve tissue, connective tissue, epithelial cells of the gut, subepidermal tissue, exocrine and cement glands, as well as the testis, vas deferens and spermatophore sac of male L. salmonis and the ovaries and oocytes of females. Viral RNA was detected in both the cytoplasm and the nucleoli of infected cells, and putative nuclear export and localization signals were found within the ORF I, III and L proteins, suggesting nuclear replication of LsNSRV-1. RNA interference (RNAi) was induced twice during development by the introduction of a double-stranded RNA fragment of ORF I, resulting in a transient knockdown of viral RNA. A large variation in the knockdown level was seen in adult males and off springs of knockdown animals, whereas the RNA level was more stable in adult females. Together with the localization of viral RNA within the male spermatophore and female oocytes and the amplification of viral RNA in developing embryos, this suggests that LsNSRV-1 is transmitted both maternally and paternally. Small amounts of viral RNA were detected at the site where chalimi were attached to the skin of Atlantic salmon . However, as the RNAi-mediated treatment did not result in LsNSRV-1-negative offspring and the virus failed to replicate in the tested fish cell cultures, it is difficult to investigate the influence of secreted LsNSRV-1 on the salmon immune response.The complete genome sequence of a novel mononegavirus, Lepeophtheirus salmonis negative-stranded RNA virus 1 (LsNSRV-1), obtained from a salmonid ectoparasite,  Lepeophtheirus salmonis), is a marine ectoparasite feeding on mucus, skin and blood of salmonids in the northern hemisphere [Salmo salar) has led to an increase in host availability and density [The salmon louse  exhibit the gene order 3′-UTR – N gene – ORF II gene – P gene – ORF IV gene – G gene – L gene – 5′-UTR. The ORF II protein of nyaviruses is a negative regulator of the polymerase activity, and ORF II and ORF IV are suggested to form a two-complex matrix protein [In the last few years, there has been a dramatic increase in the number of mononegaviruses discovered in arthropods, as new techniques for virus detection have been developed –12. The oviridae , 14. Theoviridae . For boroviridae –18. Withoviridae , 20 and oviridae , 22. In  protein . The mym protein .Caligus rogercresseyi: Caligus rogercresseyi rhabdovirus Ch-01 (CrRV-Ch01). CrRV-CH01 clusters phylogenetically with the two other caligid rhabdoviruses to form the newly created genus “Caligrhavirus” (awaiting ratification by the ICTV) within the family Rhabdoviridae. CrRV-Ch01 differs from LSRV-No9 and LSRV-No127 by having an additional ORF with unknown function [L. salmonis, Lepeophtheirus salmonis negative-stranded RNA virus 1 (LsNSRV-1), which shows similarities to artoviruses.In 2014, Økland and colleagues described two rhabdoviruses infecting salmon lice: Lepeophtheirus salmonis rhabdovirus No 9 (LSRV-No9) and Lepeophtheirus salmonis rhabdovirus No 127 (LSRV-No127). These viruses are present in the glandular tissue of the louse and have a high prevalence in all developmental stages. Viral RNA is also present in the skin of the salmon surrounding the site where chalimi were attached, but the viruses do not replicate in selected fish cell cultures . Virus-fThe complete description of the materials and the methods for Illumina sequencing and cell culturing systems has been reported elsewhere .In short, a pooled sample of total RNA from five adult lice collected from different locations on the west coast of Norway was sequenced by BaseClear  using Illumina next-generation sequencing. BF-2 (ATCCCCL91), ASK , CHSE-21L. salmonis from nine salmon farming sites in western Norway and from wild Atlantic salmon in the Oslofjord, 22 C. rogercresseyi from three salmon farming sites in Region X in Chile, and two Caligus elongatus from farmed Atlantic salmon in western Norway were tested for the presence of the LsNSRV-1 genome. To study the tropism of the virus, 16 L. salmonis were cut into five  or six  pieces: the anterior part of the cephalothorax, the middle part of the cephalothorax, the posterior part of the cephalothorax, the genital complex, the abdomen and the egg strings.A real-time RT-PCR assay (TaqMan probes) based on the putative L protein ORF of LsNSRV-1 was designed for relative quantification (Table ® (Sigma-Aldrich) according to the manufacturer’s protocol with a few modifications: the tissue was homogenized for seven minutes at 50 Hz using a Tissuelyser LT (QIAGEN) and a 5-mm bead, and an additional washing step with 1 ml 100% ethanol was included before air drying and elution with 50-100 µl of DEPC-treated water. An AgPath-ID™ One-Step RT-PCR Kit (Applied Biosystems™) and Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems) were used for real time RT-PCR analysis with the following reactions: 1X RT-PCR buffer, 800 µM forward and reverse primer, 176 μM probe, 0.5 × RT-PCR enzyme mix, 2.0 μl template, and RNase-free water to a total volume of 12.5 μl. The reaction was run according to the standard protocol for the AgPath-ID™ One-Step RT-PCR kit (Applied Biosystems™).All samples were stored at – 20 °C. RNA was extracted using Tri ReagentRNA from infected lice was ligated to allow circularization and sequencing of the LsNSRV-1 genome termini. Total RNA was extracted from 5-7 lice using Tri Reagent (Sigma-Aldrich). To increase the efficiency of RNA ligation, the 5’ triphosphate residues of the RNA were removed by incubating 5 μg of total RNA with 5 units of 5’ RNA pyrophosphohydrolase  in 40 μl of 1 × NEBuffer 2 for 30 min at 37 °C . RNA cleIn situ hybridization was performed on adult female and male lice according to Dalvin et al. [n et al.  with modn et al. . Digoxigr) of the putative proteins were calculated using ProtParam [The theoretical isoelectric point (pI) and molecular mass  according to supplier’s instructions. RNA interference (RNAi) was performed as described previously by soaking of nauplii [The 3′ end of ORF I was amplified by PCR using Q5 high-fidelity DNA polymerase (New England Biolabs) according to the supplier’s instructions, using the same T7 overhang primers as were used for the on Table . The res nauplii  or injec nauplii .The dsRNA was further applied to a strain of LsRV-negative lice (LSOslo)  in an atIn the second approach, the concentration of dsRNA was increased. Nauplius I larvae from three pairs of egg strings were divided into four groups, where one group was treated with elution buffer while the other three groups were treated with 13, 20 and 27 ng of dsRNA per µl, respectively. Again, fish kept at 12 °C were infested with copepodids that were given 20 ng of dsRNA per µl at 7 days post-immersion (dpi), and 1000 ng of dsRNA per µl at the pre-adult I♀/II♂ stage (18 dpi). The lice were put back on the fish, and the egg strings were collected from the adult lice at 34 dpi and hatched in a single well flow-through system . SamplesL. salmonis. The sequence was confirmed by Sanger sequencing, and the 5′- and 3′-terminal sequences were determined after circularization of the genome (GenBank accession number: MG489864). The complete 12,434-nucleotide (nt)-long negative-sense genome of the virus contains five ORFs with putative transcription initiation and termination sites in the order 3′-ORF I-ORF II-ORF III-G-L-5′. The G gene is in reading frame 1, while the ORF I, ORF II, ORF III and the L gene are in reading frame 3. The genome has a 173-nt-long 3′ leader region and a 129-nt-long 5′ trailer region. The first 29 nt of the 3′ leader region shows 86.7% reverse complementarity to the last 29 nt of the 5′ trailer region . The protein has a molecular weight of 73.9 kDa and a pI of 6.5. A Blastp search of the putative ORF I protein found sequence similarity to hypothetical protein 1 of Běihǎi rhabdo-like virus 2 (YP009333446), hypothetical protein 1 of Běihǎi barnacle virus 8 (YP009333182), and a hypothetical protein of Pteromalus puparum negative-strand RNA virus 1 (PpNSRV-1) (APL97663). Aligning them to the ORF I protein sequence revealed 23%, 25% and 25%, amino acid sequence identity, respectively. Additionally, the ORF I protein shares similarities with an uncharacterized Daphnia magna protein (KZS21910) and an uncharacterized protein from Dendroctonus ponderosae (XP019755411), both with 24% amino acid sequence identity. LocNES, NESsential and NESmapper predicted the NESs 24TMARALPERIGTLTL38 (score 0.513), 33IGTLTL38 (score 0.65) and 26ARALPERIGTLTLI39 (score 5.85), respectively. A proline-rich region (634PVVPAPAIRPPGPQLPPQNDGPPQDPNE661) was identified at the C-terminal end of the hypothetical protein, and a possible late domain was identified at amino acid position 221 (221YPDL224).The hypothetical ORF I gene is 2086 nt long and contains an ORF of 1986 nt encoding a putative protein of 661 amino acids (aa) (accession no: AUZ99695). The gene possesses a presumptive transcription initiation signal (TIS) (GAAACAA) and a transcription termination/polyadenylation signal (TTS) (TAAT(A)5). The 5′-UTR of the ORF II gene overlaps the 3′-UTR of the ORF I gene by 33 nt. ORF II encodes a putative protein of 66 aa with a pI of 6.8 and a molecular weight of 7.6 kDa (accession no: AUZ99696). A Blastp search revealed no significant similarity to any known viral proteins. However, the ORF II protein shared slight sequence similarity with the condensation domains of two hypothetical proteins from the plant pathogenic fungi Bipolaris victoriae (XP_014554506) and Bipolaris zeicola (XP_007711178).The small putative ORF II gene is 337 nt long from its TIS (GAAATAA) to its TTS (TAACTT(A)5). The 1173-nt-long ORF III encodes a putative protein of 391 aa with a molecular weight of 42.9 kDa and a pI of 5.7 (accession no. AUZ99697). No similarity to other proteins was revealed using Blastp. c-NLS mapper predicted a bipartite NLS at amino acid position 15 (15KSGVKIIQTDVLDHLSESILEYDKKLKATKEP46) with a score of 7.7 [360PFSAP364 and 381LDRLF385.The hypothetical ORF III gene encompasses 1327 nt from the putative TIS (GAAACAA) to the TTS (TAAG(A)e of 7.7 . Using t5). The 1794-nt-long ORF encodes a protein of 598 aa with a molecular weight of 66.9 kDa and a pI of 6.7 (accession no: AUZ99698). The putative G protein shows 24% amino acid sequence identity to a putative glycoprotein from PpNSRV-1 (APL97666) and a hypothetical protein from Húběi rhabdo-like virus 6 (APG78704). Additionally, a lower level of similarity was identified between the G protein and two uncharacterized arthropod proteins of similar size . A Phobius search of the G protein predicted a signal peptide and a transmembrane region at amino acid position 1-20 and 520-542, respectively. The putative glycoprotein contains eight predicted O-linked and three predicted N-linked glycosylation sites.The putative glycoprotein (G) gene is 1860 nt long from the TIS (GAAATAA) to the TTS (TAAT(A)5, respectively. The putative L protein has a molecular weight of 247.5 kDa and a pI of 8.8. A Blastp search revealed sequence similarity between the putative LsNSRV-1 L protein and L proteins of the artoviruses PpNSRV-1 , Běihǎi barnacle virus 8 , Húběi rhabdo-like virus 6  and Húběi rhabdo-like virus 8 , and Borna disease virus , Nyamanini nyavirus , and S. sclerotiorum negative-stranded RNA virus 2 .The putative L gene is 6556 nt in length, containing a 6534-nt-long ORF encoding a protein of 2178 aa (accession no. AUZ99699). The TIS and the TTS are assumed to be GAAACAA and TAAG(A)Mononegavirales revealed that block III is the most conserved domain, while block VI is the least conserved  by cNLS-mapper with a score of 7.5, and a possible bipartite NLS was detected by Motif scan at position 451 (451RKEWLLTPS-IKSDRR466). In block V, the sequence GSGT-72 aa-HR may correspond to the motif GSxT-(60–70 aa)-HR, which is essential for mRNA capping in nonsegmented negative-stranded RNA viruses [The L protein is the most conserved protein of the mononegaviruses, composed of six conserved blocks containing essential motifs for the structure and function of the polymerase . Pairwised Table . In blocognition . The subognition . A monop viruses .Table 2PArtoviridae [Artoviridae clade has a bootstrap support value of 100, and clusters together with members of the families Mymonaviridae and the newly established family Lispiviridae with a bootstrap value of 42. The clade corresponding to the families Artoviridae, Mymonaviridae and Lispiviridae is separated from the remaining mononegaviral families in a larger clade with members of the families Nyamiviridae, Bornaviridae and Xinmoviridae with a support value of 79.The phylogeny inferred from comparison of L protein sequences of 73 mononegaviruses showed LsNSRV-1 clustering with members of the newly established mononegaviral family oviridae . This faC. elongatus samples tested positive for the presence of viral RNA by real-time RT-PCR. However, it is unknown if this is the same virus, as no attempts have been made to sequence LsNSRV-1 from C. elongatus.Viral RNA from LsNSRV-1 is abundant in both male and female adult salmon lice and is detected in all stages of the louse life cycle, including developing embryos within egg sacs. The prevalence of the virus is approximately 97% (152 of 157). Tropism studies revealed that viral RNA is abundant throughout the body of the louse. Gill and kidney samples collected from six Atlantic salmon that were heavily infested with LsNSRV-1-positive salmon lice tested negative for the presence of viral RNA, while skin samples at the attachment site were weakly positive. Two In situ hybridization revealed genomic RNA and expression of the ORF I gene in many L. salmonis tissues .Opposite to what was previously shown for the LsRVs , the firAfter a second round of RNAi by injection, the adult males had an average knockdown of 38.2% and the females had an average knockdown of 83.5% , nyaviruses and bornaviruses replicate in the nucleus [Culex tritaeniorhynchus rhabdovirus [L. salmonis genome with no exogenous phase should only be present in the cytoplasm as mRNA, and not as both mRNA and genomic RNA, as demonstrated by in situ hybridization. Moreover, the successful ligation and complementary termini of the putative viral genome strongly suggest that it is not incorporated in the host genome.Nucleorhabdoviruses and dichorhaviruses (family  nucleus , 71, 72, nucleus . The nuc nucleus . In born nucleus –78. NLSsbdovirus –81. One bdovirus , 83. Leubdovirus –86. Our bdovirus –88. Howebdovirus , the preArboviruses rely on horizontal transmission, mainly through feeding and infection of the arthropod’s vertebrate host , 90. TheL. salmonis could be vital for the control of this parasite. Indeed, LSRV-No9 and LSRV-No127 infection enhances the parasitic success of L. salmonis [Apis mellifera. It is therefore possible that viruses in the blood-feeding salmon lice could pose a risk to Atlantic salmon. Surveying and characterization of the virome of salmon lice could thus be of value for the fish farming industry. Clearly, more research is needed to clarify the effect of LsNSRV-1 on its host and to assess the risk of a host shift to Atlantic salmon.Understanding the role of the viruses infecting salmonis . LsNSRV-salmonis . Such evsalmonis , and hossalmonis . Howeversalmonis  that the


In particular, we found that development HRM bundles may also increase job demands, which, in turn, may result in lower levels of beneficial employee outcomes. These empirical outcomes demonstrate the strength of the driving power eliciting from job resources preceded by any HRM bundle. Moreover, this effect appears to apply to employees of all ages. Our moderated-mediation model appeared robust for several control variables. Overall, this study provides an extension of the well-known Job Demands-Resources model by including maintenance and development bundles of HRM practices used by employees that have a differential effect on job demands and job resources which in turn have an impact on employee outcomes.Using the Job Demands-Resources model literature and the life-span theory as scholarly frameworks, we examined the effects of job demands and job resources as mediators in the relationship between bundles of used HRM practices and employee outcomes. In addition, we tested for age differences in our research model. Findings confirmed the hypothesized original 2-factor structure representing maintenance and development HRM practices. Structural Equation Modeling analyses showed that the maintenance HRM bundle related directly and negatively to employee outcomes, without moderating effects of age. However, job resources appeared to mediate this relationship in a positive way as it also did for the development HRM bundle. Whereas this study showed the ‘ After asee also . For thasee also , the Jobsee also  is used The aforementioned relationships occur in the context of an aging labor force. According to life-span theory, aging is generally associated with both gains and losses , which amediating mechanisms, such as job demands  and job resources , that are provoked by HRM practices, and that ultimately give rise to employee outcomes. Secondly, findings from life-span developmental psychology have identified various systematic age-related changes in human functioning  . Accordiwork engagement is also a central variable in the motivation process of the JD-R model that is expected to affect employee performance  enhance employee outcomes, such as work engagement . In thisstatus quo. Examples are part-time work and attention for health. The second category is called the development HRM bundle and consists of practices striving to reach higher levels of employee functioning. Regardless of the content of the specific HRM practices, all practices in this bundle are focused on growth, learning new tasks, and extending the employee’s horizon. Examples are continuous development and task enrichment.The current empirical study examines the impact of two categories of bundles of HRM practices. We used an existing list of HRM practices  that comuse of HRM practices by employees, rather than on employees’ perceptions on intended or available HRM practices. We argue that it is insufficient to only shed light on employees’ perceptions about HRM , given the reduced presence at the workplace for the specific employee, and, in turn, impacts employee outcomes negatively.Third, the so-called development HRM bundle contains a group of interconnected HRM practices that have the capacity to make work more resourceful . For insFourth, this development HRM bundle may also make the employee’s work more challenging and intense . For exaHypothesis 1a: The positive relationship between the use of the maintenance HRM bundle and employee outcomes is  mediated by job demands.Hypothesis 1b: The positive relationship between the use of the maintenance HRM bundle and employee outcomes is  mediated by job resources.Hypothesis 1c: The positive relationship between the use of the development HRM bundle and employee outcomes is  mediated by job demands.Hypothesis 1d: The positive relationship between the use of the development HRM bundle and employee outcomes is  mediated by job resources.Since employees have to work longer, due to an increased retirement age , it is oSelect subjectively their goals, Optimize their strategies for goals attainment, and Compensate for age-related losses. This process of adaptation is a dynamic, life-long development in which people and their environment mutually influence each other  mediated relationship between the use of both the maintenance and the development HRM bundles respectively, on the one hand, and employee outcomes, on the other hand, through job demands and job resources, is moderated by age, such that the higher the age, the stronger the relationship in case the maintenance HRM bundle is the predictor (2a), and the weaker the relationship in case the development HRM bundle is the predictor (2b).2(1) = 0.13, p = 0.72; χ2(1) = 0.50, p = 0.48; χ2(1170) = 0.31, p = 0.76]. However, the category of respondents contained relatively more higher educated employees compared to the group of excluded ones . The mean age of the respondents was 46.9 years (SD = 10.2) and their mean job tenure was 8.54 (SD = 8.88). Please, see The data collection was done by means of an on-line questionnaire among 6,000 employees working in three Dutch working organizations from three different occupational sectors: transport, healthcare, and research and education. The questionnaires were distributed using the web-based tool 1 of 0.79, 0.81, and 0.81, respectively. We computed an overall work engagement score, as was recommended by The Utrecht Work Engagement Scale (UWES) developed by The Employability instrument developed by general health perception. The 5-item questionnaire  was rated on a five-point Likert-scale ranging from 1  to 5 ‘definitely true’). The SF-36 Health–scale has shown an acceptable level of internal consistency with a McDonald’s omega of 0.74.The SF-36 Health-scale developed by The Mental load and Emotional load, and the Pace and amount of work scales developed by The scales Independence in your work, Opportunities to learn, and Variety in your work developed by To identify commonly examined HRM practices that are in use by employees in the involved organizations, we first identified 21 relevant age-related HRM practices, in line with We used job duration (in years), gender , sector , and management position (yes or no) as control variables in our analyses.The statistical analyses were performed with IBM SPSS Statistics  used for testing the hypotheses. In that process, we tested several models to decide which was the best final models taking into account the covariance between the latent factors . We star2/df) and the Standardized root mean residual (SRMR), the approximate fit index Root mean square error of approximation (RMSEA), and the comparative fit indices Comparative fit index (CFI) and Tucker-Lewis Index (TLI). The following cut-off values of these fit indices were used to assess the model fit: χ2/df < 2 or 3, SRMR and RMSEA < 0.08, CFI and TLI > 0.90 or 0.95. In addition, the Akaike information criterion (AIC) was used to evaluate different models in which smaller values indicate a better fit. In addition, we assessed convergent and discriminant validity based on the following criteria  and carried out descriptive analyses and Pearson correlational analyses and partial correlations controlling for age.Fourth, to test the hypothesized mediation effects for job demands and job resources in the relationship between maintenance and development HRM bundles, on the one hand, and employee outcomes, on the other hand (H1a-d); and the moderation effect of age in these relationships (H2a-b), we computed a moderated meditation SEM model. Given the issues with the sample size to variables ratio, we reduced the complexity of the hypothesized SEM model  by using manifest variables see . To studFifth, multigroup SEM analyses  were empp. 699) and the fit could not be established. All standardized factor loadings were statistically significant (p < 0.001), ranged between 0.30 and 0.87, and 11 (out of 24) were 0.50 or higher  loaded significantly on their intended dimension, the maintenance and development HRM bundles. Four items loaded significantly on the maintenance bundle: Flexible work, telecommuting, reduced workload, and attention for health. Nine items loaded significantly on the development HRM bundle: job development interview, career planning, continuous development, regular training, promotion, sideways movement, task enrichment, second career, and participation-in- decision-making.In a series of models we tested the combined factor measurement model . The maintenance HRM bundle was not significantly related to job demands, but was significantly negatively directly related to employee outcomes . The maintenance HRM bundle was also indirectly positively related to employee outcomes through job resources . The development HRM bundle was positively related to both job demands  and job resources , but did not have a direct significant effect on employee outcomes. This established a complete mediation effect of the development HRM bundle on employee outcomes .Hypothesis 1a. Next, there was a direct negative effect as well as an indirect positive effect of the maintenance HRM bundle, via job resources, on employee outcomes. The latter implies that no support for Hypothesis 1b, which assumed a positive relationship, was found in our data. The effects of the development HRM bundle appeared to be completely and positively mediated by job demands and by job resources, herewith supporting Hypothesis 1c and d.The moderated mediated model showed that the maintenance HRM bundle did not have a significant direct on job demands and therefore could not establish mediation which means rejection of Hypothesis 2a. However, we did find moderating effects of age relating to job demands , and positively between job demands and employee outcomes . As age did not affect the relationship of the development HRM bundle, via job resources, on employee outcomes, Hypothesis 2b was partially supported.Regarding the moderating effects of age in our assumed mediation model outlined above, we found some interesting results as well. The relationship from the maintenance HRM bundle, through job resources, to employee outcomes was not moderated by age, and therefore there was no evidence for see also . More sp2By and large, the outcomes pointed to fairly robustness of the moderated mediation model . Regardidriving power’ of both maintenance and development HRM through job resources. However, from our analyses, we may conclude that the use of development HRM increases job demands as well, which, in turn, results in lower employee outcomes. This might refer to the so-called ‘dark side’ of HRM  for aging workers. Moreover, the so-called ‘dark side’ effect of HRM that was found for the impact of the development HRM bundle on job demands was weaker for the older workers as well. Apparently, a high amount of job demands are a risk factor for all employees, and as employees age, the use of development HRM bundle may have an increasingly supportive function. As regards the third and fourth contribution of this study it is unique in its integration of the aforementioned moderating mediating relationships together with the validation of an up to now conceptually distinction of HRM bundles, and the actual use of these. So, we go beyond the perceived availability of HRM, and investigated the actual behavior, the actual use of HRM and its’ relationships. Moreover, with this study we validated the conceptual distinction of To conclude, our results show a rather ambiguous effect of age on the relationship between development HRM and job demands e.g., . HoweverThe present study has some limitations. First, all data were collected using questionnaires, that may have resulted in biases like response set and common method variance. Second, all data were collected at one point in time, that is, the study was cross-sectional. Further research is therefore needed to address the issue of causality and reciprocal effects. As From a practical stance, the role of HRM in retaining employees’ well-being appears crucial in the realm of a continuously demanding working environment e.g., . TherefoThis study shows that age only plays a significantly distinctive role in relation to the effect of job demands in the modeled relationships. The predominant association between the both HRM bundles and employee outcomes, through job resources, turned out to be strongly positive, regardless of age. This outcome implies that, for all age categories of workers, the use of particularly development HRM bundle is highly important and should be stimulated by (HRM) managers. However, the unexpected direct negative relationship between the maintenance HRM bundle and employee outcomes was counterbalanced in case job resources were included as a mediator in this relationship.All in all, this study indicates that stimulating the role of job resources , whether they are preceded by the use of a maintenance or by a development HRM bundle, is rewarding for employees of all ages. In addition, after controlling for job duration, gender, sector, and having a management position (yes or no), we found that the process of ‘driving power’ applies more to employees with a job duration longer than 5 years, for men, for the education and research sector, and for managers. The ‘dark side’ of HRM seems to be perceived more by the counterparts: employees with job duration shorter than 5 years, women, healthcare sector, and employees without a managerial position. Our study points out that it is highly relevant to understand which and how HRM bundles influence particular employees, and employee outcomes. Hence, this study questions the contribution of age-related HRM policies. However, HRM policies focused on particular employee groups like being a non-manager, or female, turns out to be important. Managers should use the job development interviews to deepen the understanding of how the dark side can be enlightened. They should find out what exactly contributes to the dark side, and what can be done to facilitate employees. As such, our approach may establish a foundation for further theory development, and empirical research on the topic of employee well-being from the perspective of HRM.The datasets generated for this study are available on request to the corresponding author.KV: overall text and research. HK: methodology and statistical analyses. BV: overall text and research. BE: theoretical foundation. AL: overall text.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


O-ribose methylation is an abundant post-transcriptional modification mediated by small non-coding nuclear RNAs known as box C/D modification guide RNAs. The minimal structural requirements for these guide RNAs to function in higher eukaryotes are still unclear. To address this question, we generated a series of mutant variants of Drosophila box C/D scaRNA:MeU2-C28 and tested their modification guide activities in the Xenopus oocyte system. Our data suggest that box C/D guide RNA function requires either a terminal or an internal consensus kink-turn structure. We identified the minimal functional box C/D guide RNA. It consists of a single-domain molecule with (i) a terminal stem with a consensus kink-turn domain, (ii) one box C and box D connected by a 14-nucleotide antisense element and (iii) a one-nucleotide spacer between the box C and the antisense element. In this single domain RNA, the sequence of the spacer is more important than its length. We suggest that the secondary structure of box C/D RNAs, essential for guide RNA function, is more complex than generally supposed. At the same time, the expression of functional extremely short single-domain box C/D RNAs is possible in higher eukaryotes.Site-specific 2’- Small nucleolar RNAs (snoRNAs) are non-coding RNAs that may function as modification guide RNAs. Specifically, box C/D snoRNAs mediate site-specific post-transcriptional ribose methylation. The C and D boxes are two distinctive short motifs—‘RUGAUGA’ and ‘CUGA’, respectively. They localize close to the ends of a snoRNA molecule and participate in the formation of a specific terminal structure called the kink-turn, or K-turn. This structure consists of a canonical ‘stem I’ formed by 5’ and 3’ termini complementary to each other and a noncanonical ‘stem II’, which contains one U-U and two A-G pairs formed by the C and D boxes. Stems I and II are separated by an asymmetrical 3-nt bulge. Additional slightly diverged copies of the C and D boxes, the so-called C’ and D’ boxes, form an internal stem which is somewhat similar to the terminal one. Single-stranded sequences between the C and D’ boxes and the C’ and D boxes represent regions of complementarity with substrate RNAs targeted for modification. They are usually termed antisense elements (ASEs). The fifth nucleotide upstream of box D or D’ targets the position in the base-paired substrate RNA for modification. The typical structure of a box C/D snoRNA is depicted in O-ribose methylation in eukaryotes [O-methylation activity [Since the discovery of the snoRNA-guided mechanism for 2’-karyotes ,5,6, quikaryotes ,11,12,13activity ,11. BothXenopus oocytes, to further explore box C/D RNA guide activity. This system has already been used successfully for verification of modification guide RNA activities [Drosophila guide RNA for 2’-O-methylation of U2 snRNA at position 28 [Drosophila scaRNA:MeU2-C28 and tested the mutant RNAs for modification activity on U2 snRNA. In general, our data confirm the results of previous studies [One could argue that the RNA-guided modification machinery of higher eukaryotes, especially vertebrates, might have specific features different from those of yeast. Here we use a vertebrate cell system, tivities ,13,14,15ition 28 ,17,18, t studies . MoreoveXenopus laevis females were anesthetized in 0.15% ethyl 3-aminobenzoate methane sulfonate , and a piece of ovary was removed surgically. Oocytes were maintained in OR2 medium [2 medium  for up tDrosophila U2 snRNA and scaRNA:MeU2-C28, both wild type and mutants that lack the CAB-box [Drosophila scaRNA:MeU2-C28 were made by PCR-based mutagenesis and primer annealing-and-extension. Capped Drosophila sense-strand RNAs were transcribed in vitro from linearized plasmids using T3 RNA polymerase. DNA was removed by DNase I treatment followed by DNase I inactivation. RNAs were then purified on MicroSpin G25 columns . The integrity of the purified in vitro-transcribed RNAs was controlled by denaturing RNA gel electrophoresis. Small RNA and low range single-stranded RNA ladders (New England Biolabs) were used to estimate size of the in vitro-transcribed RNAs. Only RNA molecules that migrated on denaturing gels as one sharp band of the expected size with no sign of degradation and/or early termination were used for experiments. RNA concentration was measured with a NanoDrop spectrophotometer .DNA constructs were previously generated to make in vitro-transcribed  CAB-box . AdditioXenopus oocyte system [Xenopus oocytes, followed by 18.4–23 nL (0.5 pmol) of Drosophila U2 snRNA into the cytoplasm. As a control, we injected U2 snRNA alone into the cytoplasm. Oocytes were incubated overnight in OR2 medium at room temperature and nuclei were then manually isolated in 5:1 KCl-NaCl isolation medium [Drosophila U2 snRNA was tested for 2’-O-methylation induced by the injected guide RNAs.We previously described in vitro modification assays using the e system . Brieflyn medium . RNA wasDrosophila U2 snRNA alone or U2 snRNA supplemented with a guide RNA. RNA was extracted after 6–12 h of incubation and analyzed for 2’-O-methylation.In the second type of in vitro modification assay, 150 nuclei were manually isolated and collected in mineral oil. The nuclei were centrifuged to disrupt the nuclear envelope and release the nuclear contents. The resulting nuclear extract was treated with micrococcal nuclease (New England Biolabs) followed by addition of EGTA to inactivate the nuclease. In vitro-transcribed RNAs were added to the nuclease-treated nuclear extract. RNAs were either O-methylation was analyzed by a fluorescent primer extension-based method. We previously described a 6-FAM-labeled oligonucleotide specific for Drosophila U2 snRNA [O-methylation, we performed primer extension with a low concentration of dNTP (0.004 and 0.01 mM) along with control reactions at a high concentration of dNTPs (0.5 mM). AMV reverse transcriptase (New England Biolabs) was chosen to perform 2’-O-methylation analysis. Based on our previous experience [Xenopus oocytes. Fragments were separated on a capillary electrophoresis instrument  using parameters suggested by the manufacturer. To align fragments from different samples we added the Gene Scan-500 Liz Size Standard (Applied Biosystems) to each sample. We determined the exact positions of modified nucleotides with RNA sequencing products. In vitro-trans, cribed Drosophila U2 snRNA was used as a template. For chain termination, one dNTP was mixed with acyNTP (New England Biolabs) at a 1:3 (dNTP:acyNTP) ratio. Data were analyzed and visualized using GeneMapper software (Applied Biosystems).2′-U2 snRNA . To deteperience , this enO-methylation analysis are not truly quantitative and modification efficiency in Xenopus oocytes often depends on the frog used for assays. Therefore, we always injected experimental and control RNAs into oocytes from the same batch and performed enzymatic reactions on control and experimental RNAs simultaneously. For each experiment, we performed several replicates and ran serial dilutions of each sample on capillary columns.Reverse transcription-based methods of 2’-Drosophila Stt3A gene was amplified from genomic DNA and cloned into the pCS2-GFP vector under a CMV promoter. This fragment contains introns 3 and 4, which encode snoRNA:Me28S-U3344a and scaRNA:MeU2-C28, respectively. In addition, two mutant variants were made in which scaRNA:MeU2-C28 was replaced with either an extremely short but still functional single-domain mutant or the same mutant with C in the 2-nt spacer between ASE and box C . The transiently transfected cells were collected after 48 h cultivation and RNA was extracted using the TRIzol reagent. RNA was purified with the Direct-zol RNA MiniPrep Kit .Drosophila snoRNA:Me28S-U3344 and to the 3’ half of Drosophila scaRNA:MeU2-C28. Hybridization was performed at 40 °C for Me28S-U3344 detection and at 34 °C for MeU2-C28 detection in DIG Easy Hyb buffer (Roche). An anti-Dig antibody conjugated with alkaline phosphatase and the chemiluminescent substrate CDP-Star (Roche) were used to visualize the hybridized probes.Total RNA (3 μg per sample) was separated on 8% polyacrylamide–8M urea gels and transferred onto a nylon membrane . The expression of exogenous guide RNAs in HeLa cells was detected with digoxigenin-labeled probes. The probes were specific to O-methylation. For this purpose, we chose scaRNA:MeU2-C28, a Drosophila box C/D guide RNA. Earlier, we showed that this guide RNA is active in the Xenopus oocyte system [The major goal of our experiments was to determine the structural elements in box C/D guide RNAs that play critical roles in RNA-guided post-transcriptional 2’-e system . Here, wDrosophila MeU2-C28 on U2 snRNA was completely eliminated by a point mutation in the ASE at the position targeting U2-C28 for modification  and snoRNA:Me28S-U3344 . We transfected HeLa cells with these constructs and analyzed the expression of Drosophila box C/D snoRNAs by northern blots. We found completely normal processing for both full length and extremely short single-domain MeU2-C28. An extremely short non-functional variant with a CC-spacer between the box C and the ASE (single-domain guide with mutations 9 and 10) accumulated at much lower levels, but otherwise was fully processed (So far, our data confirm 2’-equences . Remarkanstructs A with a rocessed B. From ofunctional eukaryotic box C/D snoRNPs is still challenging, detailed structural analysis of functional and non-functional variants is not feasible. Archaeal box C/D RNP complexes are accessible for the structural analysis. However, a symmetrical two-domain box C/D RNP is essential for function in Archaea [It was previously shown that all four core proteins of box C/D guide RNPs bind normally to a chimeric H/ACA snoRNA that contains only half of the box C/D domain . Here, w Archaea ,26,27,28Our data strongly suggest that there are structural and functional interactions between the terminal and internal K-turn domains of box C/D guide RNAs. When one of the K-turns is mutated, the other K-turn protects the full-length guide RNA from loss of modification activity. At the same time, extremely short single-domain box C/D guide RNAs can also exist and function in the cell, although they are very sensitive to minute changes in their structure. What is the advantage, if any, of having such extremely short and vulnerable guide RNAs? How does a functional guide RNP particle assemble on these very short RNAs? Are there other functions of the short single-domain guide RNAs for which this size is crucial? These and other questions arising from our study still remain open.