With the exception of the Republic of South Africa, DRC and Senegal, most reports on ATL and HAM/TSP in African patients have been published by European and American clinicians and involve immigrants or medical returnees to Europe (France and the UK) and the United States. There is clearly a huge underreporting of these diseases on the African continent. The genetic diversity of HTLV-1 is greatest in Africa, where six distinct genotypes  have been identified. The most frequent genotype in central Africa is genotype b. The other genotypes found in central Africa  are very rare. The vast majority of HTLV-1 strains from West and North Africa belong to genotype a, the so-called ‘Cosmopolitan’ genotype. These strains form five clades roughly reflecting the geographic origin of the infected individuals. We have recently shown that some of these clades are the result of recombination between a-WA and a-NA strains. Almost all sequences from southern Africa belong to Transcontinental a-genotype subgroup.The African continent is considered the largest high endemic area for the oncogenic retrovirus HTLV-1 with an estimated two to five million infected individuals. However, data on epidemiological aspects, in particular prevalence, risk factors and geographical distribution, are still very limited for many regions: on the one hand, few large-scale and representative studies have been performed and, on the other hand, many studies do not include confirmatory tests, resulting in indeterminate serological results, and a likely overestimation of HTLV-1 seroprevalence. For this review, we included the most robust studies published since 1984 on the prevalence of HTLV-1 and the two major diseases associated with this infection in people living in Africa and the Indian Ocean islands: adult T-cell leukemia (ATL) and tropical spastic paraparesis or HTLV-1-associated myelopathy (HAM/TSP). We also considered most of the book chapters and abstracts published at the 20 international conferences on HTLV and related viruses held since 1985, as well as the results of recent meta-analyses regarding the status of HTLV-1 in West and sub-Saharan Africa. Based on this bibliography, it appears that HTLV-1 distribution is very heterogeneous in Africa: The highest prevalences of HTLV-1 are reported in western, central and southern Africa, while eastern and northern Africa show lower prevalences. In highly endemic areas, the HTLV-1 prevalence in the adult population ranges from 0.3 to 3%, increases with age, and is highest among women. In rural areas of Gabon and the Democratic Republic of the Congo (DRC), HTLV-1 prevalence can reach up to 10-25% in elder women. HTLV-1-associated diseases in African patients have rarely been reported  AlmosIn this analysis of the available data for HTLV-1 in Africa, we were confronted with seven critical issues:Data are lacking for several regions. HTLV-1 has been little studied, if at all, in many African countries. This is the case, in particular, for some heavily populated regions of North and East Africa including the Horn of Africa. There is a critical need to perform studies in these areas.The major epidemiological features of HTLV-1 infection include an increase in prevalence with age, and a higher prevalence in women than in men . An abseA large proportion of the epidemiological studies on HTLV-1 performed in Africa was conducted on relatively small groups of people and focused on either blood donors or pregnant women. These two populations are not representative of the general population and are not comparable. Indeed, studies of the population of pregnant women provide information exclusively on the prevalence in infection with HTLV-1 in young women. Similarly, in West and Central Africa, blood donors are often young men, university students or soldiers, or, in some cases, relative of hospitalized patients requiring transfusions , 8. We win situ, in hospital wards, by local physicians. With the exception of the Republic of South Africa, the Democratic Republic of the Congo (DRC) and Senegal, most reports on ATL and HAM/TSP in African patients have been published by European and American clinicians and relate to immigrants or medical returnees in Europe (France and the UK) and the USA. There are many reasons for local underreporting of ATL: a lack of awareness of these rare clinical entities among most clinicians, including many hematologists, the rapid progression observed in the absence of effective treatment, a lack of availability of the diagnostic tests for HTLV-1, immunostaining for pathology purposes , and tests for molecular T-cell clonality. The clinical and epidemiological features of ATL, HAM/TSP and ID therefore remain virtually unknown in many parts of Africa.HTLV-1-associated diseases in African patients have rarely been reported Most epidemiological studies on HTLV-1 are based on serological methods, which can be difficult to interpret in samples from sub-Saharan Africans. Indeed, in many tropical regions, particularly in sub-Saharan Africa, many ELISA-positive results have been found to be false positives. Epidemiological studies based on ELISA alone therefore overestimate the prevalence of HTLV-1, by a factor two to six, depending on the study. Historical publications 1984–1989) were valuable, as they drew attention to the high prevalence of HTLV-1 in certain regions/countries of Africa, but in most of the cases they largely overestimated HTLV-1 seroprevalences, and cannot, therefore, be included in a rigorous epidemiological review . Thus, a84–1989 wIndeterminate seroreactivity patterns, with partial immunoreactivity against HTLV-1 on Western blots (therefore not corresponding to the defining criteria of the confirmatory kits), are often observed with samples from Central Africa. In a few cases, these indeterminate patterns correspond to individuals infected with defective HTLV-1 viruses or with a low proviral load. These individuals are unlikely to transmit the virus, and the importance of their detection in terms of public health is debatable. In some cases, partial immunoreactivities may correspond to ongoing seroconversion in the individual concerned, as demonstrated by some longitudinal serological studies . IndeterPlasmodium falciparum in Cameroon  or N (for New) profiles, have been shown to be frequent in Central Africa –18. TheyCameroon , 18. In Cameroon .7. Small clusters of high HTLV-1 prevalence are often observed, with a lower prevalence of the virus in neighboring regions. This has been clearly shown in southern Japan , some re22.1We searched PubMed for studies published since 1983 on HTLV-1 prevalence and the two main diseases associated with HTLV-1 infection found in individuals of African origin: adult T-cell leukemia and tropical spastic paraparesis or HTLV-1-associated myelopathy. We also included most of the book chapters and abstracts published from 20 international conferences on HTLV and related viruses held since 1985. The results of recently published meta-analyses regarding the situation of HTLV-1 worldwide and in particular in West and sub-Saharan Africa, have also made it possible to find some original articles that have been used in this study –28.Keyword searches were also performed to identify articles from gray literature sources, including Google Scholar and Web of Science. Restrictions were imposed regarding language  and study design  or molecular (PCR) tests).Medical Subject Heading (MeSH) terms and keyword searches were performed on PubMed and other databases (Google Scholar). Additional studies were identified through manual searches of the references of relevant studies and reviews. The search terms were combined with ‘and’ statements and searches were performed on article titles, abstracts and subjects.The PubMed search terms used were: “HTLV-1” OR “HTLV1” OR “Human T-cell Lymphotropic Virus” OR “Human T-cell Lymphotropic Virus Type 1” OR “Human T-leukemia Virus Type 1” AND “ATL” OR “ATLL” OR “Adult T-cell leukemia” OR “Adult T-cell leukemia/lymphoma” OR “leukemia-lymphoma” OR “HAM/TSP” OR “Tropical spastic paraparesis” OR “HTLV-1-associated myelopathy” OR “Tropical spastic paraparesis/HTLV-1 associated myelopathy” OR “Infective dermatitis”. All these query items were associated with the name of a country or region in Africa  and territories of the Indian Ocean region . The former names of countries were also used .The database searches were updated regularly, with the last update on August 3, 2022.The titles and abstracts of the publications identified were screened independently by three reviewers , with any publication deemed potentially relevant by either reviewer carried forward for full-text evaluation. Disagreements during full-text review were resolved by consensus.2.2We identified 220 references relating to epidemiological studies on HTLV-1 performed on the African continent. Most were published in the 1990s Table S1The vast majority of the studies were carried out in Central and West Africa, leaving much of North and East Africa with little information available on the presence of HTLV-1 Figure 32.2.1The prevalence of HTLV-1 in pregnant women ranged from 0.2 to 7.7% in West Africa, 0.6-6.8% in Central Africa, 0% to 1% in East Africa, and 0.2 to 0.6% in Southern Africa. No HTLV-1 infection was detected in the only study performed in North Africa. The highest prevalences were reported in Gabon (2.1-6.8%), DRC (5%), Nigeria (3.3-7.7%), and Guinea-Bissau (2.2-3.3%) Table 1.2.2.2The prevalence of HTLV-1 in blood donors ranged from 0 to 0.1% in North Africa, 0 to 2.6% in West Africa, 0.7 to 6% in Central Africa, 0 to 1.1% in East Africa, and 0 to 0.1% in Southern Africa. As for pregnant women, prevalences were highest in Central African countries, reaching 6% among blood donors from East Gabon, 4% in DRC, and in 2.6% in some Nigerian blood donors (West Africa) (2.6%) Table 2.via contaminated blood (As indicated above (point 3), populations of blood donors are not representative of the general  population. In Gabon, the age- and sex-adjusted prevalence of HTLV-1 was four times higher in the rural population than in blood donors . Howevered blood .2.2.3Only one study was conducted on a rural population of adult outpatients in North Africa: it reported a prevalence of 0.06% in Egypt. In West Africa, the prevalence of HTLV-1 in the rural adult population generally ranged from 0.7 to 1.7%, but was much higher in Benin and Guinea-Bissau, where it ranged from 1.5 to 4.5% and from 4.6 to 5.2%, respectively. In West Africa, HTLV-1 prevalence was lower in urban populations, ranging from 0.5% in Nigeria to 2.7 to 4.4% in Guinea-Bissau Table 3.As reported for other endemic areas endemic in the world, HTLV-1 infection is unevenly distributed. Prevalences in rural areas tend to be higher than in urban populations, but geographic variation is often observed in countries with high levels of endemicity for HTLV-1. Thus, in Gabon, HTLV-1 prevalence varies between regions, ranging from 5% to 14% , 98, 99.2.3As exemplified above, it is difficult to estimate the overall prevalence of HTLV-1 prevalence for individual countries or regions of Africa due to the considerable variation of HTLV-1 prevalence between studies. Some of these variations may be due to the differences in the nature of the confirmatory assay used or evolutions in these same assays over the years, limiting the comparability of HTLV-1 prevalence values over time. Another significant variable is the demographics of the study population, as the mean age distribution and sex ratio of the population can have a major impact on prevalence . Age- and sex-adjusted prevalence values would provide a more accurate comparison, but it was not possible to extract these data, as age and sex data were missing in several studies. Moreover, some surveys performed on small study populations (fewer than 200 people) were included as no other studies were available for the countries concerned, but the prevalence values obtained in such small studies must be interpreted with care. There is a critical need for studies in areas for which few data are available.3Few epidemiological studies have assessed the risk factors for HTLV-1 infection in Africa. In most studies, the principal limitation is the absence of a multivariable approach. As a result, the proportion of transmission attributable to each mode of transmission and the associated risk factors remain largely unknown in Africa.3.1By contrast to other endemic areas, such as Japan, the USA and the Caribbean region, the sexual transmission of HTLV-1 has rarely been investigated in Africa, even though this mode of transmission is probably the most relevant Table 4.3.2Mother-to-child transmission is also a major mode of HTLV-1 transmission, given the high prevalence in pregnant women in most African countries and the lack of alternatives to breast milk. The rate of mother-to-child transmission has been estimated at 17.5% in Gabon , 22% in 3.3via contaminated blood products, during blood transfusion, organ transplantation or the intravenous injection of drugs. The lack of systematic screening for HTLV-1 infection in African blood banks and the observed prevalence among blood donors indicate that there is a significant risk of contamination via blood. An association between blood transfusion history and HTLV-1 infection has previously been reported in Central African Republic (CAR) (HTLV-1 can be transmitted ic (CAR)  while inic (CAR)  (Table ic (CAR) . In Soutic (CAR) .Despite the risk of HTLV-1 transmission and secondary HAM/TSP development, routine screening is not widely implemented in Africa due to its low cost-effectiveness in countries with high endemicity but low-income .3.4In addition to blood transfusion, a history of hospitalization, surgery or injections has also been shown to be independently associated with HTLV-1 infection in various studies in Central Africa Table 4.3.5The zoonotic transmission of STLV-1 from non-human primates (NHPs) has only been demonstrated clearly in Central Africa, where bushmeat hunting is frequent and the presence of STLV-1 in monkeys and apes appears to be high. Zoonotic transmission seems to occur primarily through bites from NHPs, the risk being particularly high for gorilla bites , 114. NH3.6HTLV-1 prevalence can differ between ethnic groups. For example, it is higher in Pygmies than in Bantus in Central African countries , 99, 11444.1We know very little about adult T-cell leukemia in Africa. Indeed, only a few cases in African patients have been reported to date S2. ThisThe few cases of ATL identified locally were mostly performed in South Africa, Senegal, Nigeria, Gabon and, to a lesser extent, in Mali Table 5.Little is known about the situation in East Africa, but because some patients suffering from other types of cutaneous T-cell lymphomas have been reported in Rwanda  and TanzThe vast majority of cases of ATL in patients from Africa reported in the medical literature were diagnosed in France, England and the United States, in immigrants and patients repatriated for medical reasons S2. In FInterestingly, the mean age of ATL cases in Africans is about 40-50 years, similar to that of patients in Brazil and the Caribbean, but very different from that in Japan, where the mean age of ATL cases is 60-70 years old . This diThe published cases of ATL in patients of African origin are listed in the 4.2Data for HAM/TSP in African patients are also very fragmentary, with both a significant lack of awareness of this disease among local clinicians and a lack of means for diagnosing HTLV-1 infection. Moreover, when considering neurological diseases, the scarcity of neurologists and of biological and technical means, such as radiological explorations (scans and MRI) in several African countries represents a major handicap. Indeed, it is crucial to differentiate the diagnosis of HAM/TSP from other spastic paraplegia-like syndromes, which are quite common in Africa. Finally, many patients with progressive gait disturbance do not, or cannot seek medical attention. The rare studies on HAM/TSP were performed in capital cities, and there are almost no data available for rural populations. This may account for the considerable underreporting of HAM/TSP in Africa, despite its chronic nature.Only a few relatively large series of HAM/TSP cases in African patients have been published by or in association with local African physicians S3. ThesThe first of these studies was performed in RSA, on a series of 24 patients, mostly women, aged between 40 and 50 years, from the Durban area of Natal Province . A subseThe second study described a series of 25 HAM/TSP patients hospitalized in Lisala, Equateur Province in DRC. Most of the patients were women, aged 30-40 years at onset . AnotherThe other four countries in continental Africa in which studies on smaller series of HAM/TSP cases have been performed locally are Côte d’Ivoire, Senegal and Togo in West Africa, and Gabon in Central Africa S3. TropIn Southern Africa, besides the cases reported in RSA, a few cases have been reported in Mozambique, Zimbabwe, Zambia and Eswatini (formerly Swaziland).Most of the other reported cases of HAM/TSP in African patients were diagnosed in France or England, in immigrants or repatriated patients Table S3We also included studies from the Seychelles, an archipelago in the Indian Ocean with a history and settlement closely linked to the eastern coast of the African continent, in this review. In 1987, a cluster of 17 cases of HAM/TSP was reported in patients living in the Seychelles . Finally4.3Infective dermatitis (ID) is a rare dermatological condition associated with HTLV-1 infection that was initially described in Jamaican children in 1990 . It has We have reported the first cases of ID in African patients in 2004 . Over a Several cases of HTLV-1-associated uveitis and myositis have been reported in Sierra-Leone, Togo, and Côte d’Ivoire , 217.Unfortunately, very little progress has been made over the last two decades towards understanding the clinical and epidemiological aspects of HTLV-1-associated diseases in Africa. The prevalence, incidence, and geographic distribution of these diseases, which are globally neglected in Africa, remain almost unknown to this day. Only major efforts to inform and sensitize local medical staff — both clinicians and the personnel of biology and virology laboratories — can improve our knowledge of this virus and associated diseases on this continent.55.1The genetic diversity of HTLV-1 is the greatest in Africa, where six distinct genotypes  have been identified . These gThe most frequent genotype in Central Africa is genotype b Figure 8The vast majority of HTLV-1 strains from West and North Africa belong to genotype a  , 225. ThWhen considering Southern Africa, many strains have been sequenced, but they are almost exclusively originate from South Africa  and Moza5.2via several routes in monkey colonies in natura: sexual transmission, mother-to-offspring transmission, through wounds acquired during fights for sexual dominance, and during the consumption of infected prey  and apes , are naturally infected with STLV-1 (C. nictitans (Allenopithecus nigroviridis) to humans, particularly after contact with NHPs . Indeed, a low proviral load , 240 or 5.3What is the origin of the cosmopolitan HTLV-1a genotype? No closely related STLV-1 strain has ever been described. This raises several possibilities. STLV-1a could still exist, possibly with a low prevalence, in one or more simian species that have not yet been studied. The natural host of STLV-1a may have disappeared or the virus may have been cleared from the original host. When considering a molecular clock model, the choice of either hypothesis would generate very different HTLV-1a spillover and diversification dates (see part V).env sequences are available; these viruses are not, therefore, represented in in vivo depends on the expression of auxiliary proteins: P12/8, P13 and P30  in 16 vertical transmission chains  revealed that 10 related individuals carried an identical virus, with no mutation (in a 755-nt segment of the LTR) . In thisn chains . From thTherefore, the apparent HTLV-1 mutation rate (substitution/site/year) increases with the number of transmission chains (human-to-human transmission) per period. Therefore, in conditions where the virus is frequently transmitted between different individuals, e.g. intravenous drug usage, or nosocomial transmission, or through ritual practices (such as scarification), the mutation rate per year will be higher than in conditions where the number of transmission rates is lower . Of note, due to the limited genetic variability within an individual, as stated above, prolonged breast feeding or recurrent intercourse between two individuals should be considered as a single transmission chain.Differences in apparent mutation rates depending on the modes of transmission have been previously demonstrated for HTLV-2: HTLV-2 mutation rate was found higher among intravenous drug users then when considering populations where the virus was transmitted vertically or by sexual transmission .via continuous accumulation of mutations. The model rarely accounts for saltatory evolution processes, such as recombination.Phylogenetic studies have estimated the apparent mutation rate at 2.1E−7 and 8E−7 subst/site/year , 263. Th6.2Until recently, it was believed that HTLV-1 evolution occurs only through genetic drift and mutation. Recombination was ignored in HTLV-1 evolution. This was further comforted by a study on 10 patients, which reported that HTLV-1 infected T-cells contain a single integrated provirus .However, in some cases, proviral load is higher than 100% in ATL patients –267, evienv gene. It remains unclear why the only recombinant groups identified to date arose through recombination between a-WA and a-Sen strains. It is possible that recombination occurred between these two groups because the infected populations are more mobile and have ended up encountering each other more frequently. Alternatively, not all recombination events may generate functional viruses. The fitness of the generated clade may be low, leading to the disappearance of the other recombinants over time. It seems most likely that the detection of recombination requires the parental groups to be sufficiently divergent. If they are too alike, it may prove difficult to differentiate between strains obtained by mutation and those obtained by recombination. In Africa, there are no clear clades with significant bootstrap values among a-TC strains. It appears likely that recombination has occurred but that there were so few differences between the parental strains that we cannot trace the recombination back to the parental strains. For the identification of such recombinants, we would need more informative positions. The generation of complete genome sequences would, therefore, be the key to identifying recombinant groups.Indirect evidence of multiple infection with different HTLV-1 strains was brought by phylogenetic studies. Indeed, we have identified two viral clades that have emerged through recombination . Some st6.3Several previous studies used the molecular clock hypothesis to date speciation between the different HTLV-1 genotypes , 269–271First, as mentioned above, evolution rates depend on the mode of transmission . Molecular clock methods should be applied only to strains with identical modes of transmission . MoreoveSecond, molecular clock models are unable to take saltatory evolution phenomena, such as recombination and deletion/insertion into account correctly. We have now demonstrated that recombination occurs in HTLV-1, and may be frequent, rendering the molecular clock impossible to implement within the HTLV-1a genotype.Third, it is unclear whether STLV has the same evolution dynamics in simian and human hosts. Therefore, when studying genotypes present in both NHPs and humans, the estimated rate may correspond to a weighted average between the rates in the different species.In light of all these issues, the estimated date of divergence between HTLV-1a and HTLV-1c ranges from 10 to 120 thousand years ago , 271. WeThe African continent is currently considered as a highly (if not the highest) endemic area in the world for HTLV-1, with a few million people infected. The distribution of HTLV-1 which remains poorly understood on this continent, appears to be very heterogenous, with high prevalence of HTLV-1 in the central African regions and low prevalence in the eastern and northern regions. There is a huge underreporting of HTLV-1 associated diseases in Africa, including ATL and HAM/TSP.The number of African people infected with HTLV-1 and developing associated diseases is likely to increase over the next century due to rapidly growing population and the aging of populations , as well as the lack of preventive measures to reduce the spread of HTLV-1.In order to improve the situation (current and future) and reduce the burden of HTLV-1 in Africa, the priorities are clear and will require both inter-African and North/South collaborations and specific financial support to:Raise awareness through major information campaigns, as has been developed in other endemic areas, such as Brazil and Japan. These campaigns should primarily target healthcare professionals  to improve knowledge about HTLV-1 and related diseases, which are still largely neglected by the medical community in many parts of Africa.Promote large-scale studies in general (representative) populations and in hospitals. Such comprehensive studies are essential to assess the prevalence of HTLV-1 infection and the real situation regarding associated diseases.Develop public health measures in areas of high endemicity, with particular emphasis on blood banks to prevent the spread of HTLV-1 by transfusion, which may be associated with the development of HTLV-1 neurological diseases.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Trans. Camb. Philos. Soc., 8, 441–455.), and exactly via the Generalized Lagrangian Mean, is divergent even in an incompressible fluid. We show that the Stokes velocity can be naturally decomposed into a solenoidal component, J. Fluid Mech., 661, 45–72. (doi:10.1017/S0022112010002867)) which we specialize to surface gravity waves and implement effectively using a Lie series expansion. We further show that the corresponding Lagrangian-mean momentum equation is formally identical to the Craik–Leibovich (CL) equation with The Stokes velocity This article is part of the theme issue ‘Mathematical problems in physical fluid dynamics (part 1)’. This ressible . Expansiximation ,11 to thMcIntyre  shows fry, uE in , is incoIn et al. )right of  is conveows that  assumes ows that  and in mows that .In this paper, we revisit the concept of Stokes velocity. For surface gravity waves, we exhibit a natural Helmholtz decomposition of uS in  and arguiar form  of the SGLM . Consider a bucket of initially motionless water. If the water is agitated, for example, by pressure forcing at the surface, then the potential energy of the water is increased, or equivalently the centre of mass of the water is elevated above its initial height. The fluid at the bottom of Bühler’s bucket, however, cannot move in the vertical and so any definition of ‘Lagrangian mean’ that tracks the position of the centre of mass of the fluid—such as GLM—will be divergent in this situation, even though the velocity of the water is entirely incompressible. Conversely, any definition of ‘Lagrangian mean’ resulting in a strictly solenoidal Lagrangian mean velocity—such as glm—cannot track the centre of mass of the fluid.The plan of the paper is as follows. In §2, we sketch the derivation of the standard form  of the S. 2 (a)We start by recalling the traditional derivation of the Stokes velocity in . The posansatzb) is ambiguous because requiring only that the To average the fast wave oscillations in , we consb) and (b) by requiring that b) produces the familiar resultSubstituting (2.3b) and  into (2.b) and  and 2.2b and a) to (2.8b), we have used wave incompressibilityb),Let us examine the divergence of velocity  in many b) with b) then results inThe solenoidal Stokes velocity arises naturally if a small change is made in the derivation in §2(a): suppose we decide from the outset to change the definition of ‘Lagrangian mean’ so that uL in  is solenn In addition to incompressibility in , surfacezero and  simplifi ⋅ uS in , but resThe curl of the Stokes velocities does not vanish:. 3Discussions of the deep return flow associated with a surface-gravity wave packet argue  that:‘the return flow Similar sentiments are expressed in ,24. With (a)b) is consistent with the idea that horizontal convergence within the wave-active zone pumps fluid downwards, out of the wave-active zone, with the vertical velocity We start with the easy solenoidal case, with Stokes transporta) and (3.3b) are order a) and (3.3b), the solenoidal pumping can be expressed entirely in terms of surface quantities:Vertical integration of  (b)We turn now to the traditional definition of the Stokes velocity. Define the Stokes transport viab). Instead, integrating (b), (3.3a) and (3.3b).Because egrating  over the (c)A widely used expression for the Stokes transport is. 4We return to the definition of the Stokes velocity and show how the glm theory of Soward & Roberts  rationalent (see  for othe (a)To introduce glm, it is convenient to rewrite equation  governinEquation  becomesThe defining condition of glm is expressed as follows. The small parameter ondition , 4.6) i iϵ is reEquation  generaliξ2¯ in  which le solving  by Tayloso that  implies so that . In the  (b)The glm formalism can be regarded as an instance of classical perturbation theory, which approximates solutions to the ordinary differential equation  by perfoe series ,20 proviIntroducing the decomposition  into 4. and usinrding to . We impoondition .Expanding q as in  we relatq as in  repeatedrivative , is4.14a) since Introducing  and 4.1 into 4. gives aton using . This pr side of  to order (c)A Lagrangian-mean momentum equation, analogous to Andrews & McIntyre Theorem I , can be ϵ ,21. We sm 1-form ,274.22ν scalars ; see appWe show in appendix A that , togetheondition  recoversondition  to obtairoducing  and expaThe Lagrangian-mean momentum equation greatly simplifies for surface gravity waves: typical wave frequencies  Lq12 in  against ondition , we compocity in  (see [26. 5This paper examines a problematic aspect of the Stokes velocity iar form  of uS: wThe main point of the paper, however, is that the Stokes velocity, understood as the difference familiar . The nonuL exist . One of uL exist , leads tic form, . We givequations ,4, widelWe stress that, in general, the coordinates associated with a Lagrangian-mean trajectory We conclude by noting that the assumption


Silicon is a beneficial element for plant growth and production, especially in rice. Plant roots take up silicon in the form of silicic acid. Silicic acid channels, which belong to the NIP subfamily of aquaporins, are responsible for silicic acid uptake. Accumulated experimental results have deepened our understanding of the silicic acid channel for its uptake mechanism, physiological function, localization, and other aspects. However, how the silicic acid channel efficiently and selectively permeates silicic acid remains to be elucidated. Recently reported crystal structures of the silicic acid channel enabled us to discuss the mechanism of silicic acid uptake by plant roots at an atomic level. In this mini-review, we focus on the crystal structures of the silicic acid channel and provide a detailed description of the structural determinants of silicic acid permeation and its transport mechanism, which are crucial for the rational creation of secure and sustainable crops. Oryza sativa (Os)Lsi1 is localized mainly in roots and involved in silicic acid loading, whereas OsLsi6 is localized mainly in nodes and is involved in silicic acid unloading from the xylem vessels  has a significantly reduced silicon uptake capacity compared to the wild type, and the yield of mutant rice is lesser than the wild type due to the reduction in the uptake of silicon , 2006. T) belong . Rice ha vessels . Cellula vessels . In rice vessels . Two genentified . OsLsi2 entified . OsLsi3 entified . Therefoiosperms . In EquiLsi1 gene revealed that Lsi1 belongs to the NIP subfamily of the AQP family AQP1, Recently, structures of Lsi1 from rice  have been reported by two research groups . Both X-Arabidopsis thaliana, AtNIP5;1, did not confer silicic acid permeability  force profiles show that the SF requires the most energy to pass silicic acid through the channel, indicating that the SF is most important for silicic acid selectivity . Mutatioeability . These eeability . Howevereability . A planteability . Another65 of SF . The funvia the Grotthuss mechanism by exchanging a covalent bond between H and O with the neighboring water molecules , JP17H06879, JP19K16056, and JP21K15029 (YS) from MEXT, Japan.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In 2019 the International Retrovirology Association (IRVA) established November 10th as HTLV World Day with the aim to increase awareness about this infection. In 2020, HTLV Channel was founded with the same primary objective. HTLV Channel is an interactive platform that uses social media to disseminate research advances and basic knowledge about HTLV. In addition, this platform aims to support patients and to foster research collaboration, mainly in Latin America. In the year of its foundation, HTLV Channel celebrated the World HTLV Day with a webinar co-organized with the Brazilian Ministry of Health. In 2021, the Pan American Health Organization/World Health Organization (PAHO/WHO) joined forces with HTLV Channel to produce the HTLV World Day 2021 Webinar. Four decades after the discovery of HTLV (https://youtu.be/pbpruvW5JA0 and https://youtu.be/g6DUXoqIH9A).Human T-cell lymphotropic virus type 1 (HTLV-1) is a neglected retrovirus that causes severe diseases worldwide. It is a blood borne virus that can also be transmitted by unprotected sex and from mother-to-child, predominantly  of HTLV . The WebWe should be ashamed because we are still with these diseases. We are supporting health inequities. So, we should do something as a world community” Prof. Patricia Garcia.“Although the amount of research compared with other diseases is small, most HTLV clinical research has been on HTLV-1-associated myelopathy (HAM) and adult T cell leukemia lymphoma (ATL), despite which patient care has improved little and both diseases still have poor outcome. The causative link between HTLV-1 and a multitude of inflammatory conditions, e.g., infective dermatitis, uveitis, pulmonary disease, and its impact on co-infections is starting to be better recognized and this was clearly presented at the Webinar by Dr. Fabiola Martin and Prof. Eduardo Gotuzzo. Indeed, the association of HTLV-1 with a 57% increase in the adjusted mortality rate revealed in a recent meta-analysis  significHTLV-1 infects mainly those with low-income, limited formal education, females, and vulnerable groups. HTLV-1-associated diseases and the stigma that is commonly associated with this infection impact patients' work capacity, reducing their income and accentuating health inequities , 6. FamiIf we do not look, we will not find it” Dr. Noreen Jack.“HTLV-1/2 infection is considered a silent infection. Most patients are still considered asymptomatic, despite the clear impact of HTLV-1 on quality of life, mental health, and socio-economic status of those infected individuals who have not developed HAM or ATL. These two well characterized diseases that are associated with HTLV-1, HAM and ATLL, have a long incubation time, and their onset is usually late in adulthood. Therefore, there is a need for active surveillance to identify those infected. Until widespread screening is available targeted testing should be offered and include sexual partners and family members of PLHTLV, and those at higher risk of acquiring STIs and blood borne viral infection, such as patients with STIs, those that take Pre-Exposure Prophylaxis (PrEP) to prevent HIV infection, sex workers, and people who inject recreational drugs.Most countries screen blood donors for HTLV-1/2 infection. In the Americas, the uptake of this policy is high. In 2016 and 2017, 90% of blood units collected in Latin America and the Caribbean were screened for HTLV-1/2 . HoweverThe development of a low-cost, rapid, point of care test for HTLV-1/2 screening would facilitate test uptake worldwide and should be a research priority. Local and international agencies should establish guidelines for the diagnosis of HTLV infection, including the need of confirmatory testing.The lack of knowledge about HTLV is the main complicating factor” Ms Adjeane Oliveira.“The lack of awareness about HTLV-1/2, including among health care workers, was mentioned by patients' representatives and experts as a major barrier to the advance of policies to control this infection. The lack of awareness prevents adequate care, contributes to increase stigma, and impairs the inclusion of HTLV-1/2 in the international and local health agenda. In addition, it is detrimental to research and contributes to the limited interest of pharmaceutical companies into the development of new therapeutic and preventive interventions and the scarcity of research funding in general.The global community should take advantage of online platforms and social media to disseminate information about HTLV-1/2. In fact, the Webinar itself is a good example of how these tools may be useful to push for action. In Brazil, HTLV was included in an online course about STI for healthcare workers and a clinical guideline about HTLV was published. Similar guidance is under development in the UK and expected to be published during 2022. The HTLV Community should take this opportunity of renewed interest in HTLV-1/2 being shown by international agencies, such as the PAHO/WHO, to strengthen the discussion about this virus. In this regard, a strong collaboration between researchers, patients' representatives, and policy makers, as reported in Brazil and UK, is key to the implementation of effective measures to prevent and control HTLV-1.It was interesting to observe the dichotomy when comparing the view of Australia's healthcare professionals and that of the aboriginal community from Central Australia, who suffer with a high prevalence of HTLV-1 infection, affecting almost half of the adult population . AccordiWe certainly can do better to reduce mortality and morbidity” Prof. Peter Figueroa.“An opportunity to introduce policies for HTLV-1/2 at relatively low cost is to take advantage of programs that are already implemented. Most countries have local programs related to maternal health, HIV and STIs, neglected diseases, etc. PAHO/WHO recently launched a platform for the elimination of a number of infectious diseases . HTLV-1/It is important for policies to be tailored to the local setting. In this regard, particularities of indigenous communities from Brazil and Australia were shared during the Webinar. As stated above, the medical leadership‘s assumptions have been a major barrier to the implementation of policies for the aboriginal community in Australia. In Brazil, cultural, linguistic, and geographic barriers were identified as difficulties to implement policies targeting indigenous people in that country. In Brazil, the collaboration between HTLV-1/2 specialist researchers and the Special Secretary of Indigenous Health (SESAI) to increase awareness about HTLV-1/2 and the production of informative factsheets about HTLV-1/2 in different indigenous languages are examples of good practices that can be achieved at relatively low cost.We need now to institute simple programs, starting with antenatal testing and mother-to-child transmission prevention” Prof. Peter Figueroa.“There is a common belief that as there is no cure for HTLV infection, nothing should be done. However, the absence of a curative treatment for HTLV-1/2 infection reinforces the importance of implementing policies to prevent new infections. Several effective measures to prevent new infections are known. The effectiveness of avoidance or shortening the duration of breastfeeding to reduce HTLV-1 transmission is well recognized. The implementation of antenatal screening was unanimously identified as a priority. Indeed mother-to-child transmission disproportionally contributes to HTLV-1 associated diseases .The implementation of HTLV-1/2 antenatal screening integrated with other maternal and child health programs is an opportunity to identify those infected, to prevent new transmissions, to obtain epidemiological data and to verify the success of the policy. For this to succeed there is a need to increase awareness about HTLV among healthcare professionals, so they can provide proper counseling for seropositive women.Why is this virus still around and without effective surveillance, control or cure?” Dr. Fabiola Martin.“Professor Peter Figueroa's statement, reflected in the title of this manuscript “We need to translate research into meaningful health policies and programs to HTLV” summarizes the main message of the webinar. There is still a long way to go before we can start planning HTLV-1/2 elimination, but we must start paving the way now. Many different actors are involved: local governments, HTLV experts, patients' representatives and international agencies and need to start immediately to act together toward this goal. Paraphrasing a Chinese proverb: “The best time to act was decades ago. The second-best time is now.”The short-term goals, for each of the actors, drawn from this interactive webinar are presented in the CR, TA, LS, and RI conceptualized the manuscript. CR drafted the initial manuscript. CR, TA, LS, AM, RM-S, MAF, GPT, and RI wrote and critically revised the final manuscript. All authors approved the final version of the manuscript.RI is a Research Grantee (#312979/2018-5) of the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, CNPq. GPT is supported by NIHR Imperial College Trust. TA is supported by a scholarship from HCFMUSP with funds donated by NUBANK under the #HCCOMVIDA scheme.CR and TA are co-founders of HTLV Channel, a non-profit self-funded platform by HTLV Channel. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules encoded by endogenous genes, which play a vital role in cell generation, metabolism, apoptosis and stem cell differentiation. C3H10T1/2, a mesenchymal cell extracted from mouse embryos, is capable of osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. Extensive studies have shown that not only miRNAs can directly trigger targeted genes to regulate the tri-lineage differentiation of C3H10T1/2, but it also can indirectly regulate the differentiation by triggering different signaling pathways or various downstream molecules. This paper aims to clarify the regulatory roles of different miRNAs on C3H10T1/2 differentiation, and discussing their balance effect among osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation of C3H10T1/2. We also review the biogenesis of miRNAs, Wnt signaling pathways, MAPK signaling pathways and BMP signaling pathways and provide some specific examples of how these signaling pathways act on C3H10T1/2 tri-lineage differentiation. On this basis, we hope that a deeper understanding of the differentiation and regulation mechanism of miRNAs in C3H10T1/2 can provide a promising therapeutic method for the clinical treatment of bone defects, osteoporosis, osteoarthritis and other diseases. C3H10T1/2, isolated from mouse embryos, is the primary cell component of loose connective tissue. It is differentiated from mesenchymal cells in the embryonic stage . miR-335The differentiation of tri-lineage of C3H10T1/2 depended on the spatiotemporal differences in gene expression of cells, which was not only determined by the internal developmental program of cells, but also influenced and regulated by the extracellular environment, and sometimes such external control conditions or environment played a decisive role in the formation of specific cells . miRNAs In this review, we first identified the key signaling pathways and molecular factors related to the differentiation of C3H10T1/2 tri-lineage and explored how miRNAs participated in and regulated these signaling pathways and factors. In addition, the significance of tri-lineage differentiation of C3H10T1/2 in the early diagnosis of bone defects, prevention of bone loss and bone marrow fat was briefly discussed.The biological generation of miRNA is a multi-step process Fig. , startinStudies have found that the 5' end of miRNA is more conservative in contrast to the 3' end, and there is a frequently conservative A1 in the targeted mRNA 3′-UTR, adjacent to the downstream sequence of the seed site . A1 is nBased on the sequence of the 5' end of miRNAs or the 3' end of miRNAs, interactions between miRNAs and their targeted mRNAs can be forecast . In addiOsteogenic differentiation is a key step of bone formation; that is, C3H10T1/2 undergoes a complex process of osteoblast progenitor cells, osteoblast cells and finally osteogenesis . C3H10T1Recently, substantial researches have highlighted the regulation of miRNAs on osteogenetic differentiation of C3H10T1/2 , which is mainly found in subcutaneous and visceral tissues . PatholoSubstantial miRNAs have been shown to be closely related to the adipogenic differentiation of C3H10T1/2 , thus inhibiting osteogenic differentiation of C3H10T1/2 . Some miMAPK signaling pathway also plays a key role in regulating adipogenic differentiation in C3H10T1/2 cells. The differentiation of C3H0T1/2 cells into adipocytes is a complex physiological process that includes clonal expansion, growth arrest and terminal differentiation. MiRNAs indirectly participate in these adipogenic differentiation process by directly regulating the expression of MAPK family proteins. The same miRNA that acts at different times may have the opposite effect. For instance, when miR-143 is overexpressed during the clonal expansion stage, the adipogenic differentiation is inhibited, whereas the overexpression of miR-143 during the growth arrest stage or terminal differentiation stage promotes the adipogenic differentiation of C3H10T1/2 .MiRNAs can also indirectly regulate cell chondrogenic differentiation through the MAPK signaling pathway. It is noteworthy that unlike osteogenic and adipogenic differentiation, miRNAs often co-regulate cytokines on the MAPK family proteins in chondrogenic differentiation. Surprisingly, interleukin is the most common among the cytokines involved in regulation. For instance, Sun et al.  discoverWnt signaling is transmitted by a seven-channel family of transmembrane g protein-coupled acceptors (frizzled (Fzd)) and a family of arrow/Lrp co-receptors  or Ryk or Ror transmembrane tyrosine kinases . The binThe Wnt signal pathway is a conserved signaling axis involved in a variety of physiologic process like proliferative, differentiative, apoptotic, migratory, invasive and tissular homeostasis –121. RecIt has been shown that the Wnt/β-catenin signal pathway participates in the osteogenic differentiation of C3H10T1/2 cells. The stimulation of the Wnt/β-catenin signal pathway facilitates osteogenetic differentiation and the inhibition of this pathway induces osteoclast differentiation . For insRecent researches have also revealed that the Wnt signal pathway exerts a key effect on the adipogenic differentiation of C3H10T1/2 cells. Research shows that the overexpression of miR-210-3p promoted the osteogenic differentiation and inhibited the adipogenic differentiation of C3H10T1/2, which may be related to Wnt signaling pathway .At present, massive studies have focused on the role of Wnt/β-catenin signal pathway in chondrogenic differentiation of stem cell, but there are still few studies on C3H10T1/2 cells as experimental targets. The clarification of this mechanism is conducive to the innovation of clinical treatment methods for osteoarthritis and cartilage damage diseases, and the mechanism of Wnt signal pathway in chondrogenesis has more space for exploration.Bone morphogenetic protein (BMP) is an important member of TGF-β superfamily . BMPs paNumerous studies demonstrate that BMP-2, 4, 5, 7 can effectively promote the tri-lineage differentiation of C3H10T1/2  can directly or indirectly act on key transcription factors such as Runx2 or Osterix and interact with each other, thus forming an intricate regulatory network that cooperatively participates in the tri-lineage differentiation of C3H10T1/2. We believe that with the further development of relevant research, the complete molecular mechanism of osteogenic, adipogenic and chondrogenic differentiation of stem cells is expected to be thoroughly uncovered, so as to provide a new theoretical basis and therapeutic targets for the effective clinical prevention and treatment of osteoporosis, ectopic ossification and other diseases.In recent years, with the in-depth discoveries and researches of C3H10T1/2, its extensive sources, outstanding proliferation ability, multi-differentiation potential and satisfactory biocompatibility with various 3D scaffold materials display its exciting potential in the treatment of bone defects. Further researches have found that miRNAs are pivotal for C3H10T1/2 cell tri-lineage differentiation.miRNAs exert an important effect on the balance among osteoblast, adipogenic and chondrogenic differentiation, which provides a promising treatment regimen for osteoporosis, osteoarthritis and other diseases. In addition, miRNAs can also extensively participate in the regulation of Wnt signaling pathway, MAPK signaling pathway and BMP signaling pathway by acting directly on specific proteins and indirectly competing against a target gene, thus affecting the tri-lineage differentiation of C3H10T1/2. It is worth noting that when the same miRNAs act on the above three signaling pathways, they may have dual or anti-temporal effects. For instance, the Wnt signaling pathway has been evidenced to facilitate the osteogenetic differentiation of C3H10T1/2 and play a dual role in its adipogenic differentiation, whereas chondrogenic differentiation may be inhibited by such pathway. The same miRNA affecting the expression of MAPK signal pathway at the clonal expansion stage inhibited the adipogenic differentiation of C3H10T1/2, while it plays a completely opposite role in the growth arrest stage or terminal differentiation stage. In addition, BMP signaling pathway and Wnt signaling pathway often promote and influence each other during osteogenic differentiation of C3H10T1/2.Massive studies have deeply revealed the mechanisms of miRNAs with regard to the regulation of C3H10T1/2 differentiation and the balance among osteoblast, adipogenic and chondrogenic differentiation. However, the understanding of the functional miRNA biogenesis and localization, and especially the understanding pertaining to how tissue-specific expression facilitates their specific modulatory patterns in different biological processes are elusive. Moreover, though there is a certain basis for the research on the mechanism of miRNA-induced cell differentiation, few people apply this theoretical knowledge to develop miRNA-targeted molecular drugs for bone diseases. Safety and efficacy are the main challenges in the design and development of miRNA-targeted drugs and are also the main reasons for the failure of early drug development. In addition, drug resistance is more likely to occur in targeted drug therapy compared with conventional treatment regiments, and the therapeutic effect in the middle and later stages is still unclear. However, it is of great interest to apply the discoveries obtained from miRNA regulation to clinical treatments. By optimizing the route of administration and time of action, miRNA-targeted drugs may also achieve better therapeutic effects. We sincerely hope that further researches on the regulatory mechanisms of miRNAs may lay the ground for the clinical therapies of bone defect-related illnesses.


HTLV-1 is a global infection with 5-20 million infected individuals. Although only a minority of infected individuals develop myelopathy, lymphoproliferative malignancy, or inflammatory disorders, infection is associated with immunosuppression and shorter survival. Transmission of HTLV-1 is through contaminated blood or needles, mother-to-child exposure through breast-feeding, and sexual intercourse. HTLV-1 is a delta retrovirus that expresses immunogenic Gag, Envelope, TAX, and Hbz proteins. Neutralizing antibodies have been identified directed against the surface envelope protein, and cytotoxic T-cell epitopes within TAX have been characterized. Thus far, there have been few investigations of vaccines directed against each of these proteins, with limited responses, thus far. However, with new technologies developed in the last few years, a renewed investigation is warranted in search for a safe and effective HTLV-1 vaccine. HTLV-1 is prevalent in many parts of the world, including Central & South America, Caribbean Islands, Africa, northeast Iran, southern Japan, Melanesia, Australia, where endemic rates are 5-10%, but in some isolated communities endemic rates as high as 50% have been identified . HoweverDiseases caused by HTLV-1 include an aggressive CD4+ lymphoproliferative malignancy, designated adult T-cell leukemia lymphoma (ATLL) and spastic paraparesis, known as HTLV-1 myelopathy (HAM) or tropical spastic paraparesis (TSP) . AlthougTransmission of HTLV-1 is through contaminated blood or needles, sexual intercourse, and breast feeding . Rarely,After transmission, a balance between virus replication, expansion of infected cells, and immune response to the virus leads to the establishment of a proviral load “set point” after HTLV-1 acquisition. However, there have been few studies of acute HTLV-1 infection aimed at assessing the determinants and kinetics of the set point. In a study of three individuals who acquired HTLV-1 infection after organ transplantation, the proviral load set point was reached within 6 weeks . Thus, tvia cell-to-cell contacts than via free virus particles  and envelope (env) genes, and enzymes from the protease (pr) and polymerase (pol) genes that encode the viral protease, reverse transcriptase (RT), and integrase (IN). Virus infection is mediated by a receptor complex on the cell surface and the virus particle is taken into cells by membrane fusion. Virus uncoating activates RT to copy the two copies of the plus-strand viral RNA genome into a dsDNA copy that is integrated into cellular DNA by the viral IN. Transcription is mediated by cellular RNA polymerase II, and viral RNAs may be spliced or unspliced prior to export from the nucleus. Translation produces viral proteins, including the Gag, Gag-Pr, and Gag-Pr-Pol polyprotein precursors which are processed into individual components during virus budding. Envelope is synthesized and processed in the reticuloendothelial-Golgi system, transported to the plasma membrane, and incorporated into the budding virus. Regulatory proteins, are encoded from multiple spliced RNAs and include the transactivator protein, TAX, the regulator of splicing and nuclear export, REX, the helix zipper protein, HBZ, as well as proteins designated p12, p27, and p30 that are presumed to regulate virus replication and/or pathogenesis.HTLV-1 is a member of the δ retrovirus family, which also includes HTLV-2 and bovine leukemia virus (BLV) . HTLV-2 HTLV-1 encodes two oncoproteins, TAX and HBZ –24. TAX env gene encodes the gp62, 488 amino acid envelope precursor glycoprotein which is cleaved by furin-like enzymes into a 20 amino acid (AA) signal peptide, a 292 amino acid gp45 surface envelope protein (SU) and a 176 amino acid gp21 transmembrane envelope protein (TM). SU has four asparagine (N)-linked glycosylation sites , glucose transporter 1 (GLUT-1), and neuropilin-1 (NRP-1) . SubsequRetroviral envelope proteins, like those of coronaviruses, arenaviruses, filoviruses, pneumoviruses, and orthomyxoviruses are class I fusion proteins . They arPolyclonal and monoclonal antibodies to HTLV-I envelope proteins, including SU, have been demonstrated to neutralize HTLV-I infection –36. Neutin vivo  rabbits . The aniE coli-produced HTLV-1 envelope protein  nanoparticles . This prA recombinant HTLV-1 glycoprotein protein vaccine was made against the HTLV-1c glycoprotein . This suin vivo -based vaccines hold the promise to revolutionize the infectious disease prevention field by addressing current manufacturing challenges and offering novel vaccine compositions . CriticaA safe and effective vaccine preparation in animal studies will eventually be considered for clinical trials. This would include phase 1 safety and pharmacokinetics studies in volunteers. Phase 2 and 3 trials would then be targeted to a population at risk of acquisition of HTLV-1. The largest and most suitable population would likely be individuals at significant risk of sexual acquisition of HTLV-1 in an endemic region.Previous studies in rabbits and monkeys suggest that inoculation with HTLV-1 gene products may provide protection against infection. The ideal vaccine candidate and method of inoculation remains to be deciphered. In addition, immune correlates of response remain to be determined. Although some studies suggest that neutralizing antibodies against the HTLV-1 envelope protein may provide protection against infection, the role of cytotoxic T lymphocyte responses against envelope and other viral proteins remains to be fully characterized. In addition, antibody-dependent cellular cytotoxicity, known to occur in primary infection –90, coulThe WHO issued a technical report in 2020 with a strong recommendation for global strategies to eliminate transmission of HTLV-1 . Thus, fThe author confirms being the sole contributor of this work and has approved it for publication.This work was funded by PHS grants CA 252869, CA255095, CA100730, and AI156014 to LR.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


PEG supplementation increased aminopeptidase and acid protease activity after 3 days of ensiling (p < 0.05) and increased carboxypeptidase activity during the entire ensiling process (p < 0.05). The keystone bacteria changed following PEG addition . In total, 510 metabolites were identified after 60 days of sainfoin ensiling, with 33 metabolites annotated in the Kyoto Encyclopedia of Genes and Genomes database. Among the metabolites, phospholipids were the most abundant (72.7% of 33 metabolites). In addition, 10 upregulated and 23 downregulated metabolites were identified in the PEG-treated group when compared with the control group, after 60 days of ensiling (p < 0.05). Pediococcus  and Lactobacillus  were the bacteria most correlated with metabolites. The results suggested antagonistic effects between Lactobacillus and Pediococcus during ensiling. The decreased proteolysis during sainfoin ensiling was mainly attributed to the inhibition of protease activity by CT, particularly carboxypeptidase activity. In addition, proteolysis decreased partly due to CT inhibiting Pediococcus activity during ensiling, with Pediococcus being significantly and positively correlated with dopamine after 60 days of ensiling .Condensed tannins (CT) from sainfoin have a high capacity to inhibit proteolysis. A previous study reported that CT from sainfoin can inhibit lactic acid bacteria activity and decrease ammonium-nitrogen (N) content during sainfoin ensiling; however, no study has focused on the metabolome of ensiled sainfoin. The objective of the present study was to investigate the effects of CT [following supplementation of deactivated CT with polyethylene glycol (PEG)] on protease activity, keystone bacteria, and metabolome during sainfoin ensiling. According to the results, PEG amendment increased non-protein N, amino acid, and soluble protein contents significantly (in the 49.08–59.41, 116.01–64.22, and 23.5–41.94% ranges, respectively,  Lespedeza cuneata Dum.-Cours.), crown vetch (Coronilla varia L.), hedysarum , birdsfoot trefoil (Lotus corniculatus L.), and sainfoin (Onobrychis viciifolia). Among the plants above, CT from sainfoin is favored considering it exhibits the highest capacity to bind protein, and inhibits cellulose digestion by rumen bacteria the least so that it delivers a greater amount of protein to the abomasum without adverse effects on rumen microbial activity  contents . During activity .Sainfoin is widespread in North America, Europe, and the Middle East, as a perennial forage and fodder legume . CT fromPediococcus activity without affecting Enterobacter activity. Enterobacter can produce amino acid nitrogen during ensiling; however, CT decreases amino nitrogen (AN) contents during the entire ensiling process  was planted in Shihezi City, Xinjiang Province, China. Whole-plant sainfoin was harvested in July 2021 at the early flower stage. After wilting for 12 h to a dry matter (DM) content of approximately 240 g/kg fresh weight, the samples were chopped into 1— to 2-cm pieces. The samples were then sprayed with a solution of 640 g/l PEG  at a rate of 217 ml/kg DM to achieve a CT:PEG ratio of 1:2 in the samples (Sainfoin ( samples . AccordiSilage samples from separate bags for 3 (five fermentation days) × 2 (two treatments) × 3 (replicates) of each silage treatment were collected. Silage samples were dried at 65°C for 48 h and ground using a 1.0-mm in preparation for DM content determination. The NPN, neutral detergent-insoluble protein (NDIP), and acid detergent-insoluble protein (ADIP) concentrations were analyzed according to g for 10 min at 4°C. The supernatant was stored at −80°C until proteolytic enzyme activity analysis. L-leucine-p-nitroanilide was used as the substrate for aminopeptidases (APs), N-carbobenzoxy-L-phenylalanine-L-alanine was used as the substrate for carboxypeptidase (CP), and azocasein was used as the substrate for acid protease (ACP). Proteolytic enzyme activity analysis was carried out according to Samples (10 g) were obtained from each silage treatment, blended in a homogenized  50 ml of 0.1 M sodium phosphate buffer (pH 6.5), and then centrifuged at 10,000 × g at 4°C for 15 min, the supernatant was collected for ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis.After 60 days of ensiling, 50-g samples of the ensiled sainfoin treatments were obtained and metabolites extracted using 400-μl methanol:water  solution. The mixture was allowed to settle at −20°C and treated using a high throughput tissue crusher  at 50 Hz for 6 min, followed by vortexing for 30 s and ultrasonication at 40 kHz for 30 min at 5°C. Afterward, the samples were placed at −20°C for 30 min to precipitate proteins. After centrifugation at 13,000 × Chromatographic separation of the metabolites was performed on a Thermo UHPLC system equipped with an ACQUITY BEH C18 column . The mobile phases consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile:isopropanol  solution (solvent B). The solvent gradient was as follows: from 0 to 3 min, 95% (A):5% (B) to 80% (A):20% (B); from 3 to 9 min, 80% (A):20% (B) to 5% (A):95% (B); from 9 to 13 min, 5% (A):95% (B) to 5% (A):95% (B); from 13 to 13.1 min, 5% (A):95% (B) to 95% (A):5% (B), from 13.1 to 16 min, 95% (A):5% (B) to 95% (A):5% (B) for equilibrating the systems. The sample injection volume was 2 μl and the flow rate was 0.4 ml/min. The column temperature was maintained at 40°C. The mass spectrometric data were collected using a Thermo UHPLC-Q Exactive Mass Spectrometer equipped with an electrospray ionization (ESI) source operating in either positive or negative ion mode. The optimal conditions were set as follows: Aus gas heater temperature, 400°C; Sheath gas flow rate 40 psi; Aus gas flow rate 30 psi; ion-spray voltage floating (ISVF), −2,800 V in negative mode and 3,500 V in positive mode, respectively; normalized collision energy, 20–40–60 V rolling for MS/MS. Data acquisition was performed in the Data Dependent Acquisition (DDA) mode. The detection was carried out over a mass range of 70–1,050 m/z.1) in R on the Majorbio Cloud Platform.2 Principal component analysis (PCA) based on an unsupervised method was applied to obtain an overview of the metabolic data. General clustering, trends, or outliers were visualized. All the metabolite variables were scaled to unit-variances before conducting the PCA. Orthogonal partial least squares discriminate analysis (OPLS-DA) was used for statistical analysis to determine global metabolic changes between comparable groups. All the metabolite variables were subjected to Pareto Scaling before the OPLS-DA. The model validity was evaluated based on model parameters R2 and Q2, which provide information on interpretability and predictability, respectively, of the model, and minimize over-fitting risk. Variable importance in projection (VIP) was calculated using the OPLS-DA model. The p-values were estimated using paired Student’s t-test on single-dimensional statistical analysis.Multivariate statistical analysis was performed using the ropls package  was used for further analyses.Statistical significance among groups was identified with VIP values >1 and p < 0.05. The ammonium nitrogen (NH4+-N), cured protein (CP), and bacteria sequence (SRA number for bacteria: PRJNA796365) data used were obtained from our former study . Data were analyzed using IBM SPSS 22 Statistics . Significant differences between treatments were determined using Tukey’s test at er study .p < 0.05), then stabilized with the prolongation of ensiling; SOLP content was the lowest in the PEG-treated group after 14 days of ensiling, which was 7.63 g/kg DM. However, the control had the lowest SOLP content during the entire ensiling process (p < 0.05). Similar results were observed for the effect of ensiling time on NDIP and ADIP contents. PEG addition decreased NDIP and ADIP contents markedly during ensiling by up to 100%, when compared with the contents in the control (p < 0.05). NPN and SOLP content trends in the control were similar. In addition, PEG supplementation increased NPN content significantly during the entire ensiling process (p < 0.05), by up to 59.41%, when compared with the contents in the control after 60 days of ensiling. Ensiling time increased amino acid (AA) contents in both the control and PEG-treated groups (p < 0.05), by 16.43 and 35.50%, respectively, after 60 days of ensiling. Furthermore, the PEG-treated group had the highest AA contents during the entire ensiling process (p < 0.05), which were up to twofold the contents in the control group.Nitrogen distribution during sainfoin ensiling is listed in p < 0.05). PB1 content decreased to 49.61% of the initial contents after 7 days of ensiling and then stabilized over time in the control sainfoin silage. However, the PEG-treated groups still had 86.42% of the initial PB1 content after 60 days of ensiling. In addition, PEG-treated groups had higher PB1 contents than the control group during the entire ensiling process (p < 0.05). However, PB2 content was higher in the control group than in the PEG-treated group after 60 days of ensiling, which were 22.26 (% CP) and 10.08 (% CP), respectively. PB3 content decreased rapidly during 14 days of ensiling in both the control and PEG-treated silage. PB3 content was higher in the control group than in the PEG-treated group during the entire ensiling process (p < 0.05). After 60 days of ensiling, PB3 content in the control was 134% higher than that in the PEG-treated group. PC content was higher in the control group than in the PEG-treated group after 60 days of ensiling, which were 10.02 (% CP) and 7.11 (% CP), respectively.The protein fraction dynamics during sainfoin ensiling determined based on the CNCPS system are shown in p < 0.05), which increased by 188.99% and 194.13%, respectively, when compared with the levels in the control. The PEG-treated group exhibited the highest CP activity at 7–60 days of ensiling (p < 0.05), which was 38.23–69.43% of the protease activity at 0 day. AP and ACP activities both increased with an increase in the ensiling period in the control group (p < 0.05). In the PEG treatment, both AP and ACP activities were the highest after 14 days of ensiling; however, they decreased after 60 days of ensiling (p < 0.05). CP activity did not change with change in the ensiling period in the control group (p > 0.05); however, CP activity increased after 30 days of ensiling in the PEG-treated group (p < 0.05).Protease activity during sainfoin ensiling is presented in Sphingobacterium, Stenotrophomonas, Pantoea, and Cellulosimicrobium. In the PEG treatment group, the keystone bacteria were Microbacterium, Enterococcus, Sphingobacterium, and Brevundimonas.Bacterial community network analysis results are illustrated in p < 0.05), 10 upregulated metabolites and 23 downregulated metabolites were identified in the PEG-treated group when compared with the control after 60 days of ensiling, as shown in In total, 510 metabolites  were ideEnterobacter abundance and metabolites (p > 0.05). Lactobacillus were significantly and highly positively correlated with prostaglandin E1, phosphatidylglycerol (two metabolites), leukotriene E4, D-Tagatose, phosphatidate, prostaglandin C1, deoxyadenosine, dodecanoic acid, and gamabufogenin , and were significantly negatively correlated with L-methionine, dopamine, cardiolipin, phosphatidylethanolamine, taurocholic acid, and guanosine . Pediococcus were significantly negatively correlated with phosphatidylserine, phosphatidate (four metabolites), phosphatidylcholine (three metabolites), phosphatidylglycerol (two metabolites), citric acid, leukotriene E4, D-Tagatose, prostaglandin C1, and deoxyadenosine , and were significantly positively correlated with triacylglycerol, dopamine, cardiolipin, phosphatidylethanolamine, and taurocholic acid . Weissella was significantly positively correlated with prostaglandin F3a .The results of correlation analysis between bacterial abundance and metabolites in sainfoin silage are shown in Enterococcus, Stenotrophomonas, and Pantoea. Enterococcus was significantly negatively correlated with one metabolite, phosphatidate . Stenotrophomonas was significantly positively correlated with prostaglandin F3a, taurocholic acid, and guanosine . Pantoea was significantly negatively correlated with phosphatidylcholine, phosphatidate, prostaglandin C1, deoxyadenosine, dodecanoic acid, and gamabufogenin , and were significantly positively correlated with cardiolipin, phosphatidylethanolamine, and guanosine .Three keystone bacteria were correlated with metabolites, including via two steps. First, plant proteases hydrolyze proteins into peptides and free amino acids (FAA); afterward, further degradation by microbial activity occurs, usually mediated by Clostridium and Enterobacter (Clostridium is more active in moist environments with moisture levels >75% (Clostridium activity was detected during sainfoin silage with a DM content of 23% (4+-N (Enterobacter relative abundance during the entire process of sainfoin ensiling (fermentation 60 days); however, AN content was the highest in PEG-treated sainfoin silage during the entire process of sainfoin ensiling (fermentation 60 days). Furthermore, AN content increased in both the control and PEG-treated group with an increase in ensiling time . Other researchers have observed that PEG causes high levels of buffer soluble N in silage, which is attributed to the strong tannin-protein binding effect of CT in sainfoin  were less than those in the PEG-treated group  after 60 d. Therefore, CT from sainfoin could facilitate the maintenance of high-quality sainfoin silage. Among the N distributed in silage, N in peptides may be more useful for rumen microbial N synthesis, as a previous in vitro study observed that certain bacteria from rumen utilize N from peptides more efficiently than N from FAA or from ammonia . However, in the case of Megasphaera elsdenii from the rumen, which is one of the main lactate-fermenting rumen bacterial species, AA N would be more useful than peptide N (Pediococcus relative abundance) during ensiling  fractions during sainfoin ensiling, suggesting that CT can decrease protein degradation during ensiling.Remarkably, PEG addition increased the SOLP content, while decreasing NDIP and ADIP contents during the entire ensiling process. Similar results were observed by Neolamarckia cadamba leaf (NCL) had no effect on CP activity during 30 days of ensiling. The mechanisms via which CT inhibit protease activity, such as tyrosinase, including competition with substrates for active enzyme sites and binding to the enzyme substrate complex, which occur through hydrogen bonding and hydrophobic interactions (Rhododendron pulchrum leaves (Ziziphus jujuba Mill. var. spinosa [Bunge] Hu ex. H. F. Chow)  , and Pin needles  show varCellulosimicrobium is a Gram-positive anaerobic bacteria that secretes several enzymes, such as glycosidases, galactosidases, glucosidases, and esterases; it exploits different types of carbon sources  to produce organic acids, in addition to being able to secrete nitrate reductase, pyrazinamidases, and pyrrolidine acyl aromatic amide enzymes  sequestration as it was negatively correlated with CO2 production during ensiling  was upregulated following PEG addition, whereas citric acid was downregulated following PEG. Furthermore, two nucleosides, including cytosine and deoxyadenosine, were downregulated and one nucleoside, guanosine, was upregulated following PEG addition. Catechin also inhibits bacteria bacteria . Enterobensiling . The resDuring ensiling, various metabolites are produced through complex metabolic reactions, which are regulated by different metabolic pathways . The mosLactobacillus were significantly negatively correlated with six metabolites among the upregulated metabolites and were significantly positively correlated with 10 metabolites among the downregulated metabolites. On the contrary, Pediococcus was significantly positively correlated with five metabolites among the upregulated metabolites, and significantly negatively correlated with 15 metabolites among the downregulated metabolites. Additionally, phospholipids were the most affected metabolites and were correlated with both Lactobacillus and Pediococcus. The results suggested the antagonistic effects between Lactobacillus and Pediococcus during ensiling. Among the metabolites upregulated following PEG addition, dopamine is converted by tyrosine. Pediococcus was significantly positively correlated with dopamine, but Lactobacillus was significantly negatively correlated with dopamine. The cell-free supernatants of some Pediococcus (Pediococcus acidophilus) have stronger stimulation effects on dopamine production by Enterobacter (Escherichia coli) than Lactobacillus (Lactococcus lactis subsp. lactis)  . Accordivia the inhibition of protease activity, particularly CP activity. In addition, the decrease in proteolysis was partly attributed to CT inhibiting Pediococcus activity, based on the highly positive correlation between Pediococcus and dopamine. CT also affected bacteria community structure, potentially through shifts in keystone bacteria. The most distinct metabolites between the control and PEG-treated groups were phospholipids, which indicated that CT inhibited bacterial activity through decreased phospholipid synthesis during ensiling.Polyethylene glycol addition decreased NDIP and ADIP contents, with NPN contents increased, during sainfoin ensiling. CT from sainfoin decreased proteolysis mainly The original contributions presented in this study are included in the article/RH: conceptualization, methodology, and writing – review and editing. FZ: conceptualization, resources, validation, and supervision. XW: resources and investigation. CM: conceptualization, methodology, and visualization. All authors have read and agreed to the published version of the manuscript.