In Argentina, the human T-cell lymphotropic virus type 1 (HTLV-1) infection has been documented mainly among blood banks with a prevalence of ~0.02–0.046% for Buenos Aires city, 0.8% for the northeast, and 1% for the northwest; both areas are considered endemic for HTLV-2 and 1, respectively. Policies and specific guidelines for testing blood donors for HTLV are included since 2005. Screening for antibodies is performed at blood banks and confirmatory testing is performed at reference laboratories. There are no specific recommendations for the assistance of communities and individuals affected, nor referral to specialized clinics on the HTLV infection. In 2016, as a strategy of intervention, we opened a specialized clinical attendance in a referral infectious diseases public hospital for the comprehensive approach to patients with HTLV, offering follow-up and counseling for patients and their families for the early diagnosis of HTLV-1/2 and related diseases. During the study, 124 patients with presumptive HTLV positive diagnosis from blood bank, symptomatic patients (SPs), relatives, and descendants visited the unit. A total of 46 patients were HTLV positive (38 HTLV-1 and 8 HTLV-2). There were nine SPs . All patients with HTLV-1 and−2 were offered to study their relatives. Two out of 37 (5.4%) descendants tested were positive for HTLV-1. Sexual partners were studied; among 6 out of 11 couples (54.5%) were found positive (5 HTLV-1 and 1 HTLV-2). Other relatives, such as mothers (1/2) and siblings (1/6), were positive for HTLV-1. According to the place of birth among HTLV-1 carriers, 58% were born in an endemic area or in countries where HTLV infection is considered endemic while for HTLV-2 carriers, 12.5% were born in an endemic area of Argentina. The proviral load (pVL) was measured in all, patients with HTLV-1 being higher in symptomatic compared with asymptomatic carriers. In addition, two pregnant women were early diagnosed during their puerperium and breastmilk replacement by formula was indicated. Inhibition of lactation was also indicated. Our study provides tools for a multidisciplinary approach to the infection and reinforces the importance of having specialized clinical units in neglected diseases, such as HTLV for counseling, clinical and laboratory follow-up, and providing useful information for patients for self-care and that of their families. The human T-cell lymphotropic virus type 1 (HTLV-1) belongs to the Retroviridae family and the Delta retrovirus genus, together with HTLV-2 and bovine leukemia virus (BLV) and the recently discovered HTLV-3 and HTLV-4 (Human T-cell lymphotropic virus type 1 is the etiologic agent of adult T-cell leukemia/lymphoma (ATL) and may lead to the development of chronic progressive myelopathy named HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) –7. OtherIn Argentina, HTLV-1 infection has been documented mainly among blood banks with a prevalence of 0.02–0.046% for Buenos Aires city, 0.8% for the northeast, and 1% for the northwest of the country. Both areas are considered endemic for HTLV-2 and 1, respectively –21. In aThe City of Buenos Aires is a non-endemic area for these viruses and is a cosmopolitan city that receives a large number of immigrants every year from different areas from Argentina and South America, some of them from endemic areas for HTLV-1. Argentina has guidelines for testing blood donors with no specific recommendations for the assistance of communities and individuals affected by HTLV-1 infection. In 2016, as a strategy of intervention, we opened a specialized clinical attendance for the comprehensive approach of HTLV carriers to implement follow-up and counseling for patients and their families for the early diagnosis of HTLV-1/2 related diseases and to provide medical care. This study aimed to describe the results from the implementation, counseling, and follow-up of HTLV carriers attending at an infectious diseases public hospital during the period 2016–2021.Clinical attendance for HTLV carriers was implemented in the outpatient Centro Municipal de Patología Regional Argentina y Medicina Tropical (CEMPRA-MT) unit at the Hospital Muñiz, which is a centenary referral infectious disease public hospital from the government of Buenos Aires city. We started in April 2016 and patients were scheduled once a week and two medical doctors were responsible for the attendance. Blood donors were referred from blood banks and HTLV carriers were referred to our clinic from other hospitals. The first visit includes counseling, physical and neurological examination, and a laboratory test . ComplemThe protocol was approved by the institution review board of the Hospital Garrahan (Number 639). Informed signed consent was obtained from patients.®, Abbott) until 2018 and since 2019 by electroquimioluminiscence . Antibodies for Chagas disease and for syphilis were studied by quimioluminiscence  following a reverse algorithm, respectively. In all cases, assays were performed following the manufacture's instructions. For HIV testing, samples were studied by a fourth-generation assay , and reactive samples were further confirmed following the national algorithm that includes viral load.Serum samples were studied for HCV antibodies, HBsAg, and Anti HBc for hepatitis B by quimioluminiscence . Fresh and serial coproparasitological studies were requested in search of Strongyloides stercoralis, including plate culture in nutrient agar.Biochemical markers include cellular blood count (CBC) with smear, evaluated by a hematologist, lactate deshidrogenase (LDH), and calcemia which were performed on a Cobas C501®, Abbott)] until 2018 and since 2019 by electroquimioluminiscence Elecsys® HTLV I/II  and reactive samples were tested by LIA  as a confirmatory test following the manufacturer's instructions. A qualitative PCR was performed for untyped virus results. Confirmed HTLV-1 samples were further tested for pVL for HTLV-1 in peripheral mononuclear cells (PBMCs) by an in-house real-time quantitative PCR (qPCR) as previously described  and a total error accepted of 0.5 log10. Both molecular studies were performed at the Virology Unit in the Hospital Garrahan.Blood samples were collected in advance for all HTLV studies to avoid recitation of the patients and following the diagnostic algorithm described . Antibodescribed . The assSubsequent visits were planned individually: for asymptomatic carriers (ACs), annual clinical and laboratory controls, such as pVL, were assigned, and for symptomatic patients (SPs), visits were scheduled according to the needs for each patient, such as consultations, laboratory testing, and referral, to other centers if required. For ACs, they were informed to acquire an annual control through a phone number destined for it. For patients who did not assist to their medical visit, they received a call back to promote it.U-test was used to compare continuous data between ACs and SPs. A two-sided p < 0.05 was considered to indicate statistical significance. Data were analyzed using the Graphpad Prism 5 software package 4.91.Standard descriptive statistics were used to describe the baseline characteristics of the population. Results were expressed as median and interquartile range (IQR). The Mann–Whitney n = 56), SPs (n = 12), relatives (n = 19), and descendants (n = 37). Of them, 46 were enrolled for the follow up study with confirmed HTLV positive diagnosis. Furthermore, 32 patients with presumptive HTLV diagnosis were found to be negative for HTLV antibodies, 29 at the time they were retested , and three patients with a presumptive diagnosis of HAM/TSP. Molecular studies were not performed on these three individuals due to the final diagnosis, which included severe discopathy in one case, and peripheral neuropathy in the other two. During the studied period (April 2016–March 2021), a total of 124 individuals visited our outpatient unit, including those with presumptive HTLV positive diagnosis from blood bank (All 46 HTLV carriers (38 HTLV-1 and 8 HTLV-2) were clinically evaluated. Among the HTLV-1 group, 76% were asymptomatic carriers (29/38) and the source of identification was: 20 blood donors, 5 sexual partners, 1 sibling, 1 mother, and 2 descendants. Among SPs, 2 patients were diagnosed as ATL and 7 patients had HAM/TSP. According to the age at enrollment for HTLV-1, SP and AC had a similar median age of 46 years (IQR = 31–52) and 44 years (IQR = 40–51), respectively. For HTLV-2 carriers, the median age at enrollment was 46 (IQR = 40–52). According to the place of birth among HTLV-1 carriers, 58% were born in an endemic area of Argentina or in other countries where HTLV infection is considered endemic. Moreover, when we analyzed AC and SP, we found that 59 and 56% for the respective groups were born in endemic areas. For HTLV-2 infection, 12.5% of them were born in an endemic area of Argentina. tuberculosis (TB) was found in one HAM/TSP patient also with a diagnosis of syphilis , hepatitis C virus (HCV), and HIV were found positive in 2 HTLV-1 SP (2/46: 4.3%), and Chagas disease in 1 HTLV-1 SP and 1 AC (2/46: 4.3%). Coinfection with S. stercoralis. Among HTLV-2 carriers' coinfection with HIV was documented in one case  and siblings (N: 6), were tested, being 1 mother and 1 sibling positive for HTLV-1 as is shown in All HTLV-1 and−2 carriers were offered to study their relatives. The overall number of descendants were 60, with 37 (62%) were available and willing to be tested, 2 of whom were HTLV-1 positive. The remaining descendants reside outside the Buenos Aires city. Sexual partners were studied; among 6/11 couples (54.5%) were found positive (5 HTLV-1 and 1 HTLV-2). Other relatives, such as mothers . All SPs and ACs returned for medical controls, and the average number of visits was 7  as is shown in 10 (data not shown) which in all cases was inferior to the total error of the assay.A total of 72 pVLs corresponding to 38 HTLV-1 carriers were done, and 19 of them (14 ACs and 5 SPs) had more than one pVL measure. The median pVL HTLV-1 at baseline  corresponding to 29 ACs and 9 SPs was 5.25 logBreastmilk replacement by formula feeding and inhibition of lactation was started in 2 women that were breastfeeding at the time of HTLV-1 diagnosis. Both women were derived from the public maternity of Buenos Aires during their puerperium with previous positive results from blood bank. The two babies born tested negative, one of them at 3 and 12 months of age, and the other one at 24 months of age.During the studied period, none of the HTLV-1 AC evolved to a symptomatic stage.10 was observed . Subsequently, pVLs remained stable for an average period of 30 months (mean pVL: 5.34 log 10).Additionally, two of the ATL cases underwent treatment with CHOEP chemotherapy, AZT/3TC, and alpha interferon and were derived to be treated in specialized onco-hematology centers. In one of them, a chemotherapy scheme of 6 cycles was prescribed, the length of follow-up was 3 years, and pVL was performed on average every 3 months during the first 2 years. Once treatment was completed (average duration: 6 months) a decline of 0.7 logTreatment among patients with HAM/TSP included, in 5 of them, corticosteroid therapy, symptomatic treatment, and physiatry.Only one HTLV-1 carrier died, she was a patient with HAM/TSP and the cause of death was a colon cancer.Human T-cell lymphotropic virus type 1 is associated with high morbidity and mortality clinical conditions, while HTLV-2 is rarely associated with disease. Despite the worldwide distribution, the infection appears to be geographically concentrated and, except from Japan, low- and middle-income countries and ethnic minority population groups in high-income nations are the most affected . The majArgentina has an endemic area for HTLV-1 in the northwest (around Jujuy province), which is reflected in the prevalence among blood banks, pregnant women, and the high incidence of TSP/HAM . The norBuenos Aires city is a non-endemic area, with a prevalence among blood donors between 0.02 and 0.046% similar to other provinces in the center of the country . The freThe outpatient unit described here was implemented to offer medical care and counseling to individuals with previous results from blood banks and SPs referred to our hospital . We found a high proportion of HTLV-1 carriers (38/46), being the source of identification blood donors in 56/124 (45%). It is important to highlight that 52% (29/56) of them were negative at that time, were retested. Considering the sensitivity and specificity of screening assays, this data reinforces the need for confirmation of blood donors' results and the respective counseling to minimize the psychological impact for patients.Clinically, as expected all HTLV-2 carriers were asymptomatic being mainly (87.5%) born in non-endemic areas. None of them were intravenous drug users, one was born in an endemic area for this virus (Chaco province), the second had a coinfection with HIV, and the only risk factor was considered to be the sexual transmission. Three carriers received blood transfusions in 1989, 1996, and 2015, since blood bank screening started in Argentina in 2005, two of them could acquire the infection through that route. The latest two cases were from the areas where HTLV infection is limited, one from Corrientes province and the other one from Santiago del Estero. Remesar et al.  among blood donors from non-endemic areas . This haMoreover, the prevalence among blood banks at the Blood Bank National Program during 2019  was 0.22% for HTLV-1/2 . For the public health system in Buenos Aires city, during the year of 2021 , the prevalence rate for HTLV-1/2 was 0.16% of which more than 70% of the samples were confirmed . Even though Buenos Aires city has a regional blood center, this only centralizes all nucleic acid testing from public health institutions, and HTLV is not included in the molecular panel. Screening for HTLV is performed in each blood bank by antibody detection using ELISA and particle agglutination technique. Confirmatory testing for HTLV is not usually performed in blood banks in Buenos Aires. Reactive samples are referred to a few centers that performed confirmatory testing, and the blood bank returns the result to the donor.Collectively, as a consequence of migration, more HTLV carriers have moved from endemic to non-endemic areas, contributing silently to the transmission of these viruses. This has been also described by Chihara et al., describing the increase in the incidence of ATL in non-endemic areas in Japan and the United States .The pVL was measured in all HTLV-1 carriers. In concordance with previous results, we found that SP had higher values than asymptomatic patients and this was statistically significant . No asymCoinfection between HTLV-1/2 and HIV has been extensively reported. In our country, since 1990, the prevalence rate in people living with HIV (PLWH) have been reported to be between 11 and 19%. In 2015, we found a decline in the prevalence rate of HTLV-1/2 among PLWH to 5% and among those HTLV/HIV coinfected, 40% were also coinfected with HBV and HCV. In the present study, coinfection with HBV, HIV, and HCV was documented among two patients with HAM/TSP (4.2%) . As it hIt has been suggested that TB could influence in the development of HAM/TSP . We founS. stercoralis may influence HTLV-1-related disease progression because screening and treatment are performed in centers caring of people with HTLV-1. Although this association has not been reported in Argentina, we include the screening for our patients and we were not able to study all for S. stercoralis because of the complexity in sample collection. Screening techniques using urine represent a diagnosis opportunity to consider and implement because it is a sample easy to collect by patients.  over the total number of HTLV cases in our group amounts to 21.7% (10/46). This observation reinforces the importance of offering the study to family members. During medical care, it is also important to emphasize the use of condoms in serodiscordant couples or in patients without a stable partner, and avoid exposure to fluids with biological risk, such as blood.Vertical transmission of HTLV occurs primarily through breastfeeding and not transplacentally or during delivery . The risSince the beginning of the study, the number of HTLV carriers enrolled in the follow-up increased. A reduction in care for other diseases was observed for the period 2020–2021 because of the impact of the COVID-19 pandemic. Beside this, we considered that adherence to follow-up in terms of visits average and pVL performed for SP and AC is remarkable. To facilitate this, we coordinate clinical and laboratory visits on the same day with a comprehensive approach through case management.Collectively, this implementation shows that there is an unattended need for medical care of HTLV carriers in the health system. It is necessary to design operational care processes linking blood banks with specialized care centers. This will help avoid lost patients and missed opportunities in diagnosis and counseling. These findings also highlight the need for HTLV screening during pregnancy. On the other hand, our results raise the question whether these clinics reflect the situation of the community in terms of the epidemiology of HTLV-1/2 infection and related diseases.Our study provides tools for a multidisciplinary approach of the infection, and reinforces the importance of having specialized clinical units in neglected diseases, such as HTLV for counseling, clinical and laboratory follow-up, and providing useful information for patients for self-care and that of their families. Furthermore, these clinics will contribute to decrease the misdiagnosis of these infections mainly in non-endemic areas and systematically collect data on the HTLV-1-related diseases leading to a national registry with mandatory reporting.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Hospital de Pediatría Juan P. Garrahan, Comite Revisor de Proyectos de Investigación y Etica. The patients/participants provided their written informed consent to participate in this study.FRS, conceptualization, investigation, methodology, data curation, writing–original draft, and formal analysis. MBB: conceptualization, data curation, formal analysis, investigation, methodology, writing—original draft, writing—review and editing, supervision, and project administration. DM: conceptualization, investigation, methodology, data curation, and writing–original draft. MVP: conceptualization, investigation, methodology, data curation, writing–original draft, formal analysis, and writing—review and editing. LM: formal analysis, methodology, and writing—review and editing. AM: conceptualization, formal analysis, investigation, methodology, writing—review and editing, supervision, project administration, and funding acquisition. TAO: writing—review and editing, supervision, and project administration. All authors contributed to the article and approved the submitted version.All laboratory and complementary studies were supported by the Hospital de Infecciosas F.J. Muñiz . Molecular studies of HTLV-1/2 were supported by Grant N° 2010-0502 (PICT CONICET). The authors would like to acknowledge AIDS Healthcare Foundation Filial Argentina for supporting the publication fee of this paper.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.


Among the many properties of proteins, sugars, nucleic acids, membranes and other cellular components, color is not present. At the same time, we humans have a natural ability of recognizing and appreciating colors, and use them generously, with the aim of both delivering information and pleasing the eyes. In this article, I suggest how we can conciliate these two situations, with the contribution of biologists, artists, and computer graphics and perception experts. The concept can be developed in a series of initiatives involving the community, including discussion sessions, technical challenges, experimental studies and outreach activities. In the representation of biological structures the scientific community has used them in a variety of situations, to indicate the most disparate features of molecular properties. The results are often of stunning beauty, and indeed, the work of structural representation can be considered as much a scientific as an artistic task . The numIn the following, I argue that, because we are so apt at seeing and noticing colors, these could be exploited in a more scientifically sound and consistent way, to indicate specific features that are relevant for the description of biomolecules, their activities, behaviours, and of their surrounding environment.1.1An preliminary note is necessary when discussing colors: in the human population, a relevant minority of male individuals have some form of Color Vision Deficiency , due to a series of mutations that lead to the inability to distinguish one or more colors. This fact should always be considered, as many illustrators do .Many natural object are intrinsically colored , and it is perfectly natural that humans have a visual system apt at seeing and recognizing them. There must have been good evolutionary reasons for the pleasure inspired by the sight of naturally colored scenes, further strengthened by millennia of cultural evolution. As we all associate emotional experiences with colors, it makes sense that our perception and interpretations are based both on the features of our physical environment as well as on cultural traditions. Inhabitants of the polar region know and interpret colors in a different way than peoples of the desert and/or of the forests, reflected in the number of words used to express different colors: while the notion that polar populations (Eskimo or Inuit) have 50 words for shades of white seems to be a metropolitan legend, the success of the fake notion testifies of how plausible we consider such an idea.Cultural standards associated with strong emotions are perceived in an unconscious way: in a most striking example, in Europe we associate the idea of death with the color black, while in India, Japan, and other eastern cultures the corresponding color is white. If this circumstance makes it difficult to propose a color scheme that is universally interpreted, it may also mean that a new code, specifically designed for cellular and/or structural biology, could be developed independent of cultural background, thus avoiding the typical ‘cultural colonialism’ often imposed through western traditions.1.2–Protein, domain, and secondary structure identity–N- to C-terminal sequence order, conservation, mutation propensity, structural stability, (un)certainty and others–Physical properties: Electric charge, lipophilic potential, propensity for Hydrogen bonds (on aminoacids or surfaces)–Changes in condition: ATP association/hydrolysis, activation by binding with external factors, proteolytic activation and othersA colored image is so much eye-catching for us, that we all are attracted and pleased when we both paint and observe them (with exceptions). Indeed, in the field of structural biology, the major graphic tools –4 offer Clearly, there is an interest in showing each of the mentioned features, and more, and using color is an easy and fast means to guide viewers attention, even beyond the aesthetic effect of the image as a whole.We have to notice, however, that on one side the interpretation of such images is necessarily bound to reading the caption, and on the other that different authors use different color palettes, and even the same authors can use variable colors in different presentations.While most of the structural illustrations published in scientific publications are made to illustrate single or a relatively small number of proteins, in the last decade, information about cellular environment can also be included in illustrations of biology: membranes, organelles, presence of Calcium, difference in pH, and other environmental properties.A very interesting and clever development in the management of colors in dynamic multi-scale visualization was proposed by Waldin et al.  in whichIn the past, with the Scientific Visualization Group of the CNR in Italy, we have proposed a visualization method that completely avoids colors , 7, arguHowever, use of B/W only is a non-solution, and the vast amount of data which can be represented, suggests a thoughtful adoption of this communication channel.In the following I examine the varied information that can be conveyed in images, both still and animated, and suggest how a collectively agreed soft standardization may ultimately guide both illustrators and viewers interpretation.2Cells are complex structures, that can be viewed in a range of dimensions: from the atomistic details of an enzymatic reaction to the process of chromatin condensation or of viral invasion, including all the processes at the mesoscale, in which, for example, the activation of proton pumps induces a change in pH in the lysosome, thus producing changes in conformation of proteins within the organelle. In this latter example we can illustrate the atomic details of the pump, but we can also show, in the same picture, the environments: the cytoplasm, with molecules of different size and roles, the membrane, in which are embedded many other constituents beyond the pump, and the interior of the lysosome, also populated with several proteins.As mentioned, the majority of illustrations for peer communication describe a single protein, and it is supposed that viewers are familiar with the surrounding environment, or that the environment is irrelevant for illustrators and viewers. However, these are technical images, whose role is to describe specific features of a single object; as such they are more related to technical drawings than to artistic representations .2.1More and more in recent years, illustrations tend to show different proteins, their assemblies and interactions, and even complete cells , 8, 9. IIt is important to show them, because, in cells, besides its shape and intrinsic motions, what determines the activity of each protein is the relation that it entertains with other proteins and molecules, which is in turn determined by the forces acting in the protein space. Electrical, hydrophobic, Van der Waals and H-bonding forces, i.e. weak local chemical interactions, are important features, and it does make sense to show them, with colors or other visual clues. This will add informative dimensions to our capacity of reading images of cellular scenes. The idea, of course, is not new: most visualization programs can show these features, but hardly simultaneously, certainly not in intuitive way.Ionic interactions. The “standard coloring” of electric charge for biologist  is inverted relative the conventional colors used in physics and engineering (negative red and positive blue or black). However, even knowing the charge polarity, and even after many years using the convention, it is not immediately evident that opposite charges (colors) attract and equal charges repel each other. My suggestion would be that charge is represented by particles, possibly directional, like small comets or arrows, of a single color, flowing in the direction  positive to negative, along the field lines associated with the local charges.Hydro-philic and -phobic potential. This is another feature frequently available from visualizations tools, and frequently expressed in colors. Because this property can be expressed as a continuum , grades of saturation or intensity, applied to any color of choice (even white) could convey the information  and with other molecules. Since also this force is directional, some visual clue to indicate polarity should be introduced.For the three forces above, the representation proposed would also indicate the spatial range of their effect, with electric forces projecting up to several Å from the molecular surface, H-bonds in the order of about 2 Å, and hydrophobic forces working practically at the surface level.2.2Proteins do not live in the vacuum; they spend their lives, from synthesis to degradation in one or more different cellular environment. The environment of the cytoplasm is different from the environment in the nucleus or in other places, and from the extracellular space.In our real world, the picture of a subject is always inserted in a background: for human subjects it could be a sofa, a beach, a mountain, a street… We recognize these environments because they have typical features.Cellular biology gives us indications for “setting the scene” and populating the different environments: ribosomes and cytoskeletal elements in the cytoplasm; narrow spaces in the ER and Golgi; walls decorated with electron transport elements for mitochondria and for chloroplasts (in this latter case with the green chlorofill antennae); DNA in the nucleus, organized in eu- and hetero-chromatin in different zones within it; structural networks and extracellular matrix on the two sides of the plasma membrane and so on.In theory, therefore, we do not need to use colors to distinguish among places. Nevertheless, it might be possible to consider a “colored light”, that illuminating the scene, confers to the different places a typical tone, thus helping in their recognition. Colored light is an easy tool to use in 3D scenes, and interferes only slightly with the colors of other objects in the scene, if any. The added information, if used consistently, would help viewers to locate the protein.Painters in the past centuries have used colors to indicate the identity of specific characters, making them recognizable in many different circumstances. For example, Krishna is typically depicted with blue skin, and people have assimilated in their culture this property, so that the God is recognized in whatever place . Similar2.3Sometimes colors are used to highlight specific features of a protein or of a complex. But colors are only one of the many tools in the hands of illustrators: lighting and composition are extremely powerful means to direct viewer attention to specific details, and lines can be embedded to indicate sequential steps of a process. While illustrators have made very liberal use of colors, these other principles of art design have not be exploited as much. One reason might be the desire to stay faithful to the images obtained by direct observation of cells and tissues using microscopy, or to represent the exceptional crowdedness of cellular environments. However, just like we feel authorized to use colors where none exists, we can also take the liberty of reducing the crowd, for the sake of clarity. Additionally, sound effects can also be considered.2.4The reason why we all recognize iconic figures of our culture is because they are embedded in our environment, we see and hear about them from the early age, so that they have become familiar. We also identify places where we have never been, and people we have never met.The same cannot be said (yet?) for cellular biology: the first place where cells are presented to us is at school, but this is also true for Tutankhamon and Julius Caesar . The difference is that Egyptian and Roman kings and emperors have a stereotyped iconography, presented in consistent way, even when simplified to the extreme for the smallest kids. For inanimated objects, the same can be said about the pyramids, the Colosseum, the Great Wall of China and the Taj Mahal: almost no kid has seen them in person, but they do know and recognize them when depicted in drawing or photograph. It helps that they are also connected with stories.A general outline for representing stereotyped cells is available, also for young children  coloring3Cells are colorless, and the cellular world is organized following chemical and physical principles that we cannot see . In our world, and with our visual system, we are unable to see cells directly, but we use many techniques to obtain information on the cellular world. The challenge of representing them has been faced and solved with many creative tools for about 60 year, from cardboard models  to virtuThe challenge here is to put together scientific rigour and artistic freedom, where the former is supposed to be universally fixed and the latter totally arbitrary.Even in this difficult situation, however, some “constants” can be suggested, and I think that if we can accept them, it would be beneficial for those who want to represent some biology, but are not artistically gifted, for those who are, , and for viewers, that is, almost everyone.Sometimes standards develop “spontaneously”, due to the success of a new concept or word: for example, we all use the word ‘scotch’ to indicate the adhesive tape, and, in the field of structural biology we all use and easily interpret the ribbon style of protein skeleton description . In otheThese committees arose following periods of spontaneous and creative confusion, with the aim of making it easier to communicate and understand each other. In the field of visualization and structural biology, in 1987 a panel was convened to discuss Visualization in Scientific Computing ; more reI suggest that the time has come to gather the many practitioners of molecular and cellular visualization, to discuss and possibly establish a few principles about colors that may develop into standards.3.1The first issue to be discussed is about the opportunity of establishing a standardization on the use of colors in molecular graphics: this can be initially discussed at the meeting, and possibly further explored through an online survey in the community. If there is general agreement on this fundamental point, the best option for such a discussion is to organize one or more workshop (possibly in person) with the contribution of structural and cell biologists, experts of human visual perception, developers of the graphic tools, and, of course, the major practitioners of our days.While in most of the examples considered, especially for large, mesoscale visualizations, color is used to discriminate between different components, some of the discussion should also focus on if and how to represent forces and relationships. The limited suggestions made in this article can serve as a discussion basis, not assuming that they are the most convenient, considering the various scientific, artistic and technical aspects.Technical aspects have also to be considered, in particular in relation the means of delivery  committee, the proposed standards should be published on major journals and widely distributed, providing space for further discussion, before being fixed (temporarily). The efficacy of such choices could be tested in experiments involving schools up to the university level (students and teachers), general public, interested public (like patients organizations), and scientists.Implementation could be achieved by requesting the developers of CG to default selected option, and warn about changing them. Editors could help requesting that images in the scientific literature are made in agreement with the standards, unless specific reasons advise otherwise.I am looking forward to the possibility of discussing the issue with esteemed colleagues worldwide, and would be ready to contribute in preparing a proposal to request funding for such multidisciplinary project.


Osteoporosis (OP) is a systemic metabolic skeletal disease which can lead to reduction in bone mass and increased risk of bone fracture due to the microstructural degradation. Traditional Chinese medicine (TCM) has been applied in the prevention and treatment of osteoporosis for a long time. Terpenoids, a class of natural products that are rich in TCM, have been widely studied for their therapeutic efficacy on bone resorption, osteogenesis, and concomitant inflammation. Terpenoids can be classified in four categories by structures, monoterpenoids, sesquiterpenoids, diterpenoids, and triterpenoids. In this review, we comprehensively summarize all the currently known TCM-derived terpenoids in the treatment of OP. In addition, we discuss the possible mechanistic-of-actions of all four category terpenoids in anti-OP and assess their therapeutic potential for OP treatment. As a systemic skeletal disease, Osteoporosis (OP) is characterized by increased risk of bone fragility, chronic pain, and even disability, leading to decreased life quality. Especially, OP strongly affects postmenopausal women and elderly population. About 30-50% of women and those who are more than 70 years old suffer from OP-induced fractures throughout their lives –3. In heIn recent years, there has been a growing interest in traditional Chinese medicine (TCM) for the treatment of OP, such as Liu-Wei-Di-Huang Wan (formula), Morindae Officinalis Radix (herb), Longspur epimedium glycoside  . TCM hasRehmanniae Radix has been clinically used for more than 3,000 years in Chinese medicine, which has an anti-OP effect through modulating the kidney and liver functions and improving blood circulation  are vital for the anti-OP activity of Rehmanniae Radix n-based nature products and are classified into monoterpenes, sesquiterpenes, diterpenes, triterpenes, and tetraterpenes. To date, more than 40,000 different terpenoids have been obtained in nature , is commonly used in TCM for treating OP in postmenopausal women or elderly men (via modulating the expression of bone morphogenetic protein (BMP)-2/core binding factor alpha 1 (CBFA1)-mediated molecules in postmenopausal OP. Meanwhile, sweroside promoted the mineralization of MC3T3-E1 cells by activating p38 signaling pathway (Enicostemma axillere subsp. axillere (Gentianaceae)  . It was anaceae) –22.Morinda officinalis HOW (Ba Ji Tian in Chinese) has been continuously used for more than 2,000 years in China as a tonic to nourish the kidney, strengthen bones, and enhance immune function in the treatment of OP - and ovariectomy-induced bone loss, and reduced inflammatory responses in MC3T3-E1 cells via inhibiting the activation of NF-κB  Libosch. ex Fisch. et C. A. Mey. Root (Dihuang in Chinese), is clinically used for OP treatment in China  Medik  plus RANKL  Medik , 100. Anory time . Recent activity . Bakuchius RANKL . In OVX  P level . Li et a P level . In addi P level .via targeting different signaling pathways. In OBs, catapol, albiflorin, and bakuchiol can activate Wnt/β-catenin signaling pathway; paeoniflorin and sweroside stimulate PI3K/Akt and MAPK signaling pathways respectively; swertiamarin inhibits RANKL/RANK signaling pathway; monotropein and swertiamarin suppress NF-κB signaling pathway. In OCs, catapol and paeoniflorin depress NF-κB signaling pathway; bakuchiol enhances PI3K/Akt signaling pathway.Collectively, monoterpenoids can protect bone from erosion Saussurea lappa C.B. Clarke roots. A recent research showed that costunolide markedly induced bone mineralization and differentiation and increased cell growth, AP activity, and collagen synthesis in osteoblastic MC3T3-E1 cells via targeting diverse key proteins, such as estrogen receptor (ER), phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), protein kinase C (PKC), mitochondrial ATP-sensitive K+ channel, p38, and c-Jun N-terminal kinase (JNK)  . Moreovepression .Saussurea lappa C.B. Clarke has been used in clinic for decades as a TCM  (Euphorbia factor L1 (EFL1) is an active diterpenoid composition extracted from the seed oil of Chinese herb Chinese) . EFL1 inChinese) .Pimenta racemosa var. grissea which exhibits anti-obesity and anti-inflammatory activities , dendritic cell specific transmembrane protein (DC-STAMP), and c-Fos. In C57/BL6 male mice of osteolysis model induced by LPS, abietic acid significantly reduced the number of Ocs and the levels of inflammatory cytokines, including tumor necrosis factor (TNF)-α and IL-6 (Abietic acid is a bioactive diterpene isolated from tivities . In RAW2and IL-6 .Andrographis aniculate (Burm. F.) Wall. Ex Nees in Wallich (Chuan Xin Lian). According to previous study, andrographolide has extensive pharmacological activities, such as anti-inflammation, anti-oxidation, anti-platelet aggregation, immunomodulation, and potential antineoplastic properties partly by targeting NF-κB  and Salvia officinalis (common sage), displayed anti-angiogenic, anti-neoplastic, anti-oxidant and anti-HIV activities  and estrogen-related receptor alpha (ERRα) to suppress RANKL-mediated osteoclastogenesis and restrained bone loss induced by ovariectomy  that is used for the treatment of trauma and fractures in clinical according to the dispelling stasis theory of TCM  Hara (via inhibiting osteoclastogenesis and enhancing osteogenesis by inhibiting interferon-related development regulator 1 (Ifrd1) and IκBα-mediated p65 nuclear translocation. Simultaneously, in vitro study revealed that oridonin motivated osteogenesis by Wnt/β-catenin signaling pathway and suppressed RANKL-induced OC formation in BMSCs.Oridonin is an ent-kaurane diterpenoid extracted from the TCM herb l.) Hara . As a pll.) Hara , 130. Rel.) Hara , 66. In In conclusion, diterpenoids are mostly investigated terpenoids that exert superior anti-OP efficacy by affecting various signaling pathways. In OBs, andrographolide, kirenol, and oridonim activate Wnt/β-catenin signaling pathway; andrographolide inhibits RANKL/RANK and NF-κB signaling pathways; tanshinone IIA blocks NF-κB signaling pathway. In OCs, euphorbia factor L1, abietic acid, carnosic acid, crocin, tanshinone IIA, and triptolide depress NF-κB signaling pathway; crocin, tanshinone IIA, and triptolide activate PI3K/Akt signaling pathway; andrographolide inhibits RANKL/RANK signaling pathway; abietic acid, carnosic acid, and tanshinone IIA inhibit MAPK signaling pathway; kirenol, tanshinone IIA, and triptolide depress NFATc1 signaling pathway; euphorbia factor L1 and carnosic acid promote Nrf2 signaling pathway.Sorbus commixta Hedlund and Celastrus orbiculatus Thunb (2D3 and prostaglandin E2 (PGE2) via inhibiting the activities of MAPK and transcription factors . On another hand, lupeol decreased hypercalcemic mediated bone loss in C57BL/6 mice -induced hypercalcemia mouse model , Baihuasheshecao (Hedyotis diffusa), Renshen (Panax ginseng C. A. Meyer), and Sanqi (Panax Notoginseng (Burk.) F.H.Chen). Nvzhenzi has been clinically applied in the treatment of OP for over 1,000 years 2D3/CaSR, Nox4/ROS/NF-κB, and OPG/RANKL/cathepsin K signaling pathways  induced by 1α, 25-(OH)2D3 2D3 .Salvia miltiorrhiza , and consequently inactivating the NF-κB and MAPK signaling pathways in vitro (BMMs and RAW264.7 cells) and in vivo (OVX C57BL/6 mice)  .Betula pubescens Ehrh., exhibiting multiple biological effects including osteogenic activity. Betulinic acid could enhance the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 through regulating the BMP/Smad/Runx2 and β-catenin signal pathways  C. A. Meyer, which acts as a neuroprotective agent and pro-angiogenic agent. Ginsenoside Rg1 promoted the proliferation and odontogenic/osteogenic differentiation of human dental pulp stem cells (hDPSCs), stimulated the proliferation of BMSCs, and suppressed the maturation and differentiation of OCs  Ker-Gawl. and has been applied in clinical use for a long time. Ophiopogonin D suppressed ROS generation to exert anti-OP effects via the FoxO3a/β-catenin signaling pathway in both RAW264.7 and MC3T3-E1 cells. In RAW264.7 cells, ophiopogonin D decreased the expression of Osteoclastic genes and the activity of CTX1 and TRAP, which are bone degradation markers in serum. In MC3T3-E1 cells, ophiopogonin D significantly promoted cell proliferation and increased the gene levels of some osteogenic markers  Bunge, which is a TCM used for thousands of years . In BMM cell model, hederagenin depressed the formation and bone (hydroxyapatite) resorption of OC induced by RANKL. Mechanism study revealed that hederagenin reduced the production of intracellular reactive oxygen species (ROS) and the activation of MAPK signaling pathway (ERK and p38), causing decreased induction of c-Fos and NFATc1. Similar to the in vitro effects, hederagenin treatment significantly prevented bone loss in OVX mice via inhibiting RANKL-induced bone resorption and OC generation (O-(2-O-acetyl)-α-L-arabinopyranoside remarkably elevated the protein levels of BSP and osteocalcin and augmented AP activity (Hederagenin is a pentacyclic triterpenoid sapogenin extracted from neration . Meanwhiactivity .Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), is a natural pentacyclic triterpenoid, and traditionally used for the treatment of snake venoms and inflammation. Recently, it was reported that tubeimoside I could inhibit the formation and function of OCs, as well as type 2 diabetes-induced decrease of bone mass in SD rats, resulting from down-regulating IκBα degradation which subsequently suppressed NF-κB transcriptional activity  to prevent bone loss , the Guangdong Basic and Applied Basic Research Foundation (No. 2021A1515110584), Special Foundation of Guandong Educational Committe (No. 2021ZDZX2001), and Guangdong Province Science and Technology Plan International Cooperation Project (No. 2020A0505100052).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. 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At the beginning of summer 2022, my colleagues and I wanted to share some thoughts about a vaccination success story, i.e., the first messenger RNA (mRNA) vaccines against coronavirus disease 2019’s (COVID-19) [Never has a vaccine been successfully developed so quickly against an emerging disease ,4. BilliAnother challenge in the field of COVID research is to decipher all the aspects of the immune response developed after vaccination, especially the cellular immune response  which stOdocoileus virginianus) [Neovison vison) [SARS-CoV-2 is more and more frequently directly or indirectly detected in different animal species, and many reports have shown its capacity to productively infect various animal species including the white-tailed deer (inianus) ,25 and tn vison) . The potn vison) ,28,29, an vison) . Age-spen vison)  and medin vison) ,37,38,39n vison) . HoweverTo conclude, the COVID-19 crisis has been impressive and inspiring for immuno-virology and vaccine development with the official beginning of an mRNA vaccine era for mass vaccination. After a one-year battle, the virus spread is still not fully controlled, and the virus has not yet been defeated. Many challenges still need to be addressed by scientists and health specialists. Moreover, disparities and inequalities between countries are still significant barriers to disease control, underscoring the urgent need for reliable, efficient, and affordable COVID-19 vaccines in many countries.


The transition from a demographic regime of high mortality and high fertility to one with low mortality and low fertility is universal and comes along with the process of socio-economic modernization. The Spanish total fertility rate has decreased to below replacement levels in the last decades. The decline has persisted since the 1960s and is diverse across the country. Based on that diversity, the use of population forecasts, not only at national but at regional levels, for planning purposes (governments and private sector) with large horizons has become a must to provide essential services. Using a Bayesian hierarchical model we constructed probabilistic fertility forecasts for Spain at the regional level. Although this approach is already issued by the United Nations little research has been done focusing on the Spanish subnational level. Our objective is to disaggregate the national projections of the total fertility rate for Spain into regional forecasts. The results of this research will show the model fitting, first to the national level and then using a multifaceted and continuous evolution of fertility over time, at the regional level, to check its convergence. While increasing life expectancy is a symptom of social progress, it is also a challenge for governments, private pension schemes and life insurers because of its impact on health care costs and pensions. A large number of demographic and actuarial studies have recognised the problems caused by ageing populations, low fertility rates and increasing longevity, therefore attention has focused on the development of methods for forecasting and projecting fertility and mortality rates –4.second demographic transition [gender transition would be more appropriate. In fact, according to the latter, the main difference concerning the first demographic transition is that a cultural component has been added. In effect, these changes are linked to the new role of women in society and its presence in the labour market and to the transformation of the organization of demographic reproduction, traditionally based on a clear separation between women, who are confined within the family, and men, who enter the labour market. With the progressive presence of women in that market, the articulation between family and work has become a central issue of our time. In this context, [The decline in fertility has been accompanied, in developed countries, by major changes affecting the role of women and the family. Some authors speak of a ansition –8, althoansition –11, the context,  analysescontext, , study tcontext, , discussEurope is one of the continents where the demographic problem mentioned above is highlighted. The TFR  is the average number of children born to women during their reproductive years) and in most of the EU member states, is below the replacement threshold of 2.0–2.1 births per woman .The fertility replacement threshold is the TFR a nation needs to stabilize the population. In a scenario where the TFR falls below 2.0–2.1 births, as the population ages and declines, economic growth and funding for government programmes are reduced, fuelled by fewer employees available to work, pay taxes and fund government social security programmes , 17.Nomenclature of Territorial Units for Statistics and is the UE harmonised manner of collecting data in the different geographical areas) from 2008 to 2019 where it can be seen to be declining to below the replacement threshold in most countries [ountries .The situation in Spain is similar to other European countries and this behaviour of the fertility rate has been accentuated during periods of financial and economic crisis. The increase in unemployment levels, job uncertainty and, in general, the economic difficulties for families, together with the decrease in marriages, have delayed or postponed, in many cases, the first childbearing. This topic is extensively developed by  who haveHowever, the socio-economic differences within Spain and the geographical diversity between the north and south make a demographic analysis at the regional level necessary to verify TFR convergence, in this sense, , 21 haveNevertheless, very few sub-national projections of fertility in Spain, at the regional level, have been made using the methodology we propose, so in this paper, we aim to project the Spanish TFR at a regional level by adopting the approach proposed by , using aquantum and the tempo effect. The quantum component refers to the TFR that would have been observed in the absence of changes in the timing of childbearing during the period in which the TFR is measured whereas the tempo effects are distortions due to changes in the timing of birth. [Modelling fertility curves has been of interest to demographers for a long time so through a variety of mathematical models, it has been possible to describe age-specific fertility patterns with specific shapes in all human populations over the years. Even though the demographic literature is offering a wide variety of fertility measures, the TFR is a key indicator for population projections due to its simplicity, ease of interpretation and availability. For instance, the United Nations Population Division produces population projections based on this measure , 29. Howf birth.  used theDespite the limitations of the TFR mentioned above, addressed by Bongaarts and Feeney, , the choIn the remainder section, we propose a literature review on approaches that explain fertility modelling combined with works that model the age structure, we think this relationship is important because an imbalance in age structures creates important problems at institutional stability levels (e.g. schools), labour force succession or old-age social security. Shrinking class sizes, workforce shortages and payroll tax collections that are insufficient to pay for retirement benefits are all consequences of long periods of below-replacement fertility.intermediate variables model of fertility. The intermediate variables analytical system consists of the disaggregation of the process by which a person is born, which revolves around three key moments: (1) intercourse, (2) conception and (3) gestation and childbirth, giving rise to eleven fertility-related variables [proximate variables. The proximate variables proposed by Bongaarts are based on three biological variables: (1) the postpartum barren interval, (2) the fertile interval and (3) the time to full pregnancy (assuming a constant duration of 9 months) [In 1956, Kingsley Davis and Judith Blake, developed the ariables . Such a  months) . Accordi months) .Some of these fertility models mentioned above are quite effective in fitting the fertility rate distributions, for instance, , using tHowever, since Lee and Carter proposed their well-known model for forecasting mortality, , a famil“demographic transition”, i.e. the transition from a sharp decline in the birth rate that took place during the 1930s to a sharp rise in the birth rate in the post-war period. In this period, in 1960, the publication date of his pioneering work on economics and fertility, Becker constructed a theory in which a family (or couple if they had not yet children), measured the utility of their consumption by including the number of surviving children. In this way, he presented children as durable goods because they provided “psychic satisfaction” to their parents over a long period, he even argued that they could be durable goods of production in certain contexts and showed that “the theory of demand for durable consumer goods is a useful framework for analysing the demand for children”.Nevertheless, difficulties arise when an attempt is made to ascertain the causes of changes in fertility from an economic point of view (beyond quantifiable factors), in this sense, family decisions in a socio-economic and psychological framework play a key role in fertility. The economic analysis of fertility formulated by Becker in the ‘60s is important to understand this demographic variable within an economic framework and it deserves to be explained in more detail, . Becker’Economic Theory of Demand for Fertility Decisions . In it, he argues that like other durable goods, children also provide “utility” and this utility is measurable and comparable to that of other goods through their corresponding utility function or set of indifference curves. In this sense, preference for certain tastes allows for differences in fertility.Using this Economic Theory of Demand, Becker adapts it and builds his quality of children is also an aspect that influences the demand for them, in the sense that a family should not only determine the number it wants to have but also the amount it spends on them, establishing the concept of “superior quality” for those children on which parents spend more money.The income is another factor that influences the “quality” of children since it can be expected that an increase in the level of income, in the long run, should increase the amount spent on each child and consequently an increase in the quality of the children.The level of cost of children can be calculated as either positive or negative. If they were positive, the children would be a durable consumers as a whole and it would be necessary to assume that an income or psychic utility is received from them. If the net costs were negative, the children would be a durable good of production and a pecuniary rent would be received from them.On the other hand, Becker argues that the net supply in this Economic Theory of Demand for Fertility Decisions is subject to two important aspects: on the one hand, a certain uncertainty that implies distinguishing between current utility and expected utility, and on the other hand, the number of children is not only related to the level of income and prices, but also to the ability to have children. In the 1960s, the average number of live births produced by married women in societies with little knowledge of contraception was very high, so the average family was more likely to have a high number of children than a low number.Finally, the concept of Subsequently, as knowledge of contraceptive methods became more popular, the quality of children and the number of children decreased.The main conclusion and what we can extract from Becker is that family decisions, and above all when it comes to having children, are taken in a social and economic environment in which the quantity and quality of children are closely related and depend on socio-economic variables and factors. Thus, an increase in income or a decrease in the cost of children would affect both the quantity and quality of children, usually increasing both. The quality of children is very important in its own right, as it determines the education, health and motivation of the future workforce.dynastic utility function that depends on the consumption and number of descendants in all generations. To maximize this dynastic utility function, according to the authors, the first-order conditions imply that any generation depends positively on the real interest rate and the degree of altruism. The second linkage is between the effects on fertility of mortality of children, subsidies to (or taxes on) children and social security and other transfer payments to adults. The third connection links fertility and population growth to international capital markets showing that fertility in an open economy depends positively on the long-term interest rate and negatively on the rate of technological progress.In 1988, along with Barro, Becker extended his theory arguing that altruistic parents choose fertility and consumption by maximizing a dynastic utility function . In theiIn the same line, that is, the one explaining there are other components influencing fertility,  concludeSubsequently, in models built in recent years, the patterns of fertility data in developed countries show considerable variation related to the fertility curve. In this sense,  proposedCountries show inequalities concerning the age at which fertility rates peak and the varying speed with which the peak is approached from the beginning and then passed to reach the end of the fertility period, , other cα, β and γ represent the estimated parameters and x is the age of the mother at the birth of the child.Because of this heterogeneity, existing models cannot capture the modern fertility pattern. To overcome this,  propose Among the models used to represent the age-specific fertility pattern of populations that do not show high early fertility, several have been shown to provide ‘fairly’ accurate fits to fertility distributions. These include, for example, the parametric fertility model proposed by , 51, whi0, which are more parameters than necessary for efficient representation. Although the model has the virtue of having parameters that have some real-world interpretations, these parameters cannot be measured in real populations, so it is difficult to fit the model to them. Extensions to the Coale-Trussell model have been proposed by [It is therefore a four-parameter fertility model posed by –54 to masplines [Mean Parity in that cohort . Brass defined P as “the average parity (cumulative lifetime fertility) of a cohort of women up to a given age, and F is closely related to the cumulative fertility (period) up to that same age”. The ratio method expresses these two quantities about each other in the form of a ratio for each age group [The Beta and Gamma distributions are equivalent to Pearson’s Type I and III curves, respectively, proposed by , the samsplines , 56 has splines , Pearsonsplines  and the splines  have shoge group .P/F is confounded by the underlying errors in the data.The main weakness of the method is that in reality, the data are never free of error, so the hypothesized pattern of deviation from the unit ratio P/F ratio to increase [The second type of error encountered, less frequent nowadays in developed countries as births are reported by the hospitals and will not encounter these problems with this reporting system, is that women tend to omit some of their live-born children, particularly those living in other households and those who have died, with the result that the proportion omitted tends to increase with the age of the mother, causing the increase .To overcome this, Gompertz’s relational model is presented as a versatile improvement since it uses the same input data (and makes the same assumptions about errors affecting fertility data) as its precursor. However, it is important to remark that the method does not require the assumption that fertility has been constant in the past , 62.Several other authors focus their analyses on applying and fitting the models described above to specific populations and specific countries, for example,  use descOn the other hand,  approachr’ method (variable-r means ‘variable rate’) from the Preston and Coale model. This model is the second of what later became known as the Death Distribution Methods, for estimating the completeness of death reporting about a population estimate at a given point in time, although primarily used for mortality projections, the variable rate approach of Preston and Coale means that it can be used to estimate various demographic measures. Therefore and based on that method to assess the level of fertility in China using census data as well as annual population surveys from 1990 to 2000, concluded that, with the proposed methodology, fertility in the country has reached a level well below replacement level.Instead,  applies In the American continent,  presentsIf we turn out our attention to Europe, interesting is also the approach of , as theyHowever, , focus tOn the contrary,  providesPerhaps, one of the most interesting works in the last years is the one by Burkimsher who studies the evolution of the fertility curves of the 1968 to 1980 cohorts of women in 22 developed countries showing that for some countries the transition from an early to a late fertility schedule goes through a phase when the first birth fertility curve is bimodal. In other countries, a model ‘shoulder’ is apparent and concludes that the existence of a bimodal fertility curve suggests the polarization of women into one group that remains longer with an early fertility schedule and a second group that moves more rapidly on to a later schedule .For the Spanish case, , offers On the other hand, , proposeMore recently and based on a more quantitative and technical approach,  present As stated before, extensions to the LC model combined with other approaches have been widely used in demographic models within a Bayesian framework have been developed. These models work better only when incorporating information about observed data or future patterns . In thisLastly, the pandemic has also affected the behaviour of the population as it brings socio-economic uncertainties that influence childrearing decisions. Although fertility intentions and behaviour are not identical and often do not run in parallel, is beyond any doubt that the Covid-19 pandemic has affected family dynamics. Moreover, the lack of data does not draw significant conclusions yet on the long-term effects of the pandemic on fertility. However, it is worth looking into the latest research on this subject as, like any other shock  the impact on society; therefore, demography is determinant.For instance,  assess tOn the other hand, , studiedThe contribution of  is equal aimed toFinally and despite what was mentioned earlier the lack of data does not yet allow us to assess the demographic impact of the pandemic but some works already try to project the future by incorporating the effects of COVID-19. For this reason, , in the padrones municipales . This population registers records individuals by recording their nationality, place of birth, age, or marital status. Since 1996, the Spanish Padrón has been updated annually with the National Statistical Institute (INE) being responsible for quality control. The INE makes this data available under the name Padrón Continuo (continuous register) and usually took 2 years for the Spanish population registers to become reliable.The data for Spain were obtained from registers at the municipality level, the Padrones municipales on the INE website for 2018 (http://www.ine.es). Maps are based on the global administrative area database provided freely available for academic use (https://gadm.org/license.html), transformed by the authors to be consistent with INE data.To address the mapping of the TFR in Spain, in Evolution of the TFR, Phase I: a pre-transitional high fertility phase; Phase II: the fertility transition in which the TFR declines from high fertility levels towards or below replacement fertility level; and Phase III, a post-transition phase of low fertility, which includes the recovery from below-replacement fertility to replacement fertility and oscillations around fertility at that same level. The observation period for each region is divided into these different phases based on deterministic definitions of their start and end periods and then modelled separately. Thus, we define τc as the beginning of the Phase II for country c, which is given byMc is the result of the maximum observed TFR in region c, and Lc,t indicates the local maximum. The period of onset of Phase III, denoted by λc for region c, is observed within the observation period if two subsequent increases below a TFR of 2 have been observed. For these regions,c and in period t. For the rest of the regions, fc > 2005−2010As stated in the previous section, the proposed Bayesian model for fertility projection is based on , 79, theThe proposed method does not model Phase I,so it is treated as it is, however, if any region is in this phase, it is assumed to be in Phase II in the following period, so this first phase is not relevant for the projections.fc,t is the TFR over the five-year period t in the region c, dc,t is the decremental term that models the systematic decline during the fertility transition, dc,t is the decremental term that models the systematic decline during the fertility transition, varepsilonc,t is a random distortion that models the deviation from systematic decline, τc is the period of onset of fertility decline and λc is the period of onset of post-transitional Phase III defined above. The distributions of the random distortions in each period are given bymτ is the mean and sτ is the standard deviation of the distortion in the starting period. The quantity σ is the standard deviation of the distortions during the subsequent periods, given by the expressionσ0 is the maximum standard deviation of the distortions, reached at the S level of the TFR, and a and b are standard deviation multipliers to model the linear decrease for larger and smaller TFR results. The constant c1975(t) is added to model the higher error variance of the distortions before 1975 and is given bydc,t in (1) is modelled as a function of the level of the TFR as follows:g is a parametric declining function. This function specifies a five-year decline (decrement) as a function of the normal level of the TFR and the vectorθ. The decrement function is the sum of two logistic functions, i.e. a double or bi-logistic function  is given bydc is the maximum possible rate of decline, p1 = p2 = 9 are constants and The fertility transition phase is modelled by a random process with drift specified ason, 2019 ). The do decline .Uc of the fertility transition is within the observation period, the onset level Uc is set to the TFR in that period, The parameters of the declining function are estimated for each region. For regions in which the onset period Uc, the five parameters that determine the rate of fertility decline and the time it takes for the transition in region c are △c4, {△ci/(Uc−△c4):i = 1, 2, 3}, and dc.Given the starting level χ, ψ2, △4, δ4, α, δ}.To estimate the parameters in each region, we use a Bayesian hierarchical model , 96 giveμ = 2.1:ρ is the autoregressive parameter with |ρ|<1 and s is the standard deviation of the random errors. Both parameters are estimated by maximum likelihood.In the post-transition modelling phase the change in TFR is modelled by a first-order autoregressive time series model, i.e. AR(1) with a mean set approximately at the replacement fertility level Phase II are based on the model for this phase, as discussed above, using the sample posterior distribution of the model parameters.Finally, the projections of the TFR during the fertility transition for regions in Finally, according to  is imporThe probabilistic projections for the Spanish TFR at regional levels include uncertainty bounds. These bounds are remarkable for less developed regions, with less available data or where more recent observed data entries do not exist.Adjustment of the TFR projection model, for which:Phase II and the starting period of Phase III have been calculated for each region (τc and λc). This phase, in our case, is common to the whole process.The starting period of Phase II model parameters has been obtained using the MCMC algorithm.A posterior sample of the Phase III using the maximum likelihood estimation method).Future TFR trajectories have been generated (this step includes the estimation of the AR(1) model parameters in The results have been analysed using a set of functions that summarise, plot, diagnose and export the results of the previous two steps.From a computational point of view, to obtain these projections, three steps have been followed in the following order:We detail the logical sequence of execution for step 1, i.e., the TFR projection model fitting in the A summary of the simulated parameters for Spain are shown in Tables The projected simulations of the TFR are shown in ‘World Population Prospects. Revision 2019’ [The left figure shows the Bayesian hierarchical model projections of total fertility using the estimates from the on 2019’ . Note th‘World Population Prospects. Revision 2019’. As in the previous model, it should be noted that only a small selection of the double logistic probability trajectories (grey lines) is shown for illustration. The observed five-year decreases in total fertility level are shown with black dots if they refer to periods of the fertility transition defined as Phase II (i.e. from high to low fertility). The median projection is the bold solid red line, and the 80% and 95% projection ranges are shown as dashed and dotted red lines, respectively. Phase I data refer to the period before the onset of the fertility transition (if it occurred since 1950). Phase III data refer to the period after the low-fertility transition not modelled using the double logit model, but using a first-order autoregressive time series model, AR(1).The graph on the right shows the Bayesian Hierarchical Model (BHM) decline curves for total fertility that have been made also with the fertility estimates from the https://ec.europa.eu/eurostat). In addition to this, the most interesting insight is the projections made by the model, which can be seen to be approaching the rate of 1.8 births per woman, a rate that is in line with the projections made by Osés-Arranz et al., (2018) [The fit of the model simulation for Spain is quite accurate as the starting point puts the fertility rate at around 1.3 births per woman, similar to that provided by Eurostat which is 1.23   discusse‘Comunidades Autónomas’) each of them with a certain degree of autonomy but dependent on the national government itself.One of the main contributions of this work has been the fitting and projection of the TFR at the regional level. We have followed the same methodology and procedure as for the national case and computed the probabilistic projections for the regional TFR including also uncertainty bounds. Spanish national-level projections are included in the upper left chart just to compare with the regions. Although the projected trend in TFR is unequivocally upward in the long-term for all Spanish regions, the intensity and timing are not the same for all of them. There are regions which, on the one hand, showed greater precocity in the onset of the fertility decline in the past, so it is worth highlighting these peculiarities; on the other hand, it is not possible to speak of uniformity concerning the amount of the increase, since this is not homogeneous throughout the territory and, likewise, not all of them always coincide in the changes in the trend. To compare the evolution of each region with that of Spain as a whole, the provinces have been grouped by area, making them coincide with their membership in the current autonomous communities .Particularly interesting are the cases of those regions with a projected TFR close to the replacement threshold level, that is 2–2.1 births, i. e. Cataluña, Andalucía, Canarias and Murcia, especially because as a starting point they are quite below the threshold, in any case, these are the regions that contribute most to the increase in the TFR at the national level and although the Spanish population is projected to grow for the rest of the century, it tends to stabilize at the end. For these regions mentioned, immigration plays an important role as these regions concentrate higher rates of people coming from other countries, especially Andalucía, Canarias and Murcia are receiving people from African countries cities like Almería, Melilla, Ceuta, La Línea de la Concepción, Algeciras, Sanlúcar de Barrameda ; Lorca and Cartagena (Murcia); Santa Coloma de Gramenet, Rubí, Reus, Sabadell and Terrassa  are the cities with the highest number of children per woman . The migOn the other hand, the regions with lower projected TFR were Cantabria, Castilla-León, Extremadura, and Galicia; in fact, cities like Torrelavega, Santander, Gijón (Cantabria); Ferrol, Vigo and A Coruña  are the cities with the lowest number of children per woman  this is In this study TFR evolution and probabilistic projections for Spain have been implemented and analyzed; in the research section, we conducted a review of the literature on fertility and age structure models which has tried to collect those works that have contributed to the improvement in this demographic area. In the empirical section, we used a Bayesian hierarchical model to fit and project the TFR at the regional level.Fertility decline is a phenomenon that has traditionally been of interest to demographers, economists, policy-makers, politicians and, more recently, the general public. Population problems mainly refer to the progressive ageing and non-replacement of generations in developed countries as a result of the fertility decline.In Spain, we have seen that two effects play a crucial role: migration and depopulation but in addition to that, the lack of public aid, the loss of income for women after motherhood, the difficulty of reconciling working time and parental  are among the worrying factors of the phenomenon. It is a structural problem because demographic factors themselves are not strong enough to push fertility down, but an exogenous factor, industrialization, is needed to cause a profound change in the social, economic and cultural structures of a country and force the demographic system as a whole to adapt so that the demographic transition can begin, .The probabilistic model we have used, adapted to the Spanish population and used to simulate projections tens of years ahead, is a very useful tool to measure and manage longevity risk. This is an important issue for those who wish to hedge longevity risk using published mortality rates, be they governments, pension plans, insurers or banks. Moreover, longevity-based risk, while a challenge not only for demographers, statisticians and actuaries but also for government agencies, can also present a broader problem for insurance companies that use external data, such as population data, rather than their internal policy data in their reserving models. The need to quantify and reserve for any potential basis risk is receiving increasing focus, particularly under Solvency II, therefore given the continuing increases in life expectancy, the use of the best available methods is now mandatory, particularly by actuaries practising in the insurance and financial risk industry, where the ramifications of inaccurate forecasts are acute and although the magnitudes of ageing are uncertain, and forecast errors are likely to be large, ageing policies can anticipate this, uncertainty should not imply inaction and forecasts contain information that can be used in the design of social policies that will have an impact on both the present and, more importantly, the future population.Completeness of Birth Reporting (provided by the UNPD) and the biases used WPP 2019 (World Population Prospects) estimates as reference levels [However, probabilistic models based on Bayesian methods and hence the one we used here, have some limitations. First, as the Bayesian inference entails the choice of prior probabilities, the construction of hierarchical models relies on certain assumptions that results are subject to such assumptions for countries with no or limited data. In our case, this was not a problem as we had enough data but for countries more limited, before fitting the model, data are adjusted for e levels . In addiFertility models are used in a wide variety of situations, for example, to smooth observed data as inputs into population projections or other analytical exercises. It is important to note that the accuracy of models predicting demographic phenomena is important when judging the quality of population forecasts. In aspects such as information content, for example, one might ask whether the forecast predicts only the total population or also age groups. For policy purposes, on the other hand, it is relevant to discern whether the predicted trend implies immediate policy measures. However, the degree to which the forecast reflects actual trends is a key factor in assessing its quality, particularly when the forecast is used for planning purposes. For example, imagine a forecast for which the odds are one against two that will cover the actual trends. Such a forecast must be handled much more cautiously than one that can be expected to be wrong only one in five times.Predictions of demographic phenomena  have become increasingly important in recent decades as life expectancy has increased rapidly. Over the years, a large number of different approaches have been proposed to model these phenomena, ranging from the simplest (the UN model programme) to the most complicated .Studies have shown that a three-parameter model can capture most of the variation in observed fertility patterns. Models with more parameters, for most purposes, are not necessary, and difficulties may be experienced in fitting such models to a small number of data. The Gompertz relational model, for instance, is a very flexible three-parameter system for modelling fertility, which has found application in many areas of demographic work.S1 AppendixSimulations of MCMC parameters and density distribution functions.(ZIP)Click here for additional data file.


CRF gene named GmCRF4a by comparing the performance of the homozygous Gmcrf4a-1 mutant, GmCRF4a overexpression (OX) and co-silencing (CS) lines. Phenotypic analysis showed that overexpression of GmCRF4a resulted in taller hypocotyls and epicotyls, more main stem nodes, and higher plant height. While down-regulation of GmCRF4a conferred shorter hypocotyls and epicotyls, as well as a reduction in plant height. The histological analysis results demonstrated that GmCRF4a promotes epicotyl elongation primarily by increasing cell length. Furthermore, GmCRF4a is required for the expression of GmYUCs genes to elevate endogenous auxin levels, which may subsequently enhance stem elongation. Taken together, these observations describe a novel regulatory mechanism in soybean, and provide the basis for elucidating the function of GmCRF4a in auxin biosynthesis pathway and plant heigh regulation in plants.Plant height is one of the key agronomic traits affecting soybean yield. The cytokinin response factors (CRFs), as a branch of the APETALA2/ethylene responsive factor (AP2/ERF) super gene family, have been reported to play important roles in regulating plant growth and development. However, their functions in soybean remain unknown. This study characterized a soybean  Glycine max (L.) Merr.] is an economically important crop cultivated globally, providing a valuable source of cooking oil and plant protein for human consumption (Rht1 (Reduced height 1) gene of wheat (Sd1 (Semi-dwarf 1) gene of rice (D8 (Dwarf-8) gene of maize  gene in maize reduces plant height, internode length, and ear height (TRANSPORT INHIBITOR RESISTANT1/AUXIN SIGNALING F-box (OsTIR1/AFB) gene family alters plant height and yield in rice (Determinate 1 (Dt1) and Dt2 genes regulating plant height and growth habits of soybean were found (Dwarf Mutant 1 (GmDW1)  (GmLHY (Late elongated hypocotyl)  , GmAP1s petala1) , and GmLpocotyl)  genes inn plants . NeverthCRF genes, with CRF4 being one of the few whose transcription is not induced by cytokinin  showed no obvious phenotypic alterations, except for a slight increase in primary root length of CRF4OX. In addition, under low nitrogen conditions, the CRF4 overexpression significantly reduced 15NO3− uptake and plant biomass, including reduced dry weight, primary root length, and lateral root number , RAV (ABI3/VP1), Dehydration Responsive Element Binding Protein, ethylene responsive factor (ERF) and Soloist according to the number and binding sequence of AP2/ERF domains . The AP2ytokinin . The crfreatment . While ut number .GmCRF4a gene in more detail by characterizing its expression patterns, subcellular localization and by GmCRF4a mutant, overexpressing, and co-silencing lines to dissect the roles of this transcription factor in regulation of plant height. We observed that the plant height of Gmcrf4a-1 mutant and co-silencing lines were significantly decreased than that of the WT. We also observed the relative expression levels of auxin biosynthesis genes and the auxin content in the Gmcrf4a-1 mutant and co-silencing lines were significantly lower than in WT. These results suggested that GmCRF4a may directly regulate plant height by mediating key components of the auxin pathway. Moreover, the phenotype of GmCRF4a overexpression lines further confirmed the above results. Our findings suggest that the manipulation of GmCRF4a gene should facilitate improvement in plant height in soybean.In this study, we tested the function of GmCRF4a knockout mutants (Gmcrf4a-1), GmCRF4a co-silencing line (CS), and GmCRF4a overexpression lines (OX1 and OX2) were grown under 16 h light/8 h dark conditions or 12 h light/12 h dark conditions with 250–300 μmol·m−2 s−1 light at 25°C for 11 days. For the field phenotypes, the materials were grown under natural conditions (about 14.5 h-light conditions) in a field in Beijing  with two plot replications. The seeds were planted in a 3.0 m row, with 0.6 m separating rows and a space of 0.3 m between adjacent plants. The plant height and node number of main stem were investigated and photographed during the period of co-silencing line (CS) R8, and the growth period and grain status of each material were counted after harvest.The soybean cultivar Tianlong 1 (TL1) is an elite material for genetic transformation. For the seedling phenotypes, the wild type (TL1), −5 from Phytozome v13.0.CRF4 phylogenetic tree the GmCRF4a homologous genes in Arabidopsis, soybean, maize, and rice were used to constructed Maximum Likelihood tree by MEGA 7.0 software. For the protein sequence alignment and critical domain analysis were conducted by DNAMAN software.The peptide sequences of AP2/ERF in soybean and Arabidopsis thaliana were selected with the Pfam name PF00847 (ap2 domain) in a threshold e-value < 10Tissue-specific expression patterns data came from the soybean Expression Atlas.GmCRF4a was cloned into the pA7-YFP vector. GmCRF4a-YFP was transiently expressed in soybean mesophyll protoplasts as described in our previous work . To generate the CRISPR-Cas9 knockout soybean mutants, gRNAs were designed using the web tool CRISPR-P,p0645 vector. According to the previously described method  kit  was used for the determination of auxin. Please see kit description for details.The seedlings were grown under 12 h light/12 h dark conditions with 250–300 μmol·mGmActin11 was used as an internal control. Three independent biological replicates and two mechanical replicates were evaluated. Related genes and primer lists are presented in Total RNA was extracted from 13-day old soybean leaves under 12 h light/12 h dark conditions using Trizol . Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in 384-well optical plates on the QuantStudio 7 Flex  using SYBR Green RT-PCR kit . The t-test and ANOVA were performed on the data using GraphPad Prism 9 software.Two-sided AP2/ERF family gene GmCRF4a (Glyma.14G205600), which originally discovered as one of the fine-mapping candidate genes and turned out to be a non-target gene, involved in the regulation of important traits such as plant height and flowering in soybean. Therefore, we conducted further studies on this gene. GmCRF4a encodes a protein of 282 amino acid residues with a predicted molecular mass of 31.7 kDa. BLASP search of the Arabidopsis database revealed that GmCRF4a shares the highest identity with AT4G27950, Cytokinin Response Factor 4 (CRF4), which led us to designate Glyma.14G205600 as GmCRF4a, the first identified in Arabidopsis CRF4 homologue. Protein analysis of the deduced amino acid sequence revealed that GmCRF4a contains a typical AP2/ ERF domain. Further amino acid alignment analysis showed that GmCRF4a shares high similarity at AP2/ERF domain with the homologues in soybean , maize , rice  and Arabidopsis , , . Phylogeis genes . Homolog methods . ClusterGmCRF4a gene, we detected the expression level of GmCRF4a in different tissues under the background of TL1. The results showed that the GmCRF4a gene was evenly expressed in all tissues  in the exon resulting in premature termination of protein translation (35S:GmCRF4a-YFP construct and obtained two overexpression lines (OX1 and OX2) and one co-silencing line (CS) using the Agrobacterium-cotyledonary node transformation system  and was taller than TL1, while the CS line was dwarf with less MSN  content in the epicotyl of 11-day-old plants grown under 12 light/12 dark conditions. The results indicated that the total IAA content substantially declined in the Gmcrf4a-1 mutant and CS line, but significantly increased in the OX1 and OX2 lines, respectively (YUCs (YUCA) genes which play a key role in the auxin synthesis pathway  genes, the negative regulator of auxin signal transduction pathway  was down-regulated in the Gmcrf4a-1 mutant and CS line, but up-regulated in the OX1 and OX2 lines  was significantly reduced, while significantly increased in the OX lines  can be found in the article/TaZ, TuZ, and BL designed the studies and wrote the manuscript. ZX conducted the bioinformatic analyses. ZX, RW, KK, NB, AA, JL, and HL performed the experiments. All authors contributed to the article and approved the submitted version.This research was funded by the National Natural Science Foundation of China (32072091), the National Natural Science Foundation of China (32171965), Science and Technology Innovation Team of Soybean Modern Seed Industry in Hebei (21326313D), the Jiangsu Seed Industry Revitalization Project [JBGS(2021)014], and the Jiangsu Collaborative Innovation Centre for Modern Crop Production (JCIC-MCP) Program.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.