The response of kegne to microtubule-depolymerizing and -stabilizing drugs, as well as subsequent gene sequencing and segregation analysis, suggests that a defect in the α-Tubulin gene is functionally and genetically tightly linked to the kegne phenotype. In diploid species such as rice, homozygous mutations in α-Tubulin genes result in extreme dwarfism and weak stems. In the allotetraploid tef, only one homeologue is mutated, and the presence of the second intact α-Tubulin gene copy confers the agriculturally beneficial semi-dwarf and lodging-tolerant phenotype. Introgression of kegne into locally adapted and popular tef cultivars in Ethiopia will increase the lodging tolerance in the tef germplasm and, as a result, will improve the productivity of this valuable crop.Genetic improvement of native crops is a new and promising strategy to combat hunger in the developing world. Tef is the major staple food crop for approximately 50 million people in Ethiopia. As an indigenous cereal, it is well adapted to diverse climatic and soil conditions; however, its productivity is extremely low mainly due to susceptibility to lodging. Tef has a tall and weak stem, liable to lodge , which is aggravated by wind, rain, or application of nitrogen fertilizer. To circumvent this problem, the first semi-dwarf lodging-tolerant tef line, called  Eragrostis tef (Zucc.) Trotter] belongs to the family of Poaceae (subfamily Chloridoideae) and is a C4, self-pollinated, and allotetraploid plant species. As the most important cereal crop in Ethiopia, tef is cultivated annually on 2.8 million ha of land, ~29% of the area allocated to cereals ], sorghum [dwarf3 (dw3)], and Arabidopsis [twisted dwarf 1 (twd1)] (A third hormone known to regulate plant height is auxin . MutatioArabidopsis mutants affected in microtubule organization were dwarf in stature (α-Tubulin gene created the twisted dwarf 1 (tid1) mutant of rice, which also showed helical growth of leaves  and tortifolia 1 (tor1) that carry mutations in MAPs (Arabidopsis mutants indicated above experienced increased sensitivity to the microtubule-depolymerizing drug propyzamide and the microtubule-stabilizing drug taxol (Setaria viridis and Eleusine indica) confer a resistance to dintroaniline , the active compounds of several non-selective herbicides , indole-3-acetic acid , paclobutrazol, propyzamide, taxol (paclitaxel), and oryzalin were dissolved in dimethylsulphoxide (DMSO).All chemicals were purchased from Sigma-Aldrich, Switzerland except brassinozole220 (Brz220), an inhibitor of BR biosynthesis (kegne mutant was derived from an EMS (ethyl methanesulphonate)-mutagenized population of the tef cultivar DZ-Cr-37 , generated for the ‘Tef Improvement Project’ at the University of Bern. The EMS concentration used for the mutagenesis of the tef seeds was 0.2% (v/v).The 2 progeny of crosses between kegne and three improved varieties, namely Magna , Quncho (QK), and Tsedey (TK), as well as the ecotype Kay muri (KK) were investigated.The FFor plants grown on soil, a mix of 50% topsoil, 40% peat, and 10% sand was used. The plants were fertilized with compound fertilizer . Growth conditions were either short day (8h light at 22 °C and 16h dark at 20 °C), equal day (12h light at 24 °C and 12h dark at 18 °C), or long day conditions (16h light at 22 °C and 8h dark at 20 °C) with 65–75% relative humidity at all light regimes.in vitro conditions, seeds were surface sterilized for 5min using 1% (v/v) sodium hypochlorite solution plus a drop of Tween-20 as wetting agent. Following sterilization, seeds were rinsed 3–4 times with distilled water and sown on square plates with half-strength Murashige and Skoog (1/2 MS) salts, 1% (w/v) sucrose, and 1% agar. The indicated amounts of hormones were added to the autoclaved and cooled media (50–60 °C). Control plates received DMSO or ethanol in the same amounts as those with treatments. Plants were grown vertically for the indicated number of days in dark or light, in a growth chamber with 14h of light at 25 °C and 10h of dark at 21 °C. For height- or length-related measurements, plates were scanned and the pictures were analysed using the software ImageJ . For the oryzalin experiments, plants were transferred after 10 d from oryzalin-containing media to 1/2 MS media, containing no oryzalin, and grown for a further 6 d. Plant height was measured after 10 d and 16 d.For experiments under in vitro conditions, were imaged with a scanning electron microscope (SEM). The length of 15 individual cells per plant was measured. Under dark conditions, the length of six coleoptile cells of two individual plants was measured. In order to measure the twisting angle, 3-day-old, dark-grown coleoptiles were imaged with the SEM. The angle of twisting relative to both edges of the coleoptile was measured using the angle tool in ImageJ. For each line, 12 coleoptiles were imaged and the twisting angle of eight cells for every coleoptile was measured.For cell length measurements, coleoptiles of 16 plants per tef line, grown for 4 d in the light and 3 d in the dark under 2 plants, seeds were grown under in vitro conditions in the dark for 3 d. Pictures of dark-grown coleoptiles were taken with a digital microscope  and analysed using ImageJ. Plants were grouped into kegne, WT, and heterozygous based on the helical growth of the coleoptiles. After taking pictures, plants were collected in liquid nitrogen and DNA was extracted for each plant. PCR amplification and restriction digestion of the Eragrostis tef α-Tubulin 1 (EtTUA1) gene was performed as described below.For Fkegne were grown on soil under equal day conditions. Physiologically mature plants were harvested 105 d and 127 d after sowing for the WT and kegne mutant, respectively. Plants were dried at 40 °C for 10 d before biomass and seed yield were measured. The harvest index was calculated as the ratio of seed yield to the above-ground biomass. For the yield experiment, plants of the WT and kegne mutant were grown under equal day conditions in a tray (45.5×28.2×6.0cm) filled with soil. Seeds were sown in lines 2cm apart. After germination, plants were thinned out to exactly 312 plants per tray, with ~2cm space between each individual plant. Sixteen weeks after sowing, all plants were harvested and dried at 40 °C for 10 d before biomass and seeds were weighed. The harvest index was calculated as mentioned above. F2 plants were grown on individual pots for the first 3–4 weeks under long day conditions followed by short day conditions until the panicles fully emerged.For weekly plant height measurements, 10 plants each from the WT and –1 RNase A solution.RNA extraction was done using the Trizol method (Invitrogen). M-MLV transcriptase (Promega) was used for cDNA synthesis. Genomic DNA extraction was done either with the NucleoSpin 96 Plant extraction kit  following the protocol of the supplier or by grinding the leaf material with a mill  before adding 200 μl of extraction buffer containing 200mM TRIS-HCl (pH 7.5), 250mM NaCl, 25mM EDTA (pH 8.0), and 0.5% SDS. Afterwards, a chloroform/isopropanol purification was performed before the extracted DNA was treated with 3 μl of a 10mg mlα-Tubulin genes from tef. Primers were designed using sequence information of the α-Tubulin genes of E. indica . For each of the three genes, two sets of primers were designed; one pair at the 5’ (S1) and 3’ end (AS1) and the other set in the central region of the gene (S2 and AS2). In addition, a pair of primers was designed to amplify specifically copy A of the genomic EtTUA1 gene. The sequences of the primers are indicated below: Tua1S1, ACCATGAGGGAGTGCATCTCGAT; Tua1AS1, ACCCAGTAGCAAAGCGGGGAATT; Tua1S2, ATTG AGCGCCCAACCTACACCAA; Tua1AS2, AGCATGAAGTGGA TCCTCGGGTA; Tua1S copy A, CTGAGCCAAGTTCGTTGATT GCTT; Tua2S, TGGCTCTGGACTTGGTTCACTAC; Tua2AS1, TACTCTGCCAGCCATGAACAGGT; Tua2S2, TGCTGCAGGC CTTTTCAGTTCTC; Tua2AS2, CAGAGTGCTGTTCATGGTAG GCT; Tua3S1, TTCAACACCTTCTTCAGCGAGAC; Tua3S2, CTTGAACAGGCTGATCTCACAGA; and Tua3AS1, ACAATC ATGACCAACAGGGGGCT.cDNA was used for amplification of the orthologous ®-T easy vector system (Promega) with procedures performed as outlined in the Quick protocol (www.promega.com) and DH5α as competent cells were transformed. Sequencing of the inserts was done from purified plasmids. The respective amplicons were sequenced and used to develop gene copy-specific primers used on genomic DNA.For sequencing, the amplified PCR products were ligated with the pGEMTUA1 gene for CAPS (cleaved amplified polymorphic sequence) marker assays. In order to score F2 plants as homozygous wild type, heterozygous, or homozygous kegne, the PCR-amplified products for the copy-specific EtTUA1 gene were digested using the restriction enzyme ClaI (New England Biolabs). PCR was performed in a 10 μl volume using 1× buffer, 0.2mM dNTPs, 0.325 μM of each forward (Tua1S copy A) and reverse (Tua1AS1) primers, and 0.1U of GoTaq polymerase (Promega) using 33 cycles of 95 °C for 50 s, 59 °C for 50 s, and 72 °C for 2min. The amplified PCR products were evaluated on agarose gels after incubating with 2.5–5U of ClaI.Genomic DNA was used as template in a PCR to amplify the 3 progeny of crosses between kegne and three improved tef cultivars were investigated to confirm the co-segregation between kegne-related phenotypes and the EtTUA1 marker.F4, pH 7.2) with 0.05% Triton X-100 (v/v) and 5% DMSO for 1h, and rinsed three times for 10min in MTSB. Fixed seedlings were incised  with razor blades to enable diffusion of antibodies into cells. Samples were then incubated with primary antibody  for 2–3h in 37°C, rinsed twice for 10min in PBS, and incubated with secondary antibody  for 2–3h in 37 °C. After rinsing twice for 10min in PBS, the whole seedling was mounted in 1:1 PBS–glycerol with 1% p-phenylenediamine (Sigma-Aldrich). Images of cortical microtubules under the outer epidermal cell wall of a coleoptile were collected using a Zeiss (Axioskop2) fluorescent microscope with ×100 oil immersion objective.Five-day-old seedlings grown in the dark on 1/2 MS plates were fixed in 1.5% formaldehyde and 0.5% glutaraldehyde in microtubule-stabilizing buffer . The mutant was named kegne, meaning ‘right hand’ in the Ethiopian Amharic language, for the unique turning of its first leaves towards the right-hand direction  direction, whereas those in the WT tilted off the central axis in either orientation.A tef mutant with a helical phenotype was identified from an EMS-mutagenized Mirection , B. The eoptiles , D, witheoptiles . StrikinKegne plants are also semi-dwarf in height. They were shorter than the WT throughout the growing period . Cell length measurements showed that both light- and dark-grown coleoptiles of kegne were significantly shorter than those of the WT .Similar to the WT, coleoptiles of kegne to these hormones and their respective inhibitors were investigated. WT and kegne plants were grown in either the light or the dark for 3 d on 1/2 MS medium containing different concentrations of GA. The unique phenotype of kegne plants in dark and light conditions was not rescued to the WT level by GA application , suggesting that kegne is not impaired in the biosynthesis of GA. When grown for 29 d on GA-containing medium, a significant increase in the length of both shoots and leaf sheaths was observed for both genotypes (data not shown). Although the same height as the WT was not achieved, kegne plants were responding to a certain degree to GA application, indicating that the mutation in kegne is not due to GA insensitivity.Since phytohormones such as GA, BR, and auxin are known to regulate cell elongation and plant height, the responses of in vitro grown plants. WT shoots under both dark and light and roots under light conditions were significantly inhibited by increasing levels of paclobutrazol. In contrast, kegne plants were unaffected by paclobutrazol application, except for the shoots grown in light .The effects of different levels of paclobutrazol, a GA biosynthesis inhibitor, were also investigated on kegne plants to the WT level. On the other hand, high BL concentrations led to a significant decrease in plant height and root length for both genotypes grown in the light and dark, respectively. This indicates that kegne is probably not impaired in the biosynthesis of BR but is responsive to BL application .Similar to GA, the application of different levels of BL did not rescue kegne plants showed de-etiolated growth when grown on high concentrations of Brz220 in the dark, although the WT was more sensitive to the treatments .Nevertheless, BR might induce the etiolated growth of tef in the dark, as evidenced by the application of Brz220, an inhibitor of BR biosynthesis. Both the WT and In addition, auxin and NPA, a polar auxin transport inhibitor, did not significantly affect the growth of roots and shoots in both genotypes (data not shown). Since defects in auxin signalling normally result in an altered gravitropic response of the roots, a root-bending assay was performed, although no obvious alterations were detected between the two genotypes (data not shown).kegne plants, indicating that the mutant is not impaired in either the biosynthesis or signalling of these three phytohormones.In general, none of the hormones and inhibitors substantially altered the response of Arabidopsis lead to dwarfism and helical shoot growth . However, the two genotypes differed in their response to propyzamide . In contrast to the WT, kegne shoots grown in the dark were not reduced in length at 1 μM propyzamide. Only at higher concentrations were both genotypes affected. The roots of kegne were insensitive to increasing levels of propyzamide in both light regimes, except for the highest concentration tested.Mutations in microtubule-associated genes in rice and kegne mutant . The statistical test confirmed that the angle distributions for the WT and kegne were significantly different . This indicates that the microtubules in kegne were distorted compared with those in the WT.Usually twisting mutants have microtubule arrays of a specific handedness; for example, in the mutants with right-handed twisting epidermis most microtubules form left-handed helices, and entation , althougt-handed . While tα-Tubulin genes  present in goose grass [Eleusine indica (L.) Gaertn.] were used to amplify the corresponding genes from tef cDNA using PCR. As tef is allotetraploid, two genomes (A and B) and thus two copies of each gene are expected. Sequencing clones of PCR products revealed no difference between the WT and kegne for the ‘Eragrostis tef α-tubulin 3’ (EtTUA3) gene while no tef orthologue could be amplified for TUA2. A point mutation was discovered in EtTUA1 in one genome of kegne (here referred to as copy A) while the other genome (copy B) was unaltered . The use of genomic DNA reveals that the EtTUA1 gene consists of four exons and three introns with a coding sequence of 1353bp (kegne corresponds to the replacement of the polar and uncharged amino acid threonine by the hydrophobic amino acid isoleucine at position 198 (T198I) in the protein sequence.The coding sequences of three f 1353bp . A cytosArabidopsis was searched in the recently published tef genome . The region upstream and downstream of the TUA1 gene was investigated for any possible connection to plant architecture . However, none of the eight genes within 100 kbp (50 kbp upstream and 50 kbp downstream) of the TUA1 gene in the tef genome was known to regulate plant stature.The TUA1 protein sequence of α-Tubulin genes of various plants shows that the threonine at position 198 occurs in a conserved region of the alignment and that it is conserved across all plants species analysed . Changes at this position have not been tolerated in the seed plants examined, indicating that the amino acid change at position 198 from threonine to an isoleucine could be deleterious to the function of the plant.The multiple sequence alignment of kegne created a recognition site for the ClaI restriction enzyme, causing the PCR amplicon for EtTUA1 of kegne to be cleaved while the corresponding amplicon from the WT remained intact due to the absence of the ClaI recognition site. Hence, this restriction site was used as a CAPS marker to distinguish between kegne and the WT for copy A of EtTUA1.The mutation in kegne phenotype , F2 progeny from crosses between kegne and several widely cultivated and popular tef cultivars were analysed using the ClaI restriction site as a marker. The results showed that reduced plant height and ClaI restriction site were linked in all 285 F2 progeny studied, indicating that the mutation at this location is tightly linked to the kegne phenotype .enotypes . Intereskegne, is the first semi-dwarf tef line and has the potential to overcome lodging, the major yield constraint in tef. Kegne is also characterized by helical growth of the first leaf. Such helical growth is a hallmark of mutations affecting the cytoskeleton. Indeed, microtubule drug assays, microtubule immunolabelling, co-segregation of the semi-dwarfism, and the helical phenotype with a polymorphism in the EtTUA1 gene strongly suggest that the kegne phenotype is due to a single base mutation in this gene.In this study, we investigated a novel mutant of tef, a cereal crop which serves as a staple food for ~50 million people in Ethiopia. The mutant, named kegne were similar to those described for other species affected in microtubule organization. In Arabidopsis, mutations in microtubule-associated genes such as TORTIFOLIA 1 (TOR1) resulted in helical growth of roots and aerial organs  to the amino acid isoleucine (hydrophobic) at position 198 , causes the kegne phenotype. Referring to the best studied structure of the pig α-tubulin, the described mutation is sited towards the central region of the protein, just before β-sheet six . This might happen if kegne is already impaired in cell elongation due to features related to low dynamic microtubules, hence the effect of reduced GA levels caused by paclobutrazol application could be masked. As a result, kegne might be resistant to certain concentrations of paclobutrazol.The present findings show that kegne under controlled conditions prompted the assessment of kegne and respective crosses to popular tef cultivars such as Quncho (Quncho was developed from the cross between the improved varieties Magna, a variety with consumer-preferred white grain colour but low productivity, and Dukem, a high yielding variety but with low market price due to the pale white grain colour. Quncho harbours both desirable traits, namely the white seed colour and the high yield. As a result, this variety has received high acceptance by tef farmers. However, since Quncho is tall and prone to lodging, the introgression of kegne will improve the standing ability of the former and as a consequence will further increase the productivity of tef. As a general rule, after hybridization, progenies need to be grown for at least eight generations in order to achieve a level of homozygosity acceptable to the authorities that give permission for the release of a crop as a new cultivar. The variety selection mechanism involves not only researchers but also farmers in a system known as Farmer Participatory Research (kegne will boost productivity of tef in farmers’ fields since lodging, the major yield-limiting factor in tef cultivation, will be tackled.The favourable characteristics of Research . Once thkegne is the first semi-dwarf and lodging-tolerant tef line, characterized by helical growth which is probably caused by a mutation in the α-Tubulin gene EtTUA1. Although complementation of kegne with the WT allele could not be carried out at the present time due to the lack of a stable transformation method for tef, the findings from microtubule drug assays, immunolabelling, and sequencing strongly suggest that the kegne phenotype, namely the semi-dwarf stature, helical growth, and oryzalin resistance, are due to a single base pair mutation in the α-Tubulin gene that replaces the polar and uncharged amino acid threonine with the hydrophobic amino acid isoleucine at position 198 of EtTUA1. The unique mutation in this highly conserved region of the α-Tubulin 1 gene among many plant species examined suggests that it is the cause of the drastic changes in the plant architecture of the kegne mutant. The present work highlights the advantages and opportunities associated with an indigenous crop. Major yield gains will reach subsistence farmers efficiently through well-established seed multiplication and distribution channels.In summary, JXB online.Supplementary data are available at Supplementary methods.Figure S1. Height differences between kegne and the wild type.Figure S2. Effect of different levels of gibberellic acid on the shoot and root length of wild-type and kegne plants grown in vitro for 3 d.Figure S3. Effect of different levels of paclobutazol on the shoot and root length of wild-type and kegne plants grown in vitro for 3 d.Figure S4. Effect of different levels of brassinolide on the shoot and root length of wild-type and kegne plants grown in vitro for 3 d.Figure S5. Effect of different levels of brassinozole220 on the shoot and root length of wild-type and kegne plants grown in vitro for 3 d.Figure S6. Effect of different propyzamide concentrations on the shoot and root length of wild-type and kegne plants grown in vitro for 3 d.Figure S7. Phylogenetic tree of α-tubulin protein sequences from diverse plant species.Figure S8. Comparison of orthologous syntenic genomic regions between tef and rice.Figure S9. Alignment of α-Tubulin genes in plants.Table S1. Yield and yield components of 312 wild-type and kegne plants grown in a tray of 0.128 m2 under equal day conditions.


HAM/TSP is a neurodegenerative disease that develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS). Physiologically, the CNS is protected and isolated from the immune system by a structure called blood-brain barrier (BBB). Thus HTLV-1-infected lymphocytes have the capacity to cross the blood-brain barrier (BBB). The mechanisms of such crossing are still poorly understood. In the context of multiple sclerosis and neuro-AIDS, the Activated Leukocyte Cell Adhesion Molecule (ALCAM) was shown to amplify leukocyte extravasation. In the earlier, ALCAM is over expressed on the BBB endothelium, whereas in the latter ALCAM expression is increased on HIV-1-infected monocytes. We therefore studied the possible role of ALCAM in extravasation of HTLV-1 infected lymphocytes. We demonstrated by FACS that ALCAM is over expressed both on HTLV-1-infected lymphocytes cell lines and on primary cells from HTLV-1 asymptomatic carriers or HAM/TSP patients. Via transduction with a Tax-encoding lentiviral vectors, we showed that ALCAM over expression is the consequence of Tax-1-induced NF-κB pathway activation. We finally demonstrated that inhibiting ALCAM with a monoclonal blocking antibody reduces significantly the extravasation of HTLV-1 chronically infected cells through a monolayer of BBB endothelial cells (the hCMEC/D3 model). This study reports a potential role of HTLV-1-induced ALCAM overpression in HAM/TSP pathogenesis.


In addition, this molecular platform reached 100% of sensitivity and specificity. In conclusion, the developed method using one tube multiplex qPCR (confirmatory and discriminatory) for HTLV-1/2 was validated. It showed low cost and high sensitivity and specificity compared to previously described assays and to the traditional confirmatory method (WB). Therefore, this platform can be a supportive tool for the current confirmatory methods adopted.Accurate diagnostic tests are powerful tool to control the spread of the human T-lymphotropic virus (HTLV) infection. In Brazil, the currently applied diagnostic algorithm for HTLV-1/2 screening is based on serological tests (enzyme-linked immunosorbent assay followed by Western Blot). However this algorithm is unsuitable due to its high cost and the elevated rate of Western Blot (WB) indeterminate results. Nevertheless, molecular techniques such as real-time PCR (qPCR) can overlap these drawbacks because of their high sensitivity and specificity. Several studies have described different qPCR protocols for HTLV-1/2 diagnosis but they lack suitable validation. For this reason, we developed and validated a qualitative multiplex qPCR platform for simultaneous detection and discrimination of the HTLV-1/2 infection in a single reaction tube. A total of 73 HTLV-positive samples and 100 samples obtained from non-infected individuals were tested. The detection limit was one copy/reaction for HTLV-1 and 10 copies/reaction for HTLV-2. We observed a high and similar efficiency between the single and multiplex format as well as the cycle treshold and


Giardia duodenalis is a common and widespread intestinal protozoan parasite of both humans and animals. Previous epidemiological and molecular studies have identified Giardia infections in different animals and humans, but only limited information is available about the occurrence and genotypes of Giardia in cattle in China. In this study, we determined the occurrence of giardiasis and genetically characterized G. duodenalis in dairy cattle in Henan Province, central China. The overall prevalence of G. duodenalis was 7.2% (128/1777) on microscopic analysis, with the highest infection rate (22.7%) in calves aged less than 1 month. G. duodenalis assemblages and subtypes were identified with multilocus genotyping based on the SSU rRNA, β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes. Two assemblages were detected in the successfully sequenced samples: assemblage A (n = 58), assemblage E (n = 21), with a mixed E and A assemblage (n = 2). Four novel subtypes of the gdh gene and seven of the bg gene were found among the G. duodenalis assemblage E isolates. Using the nomenclature for the multilocus genotype (MLG) model, nine novel multilocus genotypes E (MLGs E1–E9) and three MLGs A  were identified. MLG AII-1 identified in this study may be an important zoonotic subtype. The dairy cattle in Henan are a potential public health concern. Giardia duodenalis  is a common protozoan in cattle worldwide. It often presents no or mild symptoms in adult cattle, but clinical manifestations of diarrhea, weight loss, and malabsorption can present in calves G. duodenalis infection in humans, contracted by the fecal–oral route or by the ingestion of contaminated food or water G. duodenalis and its potential threat to public health are of increasing concern G. duodenalis is a species complex, comprising eight distinct assemblages/genotypes (A–H) The accurate identification of parasites is central to the effective control of parasitic diseases. Molecular studies have confirmed that Giardia have been reported in rabbits (subtypes B-I and B-VIII), monkeys (subtypes A-II and B), macaques (subtypes A-II and B), sheep and goats , dogs , and humans  G. duodenalis infection in calves in Heilongjiang, China G. duodenalis in cattle in China and an even greater lack of molecular data, we determined the prevalence of G. duodenalis in dairy cattle in Henan, China, and characterized it at the molecular level, using multilocus genotyping at the SSU rRNA, β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) loci.In China, the occurrence and molecular studies of This study was performed strictly according to the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Health, China. Our protocol was reviewed and approved by the Research Ethics Committee of Henan Agricultural University. All fecal specimens were collected from animals with the permission of the farm owners. No specific permits were required by the authorities for specimen collection. The field studies did not involve endangered or protected species.G. duodenalis infections. Farm 1 is the largest dairy farm in Henan Province, located in a suburb of the city of Zhengzhou, consisting of approximately 1000 animals, including calves (<6 months old), heifers (6–24 months old), and cows (>2 years old). The farm ranked among the top producing dairy farms in Henan. All dairy cattle were housed in different age groups in free stalls indoors.From August 2008 to October 2009, 1777 fecal samples were randomly collected from dairy cattle aged from newborn to 2 years old on 15 different intensive farms in Henan Province, central China , Table 1G. duodenalis-positive samples were stored in 2.5% (w/v) potassium dichromate solution at 4°C for molecular characterization.The fresh feces were placed into clean plastic bags marked with the date, age, and geographic origin, transported immediately to the laboratory, stored at 4°C, and then examined with a Lugol-staining technique and microscopy at 400× magnification. All G. duodenalis samples using the E.Z.N.A. Stool DNA kit , according to the manufacturer's instructions. The extracted DNA was eluted in 100 µl of AE elution buffer and stored at −20°C.The samples were washed three times in distilled water with centrifugation at 3000× g for 10 min to remove the potassium dichromate. The genomic DNA was extracted from the G. duodenalis samples were determined with nested PCR amplification of the SSU rRNA, bg, gdh, and tpi genes, according to previous studies bg, gdh, and tpi loci were conducted in 25 µL reaction mixtures containing of 1× PCR buffer , 200 µM each dNTP , 0.4 µM each primer, 1 unit of TaKaRa rTaq DNA polymerase , and 2 µL of DNA sample. In the SSU rRNA protocol, 1× GC buffer II  and LA Taq DNA polymerase  were used instead of 1× PCR buffer and rTaq. For the second amplification of bg, 10 µL of the first PCR amplicon was diluted in 90 µL of water and 2 µL of that dilution was used as the template for the second amplification. DNA samples from G. duodenalis isolates from humans and distilled water were used as the controls for each target-gene-based PCR analysis. The PCR products were visualized on a UV transilluminator after electrophoresis in 1.5% agarose gels and staining with ethidium bromide.The genotypes of the G. duodenalis genotypes were identified by alignment with reference sequences downloaded from GenBank (http://www.ncbi.nlm.nih.gov) using MEGA 4 All nested-PCR amplicons were sent to Beijing Nuosai Biological Engineering Biotechnology Company for two-directional sequencing on an ABI PRISM 3730 XL DNA Analyzer . The 2 test was used to compare the prevalence of G. duodenalis between different cities, the association between infection rate, age, and sampling season, and between the assemblage distributions and age. Differences were considered significant at P<0.01.The χgdh gene, KF843932–KF843940 for the bg gene, and KF843941–KF843948 for the tpi gene.All nucleotide sequences were submitted to the National Center for Biotechnology Information (NCBI) GenBank database under the following accession numbers: KF843921–KF843922 for the SSU rRNA gene, KF843923–KF843931 for the G. duodenalis was 7.2% (128/1777); 13 of 15 farms were Giardia-positive and the infection rate (95% CI) ranged from 0% to 14.4% on different farms , whereas no Giardia cysts were detected in adult cattle older than 1 year  .The association between the sampling season and the prevalence of arm Zz-1 . The preG. duodenalis-positive samples based on four loci, and 73 SSU rRNA, 67 tpi, 77 bg, and 61 gdh gene sequences were obtained. Fifty-six samples were successfully sequenced at all four genes, whereas the remaining samples were amplified at only one to three genes , reported to infect only hoofed livestock, and assemblage A (n = 21), which infects humans and a number of other mammals.We genotyped 128 ee genes . All nucG. duodenalis among assemblage E and A was observed at the tpi, bg, and gdh loci. Six different subtypes of assemblage E were identified at the tpi locus (KF843941–KF843946), and these showed 100% similarity to sequences available in GenBank with accession numbers of AY655705, EF654683, JN162351, AB569406, EF654689, and JF792419, respectively. Seven subtypes of assemblage E were also identified at the bg and gdh loci. Four subtype sequences at the gdh locus (KF843926–KF843929) have not been reported previously, and showed similarity of 99% to assemblage E . The remaining three subtype sequences at the gdh locus (KF843923–KF843925) showed 100% similarity to isolates B7 from cattle (EF507645), SG-10 from sheep (KC960647), and Ca39 from cattle (AB569388), respectively. Subtypes AI and AII of assembly A were identified at the tpi, bg, and gdh loci, with subtypes AI and AII identical to sequences with accession numbers AF069556 (subtype AI) and AF069557 (subtype AII) at the tpi locus, AY655702 (subtype AI) and AY072723 (subtype AII) at the bg locus, and EF507642 (subtype AI) and EF507674 (subtype AII) at the gdh locus. The variations among the new subtypes at different nucleotide sites are summarized in The genetic diversity of G. duodenalis-positive samples were infected with assemblage A, while 26% were infected with assemblage E. Assemblage E was found on twelve of fifteen farms, while assemblage A was only found on three farms: two of three farms being infected with assemblage E and assemblage A, one being present as a single assemblage A on Xx-1 (The data was not shown). Fifty-eight G. duodenalis assemblage E and twenty-one G. duodenalis assemblage A isolates were assigned to different ages groups. Assemblage E was found in all age groups and the highest percentage was in 61–90-day-old cattle , while assemblage A was only observed in calves <180 days old, with the highest percentage in calves <30 days old , which is a little higher than that recently reported in same-aged cattle in Heilongjiang Province, China  G. duodenalis in this study and those in other countries, such as the United States (44%), Canada (42.0%), Galicia (30.1%), Denmark (43.6%), Belgium (31.3%), Vietnam (10.2%), and New Zealand (31.0%) In this study, the overall infection rate of G. duodenalis in cattle in the current study revealed that infection was frequent in calves younger than 6 months old, but was rare in adult cattle older than 1 year. This finding was in accordance with previous reports regarding giardiasis as a common infection in immature farm animals G. duodenalis cysts were rare in calves younger than 1 month and found the highest prevalence in 4–5-month-old animals Cryptosporidium, and so on.There was a significant association between the age of sampling and the likelihood of infection. G. duodenalis infection was noted in winter, which is identical to a previous study conducted in the Netherlands G. duodenalis shedding peaked in December and February. They explained that in this period, because the weather is cold and the animals are housed indoors, the risk of infection with serious respiratory illnesses increases, making the cattle more susceptible to all kinds of infections, including G. duodenalis, as their immunity declined. These infected animals excreted G. duodenalis oocysts and then possibly infected other cattle in the cattle sheds via another vector: caretakers, cats, or mice. Similar climates might explain the consistent observations between the Netherlands and this study.In this study, the peak G. duodenalis assemblage E (72%), whereas a minority harbored the zoonotic assemblage A (26%). These findings are consistent with previous reports from China, Belgium, United States, Canada, Denmark, Australia, and Portugal bg gene (assemblage E) were not consistent with those at the other two genes (assemblage A)  . This maCryptosporidium species and genotypes, the zoonotic species, C. parvum primarily infected calves under 2 months of age, while the species and genotypes that were not infectious for humans primarily infected post-weaned calves G. duodenalis, we observed the differences in the assemblage distributions in the various age groups. The potentially zoonotic assemblage A was only found in calves <180 days of age, whereas assemblage E was found in all age groups. The highest infection rates of assemblages A and E were found in animals of 1 and 3 months old, respectively. These results are similar to those of previous reports in which the prevalence of assemblage E was highest in post-weaned calves and assemblage A was only detected at 6 months of age In a study of G. duodenalis in humans and animals from different geographic regions. In this study, three MLGs were identified in 18 assemblage A isolates, including a known MLG AII-1 (n = 5), a novel MLG AI (n = 12), and a combination of both (n = 1). The distribution of the G. duodenalis multilocus subtypes is consistent with previous findings that subtype AI has a preference for livestock, whereas subtype AII is commonly detected in humans G. duodenalis subtype in humans and animals in China and other countries indicates that MLG AII-1 may be an important zoonotic multilocus genotype.The MLG model was developed by Caccio et al. in 2008 and used later by Sprong et al. and Lebbad et al. Giardia in dairy cattle in Henan Province, China, and provides some preliminary data on the genetic diversity of the parasite in this region using multilocus genotyping. Whether MLG AII-1 is the main zoonotic subtype must be investigated further with systematic molecular epidemiological analyses of both humans and animals.In conclusion, this study has reported the prevalence of


Following infection and integration into the genome of host CD4+ T-lymphocytes, human T-lymphotropic virus type-1 (HTLV-1) persists by driving proliferation of infected T-cell clones which are counter-selected by HTLV-1-specific cytotoxic T-lymphocytes (CTLs). HTLV-1 Tax protein is the dominant target antigen recognized by these CTLs, and Tax is thought to cause the infected T-cell proliferation. In 40% of cases of adult T-cell leukaemia/lymphoma (ATLL) Tax protein is not expressed, suggesting that the provirus has down-regulated Tax to allow immune escape and viral persistence. Here, we tested the hypothesis that the genomic site of proviral integration determines the degree of clonal expansion and therefore determines the risk of ATLL. Proviral integration sites in 242 ATLL cases and 96 asymptomatic carriers were mapped and quantified using our recently described high-throughput technique . In the ATLL cases, we identified promoter deletions predicted to cause tax gene silencing, we sequenced the tax gene and quantified promoter methylation. The results showed that 65% cases of ATLL could silence Tax by promoter deletion, tax gene nonsense mutations or tax promoter hypermethylation. High-throughput sequencing data showed that ATLL cases were not invariably monoclonal and that the provirus is typically integrated into a transcriptionally active region of the host genome. The results also suggest that transcriptional activity of the flanking host genome contributes to tax gene silencing, allowing CTL escape and clonal expansion.


Virus transmission from various wild and domestic animals contributes to an increased risk of emerging infectious diseases in human populations. HTLV-1 is a human retrovirus associated with acute T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 originated from ancient zoonotic transmission from nonhuman primates, although cases of zoonotic infections continue to occur. Similar to HTLV-1, the simian counterpart, STLV-1, causes chronic infection and leukemia and lymphoma in naturally infected monkeys, and combined are called primate T-lymphotropic viruses (PTLV-1). However, other clinical syndromes typically seen in humans such as a chronic progressive myelopathy have not been observed in nonhuman primates. Little is known about the development of neurologic and inflammatory diseases in human populations infected with STLV-1-like viruses following nonhuman primate exposure.tax gene, the proviral load in PBMC and cerebrospinal fluid cells was 12.98 and 51.68 %, respectively; however, we observed a distinct difference in fluorescence amplitude of the positive droplet population suggesting possible mutations in proviral DNA. A complete PTLV-1 proviral genome was amplified from the patient’s PBMC DNA using an overlapping PCR strategy. Phylogenetic analysis of the envelope and LTR sequences showed the virus was highly related to PTLV-1 from sooty mangabey monkeys (smm) and humans exposed via nonhuman primates in West Africa.We performed detailed laboratory analyses on an HTLV-1 seropositive patient with typical HAM/TSP who was born in Liberia and now resides in the United States. Using a novel droplet digital PCR for the detection of the HTLV-1 These results demonstrate the patient is infected with a simian variant of PTLV-1, suggesting for the first time that PTLV-1smm infection in humans may be associated with a chronic progressive neurologic disease. Virus transmission from various wild and domestic animals has contributed to the increased risk of emerging infectious diseases in human populations. Several recent endemics, such as avian flu, Human immunodeficiency virus (HIV) and ebola, originated from wild animals and which often are asymptomatic but which might induce severe diseases and a pandemic threat in humans . NonhumaHere we demonstrate the detection and sequencing of the complete PTLV-1 genome obtained from a patient with neurologic disease consistent with HAM/TSP. Phylogenetic analysis showed the virus closely clustered with STLV-1 from NHPs in West Africa. These results demonstrate that the patient is infected with the simian variant of PTLV-1, suggesting for the first time that infection of a PTLV-1 strain, clustered with STLV-1 strains from sooty mangabey monkeys, is associated with a chronic, inflammatory, progressive neurologic disease in humans.Patient NIH00261 was a 65-year old, African male who was originally from Monrovia, Liberia and immigrated to the United State (U.S.) in the 1980s. He was diagnosed with HAM/TSP for approximately 20 years. On examination, he was found to have spasticity and increased reflexes in the lower extremities, moderate lower extremity weakness, a spastic adductor gait, and decreased sensation to vibration in the lower extremities. His disability score was 6.5 in the expanded disability status scale (EDSS) and 16 in the Instituto de Pesquisa Clinica Evandro Chagas disability scale (IPEC). Both serum and CSF from the patient were reactive to HTLV-1 proteins including envelope  and Gag (p19 and p24)   was recently shown to be a precise and reliable method for HTLV-1 proviral DNA load quantification in samples with low amounts of nucleic acids and for detection of viral mutants in the target gene sequence . Using d %) Fig. a. CSF ceent Fig. a. Since tax sequences generated with the ddPCR primers (154-bp) from the PBMC of this patient. Sequence analysis demonstrated eight point mutations including one in the probe-binding region of the HTLV-1 from patient NIH00261 compared to a reference sequence from a prototypic variant of HTLV-1 cosmopolitan subtype a , polymerase (pol), env, tax, rex, p12I, and p30).To confirm the presence of genetic mutations in the HTLV-1 from patient NIH00261, we analyzed the ins Fig. b. Therefenv sequences from Cote d’Ivoire (IC) as having the greatest nucleotide identity to HTLV-1NIH00261  thymidine. The average cpm from each of the wells was plotted.Measurement of lymphoproliferation was performed as previously described . PBMCs wtax and human ribonuclease P protein subunit 30 (RPP30) primers and probes and Bio-Rad 2× Supermix, and then emulsified with droplet generator oil using a QX-100 droplet generator according to the manufacturer’s instructions (Bio-Rad) [tax: ddPCR HTLV-1 tax F: 5′-CTTATTTGGACATTTACCGATG-3′; ddPCR HTLV-1 tax R: 5′-TGAGGCCGTGTGAGAGTAGA-3′; ddPCR HTLV-1 tax probe: 6FAM-TGATTTCCGGGCCCTGC-MGBNFQ [tax/(quantity of RPP30/2)} × 100 %.HTLV-1 proviral DNA load was measured using ddPCR (Bio-Rad) as previously described . DNA wasBio-Rad) . The folC-MGBNFQ . The droC-MGBNFQ . Followi® PCR SuperMix High Fidelilty (Life Technologies) as follows: 10 min at 94 °C, 30 cycles consisting of a 30 s denaturation at 94 °C, a 30 s annealing at 55 °C, and a 60 s extension at 68 °C. PCR products were electrophoresed on 1 % agarose gel, then extracted and purified using a QIAquick gel extraction kit (Qiagen). The purified PCR product was ligated with the pCR-4™ TOPO® TA cloning vector (Life Technologies) and transformed into One Shot® Top10 chemically competent E. coli (Life Technologies). Plasmids containing each viral DNA insert were purified from transformed E.coli using QIAprep® Miniprep (Qiagen) and sequenced using T3 and T7 primers (Genewiz). The complete HTLV-1NIH00261 genome has been assigned the GenBank accession number (KU214243).Table env) and LTR regions which have the highest representation of HTLV-1 and STLV-1 sequences at GenBank and have demonstrated utility for inferring PTLV-1 evolutionary histories [env sequences was done to identify highly related reference sequences for the analysis. Reference sequences were selected based on the BLAST quality scores  and sequence length. Sequences were aligned in MEGA6 using the MUSCLE program [NIH00216 from the ML analysis using HKY + G model [env dataset was unchanged due to the shorter length of some GenBank reference sequences included in the analysis. For both alignments the Australomelanesian HTLV-1 sequences were used as outgroups and we also included some cosmopolitan HTLV-1a for comparison. The following parameters were used for the BEAST analysis: a lognormal molecular clock, the HKY + G nucleotide model, a birth–death process tree prior, and a normal distribution of substitution rate  previously inferred for HTLV-1 vertical transmission [http://beast.bio.ed.ac.uk/Tracer). All parameter estimates showed significant ESSs >4000 indicating sufficient mixing. The tree with the maximum product of the posterior clade probabilities (maximum clade credibility tree) was chosen from the posterior distribution of 9001 sampled trees after burning in the first 1000 sampled trees with the program TreeAnnotator version 1.8.2 [Nucleotide identities were determined using Geneious v8.1.5 and genome open reading frames and structure were determined by MacVector v13.5.32. Since the number of complete PTLV-1 genomes available at GenBank is limited we restricted the phylogenetic analysis to the envelope (istories –11. A BL program  followed G model . The smasmission . Two ind


Obstructive Sleep Apnoea/Hypopnoea Syndrome (OSAHS) is a common disorder in the general population but often underestimated and underdiagnosed.This questionnaire-based study evaluated the overall level of knowledge about OSAHS among Italian General Practitioners (GPs), who are frequently involved in the management of this complex disease. This represents an interesting aspect, because GPs intercept many of the patients with OSAHS, in which C-PAP could be potentially indicated. Randomly-selected GPs were provided with questionnaires, which were then returned anonymously.80 questionnaires have been validated; the participants in the sample examined were represented by 43 females and 37 males; the average age of participants was 51 years. The general knowledge on OSAHS is overall satisfactory among GPs; it is recognized by most of the GPs interviewed as pathology in constant increase, and associated with predisposing factors such as obesity. High blood pressure is perceived as an independent cardiovascular risk factor in patients with OSAHS, in line with the majority of international studies. The C-PAP has been identified as the care gold standard in patients with OSAHS, despite the lack of patient compliance in relation to this procedure, while polysomnography was found to be the main instrumental procedure used in the diagnostic workup of OSAS. The pulmonologist and a multidisciplinary team have been identified as the specialist figures of reference to which to direct the patient through the diagnostic workup. Respiratory therapists and nurses represent the role of educator in the proper management of the C-PAP in the opinion of 62% of respondents, while only 34% think that this role should be played by the GPs and/or other specialists.In conclusion, this survey about the perception of OSAHS among GPs in Italy highlighted a satisfactory overall knowledge of OSAHS and only few weak points. OSAHS is a common disorder in the general population but often underestimated and underdiagnosed . The defad hoc” and based on the guidelines and the current literature, by a panel of experts composed by General Practitioners (GPs) and Specialists . It consists of 16 items (Table The results of this study were obtained through the completion of an anonymous questionnaire, created ““Sleepiness Epworth Scale” is still poor. The latter assesses the likelihood of falling asleep or dozing off in various situations, which is a useful metric to suspecting OSAHS. 78% of the GPs surveyed did not know about it, while only 28% indicated to be aware but not to use it (Figure As a general result, a good knowledge of the definition of OSAHS emerged from the results, with 67% of correct answer to the corresponding question in the questionnaire Figure . An adeqt Figure A. 60% oft Figure B, emphast Figure A and in t Figure B the diaOSAHS is a common disorder in the general population but often underestimated and underdiagnosed. OSAHS is an independent risk factor for hypertension and cardiovascular disease and is the frequent underlying disease of secondary hypertension and resistant hypertension . Randomi


Cytokines are believed to be crucial mediators of chronic intestinal inflammation in inflammatory bowel diseases (IBD) such as Crohn’s disease (CD) and ulcerative colitis (UC). Many of these cytokines trigger cellular effects and functions through signaling via janus kinase (JAK) and signal transducer and activator of transcription (STAT) molecules. In this way, JAK/STAT signaling controls important events like cell differentiation, secretion of cytokines or proliferation and apoptosis in IBD in both adaptive and innate immune cells. Moreover, JAK/STAT signaling, especially via the IL-6/STAT3 axis, is believed to be involved in the transition of inflammatory lesions to tumors leading to colitis-associated cancer (CAC). In this review, we will introduce the main cellular players and cytokines that contribute to pathogenesis of IBD by JAK/STAT signaling, and will highlight the integrative function that JAK/STATs exert in this context as well as their divergent role in different cells and processes. Moreover, we will explain current concepts of the implication of JAK/STAT signaling in CAC and finally discuss present and future therapies for IBD that interfere with JAK/STAT signaling. Inflammatory bowel diseases (IBD) are characterized by chronic or relapsing inflammation of the intestine leading to symptoms including diarrhea, pain or hematochezia. Crohn’s disease (CD) and ulcerative colitis (UC) are considered to be the main entities of human IBD ,2. AlthoIn spite of considerable advances, however, the pathogenesis of IBD has still not been fully elucidated. Nevertheless, several pathogenetic pillars have been identified that crucially contribute to development or propagation of inflammation. As a result, IBDs are generally thought to occur in genetically predisposed patients with a weakened intestinal barrier and a reduced biodiversity of the enteric microbiota ,6,7. SubThe importance of genetic factors has been extensively studied and large numbers of genes have been identified that are associated with either CD, UC or both. Among these are several genes encoding or regulating cytokines and receptors that signal via the janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, e.g., IL-23R, IL-10, IFN-γ or IL-12B . MoreoveWeakness of the epithelial barrier in IBD is a consequence of a disequilibrium of protective and aggressive factors. For example, protective mechanisms like mucin secretion, production of defensins or intestinal epithelial restitution of small wounds are impaired ,10,11. AChronic intestinal inflammation in IBD potentially leads to the development of colitis-associated cancer (CAC). In the course of inflammation, the epithelial turnover is high, mutagens may easily cross the defective mucosal barrier, oncogenic signaling cascades are activated, and reactive oxygen species are produced. This leads to progressive accumulation of genetic and epigenetic mutations manifesting stepwise first as low- and high-grade dysplasia and later as colorectal carcinoma . This afAfter outlining the principles of JAK/STAT signaling, this review will discuss the present evidence for the involvement of JAK/STAT pathways in the pathogenesis of IBD and CAC preceded by a short summary of JAK functions and subsequently arranged according to the different members of the STAT family. The functions of the different STATs in the cellular compartments of lymphocytes, macrophages and intestinal epithelial cells (IECs) in IBD are depicted in JAK/STATs translate the signal of a large number of cytokines and interferons from the cell membrane to the nucleus. Upon binding of a particular ligand to its respective transmembrane receptor, receptors dimerize and lead to conformational changes in associated JAK molecules  Figure . This coAs there exist four different JAK molecules  and seven members of the STAT family , specific combinations of a cytokine receptor and the activated JAKs and STATs can lead to specific cellular responses . HoweverIt must nevertheless be mentioned that signaling via JAKs and STATs is not always as straightforward as outlined above. For example, JAK/STAT signaling may differ between different members of these families. Moreover, “non-canonical” pathways in which STATs are activated by other upstream signaling molecules ,26, or JAs STATs are the final effectors of JAK/STAT signaling, and each JAK is related to several STATs, most research on this signaling pathway in IBD has focused on STATs. However, there are some basic conclusions that can be drawn from reports with a general immunologic background and the limited number of studies directly assessing JAKs in colitis.JAK1- and JAK2-deficient mice die pre- or perinatally ,29,30. J−/− mice and suggested reduced Th1 and Th17 activity to be responsible for this observation [A recent study directly addressed the function of Tyk2 in experimental colitis using Tyk2 knockout mice. Such mice displayed severer experimental colitis, reduced phospho-STAT3 levels and decreased proliferation of IECs. This could be confirmed in mice with a specific deletion of Tyk2 in IECs and linked to defective IL-22 signaling . Howeverervation .de novo neoplasia and led to regression of established tumors, which correlated with STAT3 activation [In JAK3 knockout mice, a spontaneous onset of IBD-like symptoms has been reported and was connected to changes in macrophage and lymphocyte populations . Later, tivation . Taken tSTAT1 is phosphorylated by either JAK1 and JAK2 or JAK1 and TYK2 and is thought to be of special relevance in the context of signaling via the IFN-γ receptor and the related family of receptors . Howeveri.e., activated STAT1 was especially upregulated in monocytes and granulocytes [A first study of STAT1 expression in IBD reported an increase of STAT1 levels in gut tissue from both UC and CD. Phosphorylated, ulocytes . Anotherulocytes . Similarulocytes .Different suggestions can be made as to how this might be functionally relevant. While an amelioration of experimental colitis was reported in an initial assessment with STAT1 knockout mice  and uponTaken together, the precise role of STAT1 in IBD remains unclear as it is implicated in both pro- and anti-inflammatory pathways. Thus, the upregulation of STAT1 expression in human IBD could be seen either as a primary defect contributing to the development of intestinal inflammation or as the secondary attempt of the immune system to limit inflammation. However, it appears that STAT1 activation in lymphocytes rather supports IBD development, while its phosphorylation in macrophages and epithelial cells seem beneficial regarding intestinal inflammation. Variations on this theme of differential roles in adaptive and innate cells will reappear several times in the further course of the portrayal of JAK/STAT signaling in IBD.in vitro [STAT1 has also been proposed as important in CAC. Here, it seems to have a protective function as normal intestinal fibroblasts, but not activated fibroblasts or fibroblasts from patients with IBD, activate STAT1 in colon cancer cells and thereby inhibit the growth of the tumor cells . Furtherin vitro . In anotin vitro , and SOCin vitro . In conci.e., IFN-α and β) [STAT2 is predominantly involved in the signal transduction of type I interferons (α and β) . As thesα and β) , and no However, IFN-α possesses anti-tumor activity  and someIn contrast to STAT2, the essential role of STAT3 in IBD is beyond question. STAT3 signaling is a hallmark of many members of the gp130 family of cytokines , but some IFN family members like IL-10 or IL-22 also require STAT3 to exert their functions. JAK1 together with JAK2 or TYK2 activates STAT3 .In both UC and CD lamina propria T cells, STAT3 expression and nuclear translocation is induced together with downstream anti-apoptotic genes. This finding was attributed to IL-6 signaling as blockade of IL-6 trans signaling enhanced apoptosis of T cells in experimental colitis . ConsistLater, another mechanism of STAT3-mediated pathogenic contribution was detected as STAT3 was found to be essential for the differentiation of Th17 cells and Th17-dependent murine colitis ,68,69. Ti.e., lymphocytes and epithelial cells. Thus, different functions of STAT3 may be due to cell-specific signaling. The general notion is that while STAT3 controls pro-inflammatory functions in cells of the adaptive immune system, its role in innate immune cells is rather protective [Interestingly, however, several cytokines that have anti-inflammatory properties also make use of the STAT3 pathway. In concrete terms, IL-22 has been shown to induce epithelial STAT3 activation and reduce disease activity of experimental colitis in mice via recovery of goblet cells and restoring the mucus layer . This waotective . This peotective . Anotherotective . Moreoveotective , and supotective . Conversotective .However, this view of divergent functions of STAT3 in different immunologic compartments is not fully conclusive as an important role of STAT3 has also been shown for the IL-10-dependent suppressive function of regulatory T cells. Regardless of the diverse features of STAT3 in the pathogenesis of IBD, however, all these data perfectly match with the aforementioned association of mutations in the STAT3 locus with increased risk for both CD and UC .In addition to these important effects in human and murine IBD, there is growing evidence that STAT3 signaling might be an important link connecting IBD and CAC. Paralleling the previously mentioned findings in IECs, T cell- and macrophage-derived IL-6 enhanced tumor cell growth in murine colorectal cancer . This waBacteroides fragilis, a commensal bacterium of the intestinal flora, and tumor formation via STAT3 could be established [Interestingly, a non-canonical function of STAT3 in mitochondria has been proposed to mediate STAT3-dependent resistance of colorectal cancer cells to apoptosis by decreased production of reactive oxygen species . Furtherablished . Additioablished . In subsablished ,91 were As a consequence, it is not surprising that an upregulation of IL-6 and phospho-STAT3 has also been demonstrated in patients with dysplasia or cancer originating from UC . GeneralCollectively, STAT3 activation via the JAK/STAT pathway is of central importance in both IBD and CAC. Though not all findings can as of yet be easily reconciled, it can be conjectured that STAT3 acts in a rather pro-inflammatory manner in the adaptive immune compartment, while its functions in innate defense mechanisms are rather protective. Yet, the cost of the latter observation seems to be a concurrent increase of the risk of cancer development.JAK2/TYK2-dependent phosphorylation of STAT4 is virtually exclusively found in response to signaling of certain members of the gp130 family of receptors, especially IL-12R and IL-23R  that shaThe postulated involvement of STAT4 in IBD is predominantly linked to the essential role of STAT4 for the development and function of Th1 cells. Based on an enhanced expression of both Th1-related transcription factors like T-bet and STAT4 and Th1-secreted cytokines like IFN-γ and IL-2, these cells are deemed critical for the pathogenesis of CD rather than UC . IL-12 iIn experimental models, deletion of STAT4 brought about a relative protection from colitis development  and, conIn human IBD, an association of STAT4 polymorphisms and both UC and CD has been uncovered ,100,101.Only limited evidence is available concerning STAT4 in CAC. However, IL-12 has been demonstrated to be a cytokine with potent anti-tumor activity  and STATSTAT5 activation predominantly occurs in response to JAK1 and JAK3 activation by members of the γC family of receptors and JAK2 signaling originating from receptors of the IL-3 family and the single chain family .Growth hormone belongs to the latter family and has anti-inflammatory functions in experimental colitis. Reduced growth hormone signaling has been linked to impaired STAT5b activation. Interestingly, the blockade of tumor necrosis factor (TNF)-α, which is successfully used in clinical therapy of patients with IBD, was demonstrated to re-induce signal transduction via this axis . SimilarAnother protective mechanism for STAT5 in IBD also concerns the epithelium: STAT5 is critical for the proliferation of intestinal epithelial stem cells and subsequent regeneration of the crypt epithelium .Apart from this, STAT5 is involved in the regulation of T cell differentiation like other STATs; it is especially important for Foxp3 induction in regulatory T (Treg) cells as deletion of STAT5 in T cells disrupted Treg differentiation in an IL-2Rβ-dependent manner . In addiet al. showed that while IL-21-dependent STAT3 activation increased apoptosis of dendritic cells (DCs), GM-CSF-induced STAT5 signaling offers protection from apoptosis [Some interesting findings have also been reported for STAT5 signaling originating from granulocyte macrophage colony stimulating factor (GM-CSF), although the implications for colitis are not fully resolved. Wan poptosis . Converspoptosis .Taken together, it appears that STAT5 has rather anti-inflammatory properties both in the epithelium and in adaptive immunity. To the contrary, the role of STAT5 in CAC is far from being understood and can only be deduced from the poor evidence that is available for colorectal carcinoma (CRC) in general. Here, phospho-STAT5 expression is associated with higher levels of cyclin D1 and poor prognosis , and sinSTAT6 phosphorylation occurs upon JAK1 and JAK3 activation and is virtually only induced by signaling via some members of the γC family of receptors, namely IL-4R and IL-13R .Besides GATA3, STAT6 is the most important transcription factor determining Th2 differentiation. Additionally, interplay of STAT6 with PU.1 and IRF4 is implicated in the generation of the recently described Th9 subset of CD4+ effector cells. Both lineages are thought to be pathogenic especially in UC .Compatibly, STAT6-deficient mice are relatively protected from oxazolone-induced colitis, which is accompanied by a reduction of Th2 cytokines . MoreoveIn human IBD, phospho-STAT6 is increased in UC ,131, andet al. reported a shift from preferential STAT6 activation to STAT3 activation regarding non-cancerous and cancerous samples from IBD patients [In vitro evidence, however, rather favors the view that STAT6 supports carcinogenesis by promoting proliferation and preventing apoptosis of epithelial cells [Regarding CAC, Wick patients , respectal cells ,135. Conal cells . Collectper os and therefore be more convenient for patients.Although STAT molecules are the ultimate effectors of JAK/STAT signaling, currently tested therapeutic applications in IBD predominantly target JAKs, thus interfering with the signaling of a variety of cytokines . This isvs. 10%, and the side effect profile was judged tolerable [The compound that is furthest advanced in clinic trials is tofacitinib, which predominantly inhibits JAK1 and JAK3. In a phase II trial in patients with UC, up to 78% of the patients receiving tofacitinib had a clinical response after eight weeks compared with 42% in the placebo group. Remission was noted in up to 48% olerable . Meanwhiolerable . It coulMoreover, the JAK inhibitors peficitinib and GLPG-0634 are currently being tested in early-stage clinical trials .What is also worth discussing in this context due to its close relation to JAK/STAT signaling and its probable implication in CAC development is anti-IL6R or anti-IL6 treatment. Regarding the central role of IL-6 in the orchestration of immunologic events in IBD, great hopes have been placed in blocking IL-6 signaling. However, data from clinical trials are thus far scarce , and advFinally, JAK inhibition might also become an option for manifest CRC or CAC. In a mouse model of colitis-induced colorectal cancer, JAK2 inhibition resulted in reduction of proliferation and angiogenesis and down-sized established tumors . As a coClearly delineating the role of JAK/STAT signaling in IBD and CAC is difficult due to the pleotropic functions of the single molecules. However, some general conclusions concerning the pathogenesis of IBD can be made based on the above depictions. First, while most reports favor a rather aligned role of JAK/STATs for immunologic processes within the same immunologic compartment, opposing features of JAK/STAT signaling in innate and adaptive immunity are not uncommon. Second, the translation of findings from rodent models into clarification of the human situation is still in progress as most human data concern expression studies or genetic associations. This may be a direct consequence of differential JAK/STAT signaling in different cells or environments because this makes it difficult to draw straightforward conclusions from applying inhibitors of single molecules. Third, what is protective in JAK/STAT signaling in IBD seems to be rather disease-driving in CAC regarding several STATs. This is likely due to the fine line between regeneration and uncontrolled proliferation. However, detailed and coherent functional data regarding the implication of JAK/STAT signaling in CAC development are only available for STAT3. And fourth, although several obstacles are present as outlined, therapeutic application of compounds that interfere with JAK/STAT pathways are within reach. Hopefully, further advances in basic science will help to develop drugs that selectively target the detrimental consequences of JAK/STAT signaling, while clinical research will identify indications and patient groups in which interfering with JAK/STAT signaling is a promising treatment.


Intervertebral discs (IVDs) are strong fibrocartilaginous joints that connect adjacent vertebrae of the spine. As discs age they become prone to failure, with neurological consequences that are often severe. Surgical repair of discs treats the result of the disease, which affects as many as one in seven people, rather than its cause. An ideal solution would be to repair degenerating discs using the mechanisms of their normal differentiation. However, these mechanisms are poorly understood. Using the mouse as a model, we previously showed that Shh signaling produced by nucleus pulposus cells activates the expression of differentiation markers, and cell proliferation, in the postnatal IVD. In the present study, we show that canonical Wnt signaling is required for the expression of Shh signaling targets in the IVD. We also show that Shh and canonical Wnt signaling pathways are down-regulated in adult IVDs. Furthermore, this down-regulation is reversible, since re-activation of the Wnt or Shh pathways in older discs can re-activate molecular markers of the IVD that are lost with age. These data suggest that biological treatments targeting Wnt and Shh signaling pathways may be feasible as a therapeutic for degenerative disc disease. Intervertebral discs (IVD) consist of a multilayered fibrocartilaginous annulus fibrosis (AF) surrounding a semi-liquid nucleus pulposus (NP), in which cells derived from the embryonic notochord The most effective treatment of DDD would be to stimulate regeneration of the disc using the developmental mechanisms that previously controlled its normal growth and differentiation. However, these mechanisms are not well understood. We have used the murine disc to identify signaling pathways active during disc growth and differentiation, and have shown previously that the NP cells synthesize many signaling ligands during postnatal growth and differentiation in vitro and in vivo, that canonical Wnt signaling is an essential activator of Shh signaling in the disc. Furthermore, we show that both pathways become down-regulated with age, concomitant with a decline in expression levels of extracellular matrix and other differentiation markers in the disc. To test whether the age-related down-regulation was permanent, we tested the effect of activating the canonical Wnt pathway in older discs, at one year of age. This had the effect of re-activating Shh signaling and expression of its downstream targets. These data suggest that biological therapies for age-related changes in the IVD of humans may be possible in the future.Here we show, both in vitro the effects of the small molecules BIO and XAV939 on IVDs 3 or P4t5) either in vehicle only, 30 µM BIO, or 400 µM XAV939. The results are shown in We have showed that blockade of Shh signaling resulted in up-regulation of canonical Wnt signaling in postnatal day four (P4) mouse IVDs We have demonstrated that Shh signaling is both necessary and sufficient for cell proliferation in the NP during postnatal growth flx/flx mice flx/flx) and the Wls mutants (Wls−/−) at this stage. Immunofluorescence staining showed no change in the expression of SHH in the Wls−/− NPs . 3). The IVDs were washed and either snap frozen for cryosectioning, or the NP cells were dissected and processed for RNA isolation. H&E staining of mid-coronal sections through the IVD shows that, following activation of canonical Wnt signaling, the NP cells started to lose their highly compacted structure and regained the reticular structure seen during growth, whereas IVDs treated with vehicle only were unchanged , whereas the organ culture experiments were carried out during early postnatal life. Second, differences in experimental design may affect the outcome. In some studies, NP cells cultured in monolayer on plastic dishes were used In other studies, however, activation of Wnt signaling has had opposite effects. The addition of LiCl to12-week old rat NP cells in culture caused loss of cell proliferation, and reduced expression of This study also addresses the relationship between cessation of growth and changes in the Wnt and Shh pathways. The NP cells change both their morphology and gene expression patterns as they transition from the rapid growth phase of the spine during the first eight-nine postnatal weeks In conclusion, we show here that Shh signaling from the NP cells, previously shown to control the expression of cell differentiation and proliferation in the IVD during rapid postnatal growth, is controlled in turn by canonical Wnt signaling, and that feedback between the two pathways controls the level of Wnt signaling. We also show that the activities of both pathways declines in aged discs, concomitantly with age-related morphological changes in the IVD. These changes can be at least partially reversed in organ culture by re-activating either Wnt or Shh signaling. The degree to which age-related changes can occur, and the mechanisms by which Wnt and Shh signaling control each other, and are controlled by circulating (systemic) signals such as hormones to coordinate IVD growth and differentiation in the whole spine, now become important experimental problems .FVB mice were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all experiments were carried out in strict accordance with institutional guidelines under Institutional Animal Care and Use Committee (IACUC) approval at Cincinnati Children's Hospital Research Foundation . All studies were carried out using at least three IVDs in each group and repeated three times.flx/flx conditional knockout mice flx/flx mice and genotyped as previously described Wls2. Small molecule regulators of Wnt signaling pathway, Stemolecule BIO , a Wnt signaling activator 3 - t5). Vehicle-treated discs served as controls. At the end of the culture period the IVDs were washed three times in cold 1x PBS and either snap frozen in OCT molds for histological evaluation, or the NP cells were dissected and collected in RNAlater  and stored at −80°C for qPCR analysis.Lumbar spines were dissected from postnatal day-four (P4), or one-year-old (1yr) mice, and cultured as described previously Cryosections from cultured IVDs, and from the Wls mutant mice were collected at a thickness of 6 µm in the coronal plane, using a Leica cryostat. Sections were processed immediately, or stored at −20°C for later use. Histology of the vertebrae was analyzed by fixing sections in 2% paraformaldehyde (PFA) and staining with hematoxylin and eosin (H&E). Sections were photographed using a Zeiss axiovert microscope.Immunoflourescence analysis was carried out on 6 µm-thick mid-coronal cryosections of IVDs from the in vitro and in vivo experiments. The sections were fixed in 2% PFA for 2 minutes, permeabilized using 1x PBS containing 0.25% Triton-x100 for 20 minutes, blocked in blocking buffer for one hour as previously described 202 diluted in methanol to quench the endogenous peroxidase activity followed by three washes in 1x PBS. The sections were next incubated with anti-BrdU antibody provided with the kit for 2 hours at room temperature, followed by 30 minutes incubation with HRP-conjugated secondary antibody. To visualize the BrdU positive cells under the microscope, the sections were treated with the Metal enhanced DAB Substrate kit , and the nuclei were counter-stained pink with nuclear fast red . The sections were dehydrated and mounted in xylene based mounting medium. Sections were photographed using a Zeiss axiovert microscope and BrdU-positive NP cells from the total NP cells on individual section of different treatment groups were counted using the Axiovision software.Bromo-deoxy-Uridine (BrdU) assays were carried out using Amersham Cell Proliferation Kit  as per manufacturer's instructions. BrdU (1∶1000) was added to the culture medium 24 hours before the end of the culture period. The cultured IVDs were washed three times in 1x PBS and snap frozen in OCT molds and cryosections were collected at 6 µm thickness, fixed in 2% PFA and washed thrice in 1x PBS. For the BrdU staining, the sections were pretreated with 2% HGli1- Mm00494654_m1; Ptch1- Mm00436026_m1; Shh- Mm00436528_m1; Wnt4- Mm01194003_m1; Wnt5a- Mm00437347_m1; Wnt11- Mm00437328_m1; Fzd7- Mm00433409_s1; Fzd10- Mm00558396_s1; Wls-Mm00509695_m1; Lef1-Mm00550265_m1; Bra- Mm01318252_m1; Sox9- Mm00448840_m1; Krt19- Mm00492980_m1; Col2a1- Mm01148576_m1; Acan- Mm00545794_m1; Col1a1-Mm00801666_g1; Ccnd1-Mm00432359_m1). Two reference genes 18S and GAPDH were used for normalization .Changes in gene expression in the NP cells were determined by qPCR analysis as per the MIQE guidelines The means and s.d. of the values obtained from individual experiments were calculated and represented as charts. Each experiment was repeated three times with two to three biological replicates in each group. Significance was calculated using unpaired T-test or one-way ANOVA.Figure S1Shows expression of CHSO4 protein expression (red) in P4t0 lumbar vertebrae growth plate (LV-GP). The expression of CHSO4 can be seen was much higher in the disc space. The nuclei are counter stained blue with DAPI. Scale bar  =  50 µm.(TIF)Click here for additional data file.


Cultural evolutionists have long been interested in the problem of why fertility declines as populations develop. By outlining plausible mechanistic links between individual decision-making, information flow in populations and competition between groups, models of cultural evolution offer a novel and powerful approach for integrating multiple levels of explanation of fertility transitions. However, only a modest number of models have been published. Their assumptions often differ from those in other evolutionary approaches to social behaviour, but their empirical predictions are often similar. Here I offer the first overview of cultural evolutionary research on demographic transition, critically compare it with approaches taken by other evolutionary researchers, identify gaps and overlaps, and highlight parallel debates in demography. I suggest that researchers divide their labour between three distinct phases of fertility decline—the origin, spread and maintenance of low fertility—each of which may be driven by different causal processes, at different scales, requiring different theoretical and empirical tools. A comparative, multi-level and mechanistic framework is essential for elucidating both the evolved aspects of our psychology that govern reproductive decision-making, and the social, ecological and cultural contingencies that precipitate and sustain fertility decline. Over the past 200 years, people from different religious, linguistic, ethnic and cultural groups, living in economies with different histories and value-systems, are increasingly limiting their families to around two or fewer children. How has this social norm evolved? Can it last? And can we connect theories about how people make personal reproductive decisions with the evolution of a broader culture that increasingly values and rewards smaller families?For decades, researchers of demographic transition have argued that economic, cultural, ideational and sociological factors are too deeply intertwined in this process to be completely isolated from each other –5. AsserEvolutionary theorists have produced many abstract treatments of the dynamics of fertility decline at different levels of analysis. Evolutionary anthropologists have been testing aspects of these models for at least 30 years ,7–10. ThIn principle, causal models combining economic and cultural factors are a shared objective. Empirically, this is a daunting task. In practice, little work has been done to actually synthesize different conceptual approaches ,15,16, wCultural evolutionists have long been interested in the demographic transition. Founding texts in the field ,21 emphaMoreover, some conceptual overlaps between CE and HBE can make them difficult to distinguish as alternative explanatory frameworks. Perhaps as a result, CE theory has not made inroads into mainstream demographic thinking, despite a growing representation of evolutionary research in that literature . This isEvolutionary research on fertility decline needs to address the multi-level nature of human social interaction, which generates opportunities for evolutionary dynamics that both involve reciprocal causation and are not easily reduced to individual characteristics ,23,38. AWe need clearly testable predictions from CE models and clarification of conceptual overlaps that are hampering theoretical integration. HBE's commitment to the behavioural gambit —a black-a priori privileging of one over the other. I begin by reviewing some basic conceptual differences between different evolutionary approaches, noting similarities and departures from standard demographic thinking, before briefly outlining the CE work published to date. This is followed by conceptual overlaps and a critical comparison of how data on demographic transitions are interpreted by different sub-fields. Finally, I offer some new directions that can generate novel hypotheses about the dynamics of fertility decline from an evolutionary perspective.This review argues for a deeper integration of CE theory into (evolutionary) demography, as a means to develop multi-level models of fertility decline that emphasize the coevolution of economic and cultural change and not the 2.There are a number of different ways to think about fertility decline from an evolutionary perspective . Each raBroadly speaking, there are three main approaches. First, we could decide that fertility decline is a mismatch (a case of adaptive lag) . Our psy(a)HBE is generally concerned with uncovering and understanding the costs and benefits of low fertility for individuals or lineages by examining constraints on fitness maximization, in particular how fundamental trade-offs, parental investment strategies and reproductive competition drive fertility outcomes. Personal decision-making on this approach is analogous to economic decision-making and research on fertility decline often draws on economic models of the family  and exteIn all species, the energetics of reproduction is tied to resource accumulation, but humans evolved in a skill-intensive foraging niche, which requires investment in ‘embodied’ capital  to obtain resources, as well as extra-somatic and other forms of wealth . A leadiEarly models of fertility decline ,43 showeA number of researchers have proposed that long-term fitness, or the success of lineages, is theoretically increased by decreasing fertility in the short-term culture as ‘social information transmitted via teaching, learning and imitation’ [CE work relevant to understanding demographic transition has broadly focused on three different areas: (i) how individual learning biases that evolved to optimize social information transmission generate and maintain different frequencies of cultural traits in populations; (ii) how variation and interaction between groups at different cultural equilibria lead to between-group competition, selection and transmission; and (iii) how different channels (modes) of social transmission affect the dynamics of information flow in populations. CE theory takes inspiration from both population genetic models and social psychology, and offers a multi-level approach to social behaviour, emphasizing that individual strategies for adaptation within groups and competition between groups are often co-occurring . CEs comitation’ ,21, simiitation’ . Unlike Social learning mechanisms evolved to enhance genetic fitness ,21,77. CConformist bias is argued to be especially important in maintaining between-group variation, by increasing differences between groups while maintaining similarities within them . This vaCultural traits can be acquired from many other people and modified by individuals within their lifetime. They can also make their bearers more visible as social models. Moreover, since information is transmitted horizontally (peer-to-peer) and obliquely (from non-parents of an older generation) as well as vertically (from parents-to-children) , there i3.While social learning biases are somewhat integrated into evolutionary discussions of fertility decline in HBE, the almost exclusive focus on individual-level mechanisms has led to a neglect of the second two components of CE research outlined above: group-level dynamics and modes of transmission. CE models of fertility decline have focused more on trade-offs between cultural and biological success ,55 than (a)There is some debate as to whether CE can successfully explain the origins, as opposed to the spread, and perhaps maintenance of low fertility , but in (b)The most cited CE arguments on fertility decline emphasize prestige-bias in the spread of low-fertility behaviour . PrestigChanging structures of social interaction may also change how transmission biases scale up to produce low fertility patterns at the- population level. Newson and colleagues ,57 argueSuch group-level characteristics can feed back to influence individual behaviour. Cultural niche construction models are one way to address these kinds of interactions . These eRapid ecological change can exacerbate these differences. In slowly changing contexts, and especially in relatively ‘closed’ social groups, reproductive norms may be less variable and information from different people fairly consistent . Old order Anabaptists and other culturally isolated groups, who often have high fertility even when living within low fertility societies such as the USA, are prime examples . When chet al. [In the first of a series of models, Ihara & Feldman  examinedet al.  similarlet al. .et al. [Borenstein et al.  extendedet al. ,60,82. Aet al. .et al. [A few models have begun to tackle how age-specific learning patterns influence both cultural transmission dynamics and the demographic (age) structure of a population ,61,83. T(c)et al. [Other models have examined how low fertility could be maintained over the long-run An influential explanation of demographic transition is that the real or perceived costs of children increase as living standards go up, with the opportunity costs to reproduction becoming disproportionately large for the wealthy and highly educated members of a population ,10,84. I(b)Education is usually conceptualized as an economic indicator or as a proxy for embodied capital ,12,73. B(c)Prestige- or success-biases are entirely compatible with an approach to fertility decline that emphasizes inter-individual competition for parental investment payoffs. If individuals compete to obtain wealth or status, and these have fitness-relevant outcomes (which they have had for most of human history and in most pre-transition societies), then the behaviour of wealthy, high-quality parents is a useful cue to the benefits of reproductive control. It is therefore difficult to say whether the imitation of successful individuals is an indirect bias, in the sense that individuals copy other behaviours and end up with low fertility as a by-product, or a direct bias, where individuals adopt low fertility strategically. This makes it hard to distinguish them as alternative causal pathways.(d)CE processes are multi-level phenomena. Yet, it is hard to know whether differences between particular levels of aggregation represent ‘cultural’ or ‘ecological’ determinants, and they can often be interpreted both ways. Are group-level effects on fertility indicative of cultural processes or aggregations of individual decisions in relation to some unobserved variable? Identification, selection and omitted variable problems are well known in economics and sociology  and are Of course, the appropriate definition of a ‘group’ is not obvious, and no one social grouping  will be appropriate for all domains of social behaviour. Research focusing on differences and similarities between predictors at different levels of social hierarchies ,87 will (e)Theoretical models of fertility decline usually assume undifferentiated individuals with equal opportunities to access information, perfect sampling of available cultural learning models and freedom to enact their reproductive preferences. CE models do not often include individual resource constraints on reproductive options, a hallmark of HBE, even though diffusion dynamics are sensitive to individual wealth or income heterogeneity , populatCE models should also be tempered by the understanding that kin regularly have reproductive conflicts of interest and may wield significant power over reproductive-aged women. Dichotomies between kin and non-kin, implying broadly pro-natal outcomes of kin interactions, neglect the empirical evidence that kin effects on fertility are highly varied . On the CE models typically require that high-status individuals have lower fertility in order to obtain their results ,58–60,68(f)disinvestment in reproduction in favour of other aims; self-fulfillment via education, work or some other measure of cultural success. For HBE, decisions to postpone, space or stop reproduction are sensitively tuned to environmental and social cues indicating optimal behaviour in a particular context, including cues of fecundability, mortality and resource availability throughout the life-course [coitus interruptus have been and remain critical in many fertility declines [Contraceptive use could represent strategic parental investment in the number or quality of children. But it could also represent a e-course ,92 and te-course ,94. Conte-course ,95. The e-course . More bre-course , and Distinguishing between the origins, spread and maintenance of low fertility might be useful in defining the contours of different theoretical and empirical research programmes, highlighting the kinds of assumptions that models should make, and the different scales and processes that might matter. Doing so clarifies why most empirical work in HBE, focused primarily on the origins of low fertility, has been carried out in populations in the early stages of demographic transition ,13,93,98(b)The potential role of multi-level or cultural group selection  has not per capita consumption through human capital accumulation. With increasing dependence on technology and innovation, countries sharing international research and development (R&D) and bilateral foreign direct investment are more economically productive than those that do not cooperate in this way [Historical fertility declines started in the wealthier sub-strata of technologically advanced populations during the transition from Malthusian stagnation to rapid economic growth, and are associated with profound social and economic changes brought about by the Industrial Revolution. Feedback between population density and technological innovation is thought to have then created a niche for education, to sustain subsequent economic growth . An impothis way ,104. Tecthis way ,105, andthis way ,107. Ferthis way ,108.Such population-level competition and cooperation may create selective pressure for market-oriented skills and investment in embodied capital. They can also benefit all individuals in a group, not just those who reduce fertility, for example by reducing mortality and increasing lifespans (24 years have been gained over the past century ) as popu(c)Some studies have started to address how demographic and social structure complicates inferences about the mechanisms of fertility decline. HBEs have recently begun to focus on how competition within, rather than between social strata affects reproduction ,44,84, wCE models have focused on how population-structure, age-structure and network composition affect the dynamics of cultural transmission ,60,61. F(d)How do the different evolutionary schools of thought interpret the recent ‘bounce-back’ in fertility rates in the most developed countries of the world ? One dem6.Fertility decline is a difficult moving target, because no two declines are the same and because behaviour change involves multi-level processes and often, feedback. A comprehensive evolutionary approach to fertility decline must incorporate insights from CE theory if it is to fully understand this transition process. Doing so requires a greater degree of multi-level thinking and a deeper understanding of the ‘proximate’ mechanisms that are often seen as a secondary concern. Mechanistic causal models are needed because theories that are functionally equivalent are not necessarily causally equivalent . A focus