Clavus that belong to the family Drilliidae. Venom gland transcriptomes of two specimens of Clavus canalicularis and two specimens of Clavus davidgilmouri were analyzed, leading to the identification of a total of 1,176 putative venom peptide toxins (drillipeptides). Based on the combined evidence of secretion signal sequence identity, entire precursor similarity search (BLAST), and the orthology inference, putative Clavus toxins were assigned to 158 different gene families. The majority of identified transcripts comprise signal, pro-, mature peptide, and post-regions, with a typically short (<50 amino acids) and cysteine-rich mature peptide region. Thus, drillipeptides are structurally similar to conotoxins. However, convincing homology with known groups of Conus toxins was only detected for very few toxin families. Among these are Clavus counterparts of Conus venom insulins (drillinsulins), porins (drilliporins), and highly diversified lectins (drillilectins). The short size of most drillipeptides and structural similarity to conotoxins were unexpected, given that most related conoidean gastropod families (Terebridae and Turridae) possess longer mature peptide regions. Our findings indicate that, similar to conotoxins, drillipeptides may represent a valuable resource for future pharmacological exploration.Predatory gastropods of the superfamily Conoidea number over 12,000 living species. The evolutionary success of this lineage can be explained by the ability of conoideans to produce complex venoms for hunting, defense, and competitive interactions. Whereas venoms of cone snails (family Conidae) have become increasingly well studied, the venoms of most other conoidean lineages remain largely uncharacterized. In the present study, we present the venom gland transcriptomes of two species of the genus  Conus Linnaeus, 1758 , this family is among the highly diversified lineages of Conoidea as well . Transcriptomic profiling reveals a vast diversity of the putative Clavus venom components that we term “drillipeptides.” Although biological activity of these components is still to be established, their number in the transcriptomic data set notably exceeds the toxin diversity observed in individual specimens of Conus analyzed employing the similar methodology. The complement of putative toxins observed in individual specimens of Clavus, while closely parallel to Conus venoms in many respects, is clearly unique among Conoidea, most notably in the diversity of toxins found in each species analyzed.In this study, we present a comprehensive survey of the venom gland transcriptomes of two species of the genus adiation . Neverth as well   and Clavus davidgilmouri  were recovered from the same habitat—coral rubble at depths of ∼15–25 m by local fishermen and delivered to the laboratory alive on the same day, or the day after they were collected. After breaking the shell, the mantle was removed, and the body hemocoel dissected to extract the venom gland. Dissected venom glands were immediately suspended in RNAlater (Thermo Fisher Scientific). A tissue clip was taken from each dissected specimen and preserved in RNAlater for subsequent DNA extraction to confirm initial species identification. Samples in RNAlater were kept at room temperature for 24 h, and then frozen and kept at −20 °C until RNA extraction.All studied specimens were collected in the proximity of Olango Island  on August 5, 2015. Specimens of gilmouri   or BioHYTaq DNA polymerase . The PCR profile for the COI started with 5 min at 95 °C followed by 40 cycles with the denaturation at 95 °C (35 s), annealing at 50 °C (35 s), and elongation at 72 °C (1 min), with final elongation phase at 72 °C (7 min). The amplified DNA fragments were sequenced in both directions to confirm accuracy of each sequence. The sequencing was performed in the SIEE RAS molecular facility on an ABI 3500 Genetic analyzer. Alternatively, the purified PCR products encoding Clavus COI sequences were annealed to a plasmid vector and transformed into competent cells as previously described  was sequenced for the four dissected specimens of  HCO2198 . Polymerescribed . The nucClavus, including 13 specimens sequenced in the present study, and 4 records recovered from the GenBank sequencing database; all specimens originated from the Central Philippines. Sequences were aligned using ClustalW, as implemented in BioEdit 7.0.9.0 , using RAxML-HPC2 on XSEDE , with on-column DNase treatment, according to the manufacturer’s instructions. Venom glands were homogenized in Direct-zol TRI reagent using the TissueRuptor II (Qiagen) with a disposable probes to avoid sample cross-contamination. cDNA library preparation and sequencing were performed by the University of Utah High-Throughput Genomics Core Facility. Briefly, total RNA quality and quantity were first determined on an Agilent 2200 TapeStation (Agilent Technologies). A dual-indexed library was constructed with the Illumina TruSeq Stranded mRNA Sample Prep Kit with oligo (dT) selection and an average insert size of ∼220 bp. The library was validated on an Agilent 2200 TapeStation and using a quantitative PCR assay (KAPA Biosystems Library Quantification Kit for Illumina). The 125-cycle paired-end sequencing was performed on an Illumina HiSeq2000 instrument . The statistics of the obtained raw reads data are summarized in http://ccb.jhu.edu/software/fqtrim/, last accessed April 26, 2020) and prinseq-lite (version 0.20.4)   to remov 0.20.4)  with kmee-value < 10e-10). The conotoxin sequences published in 2015–2016 that were not yet uploaded into UniProt at the time of the analysis  but no transmembrane topology  does not reveal the full diversity of transcripts, particularly for those deriving from polymorphic loci  and 2) sets of orthogroups recovered for the entire precursor by OrthoFinder 2.1.2. . The 105 BLAST hits were classified in 23 clusters, and 206 orthogroups with two or more sequences in each were returned by OrthoFinder. Subsequently, an in-house Python script that implements a pair-counting approach . All-by-all NCBI-BlastN searches identified 177 and 165 rbh pairs between the two specimens of each Clavus species  and 209 rbh pairs when comparing the two species to one another  pairs among the inter/intra-specific drillipeptide nucleotide sequences. ClustalW alignment was then used to globally align the rbh pairs . The ali another .Cv. canalicularis and Cv. davidgilmouri transcriptomes, we used OrthoFinder 2.1.2  and insulins, the low TPM transcripts were also recovered from the assembly and added to the data set. To calculate correlation between the Clavus species in transcript diversity and expression levels, Pearson’s R was calculated using the numpy method (numpy.corrcoef).To detect orthogroups in the er 2.1.2  with theClavus data set, the signal peptide was characterized using SignalP 4.0 server , the Cv. canalicularis clade showed clear genetic structure, with the highest within-clade genetic distance of 3.4%. The two Cv. canalicularis specimens studied for the venom gland transcriptome, although collected from same locality, showed a genetic distance of 2.7%, which is notably higher than between the two studied specimens of Cv. davidgilmouri. Nevertheless, the inferred pairwise comparisons within each clade fall within a range of intraspecific genetic distances demonstrated for the superfamily Conoidea .A total of 1,176 unique putative toxin-like peptides were identified in the combined Typically, the identified transcripts demonstrated a canonical organization with a 16–34 AA signal sequence, followed by a pro-region of varying length , a mature peptide region and, in ∼30% of identified transcripts, a short, one or few AA post region.Clavus toxins ranged from 120 (sequence identity threshold 51%) to 244 (sequence identity threshold 75%). In all clustering schemes, the number of Cys-patterns per cluster ranged from one to nine, and the proportion of clusters with only one Cys-pattern ranged from 0.55 to 0.82 when the sequence identity threshold was set at 51% and at 75%, respectively . Conversely, only one drillipeptide gene superfamily produced BLAST hits to each of conotoxin superfamilies A, F, I3, M, O1, O2, U, Conus insulins, conodipine, and conkunitzin.Among the putative toxins with BLAST hits, the “MWIRK”-superfamily-like transcripts showed highest diversity and total expression level. Interestingly, each of the three highly expressed and diverse groups of peptides in l-RBLs in the Clavus venom gland transcriptomes suggest that they play an important role in prey capture or defense (see below). Nevertheless, additional data on the spectrum of biological interaction of Clavus spp. are needed to pose these data in ecological context. The fact that in some cases, transcripts with similar BLAST hits represent different gene superfamilies may be explained by either: 1) the signal sequence being less highly conserved compared with typical conoidean gene superfamilies or 2) putative toxins with similar mature peptides having been recruited from different genes, or finally 3) the reference sequences of conoporins and l-RBLs actually representing heterogeneous groups themselves.The high diversity and expression levels of the “MWIRK”-superfamily-like transcripts, drilliporins, and Cv. davidgilmouri (4.47 and 4. 32) compared with Cv. canalicularis (4.24 and 4.26)   online. lenavati , reporteCv. canalicularis and Cv. davidgilmouri, respectively. Comparative analysis of the putative toxin sequences between the two Cv. canalicularis specimens revealed that 89.3% of rbh pairs were >95% identical, and of these, 65.0% of sequences were >99% identical , and between the two studied specimens of each species  . The latter result cannot be attributed to artifacts of assembly . It can be, however, explained by the notably higher genetic divergence between the two Cv. canalicularis specimens, inferred from the COI sequences, suggesting that the profile of the venom gene family expression is highly volatile and may vary considerably among conspecific individuals.The most diverse gene superfamilies  were represented in each nscripts D. Overalctively)  online. compared F and G, Clavus suggest that the repertoire of putative toxin gene families expressed in venom gland remains largely conserved within species and shows divergence between the closely related, (but not sister) species. This result closely parallels intra- and inter-specific comparisons reported in two closely related sister species of Conus; Conus praecellens and Conus andremenezi  and the venom composition at the sequence level appear to be more conserved and are expected to contribute to venom variation at a higher taxonomic level.Nevertheless, the results of the RBH analysis in remenezi  that shopecimens B. AlthouClavus in comparison with published data on some Conus species. The pattern of mature peptide region lengths distribution is apparently very close between the two species of Clavus. The median length of the mature peptide sequence is at 41 AA for Cv. canalicularis and at 44 AA for Cv. davidgilmouri, and the lower quartile is at 32 AA in both species. In total, 108 and 58 unique transcripts in the venom gland of Cv. canalicularis and Cv. davidgilmouri, respectively, show a mature peptide length of <25 AA residues. The putative toxins of 20 gene families with mature toxin regions containing on average 13–30 AA residues closely match in this respect typical conotoxins , and Crassispira cerithina (Pseudomelatomidae)  suggest that drillipeptides, like conotoxins, may be promising candidates for future pharmacological investigations. Venom peptides with high expression levels can readily be explored by mass spectrometric analysis of Clavus venom, followed by chemical synthesis and activity testing. One such bioactive peptide, cdg14a, was identified by mass spectrometry and has been purified from the venom of Cv. davidgilmouri. This peptide elicits scratching and hyperactivity followed by a paw-thumping phenotype in mice . Cv. canalicularis and Cv. davidgilmouri respectively that are both highly expressed  and comprise transcripts with mature peptide regions of ≤50 AA (area shown in gray). They could be first analyzed in a search of the most relevant target for subsequent structural studies and bioactivity assays.The mature toxin regions are on average longer in other lineages of the “Turrid” major clade of conoideans : Gemmulatomidae) . The verrebridae , whereas in mice . The preClavus venom share the same canonical structure of signal sequence, propeptide region followed by a single mature toxin with most conotoxin precursors, drillipeptide sequences are quite divergent. We therefore avoid inference of sequence homology between drillipeptides and conotoxins. Nevertheless, as with conotoxins, drillipeptides tend to have an even number of cysteines  in the mature peptide region. Two of the most common canonical cysteine frameworks for conotoxins, VI/VII (C-C-CC-C-C) and I (CC-C-C), are well represented in the mature peptide regions of drillipeptides  are peptide hormones that are found throughout the animal kingdom where they play a role in regulating carbohydrate and fat metabolism and as neuromodulators of energy homeostasis and cognition . Insulinrey taxa . Cone snrey taxa .Clavus that share several characteristics with Conus venom insulins, including significant sequence diversification and high expression levels of some sequences. Clavus venom insulins are derived from a single gene superfamily as evidenced by their conserved signal sequence  suggests a role of Clavus insulins in prey capture. Interestingly, insulins sequenced from Clavus share structural similarity with worm and mollusk insulins suggesting that Clavus specializes on invertebrate prey as previously reported for snail and worm-hunting Conus  and was also detected in Cv. canalicularis n2 (drillinsulin CvCn4-tpm 435). Conversely, the cluster 3 insulins  have higher expression in Cv. canalicularis, and the cluster 2 insulin is only found in Cv. canalicularis . Two insulins, drillinsulin CvCn2 (tpm 555) and drillinsulin CvCn2ii (tpm 383) from CvCn1 and CvCn2, respectively, have one AA substitution and likely represent allelic variation of the same locus. In the reconstructed phylogeny of conoidean venom insulin precursors (Clavus insulins cluster with the insulins of the Turridae species (PP 0.93), and the Conus venom insulins form a separate, well supported cluster (PP 1), which reflects phylogenetic relationships among Drilliidae, Turridae, and Conidae , and the other one, on the contrary, only currently known in Conoidea. The presence of products of both these gene clusters in Clavus may suggest that their molecular targets  and/or action mechanisms differ from one another. This hypothesis is consistent, for example, with the distinction between the defensive and predatory porins. Further studies on the diversity of porins in Neogastropoda, and on the spatial differentiation of gene expression in the Clavus venom gland, and the spectrum of molecular activities are necessary to better understand diversity and evolution of venom porins in the Neogastropoda.Porins are a class of peptides that bind to and form pores in cellular membranes . Becauseticulata . In Cumid porins  revealedd porins . The MALConoidea , mirrorirey taxa . The phyClavus spp. Six predicted Clavus venom transcripts with expression levels above 100 tpm showed detectable similarity to the l-RBLs of the Chum salmon Oncorhynchus keta and the Amur catfish Silurus asotus. When the transcripts with lower expression levels were considered, a total of 28 transcripts encoding RBLs were identified, their tpm expression levels ranging from 5 to 15,382. The majority (24 transcripts) of the predicted RBL precursors comprised two carbohydrate recognition domains, (CRDs)  has six cysteines and a short sequence lacking the C1–C3 disulfide bond. In addition, one predicted RBL transcript , despite the overall sequence identity of the Clavus RBLs to previously identified RBLs being rather low (on average <=30%). The high diversity and in some cases very high expression levels of lectins suggest that they may play an important role in envenomation. This hypothesis for Conoidea in general is supported by the evidence of high RBL differential expression in the venom gland of Profundiconus vaubani, the most early divergent cone snail genus, and a distant conoidean relative of Clavus . The radula of Clavus, with its flat marginals and comb-like lateral teeth, cannot be used to inject venom into the prey’s body, as in Conus. However, based on the anatomical studies, drilliids use marginal teeth in a similar manner—holding a tooth at the tip of the proboscis presumably to make lacerations on the prey’s integument (Clavus in the lack of efficient mechanical venom delivery achieved in Conus by means of hypodermic radular teeth.Both lectins and porins bind to the components of cellular membranes, and both are involved in immunity, due to their demonstrated ability to disrupt cellular membranes . The higtegument . ThroughClavus demonstrate unique biochemical features: displaying a diversity of gene families unparalleled among conoideans, most of which do not show similarity to known toxin families. Despite the phylogenetic position of Clavus in the “turrid” major clade of the Conoidea (that diverged from the “conid” major clade, including the genus Conus around 130 Ma—Clavus venom peptides show detectable similarity to conotoxins. Furthermore, our data suggest that the venoms of Clavus are dominated by rather short peptides, and in this regard resemble more the venom of Conus than venoms of more closely related Turridae and Terebridae. The demonstrated high diversity of short cysteine-rich peptides encoded in the venom gland transcriptomes of Clavus suggests they may also represent a valuable resource for drug discovery. Future studies on the characterization of the physiological effects and the identification of molecular targets of these peptides will elucidate the pharmacological potential of drillipeptides. Alongside the short cysteine-rich peptides, the venom of Clavus is remarkable for the high diversity and expression levels of insulins, RBLs, and porins. Insulins are likely to disrupt energy metabolism of prey, whereas porins and lectins may exert cytolytic effects that enhance the spread of venom in the prey’s body. This is the first convincing evidence of complex biochemical adaptation leveraging the lack of efficient venom delivery apparatus in the “turrid” lineage of Conoidea. Furthermore, it is certain that venom insulins, lectins, and two gene families of porins were already present in the common ancestor of Conus and Clavus—that is, in one of the earliest conoideans. Conversely, the available data are not sufficient to infer whether some known conotoxin gene superfamilies have homologs in the transcriptome of Clavus. Thus, we reveal a novel, and in many aspects, a unique fragment of knowledge of conoidean venom evolution, which will likely inspire future studies on this remarkable, yet still poorly studied taxon.The venom gland transcriptomes of previously unstudied predatory gastropods of the genus evaa083_Supplementary_DataClick here for additional data file.


The measurements were carried out with 2 × 2” NaI(Tl) scintillation detector. The measured specific activity on natural radioactive isotopes were within the following ranges: 5.8–312 [Bq kg−1], 5.5–189 [Bq kg−1] and 109–1590 [Bq kg−1] for 226Ra, 232Th and 40K, respectively. Obtained concentrations of radioactive isotopes allowed to perform the analysis of the exposure of the humans from the ionizing radiation emitted by the granites. The determination of the exposure consisted in the calculation of absorbed gamma dose rate (D) [nGy h−1] for each sample, which fell in the range between 20 and 511 [nGy h−1], annual effective dose rate (AED) [mSv year−1] ranging between 0.10 and 2.50 [mSv year−1], radium equivalent activity (Raeq) [Bq kg−1] with values between 22 and 570 [Bq kg−1], external and internal hazard indices (Hex) and (Hin) falling in the ranges 0.06−1.53 and 0.08–2.41 respectively, as well as gamma (Iγ) and alpha (Iα), representative level indices with values 0.08–2.0 and 0.029–1.56, respectively. Moreover, obtained results were compared with the international standard values given by the European Commission (EC), the United Nations Scientific Committee on the Effects of Atomic Radiation given in UNSCEAR Reports, and the results of research from other laboratories. The measurements of the specific activity of natural radioactive isotopes of radium ( The granites exhibit very good technical and chemical properties and are primarily valued for their esthetical quality. They are exceptionally resistant to frost, weathering and abrasion, while maintaining perfect grinding and polishing properties. Thus, due to these properties, the granite rocks are widely used in bridge, road, water and lapidary constructions, mainly as construction elements, monuments, road and pavement components . Granite is magmatic igneous rock formed during the crystallization process of liquid magma inside the earth under high temperature and pressure. Granite is one of the most widely occurring rocks in the earth crust. It mainly consists of quartz with intrusions of potassium feldspar, as well as acidic plagioclase and mica. Quartz has white or grey grains, sometimes red or bluish. Moreover, the quartz grains are the final product of magma crystallization resulting in its irregular shapes. The grains of quartz usually fill the empty spaces between other minerals. The potassium feldspar appears in form of white-greyish, pink or red grains, which influences the color of granite. Both minerals (quartz and feldspar) can be distinguished by the hardness and differences in cleavage. Quartz is harder and non-cleavable, and potassium feldspars are characterized by distinct bidirectional cleavability, thus the grains of feldspar reflect the light by plain surfaces. Plagioclase is usually white or grey, sometimes red (due to the presence of iron oxide). The most common mica in granites is biotite, which is easily recognizable due to its appearance in form of dark, almost black lamellae having perfect cleavability ,3,4,5. 87Rb, 40K, uranium-radium series and thorium series elements, as well as 147Sm, 138La, 187Re are present in the lithosphere D—absorbed gamma dose rate .S−6, see Equation (2). According to the UNSCEAR 2000 Report, the annual effective dose should read: AED ≤ 1 mSv·a−1 , a—annum (year), D—absorbed gamma dose rate [nGy h−1].AED—annual effective dose rate . Therefore, when Iα exceeds the value of 1, then it would be possible that the concentration of radon released from this material might exceed the recommended value  for analyzed samples of granite, The next chart shows the values of radium equivalent activity Ra−1 to 570 Bq·kg−1. The highest Raeq was achieved for Izerski granite sample G1, and slightly lower for granite from Sweden G10. Only in these two samples the value of the radium equivalent activity exceeds the limit given in UNSCEAR 1982 Report ) cannot be rejected, whereas for radium—only when the largest value from the measurement set is rejected, see In order to estimate the relationship between the determined activity concentrations of radioactive isotopes—radium, thorium and potassium—a statistical analysis of the samples had to be carried out. It should be noted that a simple box-whiskers chart shows that for thorium and radium the largest values should be considered as the most outlying, while for potassium—the smallest and largest values, see Therefore, having the results of the Shapiro-Wilk tests, 2 ≈ 0.60. A much better model is the sigmoidal model, in which the initial linear growth is followed by saturation for higher values of radium specific activity concentration. Such approach seems to be justified at least for materials of natural origin where—in wide extent—less radioactive samples contain low concentrations of both potassium and radium, and more radioactive samples have higher specific activity concentrations. Thus, the initial linear growth is anticipated. However, potassium and radium are not connected neither by common series nor by their progenies, hence for certain specific activity concentration values distinct relationship between concentration of 40K and 226Ra cannot be expected. In other words, further increase in radium concentration should not affect potassium concentration, which is visible on the chart. The determination factor for the sigmoid model (hyperbolic tangent type) exceeded 0.9, thus the model is statistically valid. 2 = 0.75), but the saturation for high concentration of thorium is clearly seen. The argumentation is similar to the radium–potassium relationship; therefore, the sigmoidal model was fitted, and the determination factor was 0.9. However, it is worth noting that essentially the sharp drop for low concentration is due only to one point; thus, to obtain higher credibility on the relationship for low specific activity concentration of thorium and potassium, the higher number of samples should be measured. In such case the statistical reasoning would be more significant. The relationship between the specific activity concentration of thorium and specific activity concentration of potassium is similar, 226Ra–232Th correlation higher than 0.8 cannot be rejected. Thorium and radium show definitely stronger dependency, not only having higher determination factor for linear model, but also higher correlation coefficient . In some other articles the authors performed much shorter experiments on a few samples from one supplier, 1 h in  or even The research of granites both Polish and foreign are important because they allow to determine the specific activity concentration of the radioactive isotopes of potassium, radium and thorium, which are used in housing, road and monumental (lapidary) construction. Granites have very good technical and chemical properties and are valued primarily for their aesthetic qualities. They are one of the raw materials whose intensive extraction does not limit the availability of its resources. Due to their properties, these rocks are widely used in construction industry and stonemasonry. Demand for aggregates crushing from rocks, such as granites, basalts and carbonate rocks, will certainly not decrease. Also, the increase in concrete production increases the consumption of cement and aggregate. Therefore, obtaining aggregates from existing mines as well as newly opened ones is becoming an urgent challenge for concrete producers. It should be taken into account that the natural radioactivity of rocks and minerals is a phenomenon occurring naturally and conventionally in the environment. Granite aggregates due to their properties are very widely used in bridge, road, water and mineral construction as structural elements. Therefore, there is a need for constant monitoring of their physical and chemical properties, one of which is radioactivity. 226Ra, 232Th and 40K for some granites are even several times higher than the average values recommended by the European Commission . Only granite G9, originating from India is characterized by low average concentration of these isotopes, and therefore is the safest for construction industry, taking into account potential radiological exposure.The measurement results show that average specific activity concentration of eq, external hazard index Hex and gamma index Iγ, as well as alpha index Iα are—for the analyzed granites-higher than levels considered safe and given in UNSCEAR Reports [The radiological parameters calculated during the research, such as absorbed gamma dose rate D, annual effective dose rate AED, radium equivalent activity Re Reports ,23,24 an Reports ,28,29,30Admissible activity levels of radium equivalent activity and external hazard index were exceeded for two granites: G1 (Izerski) and G10 (Swedish). The internal hazard index was exceeded for most of the analyzed granite samples. Gamma index was exceeded for seven of ten samples: except G5 (Poland—Strzelin), G7 (Finland) and G9 (India) One sample of granite (G1—Izerski) exceeded alpha index. The use of granite  in housing is associated with an increased risk of exposure to ionizing radiation. The research shows that almost all granite samples can be associated with elevated radiological risk. The most significant is Polish Izerski granite, exceeding all radiological parameters, the following having the potential negative impact on human health is Swedish granite (sample G10). The remaining samples also exceed the levels of some radiological parameters according to their specific activity concentration of natural isotopes. According to UNSCEAR and European Commission recommendations, the safest granite came from India—it obtained the increased result only for internal hazard index. However, although radioactivity exceeds the standard levels in the above-mentioned cases, a suggestion that granites could be harmful or dangerous for users, goes far beyond the scope of this study. Some authors even point to the need of reconsider the limits for the radiological indicators recommended by UNSCEAR or European Comission . NonetheThe statistical analysis, in turn, suggests some kind of correlation between the specific activity concentration of radium and thorium in natural materials such as granites. The more thorough statistical analysis would be performed, if getting low activity samples. In this context, interesting is non-normality distribution of the radioactivity, as well. (1)Granites are natural rocks used in stonemasonry, road construction, equipment in dwellings, cemetery monuments etc.(2)Granites extracted from various places worldwide may possess increased radioactivity and thus higher radiation protection indicators.To conclude briefly:


Synchronized food retail prices raise seasonal peaks in the cost of nutritious diets, revealing new ways to improve food systems. Seasonal fluctuations in food prices reflect interactions between climate and society, measuring the degree to which predictable patterns of crop growth and harvest are offset by storage and trade. Previous research on seasonality in food systems has focused on specific commodities. This study accounts for substitution between items to meet nutritional needs, computing seasonal variation in local food environments using monthly retail prices for 191 items across Ethiopia, Malawi, and Tanzania from 2002 through 2016. We computed over 25,000 least-cost diets meeting nutrient requirements at each market every month and then measured the magnitude and timing of seasonality in diet costs. We found significant intensity in Malawi, Tanzania, and Ethiopia , driven primarily by synchronized price rises for nutrient-dense foods. Results provide a metric to map nutritional security, pointing to opportunities for more targeted investments to improve the year-round delivery of nutrients. High food prices limit consumption and harm well-being for low-income people  912.1 [$1.50 in 2011 USD (U.S. dollar) at purchasing power parity (PPP) prices] in Tanzania, MWK  129.6 ($1.21) in Malawi, and ETB (Ethiopian birr) 6.74 ($1.34) in Ethiopia. These costs were 2.41, 3.11, and 3.49 times the country’s average level of CoCA required for subsistence (tables S4 to S6).Seasonal fluctuation in the overall cost of all nutrients is large and statistically significant. As shown in Peak timings of the three indicators in Malawi and Tanzania were estimated to be about 3 months before the harvest season starting in May. In Ethiopia, although CoCA was estimated to peak in late August, which is about 2 months before the start of harvest season in November, CoNA and CoNA premium peaked earlier in late July and mid-April, respectively. The timing and magnitude of these peaks reflect the limited degree to which different foods can substitute for each other to deliver all required nutrients around the year. As shown in Seasonal intensity in CoNA also presents great regional variations within countries. Regional results are shown in This paper introduced a combination of techniques to characterize spatiotemporal variation in food prices across three countries in East Africa, measuring the ability of local farmers and traders to achieve year-round delivery of all essential nutrients at low cost despite climatic fluctuations. We used government file data on a total of 191 items at 170 locations in various years from 2002 through 2016, solved for the least-cost combination of foods needed to meet requirements for 21 essential nutrients and dietary energy at each of 25,522 market-months, and then applied harmonic regression to estimate seasonal intensity and peak timing of diet costs at each location. Three important findings were found:First, most individual foods have significant seasonality in retail prices, extending previous observations about major commodities to all food categories. Fruits and vegetables have the largest seasonal price variations, which averages over 20% for 7 of 21 items in Tanzania, 14 of 17 items in Malawi, and 8 of 24 items in Ethiopia. Items such as carrots, mangoes, papaya, oranges, avocado, tomatoes, green peppers, and onions are important not only for the essential nutrients they provide but also for other aspects of diet quality and local livelihoods. Foods that are more easily stored and transported, such as cereal grains and pulses, nuts, or seeds, have lower levels of seasonality than the highly perishable fruits and vegetables. We also find that seasonality in the prices of widely traded grains is lower on retail markets than previous studies had found in wholesale prices on commodity markets . Monthly retail food prices considered in this study cover 97 food items in 120 markets from 57 zones of 11 administrative regions between January 2002 and December 2016 of 15 years. Like the dataset in Tanzania, the surveys are conducted in towns and cities. To ensure the survey to be nationally representative, the CSA also assigns the number of markets in each region to be proportional to the region’s share of total urban population in Ethiopia. CSA enumerators collect three price quotations from traders, retailers, and consumers in the first 15 days of each month. They retain the median of those three prices and, before our receipt of the data, also trimmed outliers below the 1st and above the 99th percentile of each item.After assembling each country’s archival price data, we converted their units of measure to local currency per kilogram of edible matter (LCU/kg), and matched the item’s description to entries in local food composition tables  Σiaij × qi ≤ ULj.(ii) Σiaij × iq ≤ AMDRj,upper × E/je.(iii) Σiaij × qi ≥ AMDRj,lower × E/je.(iv) Σiaie × iq = E.(v) Σq1 ≥ 0, q2 ≥ 0, q3 ≥ 0,…, iq ≥ 0.(vi) ip), choosing quantities (iq) to meet or exceed the population’s estimated average requirement (EAR) for nutrient j given the quantity of nutrient j in each food ijn, within the further constraint of overall estimated energy needs (E), while remaining below upper levels (UL) for most micronutrients and the chronic disease risk reduction (CDRR) upper bound for sodium, and within a range for macronutrients determined by acceptable macronutrient distribution ranges (AMDRlower and AMDRupper) as percentages of daily energy needs (E). The reference number je is the energy density of macronutrients, which is 4 kcal per gram of protein and carbohydrate and 9 kcal per gram of lipid. In the analysis, we included 21 nutrients, including 3 macronutrients , 8 minerals , and 10 vitamins . Using this same framework, we also computed the CoCA for daily subsistence, using only starchy staples to meet the constraint of energy needs alone.The objective is lowest diet cost given the price of each food  and peak timing (P) of seasonality where ω is a constant equal to 1/12, indicating 12 months per annual cycle. T(t) is a cubic polynomial term of t, controlling the trend of time series. tY controls the fixed effect of crop years. In Tanzania and Malawi, the first month of a crop year is May, while it is October in Ethiopia  − 1}, where A is the amplitude of the seasonality. Therefore, the seasonality is comparable across different food items, price indicators, and countries, and over time. The estimates of amplitude (A) and peak timing (P) and their variances are calculated using the δ method and the equations below . Thus, the peak timing estimates can be formally compared. If the 95% CI does not contain the value of zero, then seasonality will be determined significant  by markets. If an estimation was based on observations from a single market, an ordinary least squares model was applied instead. All regression models were run in Stata/SE 15.1.


No cases under the severe category were identified. (4) Conclusion: No new risks were found in the population where this study was conducted.(1) Background: drugs provide a significant benefit for patients who require medical treatment; however, their use implies an intrinsic potential danger, with the possibility of causing unwanted effects. These effects are known as adverse drug reactions (ADRs). Post-marketing drug safety surveillance detects unknown risks that have not been identified in clinical trials, and it is necessary to monitor marketed medications under real-life practice. Due to the scarce information about fixed combination of ciprofloxacin 0.3%/dexamethasone 0.1% (SDO), we performed a drug safety surveillance study. (2) Methods: A prospective non-controlled drug safety surveillance study was conducted in Peruvian population. A total of 236 patients prescribed SDO were included derived from 12 sites. Patients’ standardized information was collected through two phone calls, including demographics, medical history, prescribing patterns of SDO, concomitant medication, and ADRs in detail. The ADRs were classified by causality and severity, followed by outcome measures to identify new risk. (3) Results: 236 patients prescribed with SDO participated in the study and 220 were included. A total of 82 ADRs/220 patients were reported after the use of SDO, presenting a ratio 0.37 ADR/patient. The most frequent ADR with SDO administration was eye irritation (30%). All ADRs were classified as non-serious, and 97.5% ( Prescription drugs provide a significant benefit by treating, preventing or diagnosing diseases; however, their use implies an intrinsic potential danger, possibly causing unwanted effects. These effects are known as adverse drug reactions (ADRs) ,2. The WThere are different combinations of antibiotic/steroid ophthalmic medications available for the treatment of ocular infections that have been used for several years . The con® (SDO)  in uncontrolled Peruvian population through drug safety surveillance.In this study we characterized the ADRs associated to the use of an ophthalmic fixed combination of ciprofloxacin 0.3%/dexamethasone 0.1% Sophixin DX OftenoA prospective non-controlled drug safety surveillance was conducted in Lima, Perú, from February 2019 to April 2020 by 12 collaborating sites. The study’s protocol and its corresponding informed consent form were reviewed and approved by an ethics committee .Patients were recruited from 27 February 2019 (first enrolled patient) to 29 April 2020 (last patient’s completion). Because this is a non-interventional study, patients who were prescribed SDO by an ophthalmologist (on his/her own initiative) were derived to a member of our team. Afterwards, they were informed about the enrollment process and invited to participate in the study. If the patient agreed to participate, the informed consent was signed.Over a period of 14 days, a member of our team conducted telephonic interviews with the patients on two different dates, days 7 and 14 after the start of the drug´s administration. On day 7, the personal data were collected  as well as the characteristics of the drug and its prescription , data from the patient’s medical history  and data from any ADR in case they appeared , existence of a similar preceding ADR with the same drug, dechallenge (if applicable), response to dose modification (if applicable), existence of other cause different to drug application that may explain the ADR (if applicable)). During the call that took place on day 14, follow up and general experience with the drug´s use was inquired.® 365 ProPlus., Washington, Redmond, USA) by Laboratorios Sophia’s Pharmacovigilance Unit and patients were classified according to their age as follows: children (0–12 years old (y/o)), adolescents, (>12–18 y/o), adults (>18–60 y/o), and geriatric (>60 y/o). If any of the patients reported any ADRs, these were classified and evaluated according to severity and causal relationship.The data collected in each of the scheduled calls was registered in paper and subsequently was recorded an Excel document  ,13. Subs® IBM Corporation 2020, Armonk, NY, USA) [TBD’s tolerability was evaluated by measuring different parameters: patient´s demographics characteristics, prescription and dose, interactions   safety s® IBM Corporation 2020 and MedicinesComplete® “Martindale Drug reference” The Royal Pharmaceutical Society 2020) [® database.An analysis was performed comparing the incidence of reported ADRs of this study with those found in two reference drug information databases (Micromedexty 2020) ,15. The Quantitative variables were expressed as the mean ± SD, and qualitative variables were described as frequencies and percentages. A binary logistic regression analysis was performed to adjust the variables associated with the dependent variable (ADR) by gender ; comorbidities (“yes” and “no”); prescription ; age group (adult and geriatric); and dose  were used as covariates; moreover, multinomial logistic regression analysis was realized to adjust the variables associated with the dependent variable (ADR) by SDO fixed combination; ciprofloxacin and dexamethasone. All the analyses were made using SPSS version 25 for Mac n = 43) on account of one of the following: unanswered call (60.5%/n = 26) or wrong registered number (39.5%/n = 17). A total of 220 patients were contacted; 122 women , none of them were pregnant; and 98 men   .The results of the study and the databases are shown in n = 145) of patients under treatment with SDO used other simultaneous treatments like artificial tears  which were the most commonly used products concomitantly with SDO, followed by ophthalmic NSAIDs , antiglaucoma drugs , glucose-lowering medications , antihypertensive medications , monoclonal antibodies , and others ; the remaining 34% of patients were not using any concomitant therapy. With those data a search of different bibliographic sources was conducted, and 21 possible systemic drug interactions with SDO (ciprofloxacin/dexamethasone) were identified: 2 patients used fluoxetine, 7 used oral glucose-lowering medications, and 12 used NSAIDs.The 66% (n = 80) and 2.5% as moderate (n = 2). Moreover, 67% of the patients who presented any ADRs improved in one min or less after the application of the product. No cases under the severe category were identified, in the same way no serious ADRs were identified.A total of 82 ADRs/220 patients were reported after the use of SDO, presenting a ratio of 0.37 ADR/patient; these ADRs were classified into 3 SOC and 9 PT groups, finding that the most frequent SOC group was eye disorders (93%) and the more frequent PT was eye irritation (75%). The most frequent causality was probable with 72% of cases, followed by possible with 15% . The ADRp = 0.008, 95% CI 1.28–4.974); however, some factors show a tendency to be associated with the increase in the incidence of ADRs. None of them were statistically significant ; but not for ciprofloxacin . Likewise, eyelid edema , eye pain , Ocular hyperemia , and Vision blurred  were found not statistically significant when comparing SDO with ciprofloxacin or dexamethasone. The incidence of the rest of reported ADRs was below expected . On the other hand, because the telephone interviews were carried out at two given times, the patients may not report all the ADRs, considering that their answers could be subject to what the patient remembered at the time of the call. This could be mainly a point to consider in the case of children and older people. However, it has some advantages, such as individualized pharmacovigilance, continuous monitoring and more detailed information on adverse events from a large number of patients.In our study, we found no increase in the incidence of ADRs related to SDO use compared to those reported in the literature for its active ingredients administered individually; also, no new risks or safety signals were observed in the population where this study was conducted. Consequentially, a good tolerability safety profile was confirmed.We identified an increase of ADR in females exposed to systemic drugs supported by the literature but limited, on this regard was available for ophthalmic drugs specifically; nevertheless, more studies are needed to assess these results.


Abelmoschus esculentus L.). The treatments each year 2017 and 2018 consisted of: rabbit manure, cow dung, poultry manure, green manure [Mexican sunflower (Tithonia diversifolia Asteraceae)], pig manure, NPK 15-15-15 fertilizer applied at 120 kg N ha−1 and a control (no manure/inorganic fertilizer). The seven treatments were laid out in a randomized complete block design with three replication. Organic manures and NPK fertilizer increased the soil organic matter (OM), N, P, K, Ca and Mg , growth, yield, minerals, protein, ash, carbohydrate and mucilage contents of okra fruit as compared with control. Organic manures improved okra yield compared with NPK fertilizer. Okra growth and yield parameters were significantly higher in 2018 compared with 2017. Control, rabbit manure, cow dung, poultry manure, green manure, pig manure and NPK fertilizer in 2018 increased the pod yield of okra by 9.7%, 35.3%, 57.9%, 36.2%, 39.2%, 45.5% and 3.2%, respectively compare with the same treatment in 2017. Amongst various organic manures, poultry manure produced significantly higher plant growth, yield, mineral and proximate composition of okra because of its high soil chemical properties which could be related to its lowest C: N ratio, lignin and lignin: N ratio. Results also showed that okra grown during high intensity rainfall has higher yield but with reduced quality except its mucilage content. Therefore, planting of okra with poultry manure under moderate rainfall will enhance the health benefit from the fruit, however, those that desire its mucilage content planting during high rainfall is recommended.Use of organic manures to meet the nutrient requirement of crop would be an inevitable practice in the years to come for sustainable agriculture since organic manures generally improve the soil physical, chemical and biological properties. Hence, field experiments were carried out in 2017 and 2018 to compare the impact of different organic manures and NPK fertilizer on soil properties, growth, yield, proximate and mineral contents of okra ( Abelmoschus esculentus L.) is an important tropical and subtropical vegetable crop grown for its fresh leaves, buds, flowers, pods, stems and seeds. The fresh pods can be eaten as vegetables in form of salads, in soups and stews or boiled. Okra fruits contain mucilage upon cooking. The mucilage has medical importance as it is used as a plasma replacement or blood volume expander and binds cholesterol and bile acid carrying toxins dumped into it by the liver1. Okra fruits contain fiber, vitamin C, folate and antioxidants2. The seeds contain oil that is edible by man as well as in soap industry3.Okra  is the basis of sustainable crop production in Nigeria and other tropical countries5. Hence, there is need to improve the fertility of the soil for continuous and increased crop production.Despite the enormous potentials of okra fruits production, its yield per hectare and quality in Nigeria had been greatly hampered by the low fertility status and organic matter contents of the soils which translate into low productivity and consequently reducing income for the farmer6. Although, in comparison to inorganic fertilizers, organic manures contain smaller quantities of plant nutrients. The use of inorganic fertilizer to increase yield has been found to be effective as a short-term solution but demands consistent use on a long-term basis. The high cost of inorganic fertilizers makes it uneconomical and out of reach to poor farmers and it is also undesirable due to its hazardous environmental effects7. Therefore, it is essential to investigate the use of locally sourced organic materials which are environment friendly, cheap and probably an effective way of improving and sustaining the productivity of soils and arable crops such as okra.In years to come, utilization of organic manure to meet crop nutrient requirement will be an unavoidable practice to enhance sustainable agriculture, this is because, the physical, chemical and biological properties of soil is generally improved by the addition of organic manures which in turn enhances crop productivity and maintains the quality of crop produce10. However, since organic and in organic fertilizers contain different chemical composition and quality they therefore may react differently when applied to the soil with respect to soil chemical properties, crop yield and quality. This aspect needs investigation especially in Nigeria where such data on the effects of different organic and inorganic amendments on the mineral and proximate contents are lacking. Available work dealt with organic amendment on the yield of okra without putting into consideration the quality of okra fruits. In Sokoto northern Nigeria10, investigated different sources of organic manure  on growth and yield of okra. Their results revealed that poultry manure promotes higher growth and yield of okra compared with cow and sheep manure.Okra yield responses to organic and inorganic fertilizers have been reported by several workers11 investigated the effects of Sheep, cow and poultry manures and their combinations on the growth and yield of okra, their result also revealed that poultry manure has the highest yield. Also in Malaysia9, six different treatments  were investigated on the growth and yield of okra, According to the study, application of poultry manure significantly increased the growth and yields performances on okra compared to other types of organic fertilizers. To have a holistic approach on the response of organic manures on soil chemical properties and okra performance, data on the quality of such okra fruit is imperative. Therefore, the study is to compare the impact of different organic manures and NPK fertilizer on soil properties, growth, yield, proximate and mineral contents of okra grown in derived savanna zone of Nigeria.Fagwalawa and Yahaya−1, 2.0 cmol kg−1 and 0.15 cmol kg−1 respectively in both cropping seasons of 2017 and 201812. Exchangeable Mg was adequate in 2017 but low in 2018. Analysis of the soil amendments used for this experiment is shown in Table The result of the pre-planting chemical and physical analysis of the experimental field Table , indicatTable The results of the response of okra growth and yield to different organic manure sources and NPK fertilizer are presented in Table The effect of different organic manure sources on proximate and mineral content of okra in 2017 and 2018 are shown in Table 14 have shown that animal manures and green manure increased soil OM, N, P and CEC and this was attributed to the availability and adequate supply of organic matter. The slightly lower pH of organic amended soil compared with the control could be due to the fact that during microbial decomposition of the incorporated manures, organic acids may have been released, which neutralized the alkalinity of the manures, thereby lowering the pH of the soil below their initial values. Adekiya et al.4 observed a similar trend in their work on organic amendments of soils. NPK fertilizer has the lowest pH as a result of leaching of bases from soil surface. Poultry manure significantly produced the highest soil chemical properties, this could be related to its lowest C:N ratio, lignin and lignin: N ratio , N, P, K, Ca and Mg contents of the soil compared with control. This result is consistent with the analysis recorded for the amendments in the present study that they contain these nutrients and also attested to the fact that the soil was deficient in these nutrients Table . These nio Table  which fa15.In both years, poultry manure increased N, P, K, Ca and Mg compared with other manures. This was due to the fact that poultry manure was low in C:N ratio, lignin and lignin/N and high in N, P, K, Ca and Mg compared with other manures. Due to high quality of this amendment, it decomposes quickly and release nutrients to the soil. The significant increase in soil nutrients especially OM, N, P and K in organic amendments compared with NPK fertilizer was due to leaching in NPK fertilizer treated plots. The reduction in Ca and Mg in NPK fertilizer plots compared with organically amended plot was due to the fact that NPK fertilizer did not contain Ca and Mg. The reduction of soil bulk density observed in both years with organic manures compared with control and NPK fertilizer could be attributed to increase in soil organic matter resulted from the degraded organic residues by soil microorganisms. Organic matter is known to improve soil structure, aeration and reduce soil bulk densityThe decreasing order of okra yield were poultry manure > green manure > pig manure > cow dung > NPK fertilizer = rabbit manure > control. The positive effect of organic manure on growth and okra yield could be due to the contribution made by amendments to fertility status of the soils as the soils were low in organic carbon content. Manure when decomposed increases both macro and micro nutrients as well as enhances the physio-chemical properties of the soil for the betterment of okra growth.16 reported that C:N ratio of organic materials markedly influences the decomposition rate and the mineralization of N because N determines the growth and turnover of the microorganisms that mineralize organic carbon.Okra grown on poultry manure performed better in terms of growth and yield compared with other sources of organic soil amendment and NPK fertilizer. This could also be related to low C:N ratio, lignin and lignin/N values These attributes of poultry manure will lead to fast mineralization and early release of nutrients to a short gestation crop like okra, hence there was a boost in the morphological growth of the plant which translate to greater yield compared with other amendments. Wolf and Snyder17.The reduced growth and yield of okra in plots treated with other manures in comparison with poultry manure could be as a result of immobilization of soil nutrients which occurs when soils are treated with animal manure of high lignin contents which results from the feed eaten by the animal18. The superior N supply by poultry manure during okra cropping in this experiment may be the reason for better growth and yield of okra in plots with poultry manure18. The obtained results corroborated the finding of19 that poultry manure increased the height of okra relative to other amendments.Higher yields were observed in poultry manure plots compared with inorganic fertilizer because, the nitrogen content of poultry manure is released to the soil gradually and steadily over longer time for the growth of the plant compared with nitrogen from NPK fertilizer which is prone to losses by run-off, volatilization, leaching and/or denitrification. Poultry manure has been said to be a better soil amendment compared with chemical fertilizers because of the greater capability of poultry manure to preserve its N20.The better performance of okra under NPK fertilizer plots compared with the control was due to release of nutrients  from the fertilizer which are absorbed by the okra plants. Okra growth and yield in second year (2018) was better than that of first year (2017). This was due to the differences in the amount of rainfall between the two years Table . Year 20P < 0.05. Poultry manure had the highest values of N, P, K, Ca and Mg in the okra fruit compared with other amendment and NPK fertilizer. This result is also consistent with the soil chemical properties and growth and yield for this treatment. The poultry manure may have improved the availability of nutrients to the crop by enhancing the mineralization and supply of readily available nutrients to the soil21.The fact that organic manures and NPK fertilizer increased okra mineral contents compared with the control was attributed to increased availability of the nutrients in soil as a result of the mineralization of the manures leading to increased uptake by okra plants. In both years, the correlation coefficient between soil and okra fruit N, soil and okra fruit P, soil and okra fruit K, and okra fruit Ca, soil and okra fruit Mg were all significant with R values of 0.83, 0.71, 0.66 0.81 and 0.88, respectively at 22. The crude protein contents in all the organically amended soils were above the critical 13–17%24. Therefore the organic materials sustained good nutritive quality of okra as opposed to NPK and control. The ash content in poultry manure treatment was significantly higher possibly because of the balanced nutrient in the manure, unlike NPK with inferior contents of N, P, and K and the control with lower concentration of nutrients. In this study, poultry manure gave the highest N, P and K concentrations of 3.04%, 11.9% and 1.16% in 2017 and 4.41% 13.4% and 1.87% in 2018  and consists of rhamnose, galactose, glucose, galacturonic acid and glucuronic acid in addition to 3.7% acetyl groups27. Ahmad et al.27 also reported that compost and NPK fertilizer application increased the mucilage of borage plant. The highest value of mucilage in poultry manure could be related to increased soil nutrients compared with other treatments. Okra produced under organic amendments has better qualities  compared with NPK fertilizer and control. This is because organic manures not only increase soil nutrients but also improves the physical by prevention of erosion and leaching of nutrients (in this experiment reduce bulk density) and biological properties28. Organic manures also contains both micro and macro nutrients unlike NPK fertilizer that contains only N, P and K. Lumpkin29 also, was of the opinion that organically produced vegetables were of higher qualities than those produced using conventional methods. Cropping of okra in 2018 reduced protein, ash, fat, carbohydrate, N, P, K, Ca and Mg contents of okra fruit compared with 2017 cropping. This could be adduced to high rainfall in 2018 compared with 2017. High rainfall has been reported to reduce the nutrient and proximate content of vegetables30. Climatic conditions have a great effect on the concentration of mineral in plants. Variation in temperature and rainfall have been reported to influence the chemical composition in plants31.The increased nitrogen to the soil by the incorporation of organic manures increased the nitrogen uptake by the okra fruit thereby increasing protein. This explains the reasons for increase in crude protein values between organic amendment and control and NPK fertilizer treatment. Poultry manure has the highest value of crude protein, this is also consistent with its high soil N level Table . NitrogeResults of this experiment showed that organic manures and NPK fertilizer increased the soil chemical properties , growth, yield, minerals, protein, ash, carbohydrate and mucilage contents of okra fruit as compared with control. Organic manures improved okra yield compared with NPK fertilizer. Amongst various organic manures, poultry manure produced significantly higher plant growth, yield, mineral and proximate composition of okra because of its high soil chemical properties which could be related to its lowest C:N ratio, lignin and lignin: N ratio. Results also showed that okra grown during high intensity rainfall has higher yield but with reduced quality except its mucilage content. Therefore, planting of okra with poultry manure under moderate rainfall will enhance the health benefit from the fruit, however, those that desire its mucilage content planting during high rainfall is recommended.2. The experimental site falls under the derived savanna agro-ecological zone of North-Central Nigeria. Weeds in the experimental soil before cultivation included Mexican sunflower (Tithonia diversifolia Asteraceae) and Guinea grass (Panicum maximum Jacq). Different sites were used for the experiment in 2017 and 2018, but the two sites were very close to each other.During 2017 and 2018 cropping seasons, okra was grown in a field experiment at the Landmark University Teaching and Research Farm, Omu-Aran, Kwara state , with altitude of 562 m above sea level. The rainfall pattern was bimodal with peaks in August (Table Tithonia diversifolia Asteraceae)], pig manure, NPK 15-15-15 fertilizer and a control (no manure/ inorganic fertilizer). Both the organic manures and NPK fertilizer were applied at the rate of 120 kg N ha−132. This was equivalent to 800 kg ha−1 for NPK 15-15-15, 11.9 t ha−1 for rabbit manure, 5.5 t ha−1 for cow dung, 4.1 t ha−1 for poultry, 4.8 t ha−1 for green manure and 5.6 t ha−1 for pig manure. The seven treatments were laid out in a randomized complete block design (RCBD) with three replications. Each block consisted of 7 plots measuring 3 × 2 m with 1 m block and 0.5 m between plots.The treatments each year 2017 and 2018 consisted of: rabbit manure, cow dung, poultry manure, green manure [Mexican sunflower  land preparation after which plots were marked out to the require plot size of 3 × 2 m. A hand-held hoe was used to incorporate the manures into the soil to a depth of approximately 20 cm. All animal manures were obtained from the livestock section of Landmark University Teaching and Research Farm. Fresh top Mexican sunflower was collected from a nearby farm and hedge containing green tender stems and the leaves. The plots were allowed for 4 week before sowing of okra seeds. Sowing of okra variety NHAe-47-4 was done late April for both 2017 and 2018 croppings. Two seeds of okra were sown per hole at inter-row spacing of 0.6 m and 0.6 m intra-row spacing manually giving a plant population of 27,778 plants ha2. Textural class of the soils were determined by the method of33. Soil organic carbon (OC) was determined by the procedure of Walkley and Black using the dichromate wet oxidation method34. Total N was determined by the micro-Kjeldahl digestion method35. Available P was determined by Bray-1 extraction followed by molybdenum blue colorimetry36. Exchangeable K, Ca, and Mg were extracted using 1 M ammonium acetate37. Thereafter, concentration of K was determined on a flame photometer, and Ca and Mg were determined by EDTA titration method. Soil pH was determined using a soil–water medium at a ratio of 1:2 with a digital electronic pH meter. At the termination of the experiment in 2017 and 2018, soil samples were also collected (on plot basis) and similarly analysed for soil chemical properties as described above. Also at the end of the experiment each year, soil bulk density were determined all plots. Five undisturbed samples  were collected at 0–0.15 m depth from the centre of each plot at random and 0.15 m away from each okra plant using core steel sampler4. The samples were used to evaluate bulk density after oven-drying at 100 °C for 24 h4.Before the start of the experiment, soil samples from topsoil (0–15 cm) were taken from random spots in the study area and were bulked together, air-dried and sieved using a 2-mm sieve and their physical and chemical characteristics were determined1.12} developed by38, where LA = leaf area, L = leaf length and W = leaf width. Edible pods were harvested at four day intervals, counted and weighed. Pod weight was evaluated based on the cumulative harvests per plot. For 2017 cropping of okra, fruits were harvested until November 2017 while for 2018 okra cropping, fruits were harvested until July 2018.Collection of data for growth (plant height and leaf area) was done at mid-flowering of okra plant (about 47 days after sowing). Plant height was determined by the use of meter rule while leaf area was determined by using the model {LA = 0.34(LW)39. One gram of each sample was digested using 12 cm−3 of the mix of HNO3, H2SO4, and HCLO4 (7:2:1 v/v/v). Contents of N, P, K, Ca and Mg were determined by atomic absorption spectrophotometry. Lignin content of the organic amendments was determined from acid-free detergent fiber using the method described by40.At harvest in 2017 and 2018, eight okra fruits of uniform sizes were randomly collected from each plot and analyzed for mineral contents according to methods recommended by the Association of Official Analytical Chemists39. Samples of okra fruits were sliced with a knife and blended. After blending, it was diluted with ten times its weight with water (1:10). The viscous solution was separated from the debris using fine cloth41. Mucilage of the extracted viscous liquid was measured using viscometer.Samples of okra fruits from each plot was taken for proximate analysis. The ash, crude protein, crude fat and carbohydrate contents of the okra fruits were determined using standard chemical methods described by Association of Analytical chemists42. N was determined by the micro-Kjeldahl digestion method. Samples were digested with nitricperchloric-sulphuric acid mixture for the determination of P, K, Ca, and Mg. Phosphorus was determined colorimetrically using the vanadomolybdate method, K was determined using a flame photometer and Ca and Mg were determined by the EDTA titration method43.About 2 g of each organic manures used was collected and analysed for N, P, K, Ca, and Mg as described by44 and treatment means were separated using Duncan Multiple Range Test (DMRT) at 5% probability level.The data collected were subjected to statistical analysis of variance (ANOVA) using the Genstat statistical package


To examine three levels of need for recovery (NFR) after work in relation to effort from work demands, demand compensatory strategies, effort-moderating or -reversing resources, and health including health behaviors. A further purpose was to examine occupational characteristics determining NFR.5000 engineers, carpenters, nurses, and home care nurses were invited to participate. NFR k-means clusters were calculated from 1289 participants. The effect from three levels of NFR regarding demands, compensatory strategies, resources at work, health, and health behaviors was examined using analysis of variance (ANOVA) and post hoc analysis. Prevalence ratios (PRs) of suboptimal health for three levels of NFR were calculated using Poisson regression. Linear stepwise multiple regression predictors explaining NFR were examined also occupation wise.NFR centroids at 5.8/33, 13.1/33, and 21.0/33 points were identified. ANOVA showed corresponding effects from NFR levels on work demands and compensatory strategies. The inversed proportion concerned levels of resources at work. Only the low NFR cluster negated regular health effects. The other two cluster groups also repeatedly worked while ill and presented PRs concerning health effects from 1.9 to 3.9 when compared to the low NFR group. Making good quality work, recovery opportunities, and thinking of work when off work were the most important predictors of NFR among 1289 participants with also occupation-wise interpretable profiles.Three levels of NFR meant corresponding levels of work demands, work-demand compensatory strategies, and unfavorable health behaviors. An inversed proportion of resources related to the same levels of NFR. Low NFR meant no regular health effects which could guide limit values regarding salutary NFR. Important predictors of NFR were resources making a good quality work, recovery opportunities, and reversely effort from rumination when off work. Occupation-wise predictors could guide interventions. The relationship between self-reported unfavorable working conditions and severe health consequences such as cardiovascular disease  instrument measures job control in terms of opportunities for recovery during work hours and in the interface between work and home  concerning a heightened level of NFR after work. A further aim was to examine the impact of diverse occupational characteristics on NFR.How are NFR cluster groups related to work demands and resources doing the work?How are NFR cluster groups related to work-demand compensatory strategies?How are NFR cluster groups related to functional health effects?How are NFR cluster groups related to health-related behaviors?What is the impact of occupationally characteristic on NFR after work?Specific research questions were:The present baseline assessment is part of a repeated measurement longitudinal epidemiological research design on how to facilitate recovery from work in three different age groups and four different occupations. The design includes also measurements of physical load, physical exertion, and pain. The examination of physical and mental need for recovery together and over time will gradually take place and be presented elsewhere.The selection of respondents was based on age and differential exposure to physical load, mental load, or physical and mental load. A further alignment was to include professions with high versus low rates of occupational injuries, where engineers represent a clear low rate of reported occupational injury (Statistics Sweden). The age groups were: 18–35, 36–45, and 46–70 years. The professional groups were engineers and architects with a 5-year university degree, carpenters working at building sites, nurses working in hospitals, and home care nurses working in the homes of care recipients. Random sampling was done based on the Swedish Standard Classification of Occupations (SSYK), using the above criteria and including individuals 19–70 years of age. Sampling was done using proximity sampling in the Västra Götaland Region or within a specified municipality in Västra Götaland Region.In a first step, Statistics Sweden sent letters of invitation to 1250 representatives in each of the four occupational groups. The invited individuals were requested to mark “other” if their current occupational group was neither engineer nor carpenter, nurse, or assistant nurse working in home care. The letter also included a numbered consent form, the survey, and a return envelope. Interested individuals sent the consent form including their name, postal address, and social security number and the completed questionnaire back to the research group. One reminder, based on a temporary, numbered list of addressees at Statistics Sweden, was sent to those who had not responded within 2 weeks. This concluded the first phase of the longitudinal study. A total of 1292 individuals had responded (25.8% response rate). The response rate was 18%, 12.5%, 43.4%, and 8.6% for engineers, carpenters, hospital nurses, and home care nurses, respectively.The total survey consisted of 115 questions.The background variables were sex, age, marital status, children, children currently living at home, type of employment, contract, and working hours (full time/part time). Concerning the number of children living at home, responses were divided into three categories: 1 = none, 2 = one or two, and 3 = three or more.Figure The Swedish version of the Copenhagen Psychosocial Questionnaire (COPSOQ) adapted by Berthelsen et al.  was usedCompensatory strategies for handling work demands  among 1280.Two work moderating resource scales were collected from Swedish version of the Copenhagen Psychosocial Questionnaire (COPSOQ) (see paragraph on demands); Social support from colleagues , and Social support from the supervisor . Aronsson et al. , when exRecovery opportunities’ (RO) scale records a load effect reversing effect. That is the degree of personal freedom regarding work shifts in terms of, for example, breaks and scheduling of shifts. In parallel to the NFR scale (see below), the RO scale has been translated from Dutch . The participants were assigned to one of the three clusters where the difference between the individual score and the cluster center was the smallest. The cluster 3 means analysis designated the after-work load reaction as a high, a low, or an in-between level of NFR. Regarding each cluster group, the means of NFR and of the successive scales or single items were transformed into descriptive profiles  of suboptimal health for each of the cluster groups was calculated through a modified Poisson regression  and Tukey Honestly Significant Difference post hoc analysis. Thereafter, the risk for effects on health in terms of health and well-being scales were dichotomized in terms of frequency of complaints . Informed consent forms were sent back to the research group together with the completed first measurement questionnaire.The four professions and the group labeled “other” were examined regarding background variables. As can be seen in Table This reason for working part time was almost exclusively seen in human service work Table .The mean NFR score for the total sample of 1289 participants was 11.6 points . The cluster analysis centroids were identified at 5.83, 13.08, and 20.96 points for the “low NFR”, “in-between NFR”, and “high NFR” groups, respectively. The cluster groups experienced the NFR load reaction “less than sometimes”, “sometimes” “and” “often”, respectively. Altogether, the recovered group (“low NFR”) comprised 40.2%, the in-between group 41.6%, and the not recovered group, 18.1%. The low, in-between, and high NFR groups had an age mean of 49, 47, and 45 years, respectively. Regarding children, 56%, 58%, and 70%, respectively, of the low, in-between, and high NFR groups had children living at home (Table The mean NFR score for architects/engineers was 9.5 (SD 5.5), for carpenters 10.5 (SD 5.3), for hospital nurses 12.2 (SD 6.0), and for home care nurses 14.2 (SD 6.7). The relative frequencies of engineers, carpenters, nurses, and home care nurses with a low NFR were 49%, 48%, 37%, and 26%. The corresponding figures concerning a high NFR were a 13%, 15%, 19%, and 30%, respectively   Table . The lowN = 1285–1287), the means and (standard deviations) were in size as follows: working more intensively 2.8 (1.0), thinking of work when off work 1.9 (1.2), skipping breaks 1.7 (1.3), lowering the quality of work 0.9 (1.0), and taking work home 0.9 (1.7). The between-group ANOVA showed a significant effect of NFR group on strategies to increase efforts concerning work when handling work demands  or sometimes experienced NFR(in-between group) and the low NFR group showed that the two former groups at two different levels reported distinctly higher demands and distinctly lower available effort-moderating and cumulative effort-reversing resources at work than the low NFR. The group less than sometimes experiencing NFR (low NFR) group presented an inverse pattern of lower demands and higher effort-moderating and cumulative effort-reversing resources. Moreover, the high NFR group and the in-between group at two different levels both contrasted to the low NFR group that more rarely used work-demand compensatory strategies such as skipping breaks to finish what needs doing at work. Concerning health effects, a salutary meaning of a low NFR could be validated, while this group negated regular health effects. In contrast, a high NFR and an in-between level of NFR value at two different levels meant experiencing symptoms on a regular basis and an unhealthy pattern of up to a fourfold magnitude concerning health effects compared to the low NFR group. During the last 12 months, the health behaviors concerning a real need for sick leave to some degree differed between the cluster groups, while the contrasting high NFR group had worked despite this need two to three times as compared to zero to one time in the other groups. A minor contrast between the groups concerned exercise habits where the low NFR group was more often physically active at an intensive level.The fifth research question concerned the impact on NFR from occupational characteristic of four narrowly specified occupational groups. In the same order of magnitude and in size, the most important impact on NFR came in the group of engineers and architects from Thinking of work when off work and Quantitative demands. Concerning the carpenters, this impact stemmed from Lack of opportunity making a good quality job, working more intensively to finish and Lack of recovery opportunities. In the group of hospital nurses, this impact comprised of Lack of opportunity making a good quality job and Thinking of work when off work and among Home care nurses from Thinking of work when off work, Lowering the quality of the job to finish, Lack of recovery opportunities, and Lack of opportunity making a good quality job. In the non-academic groups, the total of occupational characteristics explained 48–53% of the variance in NFR. The corresponding figure in the higher educated groups was 40–43%.The suggestion of Fig. NFR equals accumulation of effort and fatigue. If not nugatory, this gradually also creates a situation where fatigue compensating effort results in a successively more vulnerable states of overdraft fatigue during the day at work, NFR. In addition to being a vulnerable state of mental fatigue, compensatory effort may alter the functioning of the brain in the direction of the effort being both very costly and inefficient  by not depleting them . Regardless of the differences in the level of education, there are important similarities between the caring professions. These similarities include often working part time. The patterns of effort starting from the compensating strategy of lowering the quality of work was a very pronounced source of effort in home care nurses and concerned hospital nurses, as well, wherein both professions’ effort could be moderated by Quality of work. Thinking of work when not at work was an important source of effort in both caring professions. These tripod mechanisms may rely on the conflict in caring professions described by Pousette  above. AInterventions in the engineer/architect group need to aim at sufficient resources in terms of staffing for doing the job to discontinue quantitative overload forcing a work style marked by intensity and absorption  representing a low rate of occupational injury. We had a low response rate and more women than men participated in the study, and conclusions from this study may have to be drawn with these limitations in mind. At the same time, men and women are found to be equally sensitive to strain damage or psychological problems from physical or mental strain. S.c gender differences could instead be explained from various gender typical occupational characteristics (Swedish Research Council for Health, Working Life and Welfare . AccordiIn conclusion, high, in-between, and low NFR related to effort from three corresponding distinctive levels of work demands, stress responses of high work-demand compensatory strategies and unfavorable health behaviors such as repeatedly working while ill. Three distinctive levels of resources at work that could moderate effort or reverse cumulative effort during work were in inverse proportion related to high, in-between and low NFR. In the current result, the defined low level of NFR meant also to negate regular functional effects on health. This finding could guide the setting of limit values regarding a salutary level of NFR. In the total study group, the most important predictors of NFR were the effort moderation from being able to make a good Quality of work, effort reversal from RO, and additional effort from thinking of work when off work. Concerning the occupational groups, meaningful patterns of predictors appeared that could also guide interventions. An implication for future research on work and health could be to broaden the perspective on the spectrum of workload and resources to do the work. This could also have an educational significance in interventions.


More than a quarter of a million individuals in the US live with spinal cord injury (SCI). SCI disrupts neural circuitry to vital organs in the body. Despite severe incidences of long-term peripheral complications from SCI, the cardio-metabolic consequences and divergences in sex-related responses are not well described. We examined the effects of SCI on functional recovery, cardiac structure and function, body composition, and glucose metabolism on adult female and male Sprague Dawley (SD) rats. SCI was induced at T10 via contusion. Measured outcomes include behavioral assessment, body weight, dual-energy X-ray absorptiometry (DEXA) for body composition, echocardiography for cardiac structure and function, intraperitoneal glucose tolerance test (IPGTT) for glucose metabolism, insulin tolerance test (ITT), and histology of cardiac structure at the endpoint. There was a decrease in body fat percentage in both sexes, with SCI females disproportionately affected in percent body fat change. Left ventricular internal diameter during systole (LVIDs) was decreased in SCI females more than in SCI males. No significant differences in glucose metabolism were observed up to 20 weeks post-injury (PI). These data show significant cardio-metabolic differences as a consequence of SCI and, furthermore, that sex is an underlying factor in these differences. Individuals with SCI are known to face lifelong disability that results from ineffective motor regeneration and aberrant sensory reorganization2. An abundance of research has documented the nervous system aspects of SCI and many innovative methods are being developed to treat patients affected by these devastating injuries.Traumatic spinal cord injury (SCI) represents a major public challenge worldwide3. A group of symptoms termed metabolic syndrome (MS), which include type 2 diabetes, altered body fat percentage, hypertension, and cardiovascular anomalies, have been reported in patients suffering from chronic SCI4. Other non-MS issues, such as lung dysfunction and autonomic dysreflexia6, osteoporosis7, and cardiovascular disorders, commonly affect SCI patients8. Cardiovascular dysfunction is the leading cause of death for those that survive the initial SCI10. Despite this, there have been relatively few studies assessing the progression of cardio-metabolic aberrations that can occur as a result of SCI.It is becoming increasingly apparent that SCI also has major effects on non-nervous system tissues. SCIs disrupt the neural circuitry and signals to vital organs in the body and result in severe long-term complications outside of the central nervous system (CNS)11. Cardio-metabolic responses are known to differ significantly between males and females in the absence of SCI12. Comparisons of outcome by sex are not common in SCI animal studies, but due to differences in the reproductive system, sex hormones, and chromosomal makeup between males and females, there are baseline differences in metabolism and behavior in the absence of any injury15. For example, in animal models of obesity, females are less susceptible to weight gain and hypothalamic functions differ between the sexes16.A second factor that was included in our study was to assess sex-specific responses. SCI is more prevalent in males than females in the US, but the incidence of female injury is risingTo investigate these aspects, we studied the cardio-metabolic response to SCI in male and female Sprague Dawley (SD) rats. SD rats received an SCI through T10 contusion and were monitored by dual-energy X-ray absorptiometry (DEXA), echocardiography, intraperitoneal glucose tolerance tests (IPGTT), body weight, behavior assessments of functional regeneration, and histological evaluation of the heart after 20 weeks. Results revealed new aspects of cardio-metabolic alterations that occur after SCI in both males and females and, additionally, revealed aspects that were divergent between the sexes. The results support the assertion that metabolic alterations occur after SCI, can be studied using animal models, and that there is a significant difference in the development of dysfunction in females and males following SCI.19. At the endpoint, an intraperitoneal insulin tolerance test (IPITT) was measured.To assess the consistency of injury caused by contusion, the spinal cords of contused animals 14 d PI were labeled for glial fibrillary acid protein (GFAP) and the size of the lesion cavity was measured with ImageJ  was reduced significantly in SCI females at 4-, 8-, and 16-weeks PI and trended to decrease at 12- and 20-weeks PI. No significant reduction in LVIDs between male sham and SCI was observed. When comparing the percent LVID change between male and female SCI animals, the data indicates that the percent diameter change for female SCI animals was greater than what was observed for male SCI animals, as compared to sham Fig. . MeasureAt 8-weeks PI, the body weight of SCI males was significantly reduced compared to male sham animals and this continued until the study endpoint Fig. . Female For DEXA measurements of body composition, several distinctions were noted. Prior to SCI, the total body fat Fig.  and percIPGTTs showed glucose concentrations and area under the curve (AUC) were similar between males and females pre-injury Fig. . No sign22. For many of the processes that fall under autonomic function, such as metabolism and cardiovascular function, sexual dimorphism has been documented but not in the context of SCI14. To investigate the potential for cardio-metabolic and sexual dimorphism in SCI, we studied the cardio-metabolic effects on adult male and female rats for 20 weeks, spanning the acute and chronic phases after SCI23.The trauma induced by SCI affects spinal pathways that are essential not only for somatic but also for the function of the autonomic nervous system. Where the majority of SCI research has understandably focused on aspects associated with motor and sensory conduction, it is becoming increasingly apparent that SCI also induces a host of changes that are due either to primary damage to pathways that influence autonomic activity or are secondary to sensorimotor impairmentet al. performed mild and severe contusions in rats at T8 and found that by 3 weeks females had recovered significantly better than males, and maintained this throughout the 120 d study24. Datto et al. also performed a moderate contusion at T8 and results showed that by 5 weeks females had recovered significantly more than males and maintained this for duration of the 13-week study25. Both studies used more animals, with Datto et al. using substantially more, and performed their SCI higher up the spinal cord (at T8 as opposed to T10). While female SCI rats reached a higher BBB score more quickly in our study, the difference was not sustained as it was in these two studies. Von Frey assessment of sensory function was largely comparable for both sexes, but males did exhibit an indication of mechanical allodynia  metrics28. A key variable unaccounted for in these studies is the extent of physical rehabilitation the patients received. The rats in our study received no rehabilitative therapy, suggesting that baseline female recovery may be more rapid than in males and that females may be less prone to the development of mechanical allodynia.In general, human clinical studies assessing functional outcomes that have included sex as a variable have been mixed. Studies by Greenwald 33. In comparing the sexes, the results here show males exhibited a slight increase in LVID while females exhibited a reduction . A study by Cragg et al., however, showed a significant increase in cardiovascular dysfunction after SCI, but the contributions of sex to dysfunction in this study was considered a confounding variable due to the known differences in the risk for developing cardiovascular dysfunction between uninjured males and females36. Our data show that female animals do developed cardiovascular alterations as a result of SCI and suggests that they are adaptive whereas male alterations may be more prone to being maladaptive.For cardiac function, it is well established that male rats with SCI exhibit alterations in cardiac structure and function largely consistent with the results of our study, albeit using different injury models and a shorter timeline of analysision Fig. . This LVion Fig. . Such vence Fig. . A study37. Total lean muscle mass did not differ between groups.From the DEXA measurements, SCI caused significant differences in body mass Fig. . In femaThe lack of any fat gain in female SCI is intriguing. Common reasons for this outside of an SCI would include increased exercise, altered metabolism, and lower foot intake. Our study did not monitor activity nor dietary intake, which would be a useful addition for future studies. It is also not known whether SCI females failed to generate new fat or if they more rapidly metabolized fat as an energy source. Fasting glucose and AUC for the IPGTT indicated no difference from female sham to SCI  and randomly assigned to one of two treatment groups: sham or SCI. SCI was induced by contusion to the spinal cord as previously described20. Briefly, rats were anesthetized by 2% isoflurane inhalation and the surgical site at T10 was shaved and sterilized by iodine and isopropyl alcohol. The animal was draped, the surgical site was incised, and a partial dorsal laminectomy was performed at T10 to expose the spinal cord without penetrating the dura. To generate the SCI, a 10 g rod was dropped from a height of 35 mm onto the exposed spinal cord using an NYU-type impactor, where the rod remained in contact with the spinal cord for 3 s before removal. A small piece of adipose tissue was placed on top of the laminectomy site and the musculature and skin were sutured and closed with 6-0 sutures and wound clips. Sham animals received the same procedure and surgery of SCI groups but did not receive an impact. After surgery, animals received buprenorphine  twice daily for 3 d, and baytril  once daily for 7 d to prevent secondary infection. Bladders were expressed twice daily until animals were observed to have recovered bladder function.All animal use and procedures were performed with approval by the University of Wyoming Institutional Animal Use and Care Committee. Animals were cared for in accordance with the Guide for the Care and Use of Laboratory AnimalsTo assess the degree of injury caused by impact, spinal cords were harvested from contused male animals at 14 d PI. Animals were anesthetized with inhalation of 2% isoflurane with pure oxygen and were euthanized via perfusion with 4% paraformaldehyde (PFA) in 1x phosphate buffer solution (PBS) . Samples were placed in OCT compound  and longitudinal sections of 20 µm were sliced using a cryostat (Leica Biosystems CM3050 S). Fixed sections of spinal cord tissue were washed with 1x PBS 3x for 5 minutes each, then blocked using AB media  for 1 h. Sections were incubated with primary anti-glial fibrillary acidic protein (GFAP) antibody , diluted in AB media  at 4 °C overnight, washed 3x for 5 minutes each, and incubated with secondary antibody, Alexa flour 555 goat anti-chicken IgY antibody , in AB media  for 2 h. After being washed 3x for 5 minutes each, slides were covered with mounting media and cover slipped . Sections were imaged on a confocal microscope (Zeiss) and ImageJ software (NIH) was used to measure size of lesion cavity. A minimum of 3 sections were averaged to represent each animal, with 6 animals in total to assess consistency of SCI.21: each rat was placed in a small plastic pool and was scored by two independent observers during a 4-minute session, assigning scores from 0–21 to each hind limb, where 21 stands for perfectly normal, and 0 as completely paralyzed. The average score of the two observers was the final score of that individual animal and the average score of all animals within a group is considered the final score for that group.Injury and functional recovery post-injury (PI) were assessed using the Basso, Beattie, and Bresnahan scale (BBB) and von Frey tests. BBB was performed as previously described45. Briefly, rats were placed in small individual cages with mesh wire at the bottom. Von Frey filaments  were applied perpendicularly on the plantar surface of the front left, hind left, and hind right paws of each rat. The lightest filament was used first, incrementing to the heaviest filament. Each filament was performed on each paw 10 times. The filament was considered as threshold if a rat responded more than 5 times out of 10.Von Frey tests were performed as previously described46. Rats were anesthetized with isoflurane (induction at 3% and maintained on 1.5%) and heart rate was maintained between 300 and 350 beats per minutes. The hair was removed from the anterior chest wall and ultrasound transmission gel was applied. To visualize the cross-sectional view of the left ventricle, a linear transducer was used to examine the parasternal short axis view. Cine-loop analysis was performed to examine the systolic and diastolic phases of the heart. M-mode echocardiograph was acquired using echocardiography imaging equipment . The measurement was performed by tracing the internal diameters of the ventricle, averaged from three consecutive cycles for each animal. Several cardiac parameters were assessed, including left ventricle internal diameter (LVID), left ventricular (LV) mass, intraventricular septum (IVS), left ventricular posterior wall thickness (LVPW), and ejection fraction (EF).Echocardiography imaging and analysis were used to asses cardiac structure and function following SCI at different time points as we previously described47. Briefly, animals were anesthetized by isoflurane inhalation and positioned prone on the DEXA scanner . A low-level pencil-beam X-ray moved transversely from the head to the tail across the sedated animal. Differences in absorbance of X-ray was determined according to tissue density and fat percentage was calculated using fat and body mass48. Animals were weighed at each time point.DEXA scans were used to asses body fat percentage, lean tissue, and bone mineral density and content as previously described49. Rats were fasted for 16 h and weighed. Blood was sampled from the lateral tail vein using 24-g needles. Glucose levels in blood were quantified using a glucose meter  before and after intraperitoneal administration of 20% (2 g/kg) dextrose (Sigma. D9434) at 15, 30, 60, 90, and 120 min. IPITT was performed where insulin  was administered by intraperitoneal injection and glucose concentrations were quantified similarly to IPGTT.IPGTT was performed to assess glucose metabolism using published protocols50, where 3 sections per heart were imaged on an Olympus BX51 microscope and quantified using ImageJ .Hearts were removed and fixed in 10% formalin for 48 hours, dehydrated with ethanol, and embedded in paraffin. Sections were cut at 7 µm, with 50 µm between each section, and Masson’s trichrome staining was performed  according to the manufacturer’s instructions. Analysis and quantification of fibrosis was performed as previously describedStatistical analysis was performed using the mixed procedure in SAS  using fixed effects group*time*sex (repeated measures), repeated measures one-way analysis of variance (ANOVA), or unpaired two-tailed t-test. A p-value of <0.05 was considered significant. Data are presented as the mean, or least squares means (LSM), ± standard error of the mean (SEM).Supplementary figure 1.Supplementary figure 2.Supplementary figure 3.Supplementary figure legend.