Hirundo rustica) poses a number of fascinating scientific questions, including the taxonomic status of postulated subspecies. Here, we obtained and assessed the sequence variation of 411 complete mitogenomes, mainly from the European H. r. rustica, but other subspecies as well. In almost every case, we observed subspecies-specific haplogroups, which we employed together with estimated radiation times to postulate a model for the geographical and temporal worldwide spread of the species. The female barn swallow carrying the Hirundo rustica ancestral mitogenome left Africa (or its vicinity) around 280 thousand years ago (kya), and her descendants expanded first into Eurasia and then, at least 51 kya, into the Americas, from where a relatively recent (<20 kya) back migration to Asia took place. The exception to the haplogroup subspecies specificity is represented by the sedentary Levantine H. r. transitiva that extensively shares haplogroup A with the migratory European H. r. rustica and, to a lesser extent, haplogroup B with the Egyptian H. r. savignii. Our data indicate that rustica and transitiva most likely derive from a sedentary Levantine population source that split at the end of the Younger Dryas (YD) (11.7 kya). Since then, however, transitiva received genetic inputs from and admixed with both the closely related rustica and the adjacent savignii. Demographic analyses confirm this species’ strong link with climate fluctuations and human activities making it an excellent indicator for monitoring and assessing the impact of current global changes on wildlife.The barn swallow ( Hirundo rustica) is one of the most widely distributed bird species , H. r. savignii (Egypt), H. r. transitiva , H. r. tytleri , H. r. gutturalis , and H. r. erythrogaster (North America). Additional subspecies such as H. r. saturata and H. r. mandshurica have been postulated in north-eastern Asia, but their distinct subspecies status relative to the other Asian subspecies is debated  did not detect a genetic structure within the species, suggesting a rather recent subspecies differentiation  from eastern European and Turkish populations of the same subspecies and genomic covariance of the latter H. r. rustica populations with non-migratory H. r. transitiva specimens from Israel  (H. r. rustica) .In the last few years, whole genome sequencing data have been reported for a few subspecies (avignii) , includirustica) . RecentlND2 and CYB (H. r. tytleri) and the American one (H. r. erythrogaster) has raised the possibility of a secondary dispersal event, possibly about 27 kya, from North America back into Asia , particularly on the sequence variation of single mitochondrial genes, such as  and CYB . They co and CYB , thus sunto Asia . Finallyng mtDNA , suggestH. rustica  obtain a high-resolution mitogenome phylogeny of the species, 2) better define the matrilineal relationships and links between subspecies and their divergence times, and 3) assess demography through time.Despite the valuable genetic insights provided by these studies, the assessment of only a rather short segment of the barn swallow mtDNA limits their phylogenetic resolution and the understanding of this species’ origin and spread. A finer phylogenetic resolution can be achieved by sequencing the entire mitogenome, an approach that has been employed for humans and many other animal species  and rece rustica . Here, wH. r. rustica specimen (no. 20) from Italy (H. r. rustica Reference Sequence (HrrRS), was Sanger sequenced together with four additional H. r. rustica mitogenomes from Italy . The mitogenome is 18.143 bps in length and harbors 37 genes: 13 protein-coding, 22 tRNA, and two rRNA genes, as well as two non-coding regions, CR1 and CR2, following the GO-II gene order ; for H. r. rustica, they were from numerous sampling locations  with the highest variability in the two control regions . Consistent with previous results, the four haplogroups are included in two primary branches , and narrow or incomplete in those with CD mitogenomes .Phylogenetic analyses reveal that all 411 C, and D  online. branches  resultinbranches , this diFor all nodes in the phylogeny and the derived haplogroups and sub-haplogroups, we obtained age estimates both with maximum likelihood (ML) and Bayesian approaches. The estimates obtained with the two methods are very similar  online. According to our data, the female barn swallow carrying the HrAM lived 276.9 ± 24.3 kya, an almost three-fold age increase relative to earlier estimates . A resuln = 388) in our sample (H. r. rustica) as well as 46 of the 50 H. r. transitiva mitogenomes from Israel and one from H. r. gutturalis (no. 258) sampled in China . Three sub-haplogroups originated from its initial split, the largely predominant A1 and the rare A2 and A3, with the former harboring two very common sub-branches detected in all European locations as well as in Algeria  mitogenomes   haplogroup and was already differentiated into sub-haplogroups at the time of the initial rustica-transitiva split. Alternatively, rustica and transitiva maternal lineages underwent gene flow, possibly continuously over time. Finally, rustica and transitiva derive from the same ancestral population, but also admix; a process that is still going on, despite the (growing) differences in migratory behavior, moult strategy   online. strategy  and morpH. r. rustica, but it characterizes also H. r. transitiva: two subclades (2–3 haplotypes) with the oldest (A1a2e1a3) dating ∼11.6 ky  with the oldest (A1a1b3b) dating ∼11.4 ky; 20 in Italy (2–5 haplotypes each) with the oldest (A1a2g) dating ∼11.6 ky; one  in Switzerland dating ∼8.0 ky; two (2 haplotypes each) in Ukraine with the oldest subclade (A1a2e1a2a2) dating ∼7.6 ky; and one (3 haplotypes) in Poland (A1a2e1a1a2a1b) dating ∼6.0 ky. This feature is not exclusive to ∼11.6 ky  online.χ2  [24] = 10.276, 29.752 and 14.970; P = 0.0028, 0.0001 and 0.0032, respectively) compared to other European locations , A1a2e1a1a5 (∼11.5 ky), and A1a2f1b (∼11.1 ky) are over-represented in the Danish population . They suggest that long-distance dispersal between populations occurs, in agreement with observations concerning the behavioral flexibility and adaptability of the species .H. r. rustica populations and between H. r. rustica and H. r. transitiva can be at least in part explained when considering that even short-distance dispersal can lead to extensive and long-distance gene flow over the course of generations. Moreover, if the instances of long-distance dispersal from natal to breeding sites are confirmed, even at a low percentage, they would further speed up the loss of genetic structure in European barn swallow populations.While limited by the relatively small size of our population samples and restricted to haplogroup A mitogenomes, the complete or partial clustering of some sub-haplogroups of A would fit with the generally reported short-distance dispersal of offspring from natal to breeding sites, although this feature is less extreme in females compared to males , thus leH. r. transitiva mitogenomes already mentioned above and is dated at 18.9 ± 3.9 kya. It shares an ancestral node  with the sister haplogroup A, which is approximately 40 ky younger than the CD node (156.4 ± 18.0 kya) from which C and D derive.As for the remaining three major haplogroups, B, C, and D  online, H. r. gutturalis samples, four of the five sampled in China and is dated at 31.1 ± 5.7 kya, while the fifth is a member of haplogroup A. The detection of haplogroup A in the gutturalis individual might indicate past or present admixture with rustica, especially when considering that it was collected in the westernmost (Nujiang Prefecture) of the sampling locations in China, the closest to the breeding range of H. r. rustica.Haplogroup C includes only H. r. erythrogaster specimens from North America , in either one or the other of its sub-haplogroups (D1 and D2). Haplogroup D age provides a minimum time for the spread of H. rustica from Asia to the Americas and indicates that North America was most likely the nesting ground of the ancestors of H. r. erythrogaster since at least 51 ky ago.Finally, haplogroup D, dated at 51.1 ± 7.9 kya, characterizes all 15 ND2 and CYB gene variation of our mitogenomes with that reported in 119 barn swallow mtDNAs available from previous studies ND2 and CH. r. rustica and H. r. transitiva, with H. r. transitiva mitogenomes scattered among those of H. r. rustica in virtually all sub-haplogroups of A. Moreover, it reveals that the four haplogroup B mitogenomes observed in H. r. transitiva form a clade that is defined by the transitions at nps 14,235 and 14,243 in CYB. This branch encompasses also an additional H. r. transitiva specimen , the phylogeny confirms instead its complete subspecies specificity. It includes only H. r. gutturalis specimens: the four from China of this study (nos. 393–396), one from Japan, three from Russia, one from Mongolia  to haplogroup D: the 15 from Nebraska of this study (nos. 397–411), additional 14 from the USA . This was followed by a plateau throughout the LGM and up to the YD (12.9–11.7 kya), when the second increase began, lasting until the end of the Early Holocene Optimum (EHO) ∼6 kya (The Bayesian skyline plot of ) ∼6 kya .H. r. rustica ancestors expanding northward from the eastern Mediterranean basin, which might have acted as a refugium during the LGM and the YD. H. r. transitiva would then have mainly derived from specimens/populations that maintained their sedentary behavior, while H. r. rustica would descend from those that differentiated and re-acquired a long-distance migratory behavior while expanding northward at the end (∼11.7 kya) of the YD. These climatic changes, and possibly the increase in energy consumption associated with the re-acquisition of the long-distance migratory behavior, appear to strongly affect the extent to which selection modulates the evolution of mitochondrial PCGs . This is particularly evident for genes encoding subunits of OXPHOS complexes I and V, thus supporting scenarios linking heat production and avian flight ability with mitogenome variation . The oldest are in the Mediterranean area, A1a2g in Italy and A1a1b3b in Spain , Center , and North   close to 1 was detected, thus confirming the overall reduction of mitogenome variation from South to North, as expected in models envisioning a more recent origin of central and northern European populations.The chronological gradient from south to north suggests a history of northward expansion from the Near East or the Mediterranean basin at large. This is supported by the negative correlation between nucleotide diversity and latitude that we observed in Denmark)  online. Recolonization of Europe from refugia following glacial retreat has been documented in a variety of species . The patHirundo species most likely originated in Africa, as most of them have African distributions, including H. aethiopica and H. angolensis that are the closest relatives to H. rustica , thus supporting Africa as the ancestral continental source of H. rustica, and dates the divergence between H. rustica and H. aethiopica, the closest species, at about 493 kya  mitogenome is assessed and the sequencing survey is carried out on numerous specimens sampled throughout the species distribution range. Here we employed this “magnifying glass” approach to reconstruct the genetic history of an iconic species, the barn swallow. Mitogenome data allowed us to build a detailed phylogeny for the species, to determine its coalescence time as well as the ages of its haplogroups, and to better define the matrilineal relationships between subspecies.According to our data, the female barn swallow carrying HrAM lived 276.9 ± 24.3 kya, which is much earlier than previously thought . ConsideIn most cases, we observed complete subspecies and geographic specificity of mitogenome haplogroups, arising at different times and different places, which we employed together with estimated radiation times to postulate an overall model for the geographical and temporal spread of barn swallows . AccordiH. r. transitiva that extensively shares haplogroup A with the migratory H. r. rustica and haplogroup B, to a lesser extent, with the Egyptian H. r. savignii. We propose that rustica and transitiva derive from the same population source, which was located in the Levant and had adapted to sedentarism during the LGM. Our data indicate that the two subspecies began to split rather recently, shortly after 11.7 kya at the end of the YD. H. r. rustica would descend from individuals that re-acquired the long-distance migratory behavior while expanding northward to regions that were then becoming suitable as nesting grounds. In contrast, H. r. transitiva would derive from the Levantine component that remained in situ and maintained its sedentary behavior. Since then, however, transitiva did not remain genetically isolated, receiving genetic inputs and admixing with migratory rustica populations, as shown by the absence of significant correlations between genetic and geographic distances when assessing the shared haplogroups  was extrapolated from n = 340; H. r. rustica), Israel , China , and the USA (Nebraska) . H. r. rustica were sampled in Denmark (n = 31), Italy (n = 171), Poland (n = 35), Spain (n = 31), Switzerland (n = 20), Ukraine (n = 20), and Algeria (n = 32), ,  online. n = 32), , was perH. rustica mitogenomes in GenBank. However, none of these could be included in our analyses of entire mitogenomes due to the following sequence issues: KX398931 , MN829439 (H. r. rustica), MN830163 (H. r. savignii), MN954681, and MN840495 (H. r. transitiva), presence of NUMTs in ND5  from the PRJNA323498 BioProject (n = 22) could be employed in the phylogenetic analyses of ND2 and CYB gene sequences (see below).At the time when the analyses were performed, there were nine entire s in ND5 ; MN90972oProject . They alWe obtained genomic DNA extracted from muscle or liver with the ReliaPrep™  gDNA Tissue kit, using the standard protocol for animal tissue. We extracted and purified blood samples using phenol-chloroform. These samples were prepared by breaking 1–2 cm of the glass capillary containing the blood (∼4 μl) and placing it overnight at 56°C in a 2 ml tube containing: lysis buffer B , 250 µl of TBS buffer , and 40 µl of Proteinase K (20 mg/ml). Samples from Spain and Nebraska were instead extracted with magnetic beads on a 16 Maxwell® RSC 16 instrument using the dedicated Blood DNA Kit (Promega) and employing the “Blood DNA” protocol. Sample preparation in this case was performed by adding 1–2 μl of blood to 300 μl of lysis buffer and 30 μl Proteinase K and incubating overnight at 56°C. Genomic DNAs were eluted into TE buffer  or elution buffer (Promega).H. r. rustica mtDNA genes  available in GenBank using the Primer3Plus software (https://www.primer3plus.com/). Then, by using a primer walking approach, we designed a set of oligonucleotide pairs that allowed the amplification of the entire mitogenome in 11 overlapping Polymerase Chain Reaction (PCR) fragments  were then obtained by carrying out PCR reactions with a standard reaction mix (25 µl) containing 1X Buffer (1.5 mM MgCl2), 0.2 mM of each dNTP, 0.6U of GoTaq G2 Polymerase (Promega), 0.2 µM of each primer, and 20–30 ng of DNA, using the following PCR conditions: 94°C (2 min); 35 cycles of 94°C (30 s), 55°C (30 s), 72°C (2 min), and a final extension of 72°C (10 min). PCR products were visualized on a 1% agarose gel and amplicons were sequenced with standard dideoxy sequencing using BigDye v3.1 Chemistry (Applied Biosystems) on 3730xl and 3130xl Genetic Analyzer (Applied Biosystems) following the manufacturer’s protocol. Mitogenome no. 20 (MZ905359) was employed as the HrrRS. We then obtained the locus map of the barn swallow  were Sanger sequenced. We designed an initial set of oligonucleotide pairs (not shown) using partial sequences of software   and length (20 nt)   online tThe raw MiSeq sequencer data were output in BCL format, demultiplexed, and converted to FASTQ format with the Illumina® bash package, bcl2fastq2 Conversion Software v2.20, and trimmed with Trim Galore! v 0.6.4  to removHirundo angolensis (NC_050287) and Hirundo aethiopica (NC_050293) reference mitogenomes, and its topology was also confirmed by using the software Molecular Evolutionary Genetics Analysis X (MEGAX) (We built a maximum parsimony (MP) tree by manually programming the software mtPhyl v 5.003  for the  (MEGAX)  and 2 (K (MEGAX) .n = 411) coding-region sequences . We performed ML estimations using the BaseML package present in the PAMLX 1.3.1 software (Progne (not shown) and Hirundo at 9.34 Mya (95% CI: 5.8–13.2 Mya)  , thus em−8 base substitution per nucleotide per year (or one mutation every 2616 years), derived from the rate calculated in the ML method. The chain length was established at 100,000,000 iterations, with samples drawn every 1,000 Markov chain Monte Carlo steps after a discarded burn-in of 10,000,000 steps  2.6.0  under th00 steps . We perf00 steps  and ExceND2  and CYB  gene sequences (H. aethiopica (NC_050293) and H. angolensis (NC_050287) reference mitogenomes.To assess the subspecies specificity of major haplogroups in a wider sample encompassing also previously published sequences, we built a MP tree based on equences , for a thttps://www.tableau.com/).To identify a potential correlation between mitogenome diversity (entire mitogenomes) and latitude, we measured both haplotype diversity (HD) and nucleotide diversity (Pi) for the most represented haplogroups (A1 as a whole as well as its major sub-haplogroups A1a1 and A1a2) using DNAsp 6.12.03 . Both inhaplotypes (geodist R package (ade4 (stats package (ggplot2 (To assess isolation by distance (IBD), genetic distance matrices were created based on PhiSt, which was computed using the R package plotypes , while p package , applyin package . We test package . The Mange (ade4  with 999 package . We gene(ggplot2 .msac113_Supplementary_DataClick here for additional data file.


Supplemental Digital Content is available in the text. Postpartum depression (PPD) is a multifactor disorder caused by psychological, social, and also biological factors. 5-HTTLPR polymorphism in the promoter region of serotonin transporter gene seems to influence PPD onset. In this study, we examined the effect of 5-HTTLPR polymorphism on prenatal and postnatal symptoms of depression and posttraumatic stress in women.A longitudinal design with three points – time 1 (32–40 weeks gestation); time 2 (2 or 3 weeks after birth), and time 3 (3 months after birth) – was made. A total of 141 women were recruited during childbirth preparation courses. At time 1, women completed the Beck Depression Inventory (BDI) and the Los Angeles Symptoms Checklist (LASC). At time 2, they fulfilled BDI and Edinburgh Postnatal Depression Scale (EDPS), LASC and the Perinatal Posttraumatic stress disorder (PTSD) Questionnaire (PPQ); midwives and nurses collected biological test tubes by blood sampling for the genetic analysis. At time 3, the women were reassessed for BDI, LASC, EDPS, and PPQs. Analysis of variance and moderation analysis were used to correlate genotype and psychological investigations.Results showed that, compared with LL/LS genotypes, SS genotype moderated cognitive depressive symptoms onset at T2 and T3. Moreover, this genotype correlated, directly or indirectly, with PTSD postpartum aspects .Findings revealed that a lower expression of serotonin transporter gene, associated with SS genotype, seems to render women more vulnerable to depressive and PTSD symptoms after childbirth. Pregnancy has always been considered as one of the most awaited periods in a woman’s life; nevertheless, for some women, it could be combined with mild and severe disorders. Postpartum depression (PPD) is defined as an episode of major or minor depressive disorders encountered in women during the postpartum period. The Diagnostic and statistical manual of mental disorder (DSM)-IV-TR has labeled PPD ‘with onset in postpartum’ as a specific disorder that starts within 4 weeks after delivery, and in the DSM-5, it was extended to within 6 months. Literature demonstrated that PPD concerns 19.2% of women in the first 3 months, with a drop of 12.9% during the first year after birth  after childbirth. The estimated percentage of PTSD after a typical delivery ranges from 2.8 to 5.6% at 6 weeks postdelivery and 1.5% at 6 months , accordiIn summary, PPD and PTSD might be counted as severe mental health problems, and for these reasons, many studies have analyzed significant risk factors behind them. Notwithstanding this, the etiology of these disorders is not adequately explained, but it is hypothesized that both environmental and biological factors may play a part. Despite several associations were highlighted with different polymorphism, most studies have focused their attention on serotonin (5-HT), a fundamental neurotransmitter in the central nervous system  plays a central role in the reuptake of the serotonin  at 32nd to 40th weeks gestation, time 2 (T2) at 2/3 weeks after labor, and time 3 (T3) at 3/4 months. Ethical approval was obtained from the Ethical Commission of the Catholic University of the Sacred Heart of Milan. We recruited women throughout birthing classes within the 32nd and 40th weeks of gestation in one hospital of northern Italy. Participants completed several questionnaires assessing PTSD and depression postpartum.The inclusion criteria to participate in the study were as follows: an adequate knowledge of the Italian language, an age 18 years upward, and Caucasian ethnicity.One hundred fifty-five participants joined the research. One hundred forty-one women constituted the total sample at T1. At T2, the number of women active in the research was 127, and at T3, 110 women.The frequencies of the genotypes were as follows: 36 for LL, 57 for LS, and 19 for SS.M = 33.37, SD = 4.64), who are involved in all the three phases of the study. An estimated 81.7% had a stable occupation, and 84.5% had a medium-high level of education. Of the women participants, 77.7% were maiden, 14.6% were married, 6.8% were common-law wives, and 1% divorced. The majority of them were primipara (87.3). In total, 70.6% women had spontaneous natural childbirth without epidural analgesia, 14.7% planned cesarean, and 14.7% of women had diastolic delivery, while 11.7% had complications during pregnancy. At birth, most of the newborns were healthy: the Apgar score at minute one after delivery was 9.67 (SD = 0.89) while at 5 min was 9.93 (SD = 0.29). The dropout rate of the study is 19%.The age of the participants was 23–42 years  = 2.39, P = 0.301; civil status χ2 (2) = 2.43, P = 0.296; education χ2 (2) = 3.07, P = 0.216; number of child χ2 (2) = 2.96, P = 0.227; type of birth χ2 (2) = 1.18, P = 0.552.Supplementary Table S1, Supplemental digital content 1, http://links.lww.com/PG/A257 presents the descriptive statistics for PPD and PTSD among the different phases of the research.Supplementary Table S2, Supplemental digital content 1, http://links.lww.com/PG/A257 present the descriptive statistics for PPD and PTSD divided by genotype’s groups and phases.Supplementary Tables S3, S4, and S5, Supplemental digital content 1, The first phase of this research (T1) was conducted during childbirth preparation courses between the 32nd and 40th week of pregnancy. Participants filled out questionnaires assessing their demographic characteristics, depression  (The second phase (T2) took place weeks after delivery. Questionnaires assessing childbirth’s characteristics were administered: BDI and Edinburgh Postnatal Depression Scale (EDPS) (In the third phase of the research (T3) at 3 months after childbirth, the participants were reassessed with the BDI, the EDPS, the PPQ, and the LASC in order to re-evaluate target symptoms of depression and PTSD.α = 0.865; T2-Cronbach α = 0.898; T3-Cronbach α = 0.597).The BDI is a self-assessment questionnaire focusing on cognitive, motivational, affective, and behavioral symptoms of depression .The EDPS is a self-report tool consisting of 10 items with a four-step Likert scale response (0–3) .The LASC is a self-report instrument assessing the presence of PTSD symptoms, according to DSM-IV .The PPQ is a measure that evaluates specifically the distress of women who are experiencing perinatal symptoms of posttraumatic distress. It is composed of 14 items ranged on a five-point scale from 0 points  to 4 points (very often). Scores of 19 or higher indicate significant distress , according to the manufacturer’s instructions. PCR analysis was carried out as described previously  was used to test the equilibrium in the sample. A test of goodness of fit was also performed to highlight any possible statistical differences between groups. Third, the assumptions of normality of the variables were verified by analyzing asymmetry, kurtosis, and normality graphs. Inferential tests, such as the Kolmogorov–Smirnov or the Shapiro–Wilk test, were also used. Preliminary analysis showed a non-normal distribution of some variables. In particular, the following variables were found out of normality: the BDI somatic scale at T2, the cognitive scale and the total at T2 and T3; the EDPS score at T3; the LASC intrusive symptoms, hyperarousal, severity and distress at T2 and the avoidance symptoms at T2 and T3; the category B symptoms of PPQ at T2. For this reason, logarithmic transformations (Log10) were used. Finally, Univariate analysis of variance (ANOVA) and moderation modeling were performed using the statistical software SPSS 20 and a macro-program PROCESS 2.1.ANOVA analysis and moderation models had as dependent variables clinical symptoms and as independent variables or moderators, the genotype coded on three levels .P > 0.05).Descriptive statistics for PTSD and depressive symptoms are presented in the online resource Table . 5-HTTLPP > 0.05). In particular, at T1, we did not find any significant effect for the total score of BDI-II, F (2.109) = 0.853, P = 0.429, η2P = 0.015. Similarly, at T2 for the EDPS score, F (2.106) = 0.336, P = 0.716, η2P = 0.006; the BDI-II somatic scale, F (2.107) = 1.145, P = 0.322, η2P = 0.021; the BDI-II cognitive scale F (2.106) = 0.057, P = 0.945, η2P = 0.001 and the BDI-II total F (2.106) = 0.392, P = 0.677, η2P = 0.007. Also, at T3, there was not any significant effect for the EDPS score, F (2.103) = 2.366, P = 0.099, η2P = 0.044; the BDI-II somatic scale, F (2.95) = 0.450, P = 0.639, η2P = 0.009; the BDI-II cognitive scale F (2.103) = 0.018, P = 0.983, η2P = 0.000 and the BDI-II total F (2.103) = 0.559, P = 0.574, η2P = 0.011.A univariate ANOVA was carried out to verify the effect of the genotype on depressive symptoms at T1, T2, and T3. Results did not show any significant effect for the different phases (F (2.107) = 3.597, P = 0.03, η2P = 0.063 and with hyperarousal symptoms at T3 evaluated with PPQ, F (2.90) = 3.343, P = 0.040, η2P = 0.071. Post hoc with LSD test indicated that the mean score of SS genotype was higher at T2  and T3 .Results showed that there was a significant effect of the genotype on intrusive postpartum symptoms evaluated with LASC at T2, F (5.90) = 3.19, P = 0.01, and the interaction for a significant 8% of the variance F (2.90) = 4.30, P = 0.016. Unstandardized regression coefficients and SEs are presented in Table Interaction between cognitive symptoms at T2 and T3 was found. The overall moderation model accounted for a significant 38% of the variability in depressive scores, R2 = 0.38, b = 0.7426, SE = 0.2077, P = 0.006).Simple slopes analyses were then performed. A significant, positive relationship was identified between SS genotype and cognitive symptoms of depression at T3  = 4.07, P = 0.002, and for 10% in the variance of the interaction F (2.89) = 5.97, P = 0.03, R2 = 0.10.The overall moderation model accounted for a significant 43% of the variability in PTSD scores, R2 = 0.43, b = 0.6714, SEb = 0.1601, P = 0.001).It was demonstrated that only the SS genotype predicted the intrusive symptoms at T3  = 4.47, P = 0.01, R2 = 0.18 and of the interaction explaining the 4.5% (DR2 = 0.0456) of the variance. Only the SS genotype predicted the intrusive symptoms of the postpartum .The regression result showed the significance of the general model F (5.89) = 9.84, P = 0.00, R2 = 0.59. The interaction was statistically significant F (2.89) = 11.10, P = 0.00, R2 = 0.16 adding 16% of the variance (DR2 = 0.016). In particular, the SS genotype was significant in predicting avoidance symptoms at 3 months.For avoidance symptoms, the first set found an interaction between symptoms at T1 and T3, F (5.87) = 4.56, P = 0.001, R2 = 0.45 and the interaction F (2.87) = 3.81, P = 0.02, R2 = 0.06 were significant. Unstandardized regression coefficients and SEs are presented in Table Finally, the third set of regression evaluated the relationship between symptoms at T2 and at T3. The general model b = 0.4134, SE = 0.1243, P = 0.013) and the LS genotype  were significant in predicting avoidance symptoms at 3 months.In particular, the SS genotype  = 6.31, P = 0.00, R2 = 0.51 and the interaction F (2.89) = 3.13, P = 0.04, R2 = 0.05 were significant. In particular, the model showed how the SS genotype  predicted the symptoms of hyperarousal at T3.The general model Pregnancy and the postpartum period are essential phases in women’s life delineated by biological and psychological changes. The main aim of our study was to examine the association of 5-HTTLPR polymorphism and postpartum disorders, directly and indirectly. In particular, we explored the influence of 5-HTTLPR polymorphism on depressive and PTSD symptoms immediately and at 3 months after childbirth. Compatible with previous research, we found no association between the polymorphism and the depressive symptoms .As other 5HTTLPR polymorphic lengths were reported in the literature  . Previous studies, in fact, showed how SS genotype might trigger the appearance of intrusive thoughts, with vain attempts by subjects to suppress them . Taking et al., 2002). By using noninvasive neuro-imaging sensitivity such as functional MRI, Hariri et al. (2002) evaluated neural activation in subjects with SS genotype during perceptual processing of facial expressions such as fear and anger. During the task, individuals with SS genotype showed almost five times more amygdala activity than subjects with LL genotype. However, the precise molecular and cellular basis of the effects of 5-HTTLPR on amygdala reactivity remains to be determined.Further, literature had also confirmed an association between SS genotype and hyperarousal symptoms in the postpartum period, as identified in our study. Further investigations are necessary for the postpartum period to better understand the association between posttraumatic symptomatology and 5-HTTLPR polymorphism. Moreover, it would be of interest to investigate also other polymorphisms that have been demonstrated to play a role in PTDS (not related to delivery). To our best knowledge, this is the first study in which a correlation between PTSD (observed during the postpartum period) and genetic polymorphism (5HTTLPR) has been found. It is known that PTSD is most significantly  correlated with major depression, but also with schizophrenia, both of which have genome-wide significant loci implicated in brain function (Our findings offer a preliminary contribution to the growing body of research on the influence of 5-HTTLPR on postpartum disorders and might have implications for the development of intervention programs to prevent women’s mental health during childbirth and the postpartum period.There are no conflicts of interest.


The highest concentrations of macrominerals were observed in Ca, K, P, and Na, and trace minerals in Fe, Zn, Mn, and Cu. Certain homogeneity only to trace mineral contents was observed when analyzing inter-batch values from same manufacturers. In general, all phase 1 and phase 2 infant formula brands and batches met or exceeded Fe, Zn, Cu, Mo, and Se contents when compared to maximum limits established by Codex Alimentarius. In addition, Zn contents in eight phase 1 and in four phase 2 infant formulas were above the contents established by the tolerable upper intake level for children aged 0–6 and/or 7–12 months, respectively. These findings highlight the need to expand regular infant formula inspection concerning nutritional quality, as some composition aspects of these foods must be improved to follow international guidelines, since ideal requirements for infant formula composition, quality, and safety interfere in child development and adult health.Infant formulas are the main nutritional source for infants when breastfeeding is not possible or recommended. The daily need for specific nutrients, such as essential minerals, in early stages of a child's life is high because of rapid infant growth and development, which impose metabolic flux increases on these pathways to support growth, physical activity, and defense against infections. In this context, this research aimed to determine macromineral and trace mineral contents in starting (phase 1) and follow-up (phase 2) infant formulas marketed in Brazil ( Breast milk is indisputably the ideal food for infants due to its specific nutritional characteristics and optimal nutrient balance, being always available at the ideal temperature, and protection from pathogen contamination, while also displaying numerous immunological and psychological advantages in reducing infant morbidity and mortality , 2. BecaBreast milk, almost complete food and a primary nutrient source for infants and children, is composed of water, macronutrients such as fats, carbohydrates and proteins, and micronutrients, including vitamins and minerals . The maiCodex Alimentarius standards to complement or replace breast milk is the best alternative. Most commercialized infant formulas use cow milk as the main basic raw material, since it is easy to obtain and has a low cost compared to milk from other species . Because of ethical reasons, manufacturers and brands are not disclosed, and samples are coded, where brands are represented by two capital letters, followed by the recommended phase (1 or 2), and batches of each brand are identified using capital letters  all infant formulas analyzed should be composed essentially of cow milk; (2) unhydrolyzed cow milk proteins; (3) with lactose as the main carbohydrate, and (4) supplemented with DHA and ARA for children aged 0–6 months (phase 1 starting formulas) and for children aged 6–12 months (phase 2 follow-up formulas). Infant formulas containing protein sources other than cow milk, such as soy or wheat protein, and infant formulas designed for specific needs, such as lactose-free or hydrolyzed, were not included in this study. Considering the products sold in the Brazilian market that meet the inclusion criteria, 10 formulations produced by three different manufacturers were evaluated. Thus, five phase 1 and five phase 2 formulas comprising three distinct batches of each brand were selected, totaling 30 samples  was obtained from a Milli-Q® INTEGRAL 10 system . Nitric acid  was purified by sub-boiling followed by distillation in a quartz still . The multi-element standard solution Merck IV , comprising 29 elements in diluted nitric acid, was employed to prepare standard analytical curves. Two reference materials, certified Skimmed Milk Powder ERM®-BD150  and Milk Powder 1549  were used to assess method accuracy.Essential mineral concentrations were determined according to de Oliveira et al. , followi−1 were used to assess linearity. The respective correlation coefficients are in the range 0.97–1. LOD (limit of detection) and LOQ (limit of quantification) were determined for all minerals according to the Brazilian National Institute of Metrology, Quality and Technology—INMETRO  Skimmed Milk Powder ERM®-BD150  and Non-Fat Milk Powder 1549  and a five-point calibration curve ranging from 100 to 1,000 mg·L—INMETRO , as LOD The daily intake of minerals depends on both concentration of the elements in food matrix and daily food consumption. Estimated daily intake (EDI) is a concept introduced to consider these factors. Therefore, we used the following equation to estimate whether the macromineral and trace mineral intakes of phase 1 and phase 2 infant formulas are adequate for infants from 0 to 6 months and 7 to 12 months old, respectively:m is the average concentration of each element in the analyzed batches of infant formula determined by ICP-MS expressed as mg·100g−1 of formula powder; Cd is the recommended daily consumption amount stated on the label of each phase 1 and phase 2 infant formula (suggested amount to be consumed expressed in grams per day). The approximate consumption per day is described in where EDI represents the estimated daily intake of macrominerals and trace minerals, expressed as mg per day for all minerals; CThese estimated daily intakes were compared to the values of dietary reference intake (DRI) recommended by the Institute of Medicine . For infCodex Alimentarius  was performed when a significant F was found. Results were considered significant when p < 0.05. Data were expressed as means ± standard deviations (SDs). All statistical analyses were carried out using the GraphPad Prism software version 5 for Windows® .Significant differences in infant formula macromineral and trace mineral contents and their respective batches were assessed by two-way analysis of variance (ANOVA) with repeated measures. An additional Since infant formulas are similar to milk powder, the accuracy of the applied methodology was verified by analyzing Skimmed Milk Powder ERM®-BD150 and Non-Fat Milk Powder 1549 CRMs (results are shown in p < 0.01) among all the infant formulas was observed. The highest trace mineral concentrations were observed for Fe, followed by Zn, Mn, and Cu, while trace minerals detected at lower concentrations comprised Co, followed by Cr, Mo, I, and Se. The highest macrominerals concentrations were observed for Ca, followed by K, P, and Na. In addition, comparing the macrominerals in infant formulas, Mg presented the lowest concentration. In general, samples ME2 and NN2 contained the highest Ca, K, P, and Na concentrations, while NC1, DM1, and DA1 contained the lowest Ca, Na, K, P, and Mg concentrations. Furthermore, no significant difference for Mg concentrations was observed between samples NC1 and NC2, NN1 and NN2, and DA1 and DA2, and for K concentrations in samples DA1 and DA2. Also, no significant difference in P concentrations was observed between samples DA1 and DA2.Average macromineral  and trace mineral  contents in phase 1 and phase 2 formulas were highly variable for most minerals . No signNo inter-batches and brand differences were observed in almost all the trace elements, except for Fe, Zn, and Cu, where inter-batch and brand variations  were obsCodex Alimentarius  and I in ME1 , DM1 , DA1 , and DA2  were not compliant with recommendations.When the minimum and maximum or guidance upper level contents recommended by current legislations were compared with the detected macromineral and trace mineral concentrations , only Se·100g−1) are shown in ·g−1. Then, the EDI of each mineral content was calculated by considering the daily consumption recommended by the manufacturers and included on the can labels (·day−1), K (365.7–522.5 mg·day−1), and P (219.1–372.8 mg·day−1) was highest when compared with the other determined macrominerals of the phase 1 infant formulas. The highest average intake macrominerals observed in the phase 2 infant formulas were the same as that in the phase 1 infant formulas. Furthermore, the estimated average intake of Mg , Na , K , Cu (in ME2C), and I and Mn  was lower than recommendation values for AI/EAR (7–12 months).Mineral daily intakes considering the reference values of the Institute of Medicine  recommenn labels . The est·day−1. However, Zn concentrations in half of phase 1 and in four phase 2 infant formulas exceeded the tolerable limit of 4 mg·day−1 (0–6 months) and 5 mg·day−1 (7–12 months), respectively. The same results were noted for Se, where two phase 1 and three phase 2 infant formulas exceed the tolerable limit of 0.045 mg·day−1 (0–6 months) and 0.06 mg·day−1 (7–12 months).Despite being essential at low concentrations, trace minerals, such as Fe, Zn, and Se, can be harmful when daily intakes exceed recommendations. Despite growing evidence on benefits of breastfeeding on health, psychosocial, and societal aspects, current breastfeeding rates worldwide are far from optimal . Health-−1 Ca; 169 ± 17.1, 184 ± 44.6, and 192 ± 45.9 μg·ml−1 Na; 8.49 ± 1.21, 6.27 ± 2.36, and 9.3 ± 0.46 μg·ml−1 Fe; 3.49 ± 0.28, 3.21 ± 0.89, and 3.66 ± 0.79 μg·ml−1 Zn; 0.41 ± 0.06, 0.4 ± 0.14, and 0.49 ± 0.09 μg·ml−1 Cu; 0.06 ± 0.02, 0.068 ± 0.024, and 0.09 ± 0.04 μg·ml−1 Mn contents in milk-based powder infant formulas marketed in Nigeria, the United Kingdom, and the United States. The authors did not, however, evaluate K and P contents. In another assessment, Lesniewicz et al. (−1), P (ranging from 190 to 539 mg·100g−1), Fe (ranging from 3.5 to 7.4 mg·100g−1), Zn (ranging from 1.6 to 12 mg·100g−1) Cu (ranging from 0.068 to 0.502 mg·100g−1), and Mn (ranging from 0.026 to 0.374 mg·100g−1) as the most abundant macrominerals in milk-based powder infant formulas marketed in Poland and Spain. These authors did not, however, evaluate Na, K, Cu, and Mo contents. Furthermore, our results are similar to those reported by Moreno-Rojas et al. (−1), K (710.6 ± 137.4 mg·100g−1), P (294.1 ± 57.1 mg·100g−1), and Na (153.4 ± 43.6 mg·100g−1) as the most abundant macrominerals, and Fe (6.98 ± 3.04 mg·100g−1) and Zn (3.19 ± 0.86 mg·100g−1) as the most abundant trace minerals.Concerning the detected macrominerals, Ca contents were highest, followed by K, P, and Na. Regarding trace minerals, highest concentrations were observed for Fe, followed by Zn, Mn, and Cu. Macromineral and trace minerals contents were similar to those reported by Ikema et al. , of 385 −1 and 0.0007 mg·100g−1, respectively) were even higher (−1) reaches almost four times higher than in human milk (22–41 mg·100g−1), to fulfill the needs of calves that grow much faster and require more Ca to support growth (−1) would have to consume a significant extra amount of cow milk per day. Therefore, because of high and low concentrations of some minerals in cow milk, infant formula manufacturers try to adjust the concentrations to resemble breast milk and better meet child nutritional needs.Furthermore, all macrominerals and trace minerals contents found in infant formulas were higher than those found in human milk, whereas those for Mn  lower than the recommended limit . Furthermore, brands ME1, DM1, DA1, and DA2 presented an I value  lower than the recommended limit , respectively. The Se value detected in the DM2 brand  was 222% above the guidance upper levels (GUL) of 0.009 mg·100 kcal−1. The excess Se observed in this sample reflects the value observed in the calculation of the daily intake, in which Se content was shown to be above the established tolerable limit. Furthermore, a higher value than the maximum tolerable intake level was noted when calculating the daily intake for Se and comparing it with the reference value , respectively. Furthermore, infant formulas NC1A, NC1C, DM1A, DM1B, DM1C, DA1B, NC2A, NC2B, NN2A, NN2B, NN2C, DM2A, DM2B, DM2C, DA2A, DA2B, and DA2C presented K contents below the AI recommendation (400 mg·day−1 for 0–6 months and 700 mg·day−1 for 7–12 months). Breast milk is capable of adequately nourishing children in the first 6 months of life; therefore, phase 1 infant formulas must be nutritionally similar to breast milk to meet the biochemical and physiological needs of a developing child (This study verified whether consumption of infant formulas would meet or exceed the AI or EAR (Fe and Zn) for macrominerals and trace minerals analyzed. Comparing the EDI resulting from the consumption of the formulas with the reference values recommended by the Institute of Medicine , we idenng child . Howeverng child . TherefoAs observed and mentioned above, the values of K and I essential trace minerals were found to be below the recommended values for most phase 1 infant formulas. The mineral I is essential in the metabolism of thyroid hormones (thyroxine and triiodothyronine) and helps regulate basal energy metabolism and reproduction. A deficiency of I can cause severe and irreversible mental retardation, deaf-muteness, congenital anomalies, as well as the most visible clinical manifestation, such as an enlarged thyroid gland, forming a goiter . Further−1 in both the phase 1 and phase 2 formulas. However, Zn exceeded the established UL in phase 1  and phase 2  infant formulas of 4 and 5 mg·day−1, respectively. In addition, Se exceeded the established UL in phase 1 (in DM1C and DA1B) and phase 2  infant formulas of 0.045 and 0.06 mg·day−1, respectively. Long-term Zn intake over the optimal concentration can interfere in the metabolism of other trace minerals, such as Cu, reducing their absorption and causing anemia, in addition to affecting the immune system ' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.CCA and PR carried out the experiments, interpreted the data, investigated previous studies for discussion, and drafted the manuscript with support from DB and RH-D. TS, MC, KL, VP, and CC-J supervised the project and critically reviewed and corrected the manuscript. All authors contributed to the article and approved the submitted version.The authors are thankful for the financial support provided by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) Brazil, process numbers [E-26/203.049/2017 and E-26/200.891/2021] and FAPERJ, PDR-10, process number [E-26/202.254/2018], Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), process number [311422/2016-0], and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Brazil, finance code 001.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Monascus pigments (MPs) have been used in the food industry for more than 2,000 years and are known for their safety, bold coloring, and physiological activity. MPs are mainly yellow (YMPs), orange (OMPs), and red (RMPs). In this study, a mutant strain Monascus purpureus H14 with high production of water-soluble YMPs  was generated instead of primary YMPs (λmax at 420 nm), OMPs (λmax at 470 nm), and RMPs (λmax at 510 nm) produced by the parent strain M. purpureus LQ-6 through dual mutagenesis of atmospheric and room-temperature plasma and heavy ion beam irradiation (HIBI), producing 22.68 U/ml extracellular YMPs and 10.67 U/ml intracellular YMPs. WSYMP production was increased by 289.51% in optimal conditions after response surface methodology was applied in submerged fermentation. Application of combined immobilized fermentation and extractive fermentation improved productivity to 16.89 U/ml/day, 6.70 times greater than with conservative submerged fermentation. The produced WSYMPs exhibited good tone stability to environmental factors, but their pigment values were unstable to pH, light, and high concentrations of Ca2+, Zn2+, Fe2+, Cu2+, and Mg2+. Furtherly, the produced exYMPs were identified as two yellow monascus pigment components (monascusone B and C21H27NO7S) by UHPLC-ESI-MS. This strategy may be extended to industrial production of premium WSYMPs using Monascus. Monascus pigments (MPs) are polyketide secondary metabolites produced by the genus Monascus and have been used as natural edible colorants in Asian food for thousands of years (Monascus pigments (WSYMPs) and extracellular YMPs (exYMPs) have been studied extensively by researchers.of years . Generalof years . It has of years , improviof years , with grof years . Water-sMonascus genus has been widely used. The addition of Triton X-100 can change the mycelium morphology without harming cell growth in the EF of M. anka GIM 3.592, and increases exYMP production  and ARTP. Response surface methodology (RSM), EF, and immobilized fermentation (IF) were used to increase WSYMP yield and productivity.Generation of a mutant strain is difficult in industrial WSYMP production using Monascus purpureus LQ-6  was isolated from red mold rice obtained from a local market  as the parent strain. The mutant strain M. purpureus H14 was obtained through dual mutagenesis of ARTP and HIBI. Analytical-grade chemicals were obtained from Sangon Biotech Co., Ltd. . Chemical reagents of vitamins were purchased from Coolaber . Potatoes used to produce potato dextrose agar (PDA) medium were purchased from a local market .Monascus purpureus strains were cultured on PDA medium  for 7 days at 30°C in darkness. The mycelium on a PDA Petri dish containing a fully grown culture of M. purpureus strain was aseptically scraped and inoculated in a 100-ml conical flask containing 20 ml of sterile water and glass beads, and agitated for 3 min to prepare the spore suspension; the concentration was adjusted to approximately 107 spores/ml.12C6+, 80 MeV/u, and 20 keV/μm) at the Heavy Ion Research Facility in Lanzhou (HIRFL), Institute of Modern Physics, Chinese Academy of Sciences. The spore suspension (1.5 ml) was transferred into a 35-mm irradiation dish. The detailed steps were reported by Gao et al. The irradiation dose was set as 0, 50, 100, 150, 200, 250, and 300 Gy  was inoculated in a 250-ml conical flask containing 45 ml of screening fermentation broth , incubated for 10 days at 30°C, and agitated at 150 rpm in the dark.The spore suspension  was inoculated in a 250-ml conical flask containing 45 ml of fermentation broth , incubated for 10 days at 30°C, and agitated at 150 rpm in the dark.Conventional submerged batch fermentation (SBF) was conducted as follows: 5 ml of the spore suspension  using RSM to construct the extractive SBF.Recent studies  have revM. purpureus H14 were immobilized in different ratios of sodium alginate (SA) and polyvinyl alcohol (PVA) solution  instead of in 3% (wt/wt) sodium alginate solution.Immobilized fermentation was performed according to the method in our previous study, with several modifications . The spoM. purpureus H14 were determined in a 1,000-ml conical flask containing 300 ml of optimized fermentation broth. During fermentation, 10-ml samples were collected to measure metabolite concentrations.The kinetics of SBF and repeated-batch fermentation (RBF) of 4•7H2O, MnSO4•H2O, malt extract, and vitamin B5) to enhance WSYMP production after fermentation of M. purpureus H14. The Box–Behnken design (BBD) was used to optimize the components. The four independent variables used in the experimental design were ZnSO4•7H2O (0.1–0.5 g/L), MnSO4•H2O (0.05–0.2 g/L), malt extract (5–15 g/L), and vitamin B5 (0.3–0.5 g/L); three levels were used to optimize the fermentation process.Response surface methodology was used to optimize four conventional SBF broth components  in a constant-temperature incubator (adjusted to 30°C) with incandescent LED lamps; the group left in darkness (0 lx) was used as the control. WSYMP solution (5 ml) was treated in an electro-thermostatic water bath at different temperatures (25°C–90°C) to evaluate the pH, metal ions, temperature, and visible light stability.The pH of the WSYMP solution and ankaflavin-methanol solution  was regulated from 1 to 14 with 1 mol/L HCl or NaOH. A full-band scan was performed to determine the maximum absorption wavelength and measure the WSYMPs with different concentrations (1–100 mmol/L) of metal ions added to the WSYMP solution, including NaMonascus pigments were detected by ultra-high-performance liquid chromatography (Dionex Ultimate 3000 UHPLC). The mobile phase was 0.1% formic acid (20%) and methanol (80%) at a flow rate of 0.5 ml/min, with separation carried out using an Eclipse Plus C18 column  at 30°C, run time for 15 min and 5 μl of injection volume. Mass spectrometry was carried out by Ultimate 3000 UHPLC-Q Exactive  equipped with an HESI source. The parameters were set as follows: 10 ml/min of aux gas flow rate, 40 ml/min of sheath gas flow rate, capillary temperature at 300°C, 3.8 kv of capillary voltage, and 50% S-Lens. Each sample was analyzed in positive modes with a mass scan range of 100–800 m/z, and resolution was set at 70,000.Yellow The concentrations of glucose, biomass, and YMPs were measured according to our previously described methods ,b. The rF test was applied to evaluate the effect of independent variables on the RSM; the significant results were identified by a value of p < 0.05; the fitness of second-order model was evaluated by multiple correlation coefficient (R2) and adjusted R2(R2adj.). Each experiment was repeated at least three times; the numerical data are presented as the mean ± SD.For the data analysis, M. purpureus LQ-6 was 89.64% (approximately 90%) at an irradiation dose of 200 Gy. Over 250 Gy, the fatality rate exceeded 95.47%; at 300 Gy, the active M. purpureus LQ-6 spores were almost completely killed. According to modern theory of breeding andFrom e target . Thus, ae target . It remaT = 160 s (T = 180 s) in our previous study  by SBF in the screening fermentation broth, which was approximately 3.47 times greater than the concentration produced by the mutant strain M. purpureus H1. However, the primary YMPs (λmax at 420 nm), OMPs (λmax at 470 nm), and RMPs (λmax at 510 nm) were produced by the parent strain M. purpureus LQ-6 in these conditions. The ratio of exYMPs to inYMPs was 7.81, indicating that mutant strain M. purpureus H14 with high production of exYMPs was obtained through dual mutagenesis of HIBI and ARTP.From the survival rate curve for mutant strain H1 by ARTP, the mortality rate reached 90.28% at  = 160 s , which wus study , illustrMonascus has been reported to be significantly affected by glucose concentration , and the pH of the conventional SBF broth was adjusted from 2.8 to 6.8 (the raw pH was 3.8).It is known that fermentation parameters, including substrate concentration and pH, play a vital role in targeted product biosynthesis. Pigment production by the genus ntration . The coms in SBF . Based ovia SBF. The total YMP production and the ratio of exYMPs to inYMPs (exYMPs/inYMPs) in SBF for M. purpureus H14 were significantly affected by regulating the original glucose concentration and pH. YMP production and the exYMPs/inYMPs ratio were increased by 35.17 and 109.86% (to 45.08 U/ml and 4.47), respectively, when the initial glucose concentration was 20 g/L, compared to a raw concentration of 50 g/L (33.35 U/ml and 2.13). + and intracellular enzyme activity (M. purpureus and the biosynthesis of ankaflavin (a type of YMP) were favored at low pH (4.0); the growth and biosynthesis of other pigments were independent of pH , and MnSO4•H2O (0.1 g/L) significantly upregulated YMP production . In order of importance to YMP production, these can be ranked as malt extract (a representative nitrogen source), ZnSO4•7H2O, MnSO4•H2O , and vitamin B5 (representative of growth cofactors). In addition, we found that the ratio of exYMPs to total YMPs was generally approximately 70%, which was equal to the ratio of exYMPs/inYMPs at 2.33 . From the glucose and pH regulation findings, we confirmed that M. purpureus H14 was a high-production exYMP mutant strain, possibly due to the high cell permeability or the hydrophilia of the YMPs (WSYMPs). Thus, an increase in YMP production by RSM was imminent.It is known that RSM can effectively increase the yield of a target product using liquid-state fermentation (or submerged fermentation). YMP production in this study using Monascus spp. has increased. Our previous study showed that the total MPs and exMPs were increased by 33.38 and 150.32%, respectively, via SBF of M. purpureus  and parameters (Xi) in each experimental run; the following multiple nonlinear regression model , 29 experimental runs were conducted in this study , and theon model  was geneY represents the response values of YMP production, and X1, X2, X3, and X4 represent ZnSO4•7H2O, MnSO4•H2O, malt extract, and vitamin B5, respectively.where F test analysis of variance; the associated p-values were lower than 0.001 for YMPs production, indicating that the developed model and the terms were statistically significant; besides, the value of lack of fit was 0.0704, higher than 0.05, demonstrated the precision and the accuracy of the constructed models; what is more, the data show that MnSO4•H2O and malt extract were the principal factors on YMPs production (R2), adjusted R2 (R2adj), predicted R2 (R2pred), and coefficient of variation (CV%). In this study, the results of ANOVA for RSM quadratic model showed that the value of R2 was 0.9842 for YMPs production and R2adj was 0.9683, which was high and significantly close to the R2 value. These results indicated that the predicted model was appropriate. In addition, the value of R2pred (0.9138 for YMPs production) was in reasonable agreement with that of R2adj, which also demonstrated that the predicted values of YMP productions were accurate. Furthermore, adequate precision (Adeq. Pre.) of 24.113 indicated an adequate signal (greater than 4 is desirable); this model can be used to navigate the design space. Thus, from 4•7H2O, MnSO4•H2O, malt extract, and vitamin B5 were determined as 0.32, 0.1, 12.29, and 0.4 g/L, respectively. In optimized fermentation conditions for 10 days, triplicate experiments resulted in a mean YMP production of 104.67 U/ml, which was 98.26% of the predicted value. YMP production and exYMP production increased by 213.85 and 289.51%, respectively, using RSM, and the ratio of exYMPs/inYMPs increased from 2.13 to 5.41. These results indicate that RSM dramatically increased exYMP production; the yield and productivity of exYMPs were 4,417 U/g (glucose) and 8.83 U/ml/day. Subsequently, IF and EF were performed together to increase exYMP productivity.The statistical significance of the regression model equation was evaluated by the oduction . Based oMonascus pigments in submerged fermentation of Monascus spp.; the exMPs increased by 127.48% with the addition of 3 g/L Triton X-100 in submerged fermentation of M. purpureus DK  in SBF and IF; subsequently, the biosynthesis rate significantly increased in IF compared to that in SBF, with a maximum exYMP production of 139.75 U/ml at 288 h (a productivity of 11.64 U/ml/day) and 104.13 U/ml at 300 h (a productivity of 8.33 U/ml/day), respectively. In RBF without the addition of Triton X-100 for five cycles, the mean glucose consumption rate and exYMP productivity were 8.23 and 10.28 U/ml/day, respectively, not including the first batch  in the last cycle. An exYMP production of 152.08 U/ml was obtained, and the exYMP productivity was almost constant (16.89 U/ml/day). The exYMP yield and productivity increased by 570.55 and 570.04%, respectively, compared with SBF after RSM and RBF (22.68 U/ml and 2.27 U/ml/day).As shown in st batch . Howeverst batch . SimilarMonascus strains via SBF. As shown in 2O2 and dithiothreitol). The highest exYMPs production was 209 U/ml in submerged fermentation by M. ruber CGMCC 10910 after optimizing the amount of H2O2 added and the timing of the addition, with 17.42 U/ml/day of productivity  and pH (4–7) stability for tone and value  of Ca2+, Zn2+, Fe2+, Cu2+, and Mg2+. However, when the concentration reached 10 mM, the pigment value decreased by 32.60, 32.16, 36.35, 35.03, and 25.30%, and 52.62, 50.63, 50.90, 55.97, and 43.43%, respectively, at a concentration of 100 mM. The pigment tone changed to green when the Cu2+ concentration exceeded 50 mM; the YMPs solution with the addition of other metal ions maintained a yellow tone  was 1.58, 1.95, 2.16, 2.55, 7.16, and 7.59 min, respectively. However, the peaks with tR of 2.55, 7.16, and 7.59 disappeared in the WSYMPs solution , and the tR of residual three peaks occurred rearward shift within the range of recognition, which changed to 1.71, 2.01, and 2.19 min, respectively. Besides, the compounds with tR of 7.16 and 7.59 min both showed pseudo-molecular [M + H]+ ions at m/z 301.14 and 301.14, with the fragment ions in UPLC-ESI-MS2 at m/z 149.02343 and 163.03858. The fragment ions of the compound with tR of 2.55 min in UHPLC-ESI-MS2 at m/z 362.32822, 300.28851, and 265.26337. This phenomenon illustrated that some compounds or impurities (not WSYMPs) were cleared away via the adsorption–separation system with the macroporous adsorption resin LX300C. Furtherly, we analyzed the peaks with tR of 2.01 min (or 1.95 min) and 2.19 min (or 2.16 min) by UHPLC-ESI-MS. The peaks of T1-2.16 and T2-2.19 min both showed pseudo-molecular [M + H]+ ions at m/z 114.09134 . In addition, the peak of T1-1.95 min mainly showed pseudo-molecular [M + H] + ions at m/z 303.723239, 435.33057, and that of T2-2.01 min at m/z 303.723239 and 435.2188. Furtherly, the components of exYMPs solution were separated via dynamic desorption of fully saturated absorbent with glass absorbent columns of 200 mm length and 10 mm diameter. The sample with the darkest yellow color tone was analyzed by Nanjing Sheng Ming Yuan Health Technology Co. Ltd., and the result showed that the molecular weight of component was m/z at 440.1150 of [M + H]+ (17H18O5), has a weight of m/z 302 [observed m/z at 303.1235 of (M + H)+] and UV λmax value at 375 nm  observed m/z at 435.7 of [M + H]+  cells  cells . In addis) cells . These rMonascus pigments are excellent colorants, are safe, and have several physiological functions. In this study, a mutant strain, M. purpureus H14, was generated using dual mutagenesis of atmospheric and room-temperature plasma and HIBI. The physicochemical property of the produced YMP was water-soluble, and two yellow monascus pigment components were identified by UHPLC-ESI-MS. It exhibited good tone stability when subjected to environmental factors including pH, heat, light, and metal ions, but the pigment value was unstable with pH, light, and high concentrations of Ca2+, Zn2+, Fe2+, Cu2+, and Mg2+. Application of IF and EF significantly improved the exYMP yield and productivity.Yellow liujundandy@csuft.edu.cn.The data analyzed in this study is subject to the following licenses/restrictions. The data presented in this study are available on request from the corresponding author. Requests to access these datasets should be directed to JL, JL and QL designed experiments. JB, ZG, and HZ performed experiments. JB wrote the manuscript. JL revised the manuscript. MS collected important background information. FY analyzed the RSM results. JL and BZ analyzed the data. DL and XZ performed heavy ion-beam irradiation. CT analyzed the HPLC-MS data. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (no. 32101906), Hunan Provincial Natural Science Foundation (no. 2021JJ31146), Open Project Program of the Hunan Provincial Key Laboratory of Food Safety Monitoring and Early Waring (no. 2021KFJJ02), and Education Department of Scientific Research Project of Hunan Province (no. 20B619), and Education Department of Postgraduate Research and Innovation Project of Hunan Province (no. CX20210863).CT was employed by Nanjing Sheng Ming Yuan Health Technology Co. Ltd. and Jiangsu Institute of Industrial Biotechnology JITRI Co. Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Alternanthera bettzickiana ethanolic extract (ABEE). Acute oral toxicity (OECD 425) was tested in the safety evaluation. A limit test was used to identify the LD50 value. For an acute oral toxicity study a dose of 2000 mg/kg of ABEE was given orally to the treatment group, and the control group received distilled water at a rate of 10 ml/kg. Biochemical, hematological, and histopathological analyses were performed after 14 days. A formaldehyde 2% w/v solution was injected via i.p. to rats of all groups to prepare the arthritic model. Five groups were divided into control (D.H2O), standard (Diclofenac), and three groups receiving the plant extract at dose levels of 125 mg/kg, 250 mg/kg, and 500 mg/kg respectively. Treatment was continued for 10 days. Paw diameter and hematological and biochemical variables were quantified. ELISA was performed for the estimation of inflammatory cytokines. In the acute oral toxicity study, no mortality or morbidity were observed, so the LD50 of this plant was greater than 2000 mg/kg. ABEE decreased the paw diameter with the restoration of hematological and biochemical changes. SOD and CAT levels were increased while decreasing the MDA, NO, TNF-α, and IL-6 levels in arthritic rats. It is concluded that the use of A. bettzickiana has low toxicity, and it can be used for the treatment of arthritis.In many developing countries, medicinal plants have long been used for therapeutic purposes due to their low cost and toxicity. This study evaluated the safety and anti-arthritic potential of   The traditional use of herbal medicines was common before the evolution of synthetic and semisynthetic medicines and remains common . Many poAlternanthera bettzickiana belongs to the Amaranthaceae family. Amaranthaceae includes nearly 65 genera and more than 900 species. The family is widely distributed across tropical regions. Plants in this family are used for ornamental purposes, like A. salixifolius, Celosia cristata, Iresine herbtsii, etc., and some are edible and used as vegetables, such as A. hybridus, A. spinosus, A. tricolor, A. viridis, etc. , and voucher no. 520-1-13 was issued for a crude sample of UAF herbarium.Microwave extraction technology was used for the preparation of the extract. The extraction procedure involves three cycles after setting the microwave to 9,000 W. In the first cycle, 750 ml ethanol is used, while in the other two cycles, 500 ml ethanol was added to 100 g powder in three beakers. Beakers were placed into the microwave oven and cooked for 2 min. The microwave oven door remained open for a period of 30 s. This procedure was repeated five times. The same process was repeated for other cycles. The extract was filtered by muslin cloth and then by whatman filter paper to obtain filtrate. The filtrate from every cycle was collected, and the solvent was evaporated at 40°C. The extract was kept in amber colored bottles .Following the OECD guideline 425, nonpregnant female Wistar rats weighing 130 ± 30 g and aged 9–10 weeks were randomly taken. Rats were housed under standard conditions for 5 days to acclimatize them. The animal house was kept at 22 ± 3°C with 30–70% relative humidity. The light and dark cycle was 12 h long. The rats were given a standard laboratory diet with tap water.The study was carried out after taking permission from animal’s ethics committee, Govt. College University Faisalabad with Ref. No. GCUF/ERC/2143.via cardiac puncture under chloroform anesthesia. The serum was isolated for carrying out biochemical and hematological analysis. Vital organs  were removed from slaughtered rats after cervical dislocation. The weight of these organs was recorded. The organs were preserved in 10% formaldehyde solution and were implanted in paraffin wax  for 5 days to acclimatize them with new environment. The limit test dose of 2000 mg/kg dose was given. Rats were fasted 3–4 h before dosing but provided with water. Dosing was given to single rats on the basis of weight. Rats were strictly monitored for initial 30 min and then for 4 hours. Food was given 1–2 h after dosing. Four rats were given the same dose orally after survival of first rat. The same procedure was adopted for control group of five rats. The control group was administered distilled water. Both groups were observed for the toxic effects for first 6 h and then for a duration of 14 days. The surviving rats were noticed for any toxic reaction. The weight of animals was recorded as well. After 14 days rats were again weighed and blood was accumulated ffin wax .Blood from both treated and vehicle control rats was collected in EDTA-containing tubes for hematological analysis. Hemoglobin (Hb), total RBC, white blood cells (WBC) count, platelets, mean corpuscular volume (MCV), packed cell volume (PVC), monocytes (M), neutrophils (N), mean corpuscular hemoglobin (MCH), eosinophils (E), mean corpuscular hemoglobin concentration (MCHC), and lymphocytes (L) were determined using Mythic CBC analyzer. Serum was separated for biochemical analysis. Cholesterol, triglycerides, low density lipoprotein (LDL), high density lipoprotein (HDL), very low density lipoprotein (VLDL), bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, urea, blood urea nitrogen, creatinine, uric acid, proteins, albumin, globulin, A/G ratio, rheumatoid factor (RF), and C-reactive protein (CRP) were measured using Tecno 786 bio chemistry analyzer .Vital organs separated from slaughtered rats were fixed in formalin (10%) and then implanted in paraffin wax. Sections were slashed at 5 mm and stained with hematoxylin and eosin. Histopathological modifications were studied under a photomicroscope .Wistar albino rats weighing 150–180 g were used. Rats were divided into five groups of five rats each. Group 1 (disease control group): rats were injected with formaldehyde solution on the first and third day of experiment. Group 2 (Diclofenac sodium treated): rats were injected with formaldehyde solution, as in group 1, and administered diclofenac sodium daily at a dose of 10 mg/kg for 10 days. Groups 3–5 (ABEE treated) rats received formaldehyde solution as in group 1 and were administered ABEE at a dose of 250, 500, 1,000 mg/kg, respectively, for 10 days. All treatments were dissolved in distilled water and given orally; 30 min after drug administration, arthritis was developed by a sub-plantar injection of 0.1 ml of 2% w/v formaldehyde solution on days 1 and 3 of the experiment . Arthritwhere Vc is the paw diameter of disease control and Vt is the paw diameter of treated.via cardiac puncture. The hematological factors  were measured by Mythic CBC analyzer while biochemical variables  were determined with Tecno 786 bio chemistry analyzer. Next, animals were sacrificed by painless procedure by cervical dislocation under anesthesia  .The serum concentration of nitric oxide was measured using Nitric Oxide ELISA kit . Microplate ELISA Reader  was used to measure the absorbance. Serum levels of superoxide dismutase , catalase , and malondialdehyde  were determined after the study ended .n = 5), and the statistical significance was analyzed by two-way ANOVA, followed by Bonferroni’s posttest, and applied by Graph Pad Prism 5 software. p ≤ 0.05 was considered to indicate statistical significance.The results were presented as means ± SEMs  when compared with the vehicle control group. It was noted that rats showed no significant changes in LDL, VLDL, cholesterol, HDL, or triglycerides values of the treatment group relative to the vehicle control group values  in paw diameter at day 10. These results indicated that ABEE at 1,000 mg/k exhibited a peak effect, and the outcomes were prominent relative to the standard drug diclofenac sodium.p < 0.001) averted abnormal modifications of hematological variables in treatment groups altered by the formaldehyde injection. Furthermore, ALP, ALT, and AST levels noticeably increased in disease control rats and significantly decreased in animals treated with 500 and 1,000 mg/kg ABEE, while 250 mg/kg dose of extract exhibited nonsignificant decrease in ALP, ALT, and AST levels in treatment group relative to the disease control group. Similarly, the standard drug diclofenac sodium also significantly decreased ALP, ALT, and AST levels in treated group, by contrast to disease control group. Moreover, the noticeably increased creatinine and urea concentrations in disease control animals were significantly decreased with ABEE and standard drugs in treatment rats. Additionally increased serum values of CRP and RF were observed in disease control animals, while administration with ABEE and standard drug diclofenac sodium significantly decreased CRP and RF levels in treatment groups. These results indicated that ABEE at 1,000 mg/k exhibited peak effects, and outcomes were prominent as compared to standard drug diclofenac sodium.The blood and serum parameters of animals administered with ABEE and diclofenac sodium are shown in p < 0.001) was noticed in disease control rats (517.59 ± 8.355 pg/ml), although the elevation was significantly decreased by administration of ABEE at 1,000 mg/kg (273.45 ± 2.30 pg/ml), 500 mg/kg (490.00 ± 1.70 pg/ml) and diclofenac sodium (285.79 ± 2.91 pg/ml) when compared with disease control rats (p < 0.001) decreased by ABEE at 1,000 mg/kg (363.50 ± 2.31 pg/ml), 500 mg/kg (457.53 ± 5.70 pg/ml), and diclofenac sodium (377.91 ± 2.98 pg/ml) (An elevated level of TNF-α (rol rats . An abno8 pg/ml) . The mosp < 0.001) in disease control animals. However diclofenac sodium (10 mg/kg), 500 and 1,000 mg/kg concentration of plant extract increased (p < 0.001) the serum concentrations of SOD and CAT in adjuvant-injected groups, as shown in p < 0.001) in disease control animals. MDA and NO concentrations were significantly reduced by diclofenac sodium and plant extract in adjuvant-injected groups as shown in The outcomes of oxidative stress exhibited that SOD (3.93 ± 0.447 U/mL) and CAT concentrations in serum (59.31 ± 2.47 U/L) were significantly reduced . The increased formation of TNF-α leads to increase in the expression of IL-6 and IL-1β, which in turn produce the degrading enzymes responsible for osteoclast differentiation, thus stimulating the development of arthritic erosion and vasodilation in the edematous area .2O2 by changing it into water and oxygen.Reactive oxygen species (ROS) are formed as a consequence of metabolism and different environmental factors, such as cigarette smoke or air pollutants. ROS are produced at low concentrations in all tissues. In general, different antioxidant systems regulate this formation. High concentration or insufficient removal of ROS leads to oxidative stress . ROS are active and can harm cell structures, such as proteins, nucleic acids, lipids, and carbohydrates. They can change their functions. The disturbance in balance between oxidants and antioxidants is called oxidative stress. The control of redox state is important for cell proliferation and cell viability. The antioxidant system is of two types. This includes enzymatic and nonenzymatic antioxidants that are effective in neutralizing damaging effects of ROS , although in disease conditions, the antioxidant system can be overcome. Oxidative stress comes up with many disease conditions, such as cancer atherosclerosis, diabetes, asthma, and hypertension. The main enzymatic antioxidants are GSH-px, catalase, and SOD. SOD plays crucial role in free radical defense . Glutathione peroxidase and catalase inhibit the accumulation of HA. bettzickiana target biphasic reactions . Lipid peroxidation is a well-accepted mechanism of cell damage and it indicates oxidative stress in tissues and is an indicator of lipid peroxidation. Increased levels of MDA are reported in the plasma of rheumatoid arthritis patients . Nitric oxide is an endogenously formed molecule that possesses important functions in cell signaling and participates in different physiological processes. Radical species that cause oxidative activity in an inflamed joint include reactive oxygen and nitrogen species. These mediate cartilage damage . NO perfeactions .The outcomes of a previous study showed that ABEE decreased oxidative stress and mRNA expression of pro-inflammatory biomarkers , although it increased mRNA expression of immunoregulatory cytokines . These mechanisms could be accountable for the anti-inflammatory and the anti-arthritic potential of plants. The anti-inflammatory potential of ABEE may be due to the existence of many phytochemicals, such as n-hexadecanoic acid, linoleic acid, phytol, vitamin E, Ar-turmerone, squalene, and farnesol, which possess anti-inflammatory activity .Alternanthera bettzickiana ethanolic extract has low toxicity, as even a 2 g/kg dose did not showed any mortality or morbidity, and no difference was observed in biochemical and hematological parameters in comparison to the control group. Outcomes also proposed that the oral administration of ABEE in arthritic animals remarkably decreased paw thickness and inhibited abnormal changes in hematological and biochemical variables. Alternanthera bettzickiana may be used as an optimal therapy for the treatment of rheumatoid arthritis.This study concluded that


Dr Hoepner received speaker/advisor honorary from Merck, Novartis, Roche, Biogen, Alexion, Sanofi, Janssen, Bristol‐Myers Squibb, and Almirall. He received research support within the last 5 years from Roche, Merck, Sanofi, Biogen, Chiesi, and Bristol‐Myers Squibb. He also received research grants from the Swiss MS Society. He also serves as associated editor for Journal of Central Nervous System disease. All are not related to that work. Dr Kamm has received honoraria for lectures and research support from Biogen, Novartis, Almirall, Teva, Merck, Sanofi Genzyme, Roche, Eli Lilly, Janssen, Celgene, and the Swiss MS Society (SMSG). Friedli C received speaker honoraria and/or travel compensation for activities with Biogen, Sanofi Genzyme, Novartis, and Merck and research support from Chiesi, not related to this work. Dr Salmen received speaker honoraria and/or travel compensation for activities with Bristol‐Myers Squibb, Novartis, Roche, and research support by Baasch Medicus Foundation and the Swiss MS Society. She serves on the Editorial Board of Frontiers in Neurology—Multiple Sclerosis and Neuroimmunology. All are not related to this work. Dr Chan has received speakers’/board honoraria from Actelion (Janssen/J&J), Almirall, Bayer, Biogen, Celgene (BMS), Genzyme, Merck KGaA , Novartis, Roche, and Teva, all for hospital research funds. He received research support from Biogen, Genzyme, and UCB, the European Union, and the Swiss National Foundation. He serves as associate editor of the European Journal of Neurology, on the editorial board for Clinical and Translational Neuroscience and as topic editor for the Journal of International Medical Research.Data can be obtained via corresponding author.+ T lymphocytes, especially CD8+ cytotoxic T cells,+ T cell responses are discussed to be associated with COVID‐19 severity.+, CD4+, or CD8+) are different in anti‐CD20‐treated patients  with severe  or mild  COVID‐19 course.Corona Virus Disease (COVID)‐19 has a more severe course in people treated with anti‐CD20 therapies.In total, 14 patients with multiple sclerosis  or neuromyelitis optica spectrum disorder (NMOSD 3/14) receiving anti‐CD20 drugs, who had a severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV2) infection while on anti‐CD20 treatment, were identified by medical chart review . COVID‐19 was diagnosed by polymerase chain reaction (PCR) test between March 2020 and July 2021 in all cases.n = 14), and had an expanded disability status scale (EDSS) score of 2.0 . Time between last infusion and COVID‐19 infection was 4.4 months . All but one anti‐CD20‐treated patients with COVID‐19 were not vaccinated prior COVID‐19. The single patient vaccinated 2 months prior infection  was treated with Ocrevus® and managed on an outpatient basis. Further cohort characteristics are displayed in the figure. Out of 14, 7 patients were hospitalized due to COVID‐19  of whom 2/7 were treated in the intensive care unit and 1/2 required artificial ventilation. All patients recovered without sequelae.Patients were, in majority, female (9/14), 45.5 years old  28.8–52.3, p > 0.05). Fluorescence‐activated cell sorting (FACS) analysis was performed 4.0 months prior COVID‐19 . B cell populations were depleted in 11/14, and the remaining three patients had a B cell count of 1/µl, 2/µl, or 55/µl, respectively. Median number of T cell  counts is displayed in the figure without differences between hospitalized and outpatient managed COVID‐19 cases , age, sex, anti‐CD20 drug used, last administered anti‐CD20 dose (milligram), EDSS, time since last infusion (months), and overall treatment duration (years) did not differ between hospitalized and non‐hospitalized patients  having a mild‐to‐moderate disease course, 16/51 were hospitalized, 10/51 had a severe disease course, and 3/51 died.


O) at 1.5–3-Hz cycle frequencies and 1–7-mm length changes. Muscles were then fixed in formalin at LO. Fascicle lengths and sarcomere lengths were measured to calculate SSN. Intramuscular collagen content and crosslinking were quantified via a hydroxyproline content and pepsin-solubility assay. Trained rats had longer fascicle lengths (+13%), greater SSN (+8%), and a less steep passive force-length curve than controls (P<0.05). There were no differences in collagen parameters (P>0.05). Net work output was greater (+78–209%) in trained than control rats for the 1.5-Hz work loops at 1 and 3-mm length changes (P<0.05), however, net work output was more related to maximum specific force  than SSN . Therefore, contrary to our hypothesis, training-induced sarcomerogenesis likely contributed little to the improvements in work loop performance.Increased serial sarcomere number (SSN) has been observed in rats following downhill running training due to the emphasis on active lengthening contractions; however, little is known about the influence on dynamic contractile function. Therefore, we employed 4 weeks of weighted downhill running training in rats, then assessed soleus SSN and work loop performance. We hypothesised trained rats would produce greater net work output during work loops due to a greater SSN. Thirty-one Sprague-Dawley rats were assigned to a training or sedentary control group. Weight was added during downhill running via a custom-made vest, progressing from 5–15% body mass. Following sacrifice, the soleus was dissected, and a force-length relationship was constructed. Work loops (cyclic muscle length changes) were then performed about optimal muscle length (LThis article has an associated First Person interview with the first author of the paper. Summary: An investigation of adaptations in mechanical function induced by a novel method of weighted downhill running training in rats, and the connections to adaptations in muscle architecture. Skeletal muscle remodels and adapts in response to specific conditions, as observed across the hierarchy of muscle structural organisation . An examin vitro is the ‘work loop’: sinusoidal cycles of muscle shortening and lengthening with phasic bursts of stimulation meant to simulate locomotion  or PCSA between trained and control rats (P=0.14).As shown in P=0.81) (P=0.71)  C or perc(P=0.71) D betweenP<0.01, d=1.38) in trained  than control rats , implying a training-induced increase in FL (For average fascicle length (FL), 95% power was achieved with 18 control and 13 trained rats. FL was 13% longer  between trained  and control rats   in trained  than control rats  (For sarcomere length (SL), 80% power was not achieved using average SL in 18 control and 13 trained rats (Power=35%). With these sample sizes, SL did not differ ] B. A foll9,2.94)] , amounti, 2.87)] A. TherefP=0.01, d=0.99) in trained  compared to control rats  .For average SSN, 82% power was achieved with 18 control and 13 trained rats. SSN was 8% greater (808.03)] C. Fig. 4O , nor maximum absolute  or specific force . There were also no between-group differences in the width of the force-length relationship at 80% maximum , suggesting similar widths of the plateau region. Altogether, these findings indicate the active force-length relationship was not different between trained and control rats.The original data collected for the active force-length relationships are presented in p , but the coefficient b was 13% greater in trained rats , indicating a less steep passive force-length curve and lower overall stiffness. These data indicate the passive force-length relationship of trained compared to control rats was not shifted to the right, but was less steep, resulting in reduced passive tension with stretch.The original data collected for the passive force-length relationships are presented in Fig. S2). There was an effect of group on net work output in the passive work loops , with trained rats producing 0.22 J/g less negative net work than controls across all work loops [95% CI of the difference ]. However, post-hoc t-tests did not reveal between-group differences in net work output in any specific passive work loops; hence, this difference was only noticeable when viewing all work loops together. There were no effects of group on work of shortening  or work of lengthening  in the passive work loops, and there were no interactions of group×cycle frequency×length change , group×cycle frequency , nor group×length change  on any passive work loop parameters =10.43, P<0.01, ηp2=0.04], with trained rats producing on average 0.53 J/g [95% CI of the difference ] more net work than controls across all active work loops. However, there were no interactions of group×cycle frequency×length change [F(5304)=0.06 P=1.00], group×cycle frequency , or group×length change , indicating the effect of group on net work output did not differ depending on cycle frequency and length change. Post-hoc t-tests showed the effect of group was most pronounced in 1.5-Hz work loops, with trained rats producing 78% greater net work output than controls at the 1-mm length change  and 209% greater net work output at the 3-mm length change  (As shown in  d=0.78) C.Fig. 8F(1304)=0.66, P=0.42] (F(1304)=1.58 P=0.21] in the active work loops (F(5304)=0.01–0.02 P=1.00], group×cycle frequency , or group×length change .There were no effects of group on work of shortening [ P=0.42] A or workrk loops B. NeitheF(2304)=21.44, P<0.01, ηp2=0.13] and net work output, , but not work of lengthening . There were effects of length change on all these variables . Specifically, work of shortening decreased with increasing cycle frequency , and work of shortening and work of lengthening both increased with length change  (P<0.01), and with increasing length change past the optimal length change, except for between 5 mm and 7 mm   A,B. Net to 0.03) C.F=0.02, R2<0.01, P=0.89; 3 mm: F=0.06, R2<0.01, P=0.81]  , indicating the training-induced reduction in passive tension also had little effect on work loop performance. Absolute maximum isometric force was related to net work output in the 1.5-Hz, 1-mm work loop  but not the 3-mm work loop . Specific maximum isometric force, however, was related to net work output in both work loops . Altogether, adaptations in maximum specific force likely contributed most to the improved work loop performance.Linear regressions were employed to investigate which variables may have contributed to the between-group differences in net work output in the 1.5 Hz work loops at 1 and 3 mm. There were no relationships between SSN and net work output in either work loop [1 mm:  P=0.81] , suggestThis study assessed rat soleus architecture following 4 weeks of weighted downhill running training and aimed to relate muscle architectural adaptations to changes in dynamic contractile function, namely work loop performance. Our hypothesis that training would increase SSN, not change collagen content and crosslinking, and increase net work output was partly correct. Comparing trained and control rats, FL and SSN increased with training, as did SL but only when viewing every fascicle independently to increase the sample size. There were no differences in collagen parameters between groups, and increases in net work output were most pronounced in 1.5-Hz work loops at length changes of 1 and 3 mm.O is consistent with previous reports  stems from the active force-length relationship of muscle, whereby active force production is suboptimal in over-stretched positions due to limited actin-myosin binding ,b, and oThe 13% FL increase we observed with weighted downhill running training appears to have been driven by increases in both SSN (+8%) and SL (+4–5%). The increase in SL may be interpreted in two ways. First, as optimal SL is generally believed to be constant within a species , the rolO, we may have observed an increase in optimal SL, which could imply elongation of the myofilaments within the sarcomere (O (O (i.e. a longer length compared to the control muscle), they would have observed a longer optimal SL. This perspective is somewhat supported by other studies that fixed muscles at LO. O than untreated rats (2.95 µm versus 2.88 µm). O, following 3 weeks of immobilisation in a shortened position with intermittent stretching. Although they observed a decrease in SSN (due to being immobilised in a shortened position), they observed a ∼2% increase in SL compared to controls (2.2 µm versus 1.9 µm). Lastly, O and observed on average a greater SL in downhill running rats than controls (2.77±0.09 µm versus 2.75±0.11 µm). Future research might aim to assess the time course of SL and SSN adaptations during training or confirm optimal SLs via single-fibre testing to better understand these observations.On the other hand, as we measured SL at Larcomere . In thisomere  rats, on average only a 1.5%  greater LO. Therefore, like with SSN, the present study's addition of weighted vests and extra recovery days between training sessions may have amounted to a greater shift in rat soleus LO compared to body weight downhill running training, even if it was statistically insignificant.We observed on average a 3% greater Lol group . With aning limb . Rightwatraining . The dowtraining  assessedp, trained rats had a 13% greater b coefficient, indicating a less steep passive force-length curve and lower overall stiffness , which can enhance force production, and used a higher temperature (30°C versus 26°C), which may permit better force-generating ability during faster and longer excursion contractions . By doing this, we can estimate what the FL change would be for a given whole-muscle excursion . Subsequently, we can estimate the SL at that FL by dividing FL by the SSN . Assuming the width of the sarcomere force-length relationship changed proportionally to SL, we can calculate the SL excursion relative to SL at LO during the whole-muscle excursion {e.g. [(3.07–2.75 μm)/2.93]×100%=10.92%}. We can also estimate the sarcomere shortening velocity in an excursion by multiplying the sarcomere displacement by 2 times the cycle frequency [e.g. (3.07–2.75 μm)×(1.5 Hz×2)=0.96 μm/s]. Because SL at LO is different between groups, these velocities should also be expressed as SL/s for comparisons (e.g. 0.96 μm/s/2.93 μm=0.33 SL/s).In Fig. S3A,B, if SL at LO had been constant between trained and control rats [whether to achieve the observed SSN (A) or FL (B)], the trained values still would not have been much less than control values. In Fig. S3C, we also estimated whether a hypothetically very large magnitude of sarcomerogenesis  to isopropanol and incubated for 30 min at 58°C. Quantification was determined by extinction measurement of the resulting solution at 550 nm. A standard curve  was included in each assay (average linear R2=0.98), and samples were run in triplicate (average CV=0.098). Hydroxyproline concentrations in samples were determined by interpolation from the linear equation of the corresponding assay's standard curve. Results are reported as µg of hydroxyproline per mg of powdered tissue mass.The left soleus of 11 control and 12 trained rats was dissected and stored at −80°C until use in a hydroxyproline with collagen solubility assay, with pepsin-insoluble collagen quantifying the amount of collagen crosslinking . The mus2) to obtain specific values (expressed as units/cm2). PCSA was calculated as:3 . Active and passive force values were normalised to the muscle's physiological cross-sectional area . The passive force-length relationship was plotted with force in units of N/cm2 . Active force-length relationships were fitted to an asymmetric Gaussian function , normalised to PCSA, and normalised to maximum force, and plotted up to 3.5 mm on either side of LO (to match the maximum excursion of the work loops). From the fitted curves, two-tailed t-tests were used for between-group comparisons of LO, as well as the width of the curve at 80% maximum force :Per recommendations from the systematic review by t-tests were used for between-group comparisons of Lp and b.From the fitted exponential curves, two-tailed From each work loop, Aurora Scientific software calculated work of shortening and work of lengthening  and net work output of a whole cycle . Of the two active work loops in a given set, the loop that produced the highest net work was chosen for statistical analyses .t-tests were used to compare body weights between control and trained rats throughout the training period , and to compare FL, SL, SSN, muscle wet weight, PCSA, total collagen, and % insoluble collagen between trained and control rats. Since characterisation of longitudinal muscle growth was the primary objective of this study, post hoc power analyses  were performed on FL, SL, and SSN, with 80% indicating statistical power.All the following statistical analyses were conducted in IBM SPSS statistics version 26. Two-tailed t-tests were employed for comparisons between trained and control rats in specific work loops.A three-way ANOVA [group ×cycle frequency ×length change ] with a Holm-Sidak correction for all pairwise comparisons was used to compare all work loop parameters from the passive and active work loops . An effect of group defined a difference between control and trained rat work loops. Where effects of group were detected, two-tailed b from the fitted passive force-length curves, and absolute and specific maximum isometric force were performed to elucidate which training-induced adaptations contributed to improvements in net work output.Regression analyses between net work output and SSN, the coefficient α<0.05. Effect sizes from three-way ANOVAs are reported as the partial eta squared (ηp2), and from t-tests as Cohen's d  where significance was detected. Data are reported as mean±s.d. in text and mean±s.e. in figures.Significance was set at All procedures were approved by the Animal Care Committee of the University of Guelph.


In the original article, there was an error. Unfortunately, some important text in the discussion was misplaced.Discussion, “Changes Vary Between Subgroups,” paragraph one:A correction has been made to The original text, “Very-high distress in women aged 55–64 has doubled this century (from 3.5 to 7.2%) and combined high/very-high distress has increased by 50% (12.4–18.7%), both of which are highly significant findings. Very-high distress also increased in males, significantly in those aged 25–34 years. Overall, distress was greatest in women aged 18–24 years during all years; 8.0% for very-high levels and 22.1% for combined high/very-high in 2017/18. (2.1–4.0%), but overall is a more tentative finding since a significant increase did not extend to the combined high/very-high distress metric (10.6–11.5%).” has been corrected to:“Very-high distress in women aged 55–64 has doubled this century (from 3.5 to 7.2%) and combined high/very-high distress has increased by 50% (12.4–18.7%), both of which are highly significant findings. Very-high distress also increased in males, significantly in those aged 25–34 years, but this is a more tentative finding since a significant increase did not extend to the combined high/very high distress metric (10.6–11.5%). Overall, distress was greatest in women aged 18–24 years during all years; 8.0% for very-high levels and 22.1% for combined high/very-high in 2017/18 (2.1–4.0%).”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.