These patterns of genetic variation suggest recent inbreeding in a formerly large population. Currently the most contiguous assembly for a heteromyid rodent, this reference genome provides insight into the past and recent demographic history of the population, and will be a critical tool for management and future studies of outbreeding depression, inbreeding depression, and genetic load.High-quality reference genomes are fundamental tools for understanding population history, and can provide estimates of genetic and demographic parameters relevant to the conservation of biodiversity. The federally endangered Pacific pocket mouse (PPM), which persists in three small, isolated populations in southern California, is a promising model for studying how demographic history shapes genetic diversity, and how diversity in turn may influence extinction risk. To facilitate these studies in PPM, we combined PacBio HiFi long reads with Omni-C and Hi-C data to generate a de novo genome assembly, and annotated the genome using RNAseq. The assembly comprised 28 chromosome-length scaffolds (N50 = 72.6 MB) and the complete mitochondrial genome, and included a long heterochromatic region on chromosome 18 not represented in the previously available short-read assembly. Heterozygosity was highly variable across the genome of the reference individual, with 18% of windows falling in runs of homozygosity (ROH) >1 MB, and nearly 9% in tracts spanning >5 MB. Yet outside of ROH, heterozygosity was relatively high (0.0027), and historical  The design of optimal conservation strategies for the federally endangered Pacific pocket mouse, a subspecies that has undergone substantial declines and is under intensive management, relies on precise estimates of genome-wide heterozygosity, inbreeding, demographic history, and chromosomal variation. We generated a chromosome-length reference genome for this species, and found massive stretches of homozygosity across a genome with relatively high baseline heterozygosity, reflecting inbreeding in a formerly large population. The high degree of contiguity of this reference genome enabled the detection of signatures of contemporary inbreeding, highlighting the value of high-quality reference genomes for the management of endangered species, and conservation of biodiversity.Chromosome-length reference genomes are fundamental tools for addressing questions in biology, from evolutionary innovation to the underpinnings of disease (Perognathus longimembris pacificus) is a valuable model species for studying the interplay between genetic variation and extinction risk. Having been extirpated from most of its distribution along coastal southern California in the 1930s , including 28 chromosome-length scaffolds ; table 12n = 56; . The anc2n = 56;  online, 2n = 56;  online.A). This heterochromatic region was not represented among the larger contigs (>100 KB) of the DISCOVAR assembly (fig. 1DE), and short-read data mapped to the genome had markedly lower depth in this region (fig. 1B), highlighting the difficulty of assembling and mapping highly repetitive regions with short reads.Using RepeatMasker , 41% of fig. 1y fig. 1DE, and shDipodomys ordii) and banner-tailed kangaroo rat . Although not closely related to PPM . Frequent rearrangements between genomes, as demonstrated by contigs in the Dipodomys genomes that map to multiple PPM chromosomes and vice versa, is consistent with high cytogenetic variability across rodents , but 25.9% of autosomal 50 KB windows had virtually no heterozygosity  (A). Nearly half (48.1%) of homozygous windows were part of ROH >5 MB in length, including a ∼67.1 MB span of chromosome 2 (AandB). These long ROH likely stem from inbreeding within the last ten generations (B), assuming 100 MB identity-by-descent tracts are inherited per meiosis  A. Nearlymosome 2 AandB. Therations B, assumieN decreased between 150 and 100 Ka from ∼400,000 to ∼50,000, where it remained up to 10 Ka (C). Selective sweeps, which are common in large populations, can also produce signals that appear as population contraction by PSMC . Other heteromyids show similar fluctuations and large historical eN, including the federally endangered Stephen’s kangaroo rat .In contrast with these signatures of contemporary inbreeding, Pairwise Sequentially Markovian Coalescent (PSMC) estimates suggest large historical effective population sizes for PPM. to 10 Ka C. SelecteN stands in contrast with signatures of recent inbreeding in the reference individual. The ROH-based inbreeding coefficient, FROH > 1MB = 0.18, is on par with iconic endangered species such as South Asian tigers  populations  than the population average , including populations that have experienced the largest declines. Additional genomic samples are needed to estimate the level of inbreeding across the subspecies.The large historical Habitat fragmentation from urbanization has led to population declines in this and other species across southern California . This hiWe provide an overview of the methods below. Further details can be found in the A wild-born male PPM was collected in 2012 from Santa Margarita , the largest and most genetically diverse extant population . A fibroQ > 20 (23.3× genome coverage).High molecular weight DNA was extracted from ∼20 M fibroblasts, and a Sequel HiFi Library was prepared and sequenced with two SMRT cells (Pacific Biosciences) using the Sequel II Sequencing Kit v2.0. We used the Peregrine assembler  for the de novo assembly of 51.2 GB of HiFi reads with quality scores For the Dovetail Omni-C library, chromatin from ∼10.5 M fibroblasts was fixed in place with formaldehyde, extracted, and digested with DNAse I. Chromatin ends were repaired, adapter-containing ends were ligated by proximity ligation, crosslinks were reversed, and the DNA purified. Libraries were sequenced to ∼30× coverage. The HiFi-based draft and Omni-C library reads with MQ > 50 were then assembled using HiRise  from the same individual. We assembled mitogenomes from the short-read data using NOVOPlasty , retainiWe summarized statistics of the assemblies using the assemblathon_stats.pl script, and assessed the completeness of the assembly against the eukaryota_odb10 and glires_odb10 databases using BUSCO, ver 4.0.1  and GenoD. ordii) genome (Dord_2.0) and banner-tailed kangaroo rat (D. spectabilis) genome (GCA_019054845.1). We aligned chromosome-length PPM scaffolds and scaffolds >10 MB for each kangaroo rat using lastz v1.04 (https://github.com/wurmlab/flo).We evaluated synteny between the PPM genome assembly and the Ord’s kangaroo rat (tz v1.04 . We compChaetodipus baileyi) samples (SAMN03068786-9), and from five kidney samples of rock pocket mice . The mitogenome was annotated with the MITOS Web server . We trimmed adapters, mapped the reads to our assembly, removed read duplicates, and called variants using HaplotypeCaller in GATK v3.8 . We estiTo estimate historical trends in effective population size, we ran 25 replicates of PSMC , using sevac122_Supplementary_DataClick here for additional data file.


Metal elements are present in the human body, and their levels in the blood have important impacts on health. In this study, 2488 Chinese individuals were included in a genome-wide association study of 21 serum metal levels, with approximately 179,000 East Asian individuals in a bidirectional two-sample Mendelian randomization (MR) analysis, and 628,000 Europeans in a two-sample MR analysis. We identified two single nucleotide polymorphisms (SNPs) rs35691438 and rs671 that were significantly associated with serum copper levels (SCLs). The bidirectional two-sample MR analysis in the East Asian population showed that gamma-glutamyl transpeptidase levels have a causal effect on SCLs. SCLs have causal effects on six outcomes, namely risks of esophageal varix, glaucoma, sleep apnea syndrome, and systemic lupus erythematosus, white blood cell count, and usage of drugs affecting bone structure and mineralization. The two-sample MR analyses in the European population showed causal effects of erythrocyte copper levels on risks of carpal tunnel syndrome and compression fracture. Our results provide original insights into the causal relationship between blood metal levels and multiple human phenotypes. A genome-wide association study in a Chinese population identifies SNPs associated with serum copper levels. Mendelian randomization analysis reveals causal effects on multiple human phenotypes in East Asian and European populations. Toxic metal elements, such as arsenic, cadmium, and lead, usually come from environmental exposure4. The accumulation of toxic metal elements in the human body can lead to many adverse health effects and even endanger life. In addition to the environmental factors that affect metal levels in the human body, many genetic variants that are significantly related to blood metal levels (BMLs) have been discovered. Numerous genome-wide association studies (GWASs) of European populations found a large number of single nucleotide polymorphisms (SNPs) that were significantly associated with blood levels of calcium, copper, selenium, zinc, iron, magnesium, manganese, cadmium, and mercury9. However, as far as we know, only a few GWASs on multiple BMLs in East Asian populations have been reported.Metal elements are present in the human body and can be classified as essential metal elements and toxic metal elements. Essential metal elements, such as calcium, copper, iron, manganese, magnesium, selenium, and zinc, are obtained mainly through the daily diet. They play critical roles in normal physiological activities, including oxidation–reduction reactions, catalysis, metabolic regulation, and intercellular signal transduction15. However, traditional epidemiological studies are often affected by confounding factors, and therefore determining causality can be difficult. Mendelian randomization (MR) studies can avoid the bias caused by the unavoidable confounding factors in traditional epidemiological studies, thereby allowing more robust and reliable causal inferences16. The application of MR has helped to uncover the causal relationships between BMLs and human disease phenotypes. Genetically higher serum calcium levels were shown to be associated with a higher risk of coronary artery disease and myocardial infarction17. Another study of UK Biobank cohorts found that genetically increased blood levels of both copper and iron were associated with reduced risk of lipid metabolism disorders18. A two-sample MR study based on Europeans showed that bone mineral density of certain skeletal locations was influenced by serum calcium and selenium levels19. Current GWAS-MR studies of BMLs and their causal effects on human diseases focused mainly on European populations. Considering the genetic differences between European and East Asian populations, a systematic GWAS-MR of BMLs in East Asian population is necessary to provide more reliable data for the diagnosis, treatment, and basic scientific research of clinical diseases.Many epidemiological studies have shown that BMLs are closely related to human diseases20. In addition, to comprehensively study the causal effects of BMLs on multiple deep phenotypes in different populations, we used the GWAS summary statistics of erythrocytes metal levels from the study of Evans et al.6 and the GWAS meta-analysis summary statistics from UK Biobank and FinnGen to conduct two-sample MR analyses.In this study, we conducted a comprehensive GWAS of multiple serum metal levels in the Chinese population and then performed a cross-ethnic MR study to fully understand the causal effects of BMLs on multiple deep phenotypes in humans. For this, we conducted bidirectional two-sample MR analyses using our GWAS summary statistics of serum metal levels and the GWAS summary statistics from BioBank JapanPcopper = 0.08 and Pmanganese = 0.96). The basic demographic characteristics and the results of the comparison of serum levels for the same metal in two cohorts of the subjects finally included in the GWAS analysis are shown in Table We totally included 2488 subjects in our present study. After the non-parametric test on the concentration of each metal between the two cohorts, only the concentrations of copper and manganese showed no statistical difference  were found significantly associated (P < 5.0 × 10−8) with serum copper levels (SCLs)  was found significantly associated with plasma iron levels (Supplementary Table P < 1.0 × 10−5) were in Supplementary Data CP of chromosome 3, Pmeta = 1.63 × 10−9; rs671, on ALDH2 of chromosome 12, Pmeta = 2.21 × 10−10) were found significantly associated with transformed SCLs ), and both rs35691438 and rs671 still met the Bonferroni-corrected threshold. The Manhattan, Quantile-Quantile, and LocusZoom plots are in Fig. r2 = 0.99. We also conducted a GWAS meta-analysis of serum/plasma manganese level using GWAS summary statistics from FAMHES and MEWHC. However, no significant SNP was found.We performed an independent GWAS for each transformed serum/plasma metal concentration in the Fangchenggang Area Male Health and Examination Survey (FAMHES) and the manganese-exposed workers healthy cohort (MEWHC), respectively. The specific number of subjects included in the GWAS of each metal is shown in Table r2 < 0.6) were identified from the GWAS meta-analysis of SCLs via Functional Mapping and Annotation (FUMA)  reached the significance threshold (P = 0.05/17322 = 2.887 × 10−6). The number 17322 is the quantity of protein-coding genes which are mapped based on the GWAS meta-analysis summary statistics. More information for the first 30 genes with the lowest P-value of gene-based analysis were in Supplementary Table P < 0.05/15482 = 3.2296 × 10−6) gene-set via gene-set analysis. The number 15482 is the quantity of gene sets. More information for the first 30 gene-sets with the lowest P-value of gene-set analysis were in Supplementary Table Four significant independent SNPs r < 0.6 weP-value < 0.05) identified in the Mendelian randomization pleiotropy residual sum and outlier (MR-PRESSO) outlier test. Finally, six exposure-outcome pairs which could draw robust causal conclusions were put into Table P = 0.007, Power = 0.83; weighted median [WM]: OR (95% CI) 0.867 (0.771 0.974), P = 0.016, Power = 0.93; MR using the robust adjusted profile score [MR-RAPS]: OR (95% CI) 0.883 (0.809 0.963), P = 0.005, Power = 0.85); and 4 disease outcomes: esophageal varix (IVW: OR (95% CI) 1.998 (1.317 3.032), P = 0.001, Power = 1.00; MR-Egger: OR (95% CI) 6.479 (1.394 30.108), P = 0.041, Power = 1.00; WM: OR (95% CI) 1.773 (1.001 3.141), P = 0.0497, Power = 0.99; MR-RAPS: OR (95% CI) 2.060 (1.372 3.093), P = 0.0005, Power = 1.00); glaucoma (IVW: OR (95% CI) 1.107 (1.031 1.190), P = 0.005, Power = 0.89; WM: OR (95% CI) 1.110 (1.009 1.222), P = 0.032, Power = 0.90; MR-RAPS: OR (95% CI) 1.110 (1.029 1.198), P = 0.007, Power = 0.90); Sleep apnea syndrome (IVW: OR (95% CI) 1.615 (1.212 2.153), P = 0.001, Power = 0.99; WM: OR (95% CI) 1.567 (1.078 2.276), P = 0.019, Power = 0.98; MR-RAPS: OR (95% CI) 1.625 (1.191 2.218), P = 0.002, Power = 0.99); Systemic lupus erythematosus (SLE) (IVW: OR (95% CI) 2.058 (1.422 2.980), P = 0.0001, Power = 1.00; MR-Egger: OR (95% CI) 5.412 (1.344 21.788), P = 0.041, Power = 1.00; WM: OR (95% CI) 1.722 (1.070 2.770), P = 0.025, Power = 0.99; MR-RAPS: OR (95% CI) 2.110 (1.451 3.068), P = 0.00009, Power = 1.00); and 1 biomarker outcome: white blood cell (WBC) count (IVW: β (95% CI) −0.026 (−0.043 −0.008), P = 0.004; WM: β (95% CI) −0.029 (−0.053 −0.005), P = 0.020; MR-RAPS: β (95% CI) −0.027 (−0.046 −0.008), P = 0.005). The results of IVW and MR-RAPS methods for SLE are still significant with the corresponding P-value smaller than the Bonferroni-corrected threshold (0.00023); however, the results of IVW, WM and MR-RAPS methods for the other five exposure-outcome pairs and the results of MR-Egger method for esophageal varix and SLE are suggestively significant with the corresponding P-value smaller than 0.05 but larger than the Bonferroni-corrected threshold (0.00023). Their scatter plots and plots of leave-one-out analyses were displayed in Figs. In the first round of two-sample MR in East Asian population, we investigated the causal effects of SCLs on 213 deep phenotypes. Firstly, we found 21 exposure-outcome pairs might have causal relationships  identified in the MR-PRESSO outlier test for the pairs with significant horizontal pleiotropy or heterogeneity. Finally, one exposure-outcome pair which could draw a robust causal conclusion was put into Table P = 0.0004; WM: β (95% CI) 0.631 (0.194 1.068), P = 0.005; MR-RAPS: β (95% CI) 0.491 (0.210 0.772), P = 0.0006). The results of IVW, WM and MR-RAPS methods for this exposure-outcome pair are suggestively significant with the corresponding P-value smaller than 0.05 but larger than the Bonferroni-corrected threshold (0.00023). Its scatter plot and plot of leave-one-out analysis are displayed in Fig. In the second round of two-sample MR in the East Asian population, we investigated the causal effects of 213 deep phenotypes on SCLs. Firstly, we found 16 exposure-outcome pairs might have causal relationships  identified in the MR-PRESSO outlier test. Finally, two exposure-outcome pairs which could draw robust causal conclusions were put into Table P = 0.018, Power = 0.62; MR-RAPS: OR (95% CI) 1.042 (1.005 1.080), P = 0.024, Power = 0.64); compression fracture (IVW: OR (95% CI) 0.841 (0.748 0.945), P = 0.004, Power = 0.79; WM: OR (95% CI) 0.807 (0.693 0.941), P = 0.006, Power = 0.92; MR-RAPS: OR (95% CI) 0.838 (0.740 0.949), P = 0.005, Power = 0.81). The results of IVW, WM, and MR-RAPS methods for compression fracture are suggestively significant with the corresponding P-value smaller than 0.05 but larger than the Bonferroni-corrected threshold (0.00023). The results of IVW and MR-RAPS methods for carpal tunnel syndrome are suggestively significant with the corresponding P-value smaller than 0.05 but larger than the Bonferroni-corrected threshold (0.00023). Their scatter plots and plots of leave-one-out analyses were in Fig. In the third round of two-sample MR in the European population, we investigated the causal effects of erythrocytes copper levels (ECLs) on 174 deep phenotypes. Firstly, we found 16 exposure-outcome pairs might have causal relationships (Supplementary Table We identified two SNPs (rs35691438 and rs671) that were significantly associated with SCLs in the Chinese population. In the East Asian population, GGT levels had a causal effect on the SCLs, and SCLs have causal effects on six outcomes, namely risks of esophageal varix, glaucoma, sleep apnea syndrome, and SLE, WBC count, and usage of drugs affecting bone structure and mineralization. In the European population, ECLs have causal effects on risks of carpal tunnel syndrome and compression fracture.25. SNP rs671 is a non-synonymous SNP within the exonic region of ALDH2, which is widely expressed in many human tissues, especially fat and liver. The encoded ALDH2 protein belongs to the aldehyde dehydrogenase family and is involved in the synthesis of acetate from ethanol in the alcohol metabolism pathway. Though there are some researchers reported links between SCLs and alcohol consumption30, it still needs further study to clarify the intricate relationship, such as the potential association between rs671 and SCLs may be mediated by alcohol drinking behavior, between SCLs and alcohol consumption behavior on various metabolic conditions33. For rs35691438, which was significantly associated with SCLs in our study, we found no reports of associations between it and other phenotypes. The CP gene, which is close to rs35691438, is highly expressed mainly in the liver. CP encodes ceruloplasmin, a metalloprotein that binds most of the copper in plasma and is involved in the peroxidation of Fe (II) transferrin to Fe (III) transferrin35. Mutations in CP cause aceruloplasminemia, which results in iron accumulation and tissue damage and is associated with diabetes and neurologic abnormalities. To date, a study reported 2 SNPs (rs1175550 and rs2769264) on chromosome 1 significantly (P < 5.0 × 10−8) associated with ECLs6, whereas another study found no SNPs that were associated with whole blood copper levels (BCLs) at the significance threshold (P < 5.0 × 10−8)9. The finding of Evans et al.6 has not been replicated in other studies. In our GWAS, we originally planned to test two SNPs (rs1175550 and rs2769264) on chromosome 1. However, because rs1175550 had a minor allele frequency (MAF) of < 0.01 in the East Asian population and was excluded according to the quality control standards, rs1175550 was not included in the GWAS. Although rs2769264 was found in our SCLs GWAS meta-analysis results, it was not significant (P = 0.4646); the significance threshold was P < 1.0 × 10−5. We found that the MAF of rs1175550 varied widely between the East Asian and European populations in the 1000 Genomes project (http://www.1000genomes.org); 0.2% in East Asians and 21.9% in Europeans. The MAF of rs2769264 also varied widely; 28.6% in East Asians and 16.1% in Europeans. These inter-ethnic discrepancies in the MAFs and the differences between disparate biological samples (serum copper and erythrocytes copper) may explain the highly significant association of rs2769264 in Europeans and no significant association in East Asians36. Moreover, rs2235321, a synonymous SNP on the transmembrane serine protease 6 (TMPRSS6) gene of chromosome 22, was significantly associated with plasma iron levels in MEWHC. A previous GWAS reported five SNPs  that were significantly associated with serum iron levels7. We checked these five SNPs in the results of our GWAS of the MEWHC and found that only rs855791 (in TMPRSS6 in strong LD (r2 = 0.90) with rs2235321) was significant (P = 1.19 × 10−8). The number of individuals (687) in our study is quite different from the number (48972) in the study of Benyamin et al.7, which may explain why the other four SNPs were not significant in our study.We found that SNP rs671 was significantly associated with SCLs. Many studies have reported associations between rs671 and other phenotypes, such as alcohol consumption (drinkers vs non-drinkers), coffee consumption, serum alpha1-antitrypsin levels, esophageal cancer, gout39. Animal studies have shown that copper deficiency decreased bone strength40, and that the released copper stimulated bone formation41. Our results are indirectly consistent with the results of these studies.In the first round of two-sample MR analyses conducted in the East Asian population, we found that genetically higher SCLs had causal effects on lower trends for the usage of drugs affecting bone structure and mineralization (ATC code M05B), fewer counts of WBC. The genetically increased SCLs also have causal effects on higher risks of esophageal varix, glaucoma, sleep apnea syndrome, and SLE. No direct study of the relationship between SCLs and the usage of M05B Drugs has been reported so far. M05B Drugs include mainly M05BA (bisphosphonates), M05BB , and M05BC (bone morphogenetic proteins). The bisphosphonates and bone morphogenetic proteins are both conducive to bone formation42. Until now, seldom studies have directly focused on the link between SCLs and esophageal varix. However, numerous studies have shown that the accumulation of copper can lead to serious liver diseases, including hepatitis and cirrhosis, whether in Wilson’s disease or a non-Wilson’s disease48. One epidemiological study found that SCLs were increased in patients with liver cirrhosis and variceal bleeding and suggested that variceal bleeding in patients with liver cirrhosis was associated with important imbalances in blood concentrations of zinc, copper, and magnesium49. Although the specific biological mechanism between increased SCLs and the higher risk of esophageal varix is unclear, our MR finding is consistent with what was reported by Dumea et al.49 and further showed the possible causal relationship between them. Therefore, we speculate that esophageal varix may be triggered by cirrhosis, which is caused by the toxicity of copper. An in-depth study of the mechanism needs to be carried out in future work.Esophageal varix is related to cirrhosis and portal hypertension51. A study showed that unbalanced copper levels could lead to oxidative stress through the reduction of glutathione or the Fenton reaction. In a state of oxidative stress, large numbers of reactive oxygen species are generated, which induces subsequent lipid peroxidation and DNA damage52. An early epidemiological study reported that higher mean SCLs were seen in the patients with glaucoma53. Another study showed that the SCLs increased in females with primary open-angle glaucoma compared with that of males with or without primary open-angle glaucoma54. Therefore, copper ions may mediate the occurrence of glaucoma via oxidative stress. Our results are consistent with those of previous studies.Glaucoma is a complex disease with the main risk factor being intraocular pressure. However, previous studies suggested that the further pathogenic factors in glaucoma should include oxidative stress. Oxidative stress can destroy neurons and alter the structure of the trabecular meshwork, which is the principal out channel of aqueous humor, while intraocular pressure continuously increases57. Although copper is closely related to the oxidative stress state of the human body, no previous epidemiological studies have reported associations between SCLs and sleep apnea syndrome. Only one study compared complaints and disease symptoms of obstructive sleep apnea syndrome in patients with Wilson’s disease, which resulted in copper overload, and healthy controls; no significant difference was found between them58. Conversely, we found a strong causal effect of SCLs on sleep apnea syndrome, which suggests this relationship is well worth exploring in future studies.Sleep apnea is a condition in which patients have repeated bouts of shallow breathing or apnea while sleeping. It is related to oxidative stress, high blood pressure, hypercoagulation, and inflammation, and can cause episodic hypoxemia60. Higher SCLs have been found in patients with SLE63. However, another study reported that there was no significant difference in SCLs between SLE patients and healthy controls64. The results of traditional epidemiological studies on the relationship between SCLs and SLE disease activity are controversial. Yilmaz et al. found that there was a positive correlation between SCLs and SLE disease activity63, but Sahebari et al. reported that there was a negative correlation between SCLs and SLE disease activity64. Ferns et al. proposed that copper had both pro- and anti-oxidative in vivo effect65. With regard to the pathogenic effect of reactive oxygen species on SLE, previous studies generally believed that reactive oxygen species is an important pathogenic factor, but recent studies believe that insufficient production of reactive oxygen species will lead to the occurrence of SLE67. There is a sharp contrast between these research results. Our research provides clues to the current controversial epidemiological results from a genetic point of view, and further corresponding research is needed in the future.SLE is an inflammatory autoimmune disease in which the immune system destroys healthy cells and tissues throughout the body68. Copper ions have been shown to aid in the regulation of disulfiram cytotoxicity in myeloid leukemia cells. Copper can help patients with high WBC counts by restoring the sensitivity of high-density leukemic cells to disulfiram and improving the efficacy of anti-leukemic therapies that include disulfiram69. Further research on this relationship between SCLs and WBC count is well worth exploring.Although no epidemiological research on the association between SCLs and WBC count has been reported, we found some definitive evidence of such an association. Serum copper and zinc concentrations were found to be higher in users of proton pump inhibitors than in controls, and WBC counts may reduce with long‑term proton pump inhibitor therapy70. In clinical laboratories, serum GGT has long been used as a marker for excessive alcohol intake and obesity, particularly in nonalcoholic fatty liver disease, alcoholic liver disease, and chronic hepatitis71. Indeed, serum GGT is a very early and sensitive indicator of oxidative stress and inflammation72. One epidemiological study suggested that serum GGT levels were positively correlated with SCLs73. Our study is the first to describe the causal relationship between them. Although both copper and GGT are involved in oxidative stress and inflammation75, the mechanism of the causal relationship between them has not been reported so far. The specific biological mechanism is well worth discovering.To fully understand whether these 213 deep phenotypes have causal effects on SCLs, we conducted the second round of two-sample MR analyses in the East Asian population. We found that genetically high GGT levels have a causal effect on elevated SCLs. GGT is a hepatic enzyme that aids in the production of the antioxidant glutathione at the outer surface of the plasma membrane77. A recent case-control study found that the risk of SLE increased with increased GGT levels78. Another early study reported that an increase of urinary GGT activity was seen in patients with SLE79. A study of Indian children and another study of Korean adults both showed that GGT was positively associated with WBC count81. These studies showed associations between GGT levels and sleep apnea syndrome, SLE, and WBC count. We did not find relevant literature for the other diseases and traits. Currently, there are no MR studies that have directly investigated causality between GGT levels and these six phenotypes. Because of the limitation of data acquisition, we did not conduct MR analysis on these possible relationships, and did not deeply explore whether serum copper might play a mediating role or determine the extent of the possible mediating effect of serum copper in these processes. Further independent studies aimed at solving these problems are needed.On the basis of our robust first and second round MR results for the East Asians, we inferred that serum copper might have a mediation effect on the causal effects of the GGT levels on the risk of esophageal varix, glaucoma, sleep apnea syndrome, and SLE, WBC count, and the possibility of using drugs affecting bone structure and mineralization. Serum GGT levels have been reported to be associated with the severity of obstructive sleep apnea syndromeTo further study the causal effects of BCLs on these deep phenotypes of Europeans, we conducted a third round of two-sample MR analyses in European populations. We found that genetically high ECLs had causal effects on the higher risk of carpal tunnel syndrome and lower risk of compression fracture.82. Carpal tunnel syndrome is produced when the median nerve in the wrist was compressed while passing through a space-constrained osteofibrous canal83. Although no epidemiological study of the association between ECLs and carpal tunnel syndrome has been reported so far, our results provide valuable evidence that is worth exploring. The causal effect of copper on carpal tunnel syndrome be based on the neurotoxicity of copper ions and the oxidative stress induced by copper. To corroborate this relationship, independent cohort and biological function studies are needed.The most common nerve entrapment syndrome is carpal tunnel syndrome, and it has been studied extensively84, and copper has been suggested to suppress osteoclastic resorption directly85. Conversely, a national cross-sectional study in the United States suggested that high SCLs were strongly related to increased total fracture86. The differences among these studies can be attributed to the different ethnicities, sample types, and other possible confounding factors that were not controlled. Our finding that genetically high ECLs were protective against the risk of compression fracture provides a reference for subsequent epidemiological studies and a more in-depth analysis of the biological mechanisms involved.So far, we have not found epidemiological studies of ECLs and compression fracture. However, many studies have shown that copper plays a vital role in maintaining bone health. Copper eliminates bone-free radicals generated by osteoclast activity as a cofactor in antioxidant enzymesInterestingly, we found that two diseases, food allergy, and urticaria, both appeared in the European and East Asian MR results where robust conclusions could not be drawn. Although our results did not show a robust causal relationship between these diseases and BCLs, our research gives suggestive clues on the causal relationship between them. Moreover, we found that SLE and the usage of drugs affecting bone structure and mineralization both appeared in the robust results of the East Asian MR and results where we could not draw robust conclusions of the European MR analyses. We also found that compression fracture appeared in the robust results of European MR and results where we could not draw robust conclusions of East Asian MR analyses. Because we used genetic IVs related to SCLs for the East Asian MR analyses and genetic IVs related to ECLs for the European MR analyses, whether differences of significance for the same disease in different populations can be attributed to ethnic differences needs to be studied further.18. We can suggest two possible explanations for this difference. (1) We used the GWAS meta-analysis summary statistics of UK Biobank and FinnGen for iron deficiency anemia, which increased the number of cases compared with the number for the single GWAS summary statistic from UK Biobank or FinnGen. However, because using the GWAS meta-analysis summary statistics may also greatly increase the overall number, the proportion of cases in the total number for a specific disease may decrease. Such decreased proportions will lead to weaker statistical power, which may have led to our failure to detect a causal effect between iron deficiency anemia and ECLs. (2) In their study, Zhou et al. used only two IVs that were associated with ECLs, and then conducted a PheWAS-MR (phenome-wide association study and MR) analysis. After the PheWAS, some phenotypes unrelated to the selected genetic variants were excluded, and their MR analysis did not include them. However, we included 12 SNPs as IVs in the MR analysis, which increased the statistical power. This may explain the difference to a certain extent. Our significantly robust results were not referred to in their paper.We noted that another BCLs MR analysis from UK Biobank indicated that genetically increased ECLs were causally associated with a reduced risk of iron deficiency anemia, whereas this association was not significant in our study9, we have included more metals and discovered loci for SCLs. To our knowledge, this may also be the first study to use bidirectional two-sample MR analysis with hypothesis-free investigations of the causal effects between BCLs and 213 deep phenotypes in the East Asian population. We also used two-sample MR analysis with hypothesis-free investigations of the causal effects of BCLs on 174 deep phenotypes in the European population. This approach provides a more comprehensive understanding of the impact of BCLs on the health of East Asians and Europeans. Moreover, the impacts of potential weak instrumental variable bias, heterogeneity, and pleiotropic effects on the results were also considered. Possible biases caused by horizontal pleiotropy were fully taken into account in our analyses process. We used the MR-PRESSO outlier test and other robust methods for the MR analysis, and no horizontal pleiotropy was detected in the results. The PhenoScanner database or the Ensembl Project would not necessarily distinguish between vertical and horizontal pleiotropy, and the former would not bias the MR analysis87. Furthermore, because the biological mechanisms and functions of numerous genetic variants are unknown, we did not use these variants to assess the IVs associated with potential confounding factors. The genetic IVs we used explain 11.09% of the variance in SCLs and 17.06% of the variance in ECLs, which increased the statistical power to identify significant associations88. F statistics were considered to avoid weak instrument bias, and I2, which evaluates the heterogeneity scale of IVs, was used to reduce the impact of potential heterogeneity. The presence of horizontal pleiotropy was examined or corrected using MR-Egger tests and MR-PRESSO. We used the leave-one-out sensitivity analysis to ensure no results were impacted by specific variants.To the best of our knowledge, ours is the first reported systematic GWAS of 21 BMLs using two independent prospective East Asian cohorts. Compared with the GWAS of 11 BMLs conducted in the European population by Ng et al.Our study has several limitations. Although we conducted the GWAS on 21 serum and plasma metals, because of differences in the concentrations of the same metals in serum and plasma, we only did the meta-analyses of the serum copper and serum manganese. The GWASs of the remaining 19 serum/plasma metals need to be combined with other appropriate independent cohorts for further replication in the future. Because relatively small numbers of cases were used in the GWAS, it was difficult to find significant SNPs. Despite this, we found loci for SCLs; however, more research is needed to verify our conclusions. In the two-sample MR analysis, we did not find genetic IVs of ECLs or SCLs in the East Asian and European populations, respectively, which limited our comprehensive study of the causal effects of BCLs on 213 deep phenotypes in these populations. For the same reason, we separately included the genetic IVs related to the SCLs of East Asians and the ECLs of Europeans, which had an impact on the horizontal comparison of differences in the same exposure between ethnicities. Although approximately 800,000 individuals were included in the MR analyses, the number of cases of specific diseases was still small, which reduced the statistical power. Moreover, because access to data was limited, our MR results were not verified in large independent databases.89. MEWHC included 825 Chinese with their follow-up completed in 2017. More detailed information about FAMHES, MEWHC were introduced elsewhere91. Our study was approved by the Ethics and Human Subject Committee of Guangxi Medical University. All the subjects provided written informed consent before participating in the study.To conduct comprehensive GWASs of 22 serum/plasma metal levels in the Chinese population, we used the serum metal levels data obtained from FAMHES in Guangxi Fangchenggang and plasma metal levels data obtained from MEWHC. FAMHES included 4303 Chinese males aged 17–88 years old at baseline. Their questionnaires and physical examinations at baseline were completed in the Medical Center of Fangchenggang First People’s Hospital between September 2009 and December 200992. Specifically, each serum/plasma sample was diluted 20 times by using acidic solution containing 0.01% TritonTM X-100, 0.5% n-butyl alcohol and 1% nitric acid. We used ClinChek® human serum controls for trace elements  and Standard Reference Material®1640a  as standard reagent for quality assurance in serum measurement. Moreover, we used ClinChek® human plasma controls for trace elements  and Standard Reference Material®1640a for quality assurance in plasma measurement. The following 22 metals  were measured in both cohorts. The samples with concentrations less than the limit of detection were given a value equal to the limit of detection divided by √2 as concentrations. More than 20% of samples in FAMHES did not reach the limit of detection for antimony levels. Therefore, in the subsequent analysis, we included the remaining 21 metals, excluding antimony.All blood samples are stored at −80 °C until analysis. We used an inductively coupled plasma mass spectrometry  to measure the serum metal concentrations of FAMHES and plasma metal concentrations of MEWHC, respectively. Our previous research also introduced the corresponding methods93. We excluded individuals with (1) individuals call rate <0.95, (2) genotypic and phenotypic sex mismatch, (3) heterozygosity rate deviating more than three standard deviations from the mean, (4) kinship coefficient > 0.0884, (5) missingness of phenotypes or covariates. We also excluded SNPs with (1) call rate <0.95, (2) MAF < 0.05, (3) Hardy–Weinberg equilibrium P-value (PHWE) < 1.0 × 10−6. Then we used liftOverPlink (https://github.com/sritchie73/liftOverPlink) to convert genome position from hg18 to hg19. Snpflip (https://github.com/biocore-ntnu/snpflip) was used to fix the problem with reverse and ambiguous strand SNPs. Before phasing and imputation, we redid the same quality control procedure mentioned above. Eagle v2.4.1  and Minimac4 (https://github.com/statgen/Minimac4) were used to phase and impute genotyped data of autosomal chromosomes, respectively. In the procedure of phasing and imputation, we chose 1000 G Phase3 v5 EAS (GRCh37/hg19) as a reference panel. After phasing and imputation, SNPs with (1) MAF < 0.01, (2) minor allele count < 20, (3) PHWE < 1.0 × 10−6, (4) imputation quality (Rsq) < 0.7 were excluded. A total of 805 subjects were included from MEWHC and genotyped using Infinium Asian Screening Array-24 + v1.0 Kit (ASA). Because of the lack of X chromosome genotype data, we did not compare whether the genotypic and the phenotypic sex match. The remaining procedure of quality control was the same as what was done in FAMHES. Finally, before conducting GWAS, we removed the outliers whose metal concentrations were more than 4.5 inter-quartile ranges away from the median of each metal concentration. The rank-based inverse normal transformation was used to transform each metal concentration to approximate normality96.In our study, a total of 2020 subjects were chosen from FAMHES and genotyped using the Illumina Omini one platformwww.cog-genomics.org/plink/2.0/) was selected to perform the GWAS under assumptions of dosage additive effects97. P = 5.0 × 10−8 was set as the genome-wide significance threshold, and P = 1.0 × 10−5 was selected as the genome-wide suggestive threshold. Stepwise conditional analysis was conducted to find the additional independent significant signal. For replication, we planned to perform meta-analyses using GWAS summary statistics of the same metal from both FAMHES and MEWHC. However, considering the difference between serum and plasma metal concentrations, we conducted the non-parametric test on the concentrations of each metal between the two cohorts via IBM SPSS Statistics 25 (https://www.ibm.com/support/pages/downloading-ibm-spss-statistics-25). Finally, since there was no statistically significant difference in copper concentrations and manganese concentrations between the two cohorts, we respectively conducted GWAS meta-analysis on the copper and manganese levels. Because of the significant statistical difference between the concentrations of other same metals in the two cohorts, the meta-analysis was not performed in the remaining metals. The Bonferroni-corrected threshold of < 2.38 × 10−9 ) in the context of meta-analysis was considered. Since we did not find any SNP significantly associated with manganese levels in the results of GWAS meta-analysis on manganese levels, and find two SNPs significantly associated with copper levels in the results of GWAS meta-analysis on copper levels, we only conducted the subsequent analysis of blood copper.For each metal, we conducted a GWAS respectively in both cohorts using a generalized linear model adjusting age, sex, body mass index, ethnicity, first ten principal components, alcoholic drinking, and smoking status. PLINK 2.0 .To investigate the causal relationships between SCLs and 213 deep phenotypes  from BioBank Japan in the East Asian populationP < 1.0 × 10−5) with SCLs from GWAS meta-analysis as candidate IVs105. Secondly, we filtered out those SNPs in strong LD since strong linkage disequilibrium will bias the subsequent analysis. In our study, we conducted LD clumping process  using 1000G Phase3 v5 EAS (GRCh37/hg19) as a reference panel, and SNPs with r2 < 0.001, MAF > 0.01 were retained. Thirdly, we evaluated the heterogeneity of each candidate IV using I2 and P-value for Cochran’s Q statistic. F-statistic was considered to avoid weak IV bias. In this step, we included SNPs with I2 < 50, P-value for Cochran’s Q statistic > 0.05, and F-statistic >10106. Fourthly, in the harmonizing process of two-sample MR analyses, both ambiguous and palindrome SNPs would be corrected or directly excluded. Since there was no need, we did not use any proxy SNPs. Finally, a total of 11 SNPs were included as IVs in the first round two-sample MR analyses. The total variance explained by the selected 11 SNPs was calculated using the following equation , and βn referred to the additive effect94. All the 11 SNPs totally explained 11.09% of the transformed SCLs variance  with each phenotype from the GWAS summary statistics in BioBank Japan as candidate IVs. The rest of the process of selecting genetic IVs is the same as that done in the first round.To further research whether the 213 deep phenotypes from BioBank Japan have causal effects on SCLs, we conducted the second round two-sample MR analyses, which is an inverse MR analysis comparing with the first round. We selected genetic variants which were associated (https://pheweb.jp/downloads). Because of lacking serum/plasma copper levels GWAS summary statistics for European populations, we finally used the SNPs which were independently (r2 < 0.05) associated (P < 1.0 × 10−5) with ECLs as IVs6. Except that the reference panel used in the LD clumping process is 1000G Phase3 v5 EUR (GRCh37/hg19), the rest process of selecting genetic IVs is the same as that done in the first round. The details of the 12 SNPs, which were finally included as genetic IVs, are shown in Supplementary Table To comprehensively determine the causal effects of BCLs on these 213 deep phenotypes in humans, we further used the GWAS meta-analyses summary statistics of UK Biobank and FinnGen to perform the third round of two-sample MR analyses. The GWAS meta-analysis summary statistics could be downloaded at the website  in the end. More information about the 174 deep phenotypes is shown in Supplementary Data 110. Significant (P < 0.05) difference between y-intercept of MR-Egger and 0 represents that there is significant horizontal pleiotropy. P-value of Cochran’s Q statistics of IVW and MR-Egger < 0.05 represents that there is significant heterogeneity. If there was significant horizontal pleiotropy or heterogeneity, the MR-PRESSO outlier test would be used to detect the outlier, which usually leads to horizontal pleiotropy111. The NbDistribution  in the MR-PRESSO test was set to 20,000. After removing the outliers, we re-performed two-sample MR analyses with IVW, MR-Egger, and WM methods. Based on the results of the leave-one-out sensitivity analysis, horizontal pleiotropy tested via MR-Egger, and heterogeneity tested via IVW and MR-Egger, we divide the results into the section where robust conclusions can be drawn and the section where robust conclusions cannot be drawn. Then we performed MR-RAPS to re-estimate causality in exposure-outcome pairs of robust conclusions section to eliminate the estimation bias of weak IVs112. In addition, we also considered the corresponding Bonferroni-corrected threshold < 0.00023 (0.05/213 outcomes) and the suggestively significant threshold (0.00023 ≤ P ≤ 0.05) in MR analysis. Finally, we calculated the statistical power of IVW, MR-Egger, MR-RAPS, and WM methods using the methods which are based on the non-centrality parameter114. Because of the lacking variance of the exposure variable and outcome variable, we didn’t calculate the statistical power for quantitative trait outcomes. All two-sample MR analyses were performed in R version 4.0.5 using the TwoSampleMR package109. A two-sided P value < 0.05 was used to determine statistical significance.All three rounds of two-sample MR analyses followed the flow chart Fig. . FirstlyFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


Tenebrio molitor, the first edible insect approved as a novel food in the EU, is a promising candidate for alternative protein sources, implementing circular and sustainable production systems. This study aims to determine the microbiological quality and physicochemical properties of mealworm powders obtained by four different processing pathways. Contents of dry matter, protein, fat, ash, water activity (aw) and a range of microbial counts were measured and analyzed by one-way ANOVA with Tukey’s test. Results showed small differences in the proximate composition of the powder samples , except for the one that underwent a defatting step (protein 70.04% and fat 16.84%), p < 0.05. A level of water activity of less than 0.2 was reached for all pathways. Fresh mealworm samples had high total aerobic counts (8.4 log CFU/g) but were free of foodborne pathogens. Heat treatments applied during transformation were sufficient to kill vegetative cells (reduction of 2.8–5.1 log CFU/g) rather than bacterial endospores (reduction of 0.3–1.8 log CFU/g). Results were confirmed by predictive microbiology. This study validated the efficacy of a boiling step as critical control points (CCPs) of insect powder processing, providing primary data for the implementation of HACCP plans. The practice of eating edible insects (entomophagy) has been part of the regular diet for millions of people in various regions in Asia, Africa and Latin America for a long time . In EuroHowever, the insect sector is still in its early stage, especially in European countries. The report of the Food and Agriculture Organization (FAO) stated that some challenges of the insect sector, such as potential food safety issues, the establishment of suitable regulatory frameworks, research gaps, upscaling the production and negative attitudes of consumers, need to be overcome to have a more global reach . For exaSalmonella spp., STEC, L. monocytogenes, Cronobacter spp., C. perfringens, C. botulinum, S. aureus, B. cereus group. However, currently, the amount of the literature regarding the microbial quality and potential hazards of different insect species is still rather limited; more quantitative research should be carried out in order to implement an appropriate HACCP  system and progress in its risk assessment for edible insect supply chains. Recent studies have shown that edible insects generally contain complex ecosystems with marked variations in microbial load and diversity as well as stable and species-specific microbiota . High leTenebrio molitor larva  is viewed as one of the most promising species due to its nutritional value and ability to mass-produce [T. molitor or Acheta. domesticus [T. molitor, the first insect species as a novel food in the EU was issued in June 2021 [-produce ; moreovemesticus . In 2020mesticus . Soon afune 2021 . ConsequWell-designed manufacturing processes are required to produce high-quality insect products while maintaining food safety to meet the growing interest of consumers. Various technologies have been employed to process insects into non-recognizable forms, such as powders or flour, including heat treatment , drying , grinding (wet or dry) and some innovative methods mainly for protein, fat and/or chitin extraction . With rew) were measured, and the results were analyzed by one-way analysis of variance with Tukey’s test. In addition, a microbiological predictive model was used to assess the efficacy of heat treatment steps on eight significant hazards. The results of this study can provide preliminary data and proof for the application of the HACCP system in the insect food industry.Hence, the present study selected four different mealworm powder processing pathways, including boiling or freezing slaughter, oven- or freeze-drying, defatting or not and grinding. These pathways, called A, B, C and D, are described in ®, Le Gallet, France) that specialized in rearing insects for pet food. They were kept in plastic boxes and transported in hygienic conditions to the laboratory. Then, they were divided randomly into two groups: one group was immediately stored in the refrigerator at 4 °C before the first processing trial, and the other group was frozen for one week to duplicate the experimentation. Live yellow mealworm larvae (two batches), which already had a 24 h period of fasting, were purchased from a French company  with a temperature sensor. The mealworm paste, after cooking, was transferred to a centrifuge  for centrifugal separation to remove oil layer. The four processing pathways  are described in w each hour). While freeze-drying was applied to whole mealworms of pathways C and D in freeze-drying equipment . Finally, all the pathways contained an identical grinding step by a blender  to obtain different mealworm powders. Oven-drying steps were conducted in pathways A and B and stopped only when water activity was below 0.5 (Samples PA-OD and PB-OD were taken to measure a3) in stainless steel was fitted with a cellulose filter and a stainless-steel frit, both at the top and the bottom, and was filled with a sample (5 g) and diatomaceous earth (Thermo Scientific™ Dionex™ ASE™). The extraction process consisted of the following steps: loading the cell into the oven (maintained at 120 °C), filling the cell with solvent (n-hexane), heating the cell  and extracting twice. Nitrogen gas was employed to collect the remaining extract into the collection bottle (250 cm3) at the end of the extraction before unloading the cell. The oil/solvent mixture was evaporated under vacuum to dryness at 40 °C in a rotary evaporator. Carbohydrates were calculated by the difference between the total mass and the sum of crude protein, lipid content, moisture and ash.The dry matter content was determined gravimetrically by drying at 105 °C to a constant weight . Proteinw) is a measure of the availability of water to take part in chemical reactions (varies from 0–1.0), determining the limits for microbial growth [w > 0.91, and most molds need aw > 0.80; and once aw < 0.6, the product is generally considered to be shelf-stable [w) was measured on a 2–3 g aliquot of each sample using an AquaLab Pre aw meter  until aw and temperature were stable during 5–15 min. Larvae samples were ground in a blender before being analyzed. Water activity  and Tukey’s test procedures with a level of significance set to 0.05. All the statistical analyses were performed with XLSTAT software version 2018.5 (Addinsoft).Enterobacteriaceae, lactic acid bacteria (LAB), bacterial endospores, yeasts and molds count (YMC), are widely used as measures of hygienic conditions or quality of food products. The fresh mealworm samples were taken aseptically (60 g for each) and transported to the laboratory at ambient temperature and dealt with upon arrival (<10 min). All the samples from power processing trial were stored in refrigerator at 4 °C (<24 h) before analysis. The microbial analysis methods are displayed in A number of classic microbiological indicators, such as total aerobic count (TAC), ® Interscience, Saint Nom, France), the primary suspensions were stored at ambient temperature for at least 1 h. Ten-fold dilution series (from 10−1 to 10−6) were prepared and plated on different media using the pour-plate technique, according to the ISO standards. TAC were assessed after incubation on Plate Count Agar  at 30 °C for 72 h; bacterial endospores were determined on PCA plus starch at 30 °C for 72 h after a heat shock treatment (water bath at 80 °C for 10 min) of the ten-fold dilutions; LAB were incubated on de Man, Rogosa Sharpe agar  at 30 °C for 72 h. For each dilution, two replicates were performed. All microbial counts were expressed as log CFU/g. Quantification of TAC, LAB and bacterial endospores were performed in our laboratory following next steps, while others were outsourced to an accredited laboratory. Additionally, 10 g of subsamples were taken aseptically from each step during powder processing and mixed with 90 g of peptone water  in a stomacher plastic bag. After homogenization for 60 s in a paddle blender (Bagmixerref) is known, D-values can be estimated for every desired temperature (see Equation (1)) [ref is the D-value at Tref (in minutes); Tref is the reference temperature (in °C); z is the temperature increase (in °C) needed to reduce the D-value with a factor of 10.In order to assess the efficacy of heat treatments during powder processing, this study estimated log reductions of eight significant hazards based on Bigelow model. D-value is the time required at a given temperature or set of conditions to achieve a log reduction , and z-vion (1)) : logD = Reference D-value and z-value data of eight hazards were collected from the scientific literature ; they weEnterobacteriaceae and 4.0 log CFU/g for yeasts and molds. Lower numbers were observed for bacterial endospores 3.4−5.3 log CFU/g. Similar microbial counts were found in rinsed fresh mealworm samples.Average microbial counts of fresh mealworm larvae were relatively high see , with meL. monocytogenes, Salmonella spp. and C. sakazakii were not detected from any of the analyzed samples (see Staphylococci), except for sulfite-reducing anaerobes at low-level (1.0 log CFU/g), which is an indicator for Clostridium spp., mainly C. perfringens.Overall, ples see . Almost Enterobacteriacae (<1.0−3.6 log CFU/g), yeasts and molds (<1.0−3.4 log CFU/g) compared to fresh mealworms. However, the powder from pathway D (PD-P) was highly contaminated, similar to fresh mealworms   showed a better microbial quality  than raw mealworms. Likewise, lower counts were found in bacterial endospores (2.1−2.7 log CFU/g), LAB (1.8−5.2 log CFU/g), U/g) see . AdditioU/g) see .As S. aureus enterotoxin, which have a higher heat resistance  than vegetative form bacteria, while the latter all showed an enormous log reduction  (moisture content corresponding to 70.5%) as well-suitable for microbial growth and enzymatic and chemical activity [w of all samples were reduced until at very low levels .The water activities and dry matter of fresh and powdered mealworms are shown in activity . Each prThe proximate compositions of fresh and powdered mealworms are shown in Enterobacteriaceae 5.0–7.7 log CFU/g, bacterial endospores or spore-forming bacteria 0–5.3 log CFU/g, psychrotrophic aerobic count 5.9–7.6 log CFU/g and yeasts and molds 2.6–6.5 log CFU/g [Fresh or unprocessed mealworm larvae contain relatively high levels of microbial loads, which was in line with those reported in previous studies, including TAC 6.4–9.3 log CFU/g, LAB 4.9–8.3 log CFU/g, og CFU/g . MealworL. monocytogens, Salmonella spp., E. coli) were not detected in other culture-dependent analyses. However, in some studies [Staphylococcus spp. was found in a small number of samples (3.7–6.0 log CFU/g) in fresh mealworms. Garofalo et al. [Listeria spp., which was not possible to identify at the species level. L. monocytogens [Salmonella spp. [The absence of microbial pathogens in analyzed fresh mealworm samples may be due to the modern rearing techniques and GHPs of the company. To date, common foodborne pathogens  [B. cereus spores in dried and powdered insects is moderate, occurring regularly [Concerning mealworm powder, very little data on pathogenic microorganism detection are available, but spore-forming bacteria such as d dried) ,54. The egularly .In Europe, no specific microbiological criteria for insect-based products are established for human consumption currently. Some food safety authorizes—such as the Belgian Superior Health Council (BSHC), the Federal Agency for the Safety of the Food Chain (FASFC) and the Netherlands Food and Consumer Product Safety Authority (NVWA)—have composed an opinion to refer to the hygiene criteria for minced meat in EU Regulation (EC) No. 1441/2007 for edible insects ,57. For Enterobacteriaceae (4.0–5.0 log CFU/g), LAB (4.0–5.5 log CFU/g), psychrotrophic aerobic count (4.5–6.0 log CFU/g) and yeasts and molds (2.0–4.0 log CFU/g). Aside from some exceptions, these indicators are relatively sensitive to heat treatments. Klunder et al. [Enterobacteriaceae in insects, but some bacterial endospores will survive. Vandeweyer et al. [Heat treatments such as boiling, blanching, frying, roasting and other cooking methods are the most commonly applied steps for microbial inactivation (CCPs). Their efficacy in improving microbial quality depends on the combination of duration and temperature as well as the characteristics of microorganisms. There is no doubt that boiling (100 °C for 5 min) applied in pathways A, B and C and cooking (80 °C for 30 min) of pathway B are the major CCPs in our processing trials. As confirmed by the present study, similar microbial reductions after heat treatments were observed in other studies : TAC  as well as different heat-resistance of strains because they can survive mild thermal treatments (<100 °C) and exist in the final products. Garofalo et al. [C. perfringens spores had been found in marketed whole processed (boiled and dried) crickets, grasshoppers and mealworms. Caparros Megido et al. [Furthermore, the results of predictive models provided a general trend of thermal inactivation performance on significant pathogens since the parameters used are not specific to insect matrices. Particular attention should be paid to spore-forming bacteria  in this processing trial still need further research.Drying is primarily a preservation step to extend shelf life. However, it also makes food products more efficient and cost-effective to store (they do not need to be refrigerated or kept cool) and ship (powders weigh less) . The overganisms . In addiS. aureus enterotoxin when they exist in insects. Of note, the slight increase in microbial counts  for minced meat. The prerequisite for achieving desired effects is to carefully control the temperature and duration to meet the critical limits . However, these treatments are insufficient to eliminate bacterial spores and l counts  after heT. molitor larvae at low temperatures led to an insignificant log-reduction of the TAC by 0.1(±0.1). However, freezing and frozen storage have a variable effect on microorganisms; for instance, quick freezing and thawing would result in higher microbial survival rates than slow ones; relatively higher storage temperatures (−4–−10 °C) have a greater lethal effect on microorganisms than lower temperatures (−15–−30°C) [As expected, pathway D, which only contains freezing and freeze-drying, is considered inappropriate for producing microbial-safe insect powders. Bußler et al.  also rep5–−30°C) . Freeze-5–−30°C) . So, in w, from an initial value of 0.99 for fresh mealworms to very low levels (aw < 0.2), regardless of the pathway considered. This aw level is sufficient to inhibit microbial growth; however, at very low water activity (aw  <  0.2), lipid oxidation that is responsible for rancidity and the development of off-flavors can increase [All processing methods applied, as they include a drying step, significantly decreased the water activity aincrease .Regarding the proximate composition, the fat, ash and carbohydrates contents of fresh mealworms were similar to values reported in other studies, whereas the protein content  was higher than the values commonly found  ,66,67,68In terms of overall results, processing pathway D was the less efficient method for its inability to preserve protein in mealworms compared with the others processes. Although the protein content of mealworm powders seemed to be similar between processing pathways A, C and D, their effects on protein quality remain unclear, such as denaturation, modification or destruction of amino acids and the Maillard reaction. In the present study, knowledge of microbiological quality and the main physicochemical factors of fresh and powdered mealworms was obtained thanks to the combination of processing trials, analysis and predictive microbiology. The high level of microbial contamination of fresh mealworms highlights the necessity of a processing pathway involving an effective microbial inactivation, such as a boiling step considered as a CCP. Specific attention must be paid to bacterial spores when establishing the microbial criteria in insect-based products, such as insect powder. Additionally, the nutritional value of all the powdered insects remained high despite the loss of proteins in processing pathways A, C and D. Nevertheless results of this study must be considered in light of the limited number of samples analyzed. Therefore, further studies are needed to collect more data and go further with the implementation of culture-independent analysis and non-thermal inactivation methods. More insights concerning the effects of different processing steps on protein quality, such as denaturation, modification or destruction of amino acids and digestibility, are also necessary. On a broader scale, the next step in the development of the edible insect sector seems to be the enhancement of consumer acceptance by developing safe and high-quality insect food products.


A total of 3105 HIV-1 CRF01_AE gag sequences representing 17 countries from the timeline 1990–2017 were obtained. The sequences’ phylogenetic relationship and epidemic dynamics were analyzed through a Maximum Likelihood tree and Bayesian Skyline plot, respectively. Genomic variability was measured through Shannon entropy and time-dependent immunoevolution was analyzed using changes in proteasomal degradation pattern, cytotoxic T lymphocytes (CTL) epitopes, and Human leukocyte antigens (HLA) restriction profile. The most recent common ancestor of the HIV CRF01_AE epidemic was estimated to be 1974±1. A period of exponential growth in effective population size began in 1982, fluctuated, and then stabilized in 1999. Genetic variability (entropy) consistently increased, however, epitope variability remained comparable; the highest number of novel CTL epitopes were present in 1995–1999, which were lost over time. The spread of the HIV-1 CRF01_AE epidemic is predominant in countries within Asia. Population immunogenetic pressures in the region may have contributed to the initial changes and following adaptation/stabilization of epitope diversity within gag sequences.Currently, little is known about the time-dependent evolution of human immunodeficiency virus-1 (HIV-1) circulating recombinant forms (CRF) 01_AE, a dominant recombinant form associated with HIV-1 epidemics worldwide. Since  HIV remains a prevalent global health issue worldwide . HIV-1 iCRF01_AE was previously believed to have originated in Central Africa and spread to Thailand in the late 1970s . From Thwww.hiv.lanl.gov). It is found in regions including North America, Western Europe, Eastern Europe, and Central Asia, South and Southeast Asia, East Asia, Oceania, and Central Africa with the highest prevalence in South and Southeast Asia [www.hiv.lanl.gov). The gag gene of HIV-1 CRF01_AE contributes to the structural maturation of the virus by producing higher-ordered structures for the correct assembly, budding, and maturation of new infectious particles [gag protein can interfere with the processing of viral antigen, thus disrupting the generation of viable antigenic epitopes. The patient’s human leukocyte antigen (HLA) alleles represent a major host-driven selective pressure that steers the amplification of specific epitope mutations [Currently, HIV-1 CRF01_AE comprises 5.9% of total world sequences , and after removal of duplicate sequences, a total of 3105 sequences were used in the study. These sequences were deposited from the following 17 countries: Afghanistan, Cameroon, Central African Republic, China, Cyprus, Hong Kong, Indonesia, Ireland, Japan, Myanmar, Philippines, Sweden, Switzerland, Thailand, United Kingdom, United States, and Vietnam  tree was generated with 3105 software , using tgag sequences from different countries from the years 1990 to 2017 were used. Sequences were selected using the approach described by Novitsky, et al., where a limit was set to 5–6 sequences per country and year groups [8, which gave an effective sample size (ESS) of >200. MCMC sample analysis and Bayesian skyline plot construction were performed using Tracer v1.7.4 [A Bayesian Markov Chain Monte Carlo (MCMC) inference was applied to estimate the effective population size and tMRCA of HIV-1 CRF01_AE, using BEAST v1.7.4 software. For this analysis, a total of 286 r groups . If 5 orr v1.7.4 .gag sequences from each year group was calculated using an online tool available at the Los Alamos National Laboratory (LANL) HIV Sequence Database: http://www.hiv.lanl.gov/content/sequence/ENTROPY/entropy_one.html. Using GraphPad software, the mean entropy value for each year group was calculated, while the statistical significance of the difference between the mean of each year group was calculated using one-way ANOVA with Tukey’s multiple comparison test.To evaluate time-dependent changes in HIV-1 CRF01_AE genomic variability, Shannon entropy analyses were performed. Shannon entropy is a measure of the probability of acquiring mutations, including epitope-related mutations, in a given set of genomic sequences . The Shagag sequences using NetChop 3.1 software [Proteasomal degradation sites were predicted in the HIV-1 CRF01_AE software . A predigag gene sequences for each year group were created using the Consensus Maker tool, and consensus sequences were translated into amino acid sequences using the Expasy translate tool. These protein sequences were then used to predict CD8+ T cell epitopes using CTLPred software [gag epitopes was predicted using nHLAPred software [For epitope analysis, consensus software , where asoftware , where tenv sequences. For this analysis, a total of 5265 env sequences (http://www.hiv.lanl.gov/) and grouped as per the strategy described above except for the last group (2015–2018) which contained one sequence for 2018. The epitope analysis was performed as described above for the gag protein.The epitope analysis was also confirmed using HIV-1 CRF01_AE equences  were retgag sequences revealed phylogenetic clustering between the sequences from the years 2013, 2010, and 2005. Sequences from the other years were scattered throughout the phylogenetic tree, without any apparent relationship between branching topology and time of sampling  for CRF01_AE was found to be around 1974±1  betweengag variability over the study period, we performed the Shannon entropy analysis. The gag sequences were grouped into the following year sets: 1990–1994, 1995–1999, 2000–2004, and 2005–2009, 2010–2014, and 2015–2017. Shannon Entropy analysis showed a consistent increase in entropy over the years, with the highest value observed for the 2015–2017 year group  where mutations were present, while differences in proteasomal degradation scores were noted at sites 30, 82, 83, and 370  were evaluated. Analysis of HIV-1 CRF01_AE gag epitopes showed that the epitope variability increased gradually after 1990, reaching a maximum in the 1995–1999 year group (5.95%), then declined to its minimum in the 2000–2004 year group (3.87%), rose again to reach its maximum (5.95%) in 2010, and sustained till 2017 , then declined slightly and then rose again to reach its maximum (46.7%) in 2009. After a sudden decline (38.8%) in 2014, variability increased again (51%) in the 2015–2018 year group  in countries such as Thailand in the late 1980s [gag sequences between 1996–1998. It was during these years that the virus was introduced to China and began spreading in the country [gag have found similar declines in effective population size in the second half of the 1990s followed by an increase in effective population size in the 2000s [gag sequences remained stable afterward as transmission remained high.Analysis of the origin of the virus using Bayesian analysis showed that the time to the most recent ancestor of HIV-1 CRF01_AE id-1970s . After tid-1970s . In our id-1970s ) of gag te 1980s . This pe country . Previouhe 2000s . The effShannon entropy was used as a measure of time-dependent genetic evolution. It is a quantitative measurement of the uncertainty in a data set i.e. the probability of acquiring mutations in a genomic sequence, where an increase in entropy values reflects a higher probability for acquiring mutations . ShannonThe proteasome machinery degrades proteins into peptides of varying lengths, which are then sequentially processed and displayed by the HLA-I molecules to T lymphocytes . Multiplgag reached its maximum value between 1995–1999 which corresponds to the rise in effective population size during this period, followed by a decline in the 2000–2004 year group, and a gradual return to its maximum value, which was sustained after 2010 and corresponded to a stabilization of the effective population size. The selection pressure over the years may have resulted in genetic evolution which provided the virus ability to escape host response mechanisms and permeate the population [CTL epitope variability and HLA binding epitope variability were also used to understand the time-dependent genetic evolution of the virus . Epitopepulation , 36. SimHLA restriction analysis showed binding of certain HLA alleles including HLA-B*51, HLA-B*27, and HLA-B*5801 with unique or mutated epitopes . PreviouThere were a limited number of sequences available for 1990–1994, and 1995–1999 year groups. A greater number of sequences from these years would have provided further insight into the spread of HIV soon after the origin of a recombinant form. This information will be useful in understanding and responding to recombinant forms of the virus as they originate. A year-wise analysis of epitope sequences from the same populations will further help in deciphering the true nature of this phenomenon and provide a better understanding of the direction in which the HIV-1 epidemics continue to evolve. This information will be crucial in anticipating prevention and control strategies for CRF01_AE -infected patients, especially in populations where the epidemic is newly emerging.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableMutation in the epitope is represented by the color red.(DOCX)Click here for additional data file.S4 TableThe epitopes are divided in three categories: Novel epitopes (epitopes unique to one year group), intermittently recurring epitopes and mutated epitopes. Mutation(s) in epitope are underlined, while ‘-‘ in the table represents the absence of epitope in a particular year group.(DOCX)Click here for additional data file.


Acacia senegaltree, is known for its prebiotic effects. Fermentation of GA by colonic bacteria increases serum butyrate concentrations. This study was conducted to investigate if GA supplementation can increase Cathelicidin expression in macrophages.Antimicrobial peptides (AMPs) are important effectors of the innate defense system. Cathelicidins,  is one of the two major classes of AMPs in humans. The upregulation of LL-37 synthesis is a novel non-antibiotic approach to prevent or treat infectious diseases. Butyrate was found to induce Cathelicidin expression. Gum Arabic (GA), an exudate from The study was an in-vivo experiment in mice. Thirty mice were randomly divided into three groups, ten mice per group. The two intervention groups received GA dissolved in drinking water in two different concentrations (15% w/v and 30% w/v) for 28 days. The third group served as a control. Blood was collected on Day 29 to isolate peripheral blood mononuclear cells (PBMC) which were cultured to obtain monocyte derived macrophages (MDMs). The transcription level of CRAMP was determined in MDMsby qPCR.p = 0.023) in CRAMP expression in MDMs following 28 days of 15% GA supplementation, compared to the control group, but there was no significant change in the group on 30% GA supplementation (p = 0.055).We detected a significant increase (GAsupplementation can induce Cathelicidin expression in MDMs and the effect is dose dependent.The online version contains supplementary material available at 10.1186/s12906-022-03627-9. Antimicrobial peptide (AMPs), also known as host defense peptides (HDPs), act as natural broad-spectrum antibiotics and play essential roles in regulating host defense and immunity , 2. MoreEscherechia coli intracellular LL-37 induced the production of reactive oxygen species (ROS), increased the permeability of the membrane and affected the transcription of genes related to energy production and carbohydrates metabolism with the net result of inhibiting bacterial growth [LL-37 has broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as against fungi and enveloped viruses . The antl growth .Mycobacterium tuberculosis [Despite its direct microbicidal action, several line of evidence have shown that the anti-infective activity of LL-37 in vivo is mainly dependent on its ability to modulate the immune response . LL-37 brculosis .The non-microbicidal, immunomodulatory actions of Cathelicidins have attracted increasing attention and have been associated with several autoimmune diseases; such as psoriasis , systemiInduction of endogenous CRAMP/LL-37 expression has emerged as a novel approach in combating infectious diseases through alternative therapy.Short-chain fatty acids, butyrate in particular, were found to be strong inducers of LL-37 gene expression . Sodium Mycobacterium tuberculosis (Mtb) in human macrophages [An in vitro study showed that PBA induced cellular production of LL-37and inhibited the growth of rophages . Furtherrophages . In a rarophages . .Acacia senegal or Acacia seyal trees. It consists of a mixture of polysaccharides (major component) plus oligosaccharides and glycoproteins [Bifidobacteria, Lactobacteria, and Bacteriodes in the gut and is considered to have prebiotic actions [Gum Arabic (GA) is derived from the exudates of proteins , 35. Oraproteins . GA sign actions . GA is c actions , anti-ma actions  immune-m actions , 39 and  actions , 40. GA  actions . It also actions , 44. Ver actions . Also, G actions .Regarding Cathelicidin expression, several studies showed that GA ingestion increases serum short chain fatty acid concentration, mainly butyrate and propionate , 48.. MaAcacia Senegal tree with a particle size less than 210 μm . Its quality was consistent to the requirements of Food and Agriculture Organization of the United Nations (FAO) and British pharmacopoeia (BP).Gum Arabic powder used in the study was a 100% natural powder produced mechanically from the extract of 2 inhalation and blood samples were collected under aseptic condition.The study included 30 wild-type male mice aged 4 months, with an average weight of 25 g. Mice were obtained from the National Center For Research and were housed in separate cages. Mice had free access to food and water. After 5 days adaptive period, mice were randomly and equally divided into three groups as follow: The control group; 15% GA group: mice in this group received 30 g/d GA in 200 ml drinking water in a concentration of 15%w/v; and 30% GA group: mice in this group received 60 g/d GA in 200 ml drinking water in a concentration of 30% w/v. GA was provided to the two intervention groups. The lower dose of GA was selected based on previous experiments with GA , 44. TheBlood samples were collected on day 29.Mice were euthanized and blood collected by cardiac puncture and placed into EDTA tubes. Samples were centrifuged for 10 minutes at 2000 g and the plasma was stored at − 20 C°for further analysis. The buffy coat was transferred into a single tube that can contain at least twice its volume. Then it was diluted and mixed gently with equal amount of PBS.2 at 37 °C. Thereafter, the supernatant containing the non-adherent cells was removed. The remaining adherent cells in the culture plate monocyte derived macrophage (MDM) were harvested using a cell scraper and treated with Saponin 0.1% [Peripheral blood mononuclear cells (PBMC) were isolated from the diluted buffy coat using Ficoll–Paque density gradient (3 ml of Ficoll) was added to the bottom of a15 ml conical tube without touching the side of the tube. Gently the Ficoll was overlayed with the diluted buffy coat. The diluted sample was allowed to flow down the side of the tube and pool on top of the density gradient media surface without breaking the surface plane and then the tube was centrifuged at 400 g for 20 min at 18 °C. New sterile conical tubes were labeled, with the same number and same volumes as for the density gradient cell separation, which will contain PBMCs. The plasma layer was removed as much as possible with pipette. The mononuclear cells were collected from the plasma/Ficoll interface with a disposable transfer pipette and transferred into a new sterile conical tube. PBS (3 ml) was added to each tube and the tubes were centrifuged at 400 g for 10 minutes. The supernatant was removed carefully (the pellet is loose because of red blood cells) by tilting the tube and pipetting off the supernatant without touching the pellet. The cell pellet was suspended by gentle pipetting in a 1 mL PBS and the tube was then filled with PBS and centrifuged at 400 g for 10 minutes. The supernatant was discarded and cell concentration and viability were determined by using the Cellometer 2000. PBMCs were washed by using PBS and re-suspended in culture medium containing 10% autologous plasma in RPMI-1640, 1% L-glutamine, 1% sodium pyruvate, 0.5% amphotericin B and 1% penicillin-streptomycin. Cells were plated in two parallel 4-well tissue culture plates  and incubated for 5 days in 5% COnin 0.1% .RNA was extracted from MDM samples as follow: Cells were homogenized in TRI Reagent  then it was extracted with chloroform in phase lock tubes (5 Prime) following the manufactures protocol and stored at − 80 °C until processed. RNA integrity and concentration were determined on a NanoDrop 2000 (Thermo Scientific). The b-actin primers used to amplify beta actin were, BACTIN-1 (GGT-CGTCGACAACGGCTC) and BACTIN-2 (TGCCATGTTCAATG-GGGTAC). CRAMP-F1 (5′-AGGAGATCTTGGGAACCATGCAGTT-3′) and CRAMP-R1 (5′-GCAGATCTACTGCTCCGGCTGAGGTA-3′) were the primers used to amplify CRAMP . The rev.Data was analyzed using statistical package for social sciences (SPSS ver. 22). Kruskal-Wallis ANOVA on Ranks test was used to compare groups..The test was considered significant, when P. value was less than 0.05. Results were expressed as mean and standard deviation.p = 0.028) . Although the levels of CRAMP transcripts increased in the group that received 30% GA they were not statistically different from the control group (p = 0.055).Comparison between the three groups regarding the levels of CRAMP constructs revealed significant difference (28) Fig. . The groP = 0.023) in comparison with the control group. These results were expected since GA has been shown to increase circulating butyrate concentration. Matsumoto’set. al, showed that ingestion of 25 g of GA daily doubled serum butyrate level [Several cell types including macrophage are known to express Cathelicidin , 7. The te level . Butyratte level . It has te level , 30. Botte level .Studies have shown that butyrate affects LL-37 expression mainly at the transcriptional level and that LL-37 mRNA expression was directly correlated with LL-37 protein synthesis and antimicrobial activity both in vitro and in vivo . HyperacCathelicidin RNA expression in MDMs obtained from mice receiving high concentrations of GA (30% w/v.) was higher when compared to the control group, but the difference was not statistically significant Fig. In conclusion, the study demonstrated a novel effect of GA, as an inducer for cathelicidin expression. Being able to induce the expression of cathelicidin, regular consumption of GA can therefore enhance the innate immunity against bacterial, viral, and fungal infections. Hence the study asserts that GA is a promising candidate as an adjuvant treatment in infectious diseases. Effect of GA supplementation in Cathelicidine expression in humans should be investigated.Additional file 1.Additional file 2.