NAC genes in poplar, the functional characterization of drought-related NAC genes have been scarcely reported in Populus species. Here, we identified a total of 170 NAC domain-containing genes in the P. trichocarpa genome, 169 of which were unevenly distributed on its nineteen chromosomes. These NAC genes were phylogenetically divided into twenty subgroups, some of which exhibited a similar pattern of exon–intron architecture. The synteny and Ka/Ks analysis indicated that the expansion of NAC genes in poplar was mainly due to gene duplication events occurring before and after the divergence of Populus and Salix. Ten PdNAC (P. deltoids × P. euramericana cv.’Nanlin895’) genes were randomly selected and cloned. Their drought-responsive expression profiles showed a tissue-specific pattern. The transcription factor PdNAC013 was verified to be localized in the nucleus. Our research results provide genomic information for the expansion of NAC genes in the poplar genome, and for further characterizing putative poplar NAC genes associated with water-deficit.The NAC  is a large gene family of plant-specific transcription factors that play a pivotal role in various physiological processes and abiotic stresses. Due to the lack of genome-wide characterization, intraspecific and interspecific synteny, and drought-responsive expression pattern of  Petunia NAM [Arabidopsis ATAF1/2 and CUC2 [cis-element at their promoters [NAC  gene, which encodes an important regulator in ABA-dependent signaling pathway [Arabidopsis VND (vascular related NAC-domain) genes, including VND6 and VND7, are involved in xylem differentiation and cell fiber differentiation [Physcomitrella patens are related to water transport in cellular tissues [CarNAC3 gene from Cicer arietinum, can lead to an increase in proline content and antioxidant enzyme activities, and a decrease in MDA  concentration of transgenic poplar NL895 (Populus deltoides × P. euramericana cv. ′Nanlin895’) [PeNAC034, PeNAC045, PeNAC036 and PeNAC122 genes of P. euphratica, have been functionally characterized to date [Identification of NAC TFs at the genome-wide level has been documented in the genomic sequences of angiosperm species, such as 75 in a sativa , 105 in thaliana , 74 in Vvinifera , 189 in persicum . Meanwhiin yield . ANAC016 pathway . Two Arantiation . The NAC tissues . Overexp to date ,18.Populus species, as a settled and woody perennial species, may have evolved a sophisticated regulatory mechanism by which genes are regulated in response to water deficit occurring over the poplar lifetime. However, the regulating role of NAC TFs in poplar in response to drought stress remains largely unknown. This is, in part, due to the lack of the basic information of poplar NAC TFs, such as their coding sequence, physical and chemical properties, subcellular localization, drought stress-responsive expression profiling, and divergent evolutionary pattern.The frequency of extreme meteorological events and the change of rainfall pattern aggravate the degree of drought at a regional scale . Severe P. trichocarpa genome were identified, in which comprehensive bioinformatics analyses were performed, including the physicochemical attribution, chromosomal distribution, phylogenetic classification, and intraspecific and interspecific collinearity detection. Ten poplar NAC genes were randomly selected and cloned from the genome of the poplar clone NL895. Their drought-responsive expression patterns were analyzed in three tissues  of poplar NL895 using quantitative real-time PCR (qRT-PCR). Finally, the subcellular localization of PdNAC013 protein, which was encoded by one of the selected ten NAC genes, was verified in poplar NL895 protoplasts.In this research, NAC transcription factors in the whole P. trichocarpa genome. These transcripts were transcribed from 170 gene loci of the P. trichocarpa genome. The 170 NAC genes were reassigned as PtrNAC001-PtrNAC170 according to their corresponding chromosome number and physical location. Only one (PtrNAC170) of all PtrNAC genes was not located on any of the nineteen P. trichocarpa chromosomes.Here, we identified a total of 289 putative NAC (Pfam No.PF02365) domain-containing transcripts located in the entire PtrNAC genes had an average GC content of 43.89%, with a relatively wide range of from 37.84% (PtrNAC120) to 52.44% (PtrNAC055). Detailed information on these data was summarized in The PtrNAC proteins ranged from 122 amino acid (aa) (PtrNAC125) to 1524 aa (PtrNAC170) in size, with an average length of 350.71 aa. Their corresponding molecular weights were between 14.84 kDa (PtrNAC125) and 174.42 kDa (PtrNAC170). Their predicted pI (isoelectric point) varied from 4.28 (PtrNAC138) to 10.73 (PtrNAC021). The coding sequences of these PtrNAC genes, 169 were located on all nineteen chromosomes of P. trichocarpa. The 169 PtrNAC genes shared an uneven chromosomal distribution  to sixteen (Chr01). The average number of PtrNACs per 10 Mb (megabase pair) also differed among nineteen chromosomes in P. trichocarpa, ranging from 2.36 (Chr18) to 6.87 (Chr14). It seemed obvious that PtrNAC genes were not evenly distributed over a few chromosomes. Three NAC gene-rich clusters were found in Chr02 (PtrNAC025-PtrNAC029), Chr06 (PtrNAC060-PtrNAC067) and Chr14 (PtrNAC129-PtrNAC134). In addition, there were eight tandem duplicated gene pairs distributed on eight chromosomes, including Chr01 (PtrNAC011 and PtrNAC012), Chr02 (PtrNAC022 and PtrNAC023), Chr06 (PtrNAC063 and PtrNAC064), Chr09 (PtrNAC089 and PtrNAC090), Chr12 (PtrNAC112 PtrNAC113), Chr14 (PtrNAC132 and PtrNAC133), Chr16 (PtrNAC146 and PtrNAC147) andand Chr19 (PtrNAC168 and PtrNAC169).Of all 170 ribution . The totPtrNAC genes during evolutionary innovation. A total of 117 PtrNAC genes were identified to be derived from WGD events, suggesting that WGD contributed mainly to the expansion of the NAC gene family in poplar.Gene duplication events, such as tandem duplication and whole-genome duplication (WGD), were considered as a common process during plant evolutionary process, and could lead to the expansion of multigene families . In ordePtrNAC gene pairs were less than 1, demonstrating that PtrNAC members had experienced purifying selective pressure and their functions might be conserved after the expansion of NAC genes in poplar [PtrNAC pairs could be estimated based on Ks-based distribution. Their divergence times ranged from 9.5 to 208.09 million years ago (Mya). The divergence times for 55 out of all 90 collinear gene pairs were less than 31 Mya. This showed that the expansion of PtrNACs occurred after and before the split between Populus and Salix (45 Mya) [The chromosomal synteny analysis discovered a total of 90 collinear gene pairs . The synn poplar ,24. The (45 Mya) .P. trichocarpa and four other plants, including Salix purpurea, Eucalyptus grandis, Vitis vinifera and Arabidopsis thaliana  tree. These subfamilies were designated alphabetically as A through T. The number of PtrNAC genes in these subfamilies were between two (subfamily D) and twenty-two (subfamily A) (To explore the evolutionary relationships of amily A) . MoreovePtrNAC genes, we investigated the exon–intron patterns of PtrNAC genes (PtrNAC genes were transcribed at the forward (+) and reverse (−) strand of all P. trchocarpa chromosomes. Exons or introns were unequally distributed over PtrNAC genes, but most related members of the same subfamily had similar intron/exon structures. For example, all PtrNAC genes contained introns except for all members belonging to the subfamily G, five genes  belonging to the subfamily H, and PtrNAC136 in the subfamily R. In contrast, each of the six subfamilies  shared three exons pattern. The exons in the PtrNAC genes had an average number of 3.34. Two PtrNAC genes, including PtrNAC083 and PtrNAC107, possessed the maximum number of eight exons.To further understand the characteristics of the AC genes . A totalcis-elements in the upstream region of 289 PtrNAC transcripts  and G-box) [cis-elements belonging to stress-responsive element were consisted of STRE (stress response element), TC-rich repeat, MYB and MYB-like sequence. A large proportion of PtrNACs contained MYB binding sites in their promoter region, suggesting that MYB might be a major kind of transcription factors binding to PtrNACs [PtrNAC genes were implicated in abiotic stress response , and plant growth and development.Transcription factors have a close association with the regulation of gene expression via binding the promotor region of their target genes . Here wenscripts . Cis-actd G-box) , ethylend G-box) . ABA andd G-box) . The cis PtrNACs . This rePtrNAC genes under drought stress, ten PtrNAC genes were randomly selected for cloning these genes from the NL895 poplar. They were named according to the homology with their corresponding genes in P. trichocarpa. The NAC gene sequences of the NL895 poplar were compared with that of P. trichocarpa and I-69 poplar (Populus deltoides bartr. cv. ‘Lux’). Each of these genes shared a pairwise sequence identity of nearly 99% between the three Populus sepcies.Based on the RNA-seq profiling of PtrNAC genes in different tissues of P. trichocarpa. The majority of NAC genes were induced by drought stress in poplar. Among them, ten genes were selected for cloning genes from the NL895 poplar, and their drought-responsive expression pattern in P. trichocarpa had been shown in PtrNAC genes could be a marked difference among three tissues, including roots, stems and leaves.The external environment stimulus had a major influence on plant growth and development, and the plant could respond to environmental stress by regulating crucial molecular processes. The RNA-seq profiling data provided a drought-responsive expression pattern of PdNAC genes were implicated in poplar responses to drought, we adopted qRT-PCR to analyze their tissue-specific patterns under drought stress. As shown in PdNACs changed after 3% (w/v) PEG6000 treatment. The results shown that the drought-responsive expression patterns of a few PdNACs were the difference among the leaf, root and stem tissues. In the leaf tissues, drought-induced expression of all but PdNAC027 genes shared a single-pulse pattern. The expression abundance of PdNAC013, PdNAC86 and PdNAC105 genes reached the peak at 72 h after PEG6000 treatment. The expression levels of PdNAC013 and PdNAC105 genes increased by nearly 85-fold and 20-fold at 72 h compared to control group (0 h), respectively. The highest expression level of PdNAC049, PdNAC55 and PdNAC78 genes occurred at 24 h after PEG6000 treatment. In the root tissues, drought-induced expression of all but PdNAC055 genes could be clustered into three patterns, including single-peak , double-peak  and triple-peak (PdNAC078) pattern. In the stem tissues, only three genes  exhibited a single-pulse pattern in response to drought stress, and genes  showed a double-peak pattern. In short, transcriptional time series of some PdNAC genes under PEG6000 treatment might have an impulse pattern [Thus, to investigate whether the ten  pattern .PdNAC013 constructed in protoplasts of NL895 poplar, with expressing a red nuclear marker D53-mCherry protein [PdNAC013 fusion was observed microscopically only to localize in the nucleus with the red nuclear marker D53-mCherry protein  [A. thaliana (105) [V. vinifera (74) [S. lycopersicum (93) [P. trichocarpa. The intraspecific and interspecific collinearity analysis suggested that the expansion of NAC genes in poplar was caused mainly by WGD events. Recent studies also suggest that WGD event is a major driving force for expansion of multigene family in plant genome [Pyrus pyrifolia [Zea mays [PtrNACs showed diverse expression, indicating that modifications, such as point mutations, might occur in regulatory regions of duplicated genes, which affected the function and expression patterns [In this study, we identified a total of 170 iva (75) , A. thalna (105) , V. viniera (74)  and S. lcum (93) . It was t genome ,35,36. Tyrifolia  and Zea Zea mays . In addipatterns ,40.Populus and Salix are two sister lineages which shared common WGD events  happening in approximately 58 Mya [P. trichocarpa and S. purpurea were more than twice those of between P. trichocarpa and the remaining three species . Likewise, the analysis of Ks-based distribution showed that the divergence times for more than half of homologous PtrNAC pairs were less than 31 Mya. These results suggested that the expansion of PtrNACs occurred after and before the separation time of Populus and Salix (45 Mya) [PtrNACs arisen after the divergence of Populus and Salix play a role in the adaptation of Populus species to their local environment  [y 58 Mya ,42. This(45 Mya) . Additiond heat) ,44.PtrNACs under drought treatment, ten NAC (PdNAC) genes were randomly selected and cloned from P. deltoids × P. euramericana cv.’NL895’. The promoter region of these NAC genes contained many cis-acting elements related to drought stress, such as ABRE, DRE and G-box binding sites [cis-acting elements in the downstream promoter region of drought-related genes, thereby activating the expression of this gene and improving drought resistance in plants [cis-acting ABA-responsive element (ABRE elements) of TaSNAC8-6A and TaNAC48 promoter and activated their expression in wheat, thus enhancing drought tolerance of plants [ONAC066 could bind the JBS-like (JBSL) cis-element in the promoter region of OsDREB2A gene to positively regulate drought and oxidative stress response [Based on the RNA-seq profile of ng sites ,46,47. Pn plants ,49,50. Ff plants ,52. ONACresponse .PdNAC genes in three tissues of poplar NL895. Six PdNAC genes had sequence similarity to ATAF1 (ANAC002) and RD26 (ANAC072) genes, which were found to be drought-responsive NAC genes in A. thaliana [ATAF1 is an Arabidopsis NAC transcription factor that regulates ABA synthesis [OsNAC6, a member of ATAF family, can mediate the adaptation of plant root structure and up-regulate the expression of drought-resistant genes, which enhances the drought tolerance of rice plants [PdNAC055 (Potri.005G180200), PdNAC021 (Potri.002G081000), PdNAC049 (Potri.005G069500) and PdNAC078 (Potri.007G099400), shared high sequence similarity to ATAF1 from A. thaliana. It seemed obvious that the expression levels of the four PdNAC genes were induced by drought stress. PagNAC045 (Potri.007G099400), which was isolated from P. alba × P. glandulosa cv.84K, might be implicated with NaCl and ABA-mediated stresses [PdNAC genes were implicated with ABA remains unknown.Therefore, we analyzed the drought-responsive expression patterns of ten thaliana . ATAF1 iynthesis ,56. OsNAe plants ,58,59. Fstresses . HoweverANAC072 /RD26 is a positive regulator of ABA and drought stress [ANAC072 /RD26 can up-regulated under drought and salt stress, and the plants showed obvious drought resistance [PdNAC105|PtrNAC105 (Potri.011G123300) and PdNAC013|PtrNAC013 (Potri.001G404100) were two highly homologous sequences of ANAC072 /RD26. PtrNAC013 gene is overexpressed in poplar 84k , and the overexpressed plants had better salt tolerance than the wild type [PdNAC013 and PdNAC105 had significant changes in gene expression levels in leaves, roots and stems under PEG-stress treatment. PdNAC013 shared a drought-responsive single-impulse pattern in the three tissues, which reached its peak at 72 h after PEG6000 treatment. PdNAC013 protein was localized in the nucleus in the protoplasts of NL895 poplar, which was consistent with the subcellular localization of PtrNAC013 gene in tobacco [PdNAC013 and PdNAC105 may play an important role in drought stress.t stress . The gensistance ,62. PdNAild type . PdNAC01 tobacco . In concPdNAC genes were induced by drought, and might be implicated in poplar response to drought. However, the regulatory role of NAC genes in drought response in Populus species remains complex and elusive. For example, almost all PtrNAC genes had MYB binding sites (MBS) in their upstream regions, suggesting that MBS elements of a few PtrNAC genes might be bound by PtrMYB proteins. In wheat, TaMYBL1 protein can bind to two MBS cis-acting elements inserted in the promoter region of a drought-related gene TaNAC071-A, causing an increase in its expression level and drought tolerance in plant [cis-acting elements and trans-acting factors, transcriptional activation domain and DNA-binding domain of poplar NAC genes associated with drought stress.The study indicated that a few in plant . Thus, wP. trichocarpa (v3.0), Salix purpurea (v5.1), Eucalyptus grandis (v2.0) and Vitis vinifera L (v2.1) were downloaded from the Phytozome database (https://phytozome.jgi.doe.gov/ (accessed on 2 March 2022)). The GFF/GTF, transcript and protein files for Arabidopsis thaliana were retrieved from the EnsemblPlant release 53 (https://plants.ensembl.org/ (accessed on 2 March 2022)). The A. thaliana NAC (AthNAC) sequences were searched and acquired from the Arabidopsis information resource ).Genomic sequence (transcript and protein sequences) and GFF/GTF annotation files of P. trichocarpa NAC (PtrNAC) proteins, the HMM (hidden Markov model) profile of NAM domain (PF02365.15) was searched against all P. trichocarpa proteins by using HMMER (v3.3.2) with an E-value threshold of 1 × 10−3 [−5 [https://www.ebi.ac.uk/interpro/about/interproscan/ (accessed on 8 July 2022)) and Pfam (http://pfam.xfam.org/ (accessed on 8 July 2022)) service. The length, GC-content, isoelectric point (pI), and molecular weight (MW) of all PtrNACs were calculated with Biopython (v1.79).To identify the ain PF023.15 was sain PF023.15 was shttp://www.clustal.org/clustal2/ (accessed on 2 March 2022)) with default parameter. The best-fitted substitution model ‘JTT+F+R7′ was inferred from the PtrNAC MSA file by ModelFinder implemented in IQ-TREE (v1.6.12) based on minimum BIC value [Multiple sequence alignment (MSA) of PtrNAC protein sequences was performed via ClustalW ). Conserved motifs on the PtrNAC proteins were analyzed using MEME ). The MEME parameters were specified as follows: the optimum motif width was between 6 and 50 in length, and the maximum number of motifs was 15.The PtrNACs genome database and gff3 annotation file were used to investigate the exon and intron distributions of the http://cello.life.nctu.edu.tw/ (accessed on 5 May 2022)), WoLFPSORT (https://wolfpsort.hgc.jp/ (accessed on 5 May 2022)) and BUSCA (http://busca.biocomp.unibo.it/ (accessed on 5 May 2022)).The subcellular localization of PtrNAC proteins were predicted using three web tools, including Cello (P. deltoids × P. euramericana cv. ′Nanlin895′), we did the following experiments. First, the newly expanded leaves were harvested from 8-week-old plantlets, and used for the poplar protoplast isolation with Cellulase RS  and Pectolyase Y23  according to the method of Yoo et al. [PdNAC013::GFP (green fluorescence protein) was constructed according to homologous recombination method. D53-mCherry is a nuclear marker plasmid fused with rice protein D53 [2+ (PEG-4000) mediated transformation [In order to verify the subcellular localization of PdNAC013 (Potri.001G404100.1) in poplar NL895  [P. trichocarpa, S. purpurea, E. grandis, V. vinifera and A. thaliana, was carried out by BLASTp in the BLAST+ (v2.9.0) package. The resulting BLASTp outputs were used to analyze the collinearity of NAC proteins between P. trichocarpa and the other four species, by using Multiple Collinear Scan Toolkit ). The collinearity relationship of between P. trichocarpa and each of the other four species was plotted with JCVI ). To further evaluate the characteristics of the influence of evolutionary on PtrNACs family, a simple Ka/Ks calculator was used to calculate the synonymous (Ks) and non-synonymous (Ka) of PtrNAC gene pairs using ParaAT (v2.0) and KaKs-Calculator (v2.0) [−9 [The chromosomal locations of v0.4.15) . Pairwisr (v2.0) . The div2.0) [−9 ,73.P. trichocarpa genomic sequence with SeqKit (v0.8.0) [cis-regulatory elements using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 10 March 2022)).The 2 kb sequence in the front of the PtrNACs start codon (ATG) was obtained from (v0.8.0) . The 2 kP. trichocarpa under drought treatment were derived from NCBI SRA database (BioProject no. PRJEB19784). These RNA-seq data were pre-processed and trimmed with FastQC (v0.11.8) and Trimmomatic (v0.38). The spliced mapping of the trimmed RNA-seq reads against the P. trichocarpa genome (v3.0) was performed using STAR (v2.7.3a) [p-value cutoff of 0.01 [The RNA-seq data for the three tissues  of v2.7.3a) . Differe of 0.01 . The hea(P. deltoids × P. euramericana cv. ‘Nanlin895′) were grown on the 1/2 Murashige and Skoog (MS) medium (pH = 5.8). The poplar NL895 seedlings were cultivated in a greenhouse at a room temperature (23 °C) and a relative humidity of 74%. They were cultured in 1/2 MS medium for two months and then transferred to 1/2 MS medium with 3% (w/v) PEG6000 for drought treatment. Samples were harvested at 12 h, 24 h, 36 h, 72 h and 168 h (7 days) after 3% PEG6000 treatment, respectively. These samples were immediately frozen in liquid nitrogen and stored at −80 °C for subsequent RNA isolation. The total RNA was extracted from the leaf, stem and root tissues of the poplar NL895 with plant RNA extraction kit , respectively.Plantlets of hybrid poplar NL895 The total RNA was isolated from three vegetative organs  of the poplar NL895 using a plant RNA extraction kit . The concentration and integrity of the extracted RNA was determined using a combination of an ultraviolet-visible (UV-Vis) spectrophotometer  and 1% agarose gel electrophoresis. The extracted RNA was reverse-transcribed into cDNA by a reverse transcription kit . Then the cDNA was diluted 10 times for qRT-PCR experiment.TM 5α chemoreceptor cells . Finally, a single colony was screened to verify the size of bands in gel-electrophoresis, and sequenced on an ABI 3730xl DNA analyzer.Prior to qRT-PCR (quantitative reverse transcription PCR), the specific primers were designed with Oligo (v7.37) using poplar CDSs (coding sequences) as reference. The primer sequences were listed in PdNAC genes were designed by Primer Premier (v5.0) to analyze their expression patterns  genes were identified at the entire genome level. The physicochemical property calculating, chromosomal locating, phylogenetic classifying, intraspecific/interspecific collinearity inferencing, and dehydration-responsive expression profiling of these PtrNAC genes were performed. Analysis of cis-acting elements and drought-responsive expression pattern of PtrNACs indicated that these genes might be involved in drought stress response. In addition, ten NAC genes from poplar NL895 were cloned. The PdNAC013 exhibited a tissue-specific and pulsing expression pattern under drought treatment. The subcellular localization analysis suggested that PdNAC013 protein localized in the nucleus. In summary, these findings will provide a valuable clue for further screening and characterization of NAC genes in relation to poplar drought response.In the research, a total of 170


RXFP2 and TWIST1. This study identified these changes for the first time and summarized them. The results suggested that the Pc mutant locus may inhibit neural crest cell EMT generation and keratin expression, leading to failures in neural crest cell migration and keratinization of the horn bud tissue, regulating the production of the polled phenotype.The Polled Celtic (Pc) mutation locus is a genetically simple single mutation that is the best choice for breeding polled cattle using gene editing. However, the mechanism of the Pc locus for regulating horn development is unclear, so we used gene editing, somatic cell nuclear transfer and embryo transfer to obtain polled Holstein fetal bovine (gestation time 90 days) with a homozygous Pc insertion  and the wild-type 90 days Holstein fetal bovine (WH) as controls. The hematoxylin-eosin (HE) staining results showed that, compared to the WH, the EH horn buds had no white keratinized projections or vacuolated keratinocytes and no thick nerve bundles under the dermal tissue. Furthermore, DNA sequencing results showed that the Pc locus was homozygously inserted into the fetal bovine genome. A total of 791 differentially expressed genes were identified by transcriptome sequencing analysis. Enrichment analysis and protein interaction analysis results of differentially expressed genes showed that abundant gene changes after Pc insertion were associated with the adhesion molecule regulation, actin expression, cytoskeletal deformation and keratin expression and keratinization. It was also noted that the results contained several genes that had been reported to be associated with the development of horn traits, such as  Growth of horns have been a common vertebrate development, especially for bovines, where they are diverse in form and have medical and application value. However, with the rapid development of farming, horns will cause injury to other cattle and farm staff on intensive farms. The sharpness of the horns can increase the risk of physical damage to vital areas such as the udder, which can reduce the lifetime economic benefit of the cow ,2. In toMedugorac et al. 2012) first identified a single mutation associated with the polled trait in several European beef breeds, such as polled Angus and Galloway cattle [2 first iCarlson et al. in 2016, successfully produced polled calves by editing the Pc locus with Transcription Activator-like (TAL) Effector Nucleases (TALEN). Subsequently, Young et al. selected one of polled calves and crossed it with a horned cow (pp) to obtain six heterozygous (Pcp) polled calves. The Pc allele segregated in the offspring of this bull, and inheritance of either allele produced polled calves ,6. SchusUntil recently, the exact molecular mechanism through which the Pc mutation locus causes the polled trait in cattle was not known . In a prThis laboratory successfully obtained a monoclonal cell line with a Pc locus homozygous insertion in the ear margins of Holstein bull by Tild-CRISPR/Cas9 technology. Both PCR detection and DNA sequencing identified that the Pc locus was homozygous and inserted into the genome of the cell line. The homozygous mutant cell was subsequently used as a donor cell and the donor was transplanted into a stage MII enucleated oocyte by somatic cell nuclear transfer technique to prepare reconstituted embryos and perform electrofusion to blastocysts in In this experiment, the horn buds of EH fetal bovines and WH Holstein fetal bovines obtained by slaughter were identified morphologically and histologically. A distinct white keratinized projection could be observed in the WH horn bud tissue at 90 days by morphological observation, while none was observed in the EH group, seen in To investigate the regulatory mechanisms of bovine horn development, cDNA libraries of WH and EH horn bud tissues were constructed, and raw data subsequently obtained by Illumina high-throughput sequencing, with raw reads ranging from 49,208,676 to 56,395,082 for each sample. After eliminating many low-quality bases, splice-containing, and N-containing reads from the raw data, 56,135,334 (99.54%) to 48,996,840 (99.57%) clean reads were obtained in the WH and EH horn bud tissues and the Q30 was greater than 92.5% in all groups.Bos taurus, UMD 3.1) with more than 46,947,510 (91.5%) clean reads and more than 95.5% of the above reads were uniquely mapped to the bovine reference genome. Based on the expression information, the Pearson correlation coefficient between any two samples was higher than 0.987. The heat map results showed that the inter-group sample reproducibility was good in the WH group as shown in Sequence comparison showed that all groups were localized to the bovine reference genome  were screened in WH and EH as shown in The differential gene screening and analysis were performed by DE-Seq. The differential gene screening criteria were |log2FC| ≥ 1 and The GO enrichment, KEGG pathway and Reactome enrichment analyses were performed to explore the molecular mechanisms of the Pc locus regulating the polled trait in Holstein, as shown in Similarly, KEGG pathway analysis results showed that Focal adhesion (ko04510), ECM-receptor interaction (ko04512) and Calcium signaling pathway (ko04020) were also enriched significantly in http://string-db.org, accessed on 30 August 2022) for differentially expressed genes in In this experiment, protein interaction networks were analyzed using the STRING protein interaction database , laminin subunit gamma 2 (LAMC2), and laminin subunit alpha 5 (LAMA5)  . The promotility . The genigration ,25.L1CAM, LAMC2, and LAMA5, was significantly lower in EH horn buds compared to WH , actinin alpha 2 (ACTN2), actin alpha cardiac muscle 1 (ACTC1), and actin alpha 2, smooth muscle (ACTA2). The ACTA1, ACTC1, and ACTA2 genes encode one of six different actin proteins and Hu et al. demonstrated that overexpression of ACTA1 inhibited cell proliferation and migration [ACTC1 significantly inhibited cell migration [ACTA2 inhibited migration of neural stem cells by increasing Rho A expression and decreasing Rac1 expression to hinder actin filament polymerization [ACTA1, ACTN2, ACTC1, and ACTA2, was down-regulated in EH horn buds . This is a regulator of the interaction between E-cadherin and the cytoskeleton, regulating cytoskeletal remodeling and cell polarization by participating in the linkage between the two and promoting cell motility . The expThe above results suggested that the Pc locus may inhibit the development of EMT in neural crest cells by regulating the expression of cell adhesion molecules, preventing neural crest cells from initiating migration. At the same time, the insertion of the Pc locus may also inhibit the expression of actin and cytoskeletal deformation in neural crest cells, limiting the migration of neural crest cells. The neural crest cells are unable to migrate to the site of the horn bud to proliferate and differentiate into neuronal and mesenchymal cells required for horn development and due to the lack of a material basis for horn development, mutations at the Pc locus led to the creation of the polled trait.Studies of horn development found that keratin may play a key role in it. A brief white keratinized projection appears on the horn bud tissue during development in horned fetal calves and disappears later in development, whereas no keratinized projection appears on the horn bud tissue throughout the development of hornless fetal calves . It suggBased on the above experimental results, it seems that the Pc locus completed the regulation of key events in the formation of the polled trait early in fetal bovine development. The possible regulatory mechanisms include two points. First, the Pc locus may initiate the EMT event by regulating the expression of cell adhesion molecules, actin, and cytoskeletal deformation in neural crest cell migration, resulting in a decrease in the ability of neural crest cells to migrate. As a result, large numbers of neural crest cells are unable to migrate successfully to their designated sites at the horn buds for value-added differentiation. Secondly, the Pc locus may inhibit the expression of keratin at the horn bud and the process of keratinization by keratinocytes, failing to provide the material basis for later horn development. Based on the above process, we have drawn a hypothetical model of Pc regulation, see The donor cells for somatic cell nuclear transplantation were mutant cell lines with homozygous insertion of the Pc locus mediated by Tild-CRISPR/Cas9 technology in this laboratory. In previous experiments, we obtained the above cell lines carrying the Pc variant by the following steps: (1) First, we obtained the ear marginal tissue of a Holstein bull and established cell lines. (2) Second, the sgRNA was designed for the Pc locus controlling the polled in cattle, and its mutation efficiency was detected by T7El digestion and sequencing. (3) Third, the sgRNA was designed for the Pc locus controlling the polled form in cattle, and its mutation efficiency was detected by T7El digestion and sequencing. The PUC57 vector was used as the skeleton to produce homologous recombinant fragment through connecting the Pc fragment and homology length of 1600 bp (800 bp homology on each arm), then the homologous recombinant fragment was obtained by double enzyme digestion and sgRNA were co-transfected into the fibroblasts of the auricular margin in Holstein bulls. Finally, the Holstein ear margin fibroblast cell line with the homozygous Pc insertion was successfully obtained . OvariesThe results of a study by Aldersey et al. in 2020 showed that fetal bovines exhibit significant differences in horn traits at 90 days of gestation . In hornHomozygous mutant cell lines were cultured for two to three generations in 15% Dulbecco’s modified eagle medium .2 saturation humidity.Slaughterhouse ovaries were washed repeatedly with saline supplemented with penicillin and streptomycin at 27 °C. In sterile conditions, cumulus-oocyte complexes (COCs) were aspirated from 3 to 6 mm follicles using a vacuum peristaltic pump with an 18-gauge needle. Selection of COCs with homogeneous cytoplasm and dense oocytes was made using a home-made oral pipette under the microscope with a heated stage. After washing the selected COCs twice with HEPES-buffered TCM199  medium and three times with culture media, the COCs were transferred into the culture media and incubated for 22 h in an incubator at 38.5 °C and 5% CO−1 and pulses of 10 µs and two pulses. The reconstituted embryos were transferred into the embryo culture medium and incubated in the incubator for 30 min. The reconstituted embryos were transferred to the A23187 solution for 5 min, then transferred to the 6-DMAP solution and incubated in the incubator for 6 h. The reconstituted embryos were then transferred to the embryo culture solution and the blastocyst formation was counted after 7 days in the incubator. The reconstructed embryos were finally transferred into the embryo culture medium, and the blastocyst formation was counted after 7 days of culture in the incubator.The COCs cultured to maturity were digested with 1% hyaluronidase and oocytes with first polar bodies, intact morphology and homogeneous cytoplasm were selected for transfer into the manipulation solution. The microscopic manipulation aspirated the first polar body and a little of the surrounding cytoplasm. The donor cells were injected into the enucleated oocytes at the denuded pinhole and subsequently transferred into the electrical fusion medium for 1 min. The oocytes were arranged in batches in the cell electrofusion bath in a uniform manner while ensuring that the electric field was perpendicular to the contact surface of both cells. The parameters of the CF150B Cell fusion instrument  were adjusted to a field strength of 2.4 kV·cmYoung heifers of 14 to 15 months of age were selected for uniform body structure, good body condition, good genetic performance, and the presence of the corpus luteum on the ovaries on rectal examination. Recipient heifers were treated with 6 mL intramuscular cloprostenol for estrus synchronization, followed by observation and recording of estrus over 2 to 3 days.At day 7 of embryo culture, embryos were graded according to embryo quality and quality blastocysts were stored in separate 0.25 mL plastic tubes in units of one embryo for transport. The remaining blastocysts were preserved by cryopreservation by vitrification. Referring to the report of Roper et al. (2018), the best recipient cattle were selected for embryo transfer in this experiment . Ultraso2O). The PCR reaction conditions included pre-denaturation at 93 °C for 30 s, denaturation at 93 °C for 20 s, annealing at 58 °C for 30 s and extension at 72 °C for 70 s, for 33 cycles; followed by an extension at 72 °C for 2 min and storage at 4 °C. The PCR products were identified by agarose gel electrophoresis (AGE) at a concentration of 1.2% and the amplified products were sequenced by Sanger at Huada Genetics .The experiment randomly selected eight embryos labelled a to h to extract the whole genome of single cell clones using the cell tissue blood whole genome DNA extraction kit . The whole genome was used as the template and btHP-1963-F/btHP-1963-R shown in Horn bud tissue was obtained from the WH and EH Holstein male fetal bovines by vertical cutting. Tissue samples were fixed using 10% formalin and dehydrated in a graded ethanol series. They were washed in xylene and embedded in paraffin and 4 μm microtome sections were stained with hematoxylin and eosin (HE). Digital image acquisition was with a Prog Res C5 camera .The whole genome of the skin tissue was extracted separately using the cell tissue blood whole genome DNA extraction kit  and was used as a template and NEW-F/NEW-R as primers as shown in ® Reagent kit  was used to extract tissue RNA. Quality-qualified Total RNA was selected as the starting sample for mRNA sequencing. The quality required was RIN ≥ 7, the ratio of 28S to 18S RNA was greater than or equal to 1.5:1 by Agilent 2100 BioAnalyzer  and the starting volume required was in the range of 2 to 3 μg. The Qubit RNA assay kit  provided accurate quantification of starting Total RNA. The Ribo-Zero™ Magnetic Kit  was used for the isolation and removal of rRNA from Total RNA. Quality control of rRNA removed from total RNA was performed using agarose gel electrophoresis, Nanodrop microspectrophotometer , and Agilent 2100 .The TRIzolThe raw reads from the downstream machine were quality controlled using fastp  to filtep-value ≤ 0.05. Hierarchical clustering analysis was also done on the screened differentially expressed genes using R 4.1.2 software  [The clean reads were compared to the reference genomic sequence using HISAT2 2.1.0 software   with an Austria)  and genep-value < 0.05 were significantly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed using Kobas 3.0  software to obtain the enrichment of differentially expressed genes in the pathway. Differentially expressed genes were mapped to each term of the Reactome database  and the number of differential genes per term was calculated to obtain a list of differential genes with Reactome function and a count of the number of differential genes. The experiment applied a hypergeometric test to identify Reactome terms that were significantly enriched in differential genes compared to the whole background. A STRING diagram  was constructed for interaction networks of differentially expressed proteins. Visual protein interaction network maps and Hub protein interaction analysis were produced using Cytoscape software , Bethesda, MD, USA).In this experiment, Gene Ontology (GO) analysis of the differentially expressed genes were performed using R 4.1.2 software  . The GO GAPDH) expression using the 2−ΔΔCT method. The sequences of the primers used are shown in Total RNA extracted from tissues was synthesized using the Revertra ace qPCR kit  according to the manufacturing instructions. The complementary DNA (cDNA) was synthesized using a QuantStudio5 real-time fluorescent quantitative PCR system  in a total volume of 15 μL. The reaction system consisted of 10 μL 2 × SYBR green PCR buffer , 1 μL of 10 μM PCR forward primer, 1 μL of 10 μM PCR reverse primer, 1 μL of template, and 6 μL of de-RNAase water. The reaction procedure was one cycle, denaturation at 50 °C for 2 min, denaturation at 95 °C for 2 min, followed by 40 cycles of amplification, denaturation at 95 °C for 15 s, and annealing extension at 60 °C for 1 min, followed by melt curve analysis in triplicate. The expression data were normalized to glyceraldehyde-3-phosphate dehydrogenase . All experiments were repeated at least in three independent experiments.We used SAS software version 9.2  for statistical analysis. Groups were compared by one-way analysis of variance (ANOVA) followed by the Duncan post-hoc test for multiple comparisons. The present research will facilitate further studies on horn development and provide further insight into the molecular mechanisms of Pc locus regulation of the polled trait. The present research will provide ideas and a theoretical basis for exploring the mechanism of horn development and Pc locus regulation in Holstein cattle and provide methods for breeding polled Holstein bulls to improve the safety and economic efficiency of cattle farm breeding.


Population size has increasingly been taken as the driver of past human environmental impact worldwide, and particularly in the Arctic. However, sedimentary ancient DNA (sedaDNA), pollen and archaeological data show that over the last 12,000 years, paleoeconomy and culture determined human impacts on the terrestrial ecology of Arctic Norway. The large Mortensnes site complex  has yielded the most comprehensive multiproxy record in the Arctic to date. The site saw occupation from the Pioneer period  with more intensive use from c. 4,200 to 2,000 cal. years BP and after 1,600 cal. years BP. Here, we combine on-site environmental archaeology with a near-site lake record of plant and animal sedaDNA. The rich animal sedaDNA data (42 taxa) and on-site faunal analyses reveal switches in human dietary composition from early-Holocene fish + marine mammals, to mixed marine + reindeer, then finally to marine + reindeer + domesticates , with highest reindeer concentrations in the last millennium. Archaeological evidence suggests these changes are not directly driven by climate or variation in population densities at the site or in the region, but rather are the result of changing socio-economic activities and culture, probably reflecting settlers’ origins. This large settlement only had discernable effects on its hinterland in the last 3,600 years (grazing) and more markedly in the last 1,000 years through reindeer keeping/herding and, possibly domestic stock. Near-site sedaDNA can be linked to and validate the faunal record from archaeological excavations, demonstrating that environmental impacts can be assessed at a landscape scale. Significance StatementHuman impacts in the Arctic (70°N) are assessed over 12,000 years from one of the largest site complexes known using plant and animal sedaDNA, pollen, nonpollen palynomorphs , and archaeological data. From these data, it is shown that human activities rather than population levels are most important for driving vegetation changes. These activities are connected to the changing marine environment in the Holocene and a switch from marine to terrestrial resources, which, in turn, impacted the terrestrial environment. We show that combining plant and animal sedaDNA, pollen, and archaeology can provide detailed ecological reconstructions and describe shifting socio-ecological baselines, which in this case were determined by culture (including grazing/herding) associated with waves of immigration.The Arctic is warming up to three times more rapidly than any other biome, and it is critical to understand how human populations have affected and will continue to impact Arctic ecosystem biodiversity . EstimatRangifer tarandus) and moose , as well as being related to marine resources. The latter reflect water temperatures and by the earliest Holocene, the Polar Front had returned to close to Svalbard, with the 5° isotherm being close to Varanger (Gadus morhua) and haddock (Melanogrammus aeglefinus), with Arctic cold-water fish such as Polar cod (Boreogadus saida)  .−2, which is one of the lowest in Europe, although the coastal density population at over 10 persons km−2 is comparable with the Troms–Finnmark county as a whole (15 persons km−2); thus, it is not atypically low and likely a reflection of waves of inward migration . The remarkable concentration of archaeological sites in Varanger (over 100) has attracted research for over a 100 years. The fjord represents a “Maritime Core Area,” where social complexity arose in hunter–gatherer–fisher communities , and the lack of taxonomic precision . Althoug2 of raised beach, bedrock, scree, and coastal sand-flats. The archaeology covers almost all the archaeological periods and technological traditions of northern Norway/Fennoscandinavia and comprises tent-rings, semi-subterranean pit houses , middens, hearths, graves, a standing stone, and a labyrinth , and whale (Cetacea) but no reindeer (G. morhua) and some pollock (Pollachius virens) (Rissa tridactyla), guillemots (Uria), and great auk (Pinguinus impennis), whilst the more recently analyzed shellfish are dominated by black clam (Artica islandica) and periwinkle (Littorina littorea)  (Ovis/Capra), reindeer, cattle (Bos taurus), and pig (Sus scrofa). There are some seal but no whale. The fish are dominated by very large quantities of cod and haddock (Melanogrammus aeglefinus) and the birds are dominated by rock ptarmigan (Lagopus mutus), kittiwake, and guillemots. This assemblage implies both summer grazing and the collection of hay and fodder for the overwintering of the domesticated stock. This pastoral farming is combined with reindeer, boat fishing, and the use of bird-cliffs, the nearest of which is located only 1,200 m to the east of archaeological area D  dated to c. 7,600 to 5,500 cal. years BP  revealed evidence of diet, local resources, and hot-stone technology , 43. Thereindeer . A problreindeer . The fis virens) , 43. Thewere cod , revealea molva) , 44. Fewa molva) . It reveArtemisia), ribwort plantain (Plantago lanceolata), and microcharcoal from 6,900 cal. years BP. The record of cultivars fits with the archaeology with rye  first appearing c. 1,320 cal. years BP (630 CE) and then again at 880 cal. years BP, whilst barley (Hordeum) appears at 660 cal. years BP.Pollen and spore data are also available from a mire within the archaeological site, Fig.  40) and and40) aAnalysis of a 3.2 m sediment core from Lake Nordvivatnet  along with Arctic tundra shrubs  also appears in this zone in both sedaDNA (and pollen) at levels that imply it was in the lake catchment  and Ribes (currant), respectively.Zone N1 covers the Younger Dryas and is not considered further here due to it predating human occupation at this site, which at the time was a marine embayment. Zone N2 , willow-herb (Epilobium), peavine (Lathyrus), field forget-me-not (Myosotis arvensis), common buttercup (Ranunculus acris), globeflower (Trollius europaeus), and bird vetch (Viccia cf. cracca). Several of these taxa, including willow-herb, are also found in the earliest Holocene as they are pioneer, r-selected strategists suited to open, and disturbed ground, which is common under both periglacial conditions and human disturbance (In the absence of cultivars (apochores) from the lake, human impact is assessed using a combined sum of facultatively synanthropic  due to the similarity to values found in the Late Holocene when it is known that the lake was surrounded by birch-willow forest and other sites in northern Norway  and heather , which also increase at this time . An increasing proportion of pine pollen is observed and remains high until the uppermost sample at c. 500 cal. years BP. Birch, pine, and the dwarf shrubs remain dominant throughout Zone Npo3. There is a low number of facultatively synanthropic species, and they show no coherent increase over time. However, there are distinct peaks in coprophilous fungal spores with a rise in the early Holocene  during the period of birch-forest establishment with fluctuations in the middle Holocene (Npo4) and a reduction in the late Holocene (Npo5 to Npo6) except for a spike at c. 3,400 cal. years BP, which is dominated by Sporormiella sp. which suggest phases of more intense grazing around the lake  in the relatively open environment prior to human arrival due to natural fires. The microcharcoal which is supported by a peak in Neurospora (HdV 55—a genus of ascomycete carbonicolous fungus) is common in the Younger Dryas and early Holocene and its reduction in the remainder of the Holocene could be related to a reduction in natural fires due to lower summer temperatures and increase in precipitation , suggesting that Nordvivatnet was a lagoon or beach-flat at this time prior to the development of the beach barrier that today confines the lake. This would explain the rapid deposition of sediments with low organic matter and glacially scoured stones (Rangifer tarandus). Several marine taxa were detected with sporadic occurrences thereafter, which we explore in the Rana temporaria), moose , and red fox (Vulpes vulpes) from c. 9,000 to 3,500 cal. years ago, are consistent with a warmer than present climate and forest cover. Intriguingly, between c. 6,500 and 4,500, cold-adapted taxa of open habitats, such as reindeer and Norwegian lemming (Lemmus lemmus), drop out of the record and at c. 5,700 cal. years BP, we observe the only occurrence of Eurasian beaver (Castor fiber), a taxon that is absent from northern Norway today, suggesting warmth and forest cover were at their maxima over this interval. This occurrence of beaver also falls within the age range covered by the Old Stone Age house R12 from Mortensnes, which contained beaver bones  was applied to the Nordvivatnet plant sedaDNA, pollen data, and summed probability distribution (SPD) of 14C dates from archaeological sites in northern Norway  Fig. 3b3b. The pure Fig. . The plaure Fig. . The beture Fig. .Ribes), and strong evidence of reindeer. In this period, the on-site evidence is dominated by reindeer with domesticates, principally sheep, and cereal cultivation. Mortensnes records the second-most northerly, and the most easterly, occurrence of cereal pollen in Fennoscandia; the only more northerly occurrence is from Risfjorddalen, Melham (71°N), which is dated to 2,450 and 2,800 cal. years BP  superimposed on the five traditional archaeological phases, that show different ecological impacts around Lake Nordvivatnet and the Mortensnes archaeological site Fig. . In the years BP . At MortCombining the data, it is clear that the economic character of Mortensnes changed over time, from one dominated by the use of the sea and coast in the Older Stone Age to a more mixed economy using more parts of the landscape in the Younger Stone Age. This may reflect a change in hunting pattern, with early hunting occurring further inland, but also it signals a trend towards sedentism, which can also be argued from the archaeology . FinallyIn trying to interpret this temporal pattern, it is pertinent to return to the location and marine environment of Mortensnes. The importance of the Atlantic Boreal marine species increases with warmer sea temperatures and thus they became dominant in the Older Stone Age; this is happening again today with the warming of the Barents Sea . VarangeUsing the most comprehensive multiproxy paleoecological record in the Arctic to date , we show that a site with a nearly complete Holocene record of human occupation had little or no discernable effects on its hinterland until the last 3,600 years, when grazing and probably wood-cutting is evidenced but was still low. It is only in the last 2,000 years that these effects increased and in the last millennium that we get continuous and sedaDNA reads of reindeer, suggesting herding. The plant record is shown here to diverge from climatic control in the last 2,000 years, and this is ascribed to human impact. Comparison with the archaeological data suggests that it is the changing economy associated with in-migration, from marine-orientated pioneers (from west and/or east), to mixed fishing  and reindeer hunting, and finally terrestrially dominated pastoral agriculture in the Sami Iron Age and historical period, that has driven this record, rather than population per se. This implies that it is what people do, and probably where they came from, that drives environmental impact in this Arctic region, both in the past and probably into the future.Only a brief summary of the methods is given here and for more detail see the The study is part of the ECOGEN project “Ecosystem change and species persistence over time: A genome-based approach,” financed by Research Council of Norway grant 250963/F20. The publication charges for this article have been partially funded by a grant from the publication fund of UiT The Arctic University of Norway.pgac209_Supplemental_FilesClick here for additional data file.


An estimated 19 million people are infected with rifampicin-resistant/multidrug-resistant strains of tuberculosis worldwide. There is little done to prevent these individuals from becoming sick with RR/MDR-TB, a disease that is associated with high rates of morbidity, mortality, and suffering. There are multiple phase III trials currently being conducted to assess the effectiveness of treatment of infection  for RR/MDR-TB, but their results are likely years away. In the meantime, there is sufficient evidence to support a more comprehensive management of people who have been exposed to RR/MDR-TB so that they can maintain their health. We present a patient scenario and share our experience in implementing a systematic post-exposure management program in South Africa with the goal of inspiring similar programs in other high-burden RR/MDR-TB settings. Each year, more than a half million people become newly sick with rifampicin-resistant/multidrug-resistant forms of tuberculosis (RR/MDR-TB) . The pasRR/MDR-TB can have a major impact on a family. In our work in a peri-urban township located outside of Cape Town, we encountered many households struggling with the disease. As an example, the Makhanda  family struggled to meet the growing household’s most basic needs. With only one working household member, money was tight, and there was often not enough to eat. When the oldest adult brother died in a motor vehicle accident, the family matriarch, Busisiwe, had to make room for his three children—all under the age of ten years—to come and live in the already crowded shack where she was living with her daughter, her son, and four of her other grandchildren. Over the coming months, Busisiwe attributed her weight loss to the meagre food supply and the stress, but it was harder to explain away her cough. When she finally presented to the health center and was told she had RR/MDR-TB, she was devastated at the loss of her income that the daily visits to the health center for observed therapy would cause and she was worried about the family members living with her. Her son and two of the grandchildren were coughing as well, and although the nurse told her to bring all the household members to clinic for evaluation, they only had taxi fare to bring the sickest one. When she used that money to bring her grandson to clinic, she was grief-stricken to learn he was also sick and wept as they took him away to the hospital. She worried endlessly about the other children but was told there was nothing else they could do. “Just bring them to the clinic if they are sick” the nurse instructed her, as Busisiwe left the health center once again with a heavy heart, worried that she had brought this misfortune on them all. Mycobacterium tuberculosis means that a person has the bacilli living in the lungs but is not yet sick with TB disease. In this paper, we will refer to this state as RR/MDR M.tb infection. The precise data on the number of people who are infected with RR/MDR-TB are not possible to calculate since current tests for TB infection cannot differentiate between drug-susceptible and drug-resistant strains. This is because drug susceptibility testing can only be done when M. tuberculosis or its DNA has been isolated, a condition that usually defines TB disease. Modelling studies suggest that there are more than 19 million people worldwide who harbor mycobacterial strains that are resistant to rifampicin and isoniazid [M.tb infection far outstrips treatment capacity. That fact alone is reason for concerted action, but until recently, the only measure recommended to prevent RR/MDR-TB among those who were exposed was frequent clinical monitoring [Infection with soniazid  . Some of this also stems from the historical debate about the transmissibility of RR/MDR M.tb strains, with some studies suggesting that the fitness costs associated with some mutations leading to resistance could reduce the transmissibility of these forms of TB [Much of this stems from the fact that there are not yet randomized controlled trials supporting the use of a specific medication for the treatment of RR/MDR ms of TB . Severalms of TB ,9.Rapid diagnosis and prompt initiation of effective therapy are among the most effective means for preventing RR/MDR-TB since primary transmission is driving most of the burden of RR/MDR-TB in many settings in the world ,11. ThusWhen someone is newly diagnosed with RR/MDR-TB, working with them to compassionately disclose this diagnosis to other household members is key. If done in a supportive manner—as is the case with HIV and other infectious diseases —and by aBox 1Empathize with the person who was just diagnosed;Assess any physical or mental risks that might result if disclosure happens;Identify any individuals who might be able to support disclosure with other household members and share the news with that individual first;Practice the disclosure and review what is the best way to say things for the newly diagnosed person;Emphasize the concepts of “shared air” rather than contagion and blame;Offer to assist the newly diagnosed person with the disclosure if s/he would like;Stress that there are many actions that can be done to keep household members healthy;Schedule a time to follow up and review how the disclosure went.Most RR/MDR-TB transmission occurs prior to a diagnosis of RR/MDR-TB being made . Many trThe principles of what has traditionally been called preventive therapy but what should more accurately be referred to as the treatment of infection for drug-susceptible TB and RR/MDR-TB are essentially the same . The goaM.tb infection treatment, the first step is ruling out active TB disease. This is usually conducted through a symptom assessment, weight monitoring, physical examination, bacteriologic testing, and chest radiography [M.tb infection perform poorly as they require a functioning immune system and are, thus, more likely to yield false-negative results in the populations most at risk of becoming sick with TB, such as in young children or persons living with HIV [M.tb infection can be presumed if the index individual is sick with a strain of RR/MDR-TB (either confirmed or possible).For both drug-susceptible and RR/MDR iography . Tests fwith HIV . In addiwith HIV . RR/MDR M.tb infection [M.tb infection treatment were conducted several decades ago, access to drug susceptibility testing for fluoroquinolones was limited and took weeks to months to obtain. This led to the use of multidrug regimens for RR/MDR M.tb infection treatment, a strategy that increased both the pill burden and the likelihood of adverse events [M.tb infection was used in a larger cohort of persons exposed to RR/MDR-TB in the household in Karachi, Pakistan and found the treatment of infection to be effective but that the use of ethionamide resulted in poor adherence, largely mitigated by adverse events [M.tb infection, isoniaizid-based preventive therapy regimens lasting six months should be effective.What differs between the treatment of infection for drug-susceptible and RR/MDR-TB is the medications that can be used for therapy. By definition, RR/MDR-TB strains are resistant to the medications used to prevent drug-susceptible TB, including rifamycins and (for persons with MDR-TB) isoniazid. For this reason, fluoroquinolones have been the mainstay in the treatment of RR/MDR nfection . When the events . In spite events . A largee events . A combie events ,35. Of ne events  or bedaqe events . Of noteM.tb infection treatment globally have access to it. Currently, RR/MDR M.tb infection treatment is usually only implemented in low-burden TB settings. Some of this may be due to a lack of randomized controlled trials leading to a rather lukewarm policy recommendation from the WHO on RR/MDR-TB preventive therapy. However, given the notable morbidity and mortality of RR/MDR-TB disease worldwide and existing programmatic data the balance of risk/benefit ratio would likely favor wider-scale implementation of RR/MDR M.tb treatment of infection even while randomized trial data are being collected. There are three ongoing trials of RR/MDR M.tb infection treatment, two of which are assessing the role of fluoroquinolones and one of which is testing the novel nitroimidazole medication delamanid [M. tb infection, including inhaled therapies, vitamin supplementation, and nutritional support.Very few of the persons who could benefit from RR/MDR elamanid . The staMuch of what is driving the global RR/MDR-TB crisis is poverty, hunger, lack of housing, and co-morbid conditions, such as HIV, substance use disorder, malnutrition, and diabetes . EffectiAll too often, programs and providers report feeling too overwhelmed to take on TB prevention, and this may be even truer for the prevention of RR/MDR-TB. At the same time, RR/MDR-TB prevention activities are critical, since even with improved diagnostic and treatment strategies, outcomes for people living with RR/MDR-TB are far below those achieved for people with drug-susceptible form of TB. While many high-risk TB settings face unique issues and challenges, there are often cross-cutting themes of poverty, injustice, and inequity driving the transmission of RR/MDR-TB in communities. We were able to successfully implement a program aimed at RR/MDR-TB prevention in a peri-urban township outside of Cape Town, South Africa beginning in 2020. With the aim of sharing lessons learnt with policy makers and TB care providers, below is a description of the program as well as the key factors supporting its success .The Khayelitsha post-exposure management program  was implemented as a collaborative effort between Médecins Sans Frontières, the City of Cape Town, and the Western Cape Department of Health. Household contacts (particularly children and adolescents) of persons newly diagnosed with RR/MDR-TB, were assessed for signs or symptoms of TB, and provided TB counselling, either in their home or at their primary health care clinic. This was conducted by a PEP-dedicated TB nurse who worked across all 10 Khayelitsha clinics. Contacts with any suggestive signs or symptoms of TB were referred for further evaluation by a doctor and, where indicated, for chest radiograph and/or bacteriological testing. Following best practices in the South African setting, tests of infection were not routinely performed given that all these individuals were close contacts of persons newly diagnosed with TB. Contacts without clinical evidence of active TB disease would be offered 6 months of treatment of infection most commonly with a fluoroquinolone (high-dose isoniazid or delamanid was used where there was suspected or confirmed fluoroquinolone resistance in the index case).Patient-centered and differentiated care was a key aspect of this program , with persons given the option to follow up for treatment of infection in their home, at the clinic, or telephonically. Follow-up consults focused on ongoing treatment literacy/counseling support, and eliciting if there were any emerging TB symptoms or adverse events. To minimize disruptions to people’s lives, follow-up visits were conducted every two to three months, with multi-month medication refills provided. Young children were offered child friendly formulations of medication to support treatment adherence, including dispersible tablets of levofloxacin provided by the Global Drug Facility. Where socio-economic concerns were noted in the household, persons could be referred to a TB counsellor or social worker and could be provided with food parcels. Transport allowance was provided where needed for travel to attend any clinic visits.The outcomes of the initial phase of this program (112 children and adolescents on treatment of infection) are described in more detail in a recent publication. Notably, this program resulted in a high level of case detection (10% of those screened were started on treatment for TB disease), as well as high retention in care for those who received treatment for infection (80% completed treatment); therapy was well tolerated with minimal adverse events. None of those who started treatment for infection developed active TB disease (median 200-day follow-up). After the initial pilot success of this program, the work was integrated into the routine TB services offered at the clinic.It is imperative that more work is conducted to prevent RR/MDR-TB in the coming years. RR/MDR-TB is one of the most serious aspects of the global crisis in antimicrobial resistance, but efforts to prevent RR/MDR-TB have narrowly focused on resistance amplification, while ignoring other important interventions. RR/MDR-TB preventative efforts require a fundamental shift in the thinking and actions of the TB community, to a focus on providing person and family-centered care along the whole continuum. Rapid diagnosis of all forms of TB and initiation of the shortest most effective regimen is key to RR/MDR-TB prevention. So too is compassionate post-exposure care for all close contacts and members of the family coupled with supportive counselling. In this review, we have outlined key preventative strategies along the spectrum of care and shared our experiences of a family centered RR/MDR-TB preventive care model. Implementation of these strategies by committed TB programs and care providers promises to dramatically improve RR/MDR-TB prevention efforts and could, with time, reduce the implementation burdens of treating RR/MDR-TB for countries and programs. Doing so will dramatically reduce the suffering of persons, such as Busisiwe and her family.


In 2020, almost half a million individuals developed rifampicin-resistant tuberculosis (RR-TB). We estimated the global burden of RR-TB over the lifetime of affected individuals. We synthesized data on incidence, case detection, and treatment outcomes in 192 countries . Using a mathematical model, we projected disability-adjusted life years (DALYs) over the lifetime for individuals developing tuberculosis in 2020 stratified by country, age, sex, HIV, and rifampicin resistance. Here we show that incident RR-TB in 2020 was responsible for an estimated 6.9  million DALYs, 44%  of which accrued among TB survivors. We estimated an average of 17  DALYs per person developing RR-TB, 34%  greater than for rifampicin-susceptible tuberculosis. RR-TB burden per 100,000 was highest in former Soviet Union countries and southern African countries. While RR-TB causes substantial short-term morbidity and mortality, nearly half of the overall disease burden of RR-TB accrues among tuberculosis survivors. The substantial long-term health impacts among those surviving RR-TB disease suggest the need for improved post-treatment care and further justify increased health expenditures to prevent RR-TB transmission. Rifampicin-resistant tuberculosis (RR-TB) requires longer, more toxic therapy than rifampicin-sensitive disease and is associated with a higher occurrence of long-term sequelae. In this mathematical modeling study, the authors estimate that incident RR-TB in 2020 will be responsible for ~6.9 million disability-adjusted life years; 44% due to post-tuberculosis sequelae. Rifampicin is one of the key drugs in the WHO-recommended first-line tuberculosis treatment regimen, and rifampicin resistance represents a major challenge for effective tuberculosis treatment. For individuals diagnosed with resistant disease, the most commonly used treatment regimens have required an extended duration of therapy, using drugs that are both more toxic and less effective compared to the first-line regimen . As a result, individuals treated for RR-TB experience lower cure rates and higher mortality rates compared to individuals with drug-susceptible tuberculosis treated with the first-line regimen3. Moreover, many individuals diagnosed with tuberculosis are not tested for drug resistance, such that many of those with a drug-resistant strain will receive an inappropriate treatment regimen, leading to high rates of treatment failure and recurrence5. Due to these factors, many individuals who develop RR-TB will die during the disease episode or experience a prolonged period of ill health before achieving a cure.In 2021, it is estimated that 450 thousand individuals developed rifampicin-resistant tuberculosis (RR-TB)7, elevated mortality rates8, income losses9, and persistent social and psychological sequalae10. These ongoing effects contribute a substantial fraction of the overall disease burden caused by tuberculosis12. Evidence suggests that these post-TB sequelae are more common and severe for individuals surviving RR-TB14. Due to delays in initiating appropriate treatment15 and the lower efficacy of treatment regimens, individuals treated for RR-TB are likely to accumulate greater lung damage before the disease is effectively controlled by treatment. Moreover, individuals with RR-TB may receive several treatment courses before achieving a cure due to receipt of inappropriate first-line treatment or lower success rates even when appropriate treatment is available, with additional lung damage occurring during this extended disease period. The greater toxicity of drugs included in RR-TB regimens can also cause long-term sequelae. A meta-analysis of data on tuberculosis survivors found greater lung impairment among individuals surviving multi-drug resistant (MDR) tuberculosis compared to individuals with drug-susceptible tuberculosis14, and high levels of impairment among MDR-TB survivors have been reported across a range of functional domains16.Accumulating evidence indicates that tuberculosis survivors frequently experience ongoing disability1, these trends—as well as the absolute fraction of incident tuberculosis cases with rifampicin resistance—vary substantially across world regions. Similarly, national tuberculosis programs vary greatly in their capacity to identify RR-TB and provide effective treatment. The development of better diagnostics and RR-TB regimens promises to improve patient care in the future, yet the adoption of these new tools has been uneven. These differences between settings will produce differences in the magnitude of disease burden attributable to RR-TB. In this study, we synthesized evidence on the incidence, detection, and outcomes of RR-TB treatment across 192 countries and used mathematical modeling to simulate the lifetime burden of disease due to incident RR-TB in 2020, including health losses incurred during the disease episode as well as among individuals surviving tuberculosis. Based on this analysis, we report the number of disability-adjusted life years (DALYs) attributable to incident RR-TB in 2020, stratified by country, age, sex, HIV status, and disease stage.While global RR-TB incidence is estimated to be decliningIncident RR-TB occurring in 2020 was estimated to result in 6.9  million DALYs over the lifetime of affected individuals, representing 5.4%  of lifetime DALYs attributed to all incident tuberculosis in this year. The majority ) of RR-TB DALYs were estimated to result from premature mortality  million DALYs), as compared to reductions in quality of life  million DALYs). Just over half ) of all RR-TB DALYs were estimated to be incurred during the disease episode  million DALYs), with the remainder resulting from post-TB sequelae among tuberculosis survivors  million DALYs). Figure  million Table Table Supplementary Table 17 for estimating tuberculosis-attributable mortality among tuberculosis survivors and a scenario that assumed that post-TB disability weights for RR-TB were the same as for RS-TB. For both scenarios, total RR-TB DALYs were estimated to be 15–16% lower than in the main analysis.Supplementary Table We assessed the global disease burden due to incident RR-TB in 2020, including mortality and morbidity during the disease episode as well as the lifetime impact of post-TB sequelae among those surviving the disease. We found that RR-TB occurring in 2020 was responsible for 6.9  million DALYs, or 5.4% of the total DALYs due to all TB in 2020. An average of 17.3  DALYs were associated with each individual developing RR-TB, with nearly half of the total DALYs estimated for RR-TB accruing among tuberculosis survivors. While the South-East Asia Region was estimated to have the greatest absolute burden of RR-TB, former Soviet Union countries and southern African countries had the highest burden of RR-TB per population.18, with a large fraction of current tuberculosis cases experiencing resistance to rifampicin and, in some cases, many other tuberculosis drugs1. While approaches to tuberculosis diagnosis and treatment in the region have been formulated to address this challenge19, it still represents a major barrier to effective tuberculosis care, and the poor outcomes experienced by individuals with drug-resistant tuberculosis (as compared to drug-susceptible disease) are reflected in the high RR-TB burden estimates.For former Soviet Union countries, this high RR-TB burden results from historical approaches to tuberculosis treatment that produced high resistance levelsFor southern African countries, the high RR-TB burden estimates are likely related to the dual burden of tuberculosis and HIV in these countries. The relationship with HIV is notable, with HIV-infected individuals estimated to experience 40 times greater DALYs from RR-TB compared to HIV-uninfected individuals, a consequence of both elevated tuberculosis incidence for individuals with HIV and rapid disease progression and death in the absence of prompt, effective treatment. For southern African countries, the close overlap of tuberculosis and HIV epidemics may be exacerbated by poverty and health system weaknesses that make it difficult to achieve successful treatment outcomes for individuals with both HIV and RR-TB.20. While for some countries we needed to impute data gaps using other evidence, the major sources of uncertainty in this study derive from the challenge of extrapolating future health outcomes for individuals with RR-TB. This additional uncertainty is reflected in the wider uncertainty intervals reported for post-TB DALYs, as compared to DALYs estimated for the disease period. While much of the additional DALYs per RR-TB case (as compared to RS-TB cases) resulted from higher mortality during the disease episode, post-TB DALYs were also estimated to be higher for RR-TB, reflecting more prevalent and more severe sequelae following RR-TB. A substantial body of evidence now describes the elevated mortality and health burden experienced by tuberculosis survivors, motivating efforts to establish clinical standards21 and systems of care for this large cohort of individuals22. However, some part of this excess morbidity and mortality will relate to pre-existing health conditions and behaviors that co-occur with tuberculosis. Moreover, given the concentration of tuberculosis in poor and marginalized communities, the lower health outcomes for tuberculosis survivors will also partly reflect the impact of socioeconomic determinants of health that would still be present in the absence of tuberculosis. In this study, we extended a published approach to separating the causal impact of tuberculosis from these other factors12, which requires several assumptions. For this reason, additional empirical research on the causal contribution of tuberculosis and RR-TB to future health outcomes is needed.This study relies on accurate epidemiological data on drug resistance patterns across countries and population groups. This highlights the utility of efforts that have been made to develop and extend systems for monitoring drug resistance trends across many countries23. Uncertainty in pediatric estimates is compounded by limited data on post-TB outcomes for pediatric tuberculosis survivors, though the data that exist suggest persistent disease sequelae24. Second, our analysis makes assumptions about healthcare interventions and access for RR-TB survivors that may not hold in the future. In particular, the development and scale-up of effective rehabilitation interventions for post-TB lung disease could improve outcomes for RR-TB survivors25 and reduce the burden relative to the estimates in this study. Similarly, the development and deployment of better diagnostics26 and treatments27 for RR-TB may reduce the excess disease burden experienced by individuals with RR-TB relative to drug-susceptible disease. In particular, the adoption of new all-oral 6-month regimens for RR-TB could produce major improvements in RR-TB treatment outcomes in the future. An MDR/RR-TB regimen composed of bedaquiline, pretomanid, linezolid and moxifloxacin (BPaLM) was included in a 2022 update to WHO treatment guidelines2, and many countries have subsequently moved to introduce this regimen. Other short-course regimens are under development28. Given the better end-of-treatment outcomes achieved by the BPaLM regimen compared to older long-course regimens27 and the fact that cure is achieved more rapidly, it is possible that the introduction of new short-course RR-TB regimens will improve treatment outcomes and reduce post-treatment morbidity for RR-TB cohorts in the future.This research has several additional limitations. First, there is additional uncertainty around RR-TB outcomes in pediatric populations due to greater uncertainties in incidence estimates M. tuberculosis strain, clinical presentation, and treatment regimen. A second priority is interventional studies that examine approaches to protect and restore lung function during TB disease treatment and identify best practices for providing long-term rehabilitation for TB survivors. Finally, policy analyses are needed to identify the most impactful and cost-effective approaches along the care continuum  for RR-TB disease.This study suggests several directions for future research. First, empirical studies are needed that adopt quasi-experimental designs to separate the long-term effects of TB on post-TB health outcomes from the impact of comorbid conditions, health behaviors, and living conditions. These studies will be more valuable if they are able to describe how the causal effects of TB vary by individual-level factors such as RR-TB represents a major disease burden globally and a fundamental, lifelong health challenge for affected individuals. While the burden estimates reported by this study highlight the regions, countries, and population groups most impacted by RR-TB, they provide little guidance on the appropriate response to this burden. New interventions to facilitate the rapid detection and effective care for RR-TB are urgently needed, as well as concerted efforts to expand access to the best current diagnostics and treatments and policy analyses that compare actions to prevent, treat, and rehabilitate RR-TB in order to devise the most impactful approaches for reducing RR-TB burden in the future.12. This model stratifies the cohort developing tuberculosis in a single year by country, age, sex, and HIV status and tracks this cohort over the remaining lifetime of the affected individuals. Over this projection period, we recorded excess deaths and reductions in health-related quality of life causally attributable to TB or post-TB sequelae. For this analysis, we additionally stratified the model by the presence/absence of rifampicin resistance. For individuals without rifampicin resistance, we stratified the cohort according to whether the individual received or did not receive tuberculosis treatment. For individuals with rifampicin resistance, we stratified the cohort according to whether the individual (1) did not receive tuberculosis treatment, (2) received tuberculosis treatment with an inappropriate regimen (defined as the standard first-line regimen recommended for rifampicin-susceptible tuberculosis), or (3) received treatment with an appropriate regimen (defined as a WHO-recommended RR-TB regimen). Analytic inputs and methods are described below and summarized in Fig. We adapted a previously published mathematical model of future health outcomes among the global cohort of individuals with tuberculosis disease29. The fraction of cases receiving treatment was based on the number of notified tuberculosis cases within each country, sex, and age group for 2020, divided by the estimated incidence for each stratum. For countries with missing notification data, we used WHO-estimated tuberculosis treatment coverage. We removed countries with less than 10 estimated cases for 2020 .Within each stratum, we assumed that the fraction receiving any tuberculosis treatment was independent of rifampicin resistance. We estimated the fraction receiving a diagnosis of rifampicin resistance (among the population with rifampicin resistance diagnosed with tuberculosis) by dividing country-reported data on RR-TB diagnoses by WHO estimates of RR-TB incidence scaled by the fraction detected. We estimated the fraction of RR-TB initiated on second-line treatment by comparing data on RR-TB diagnoses and second-line treatment initiations for each country. Several countries had missing values for the data used to construct the study cohort. Supplementary Fig. We based assumptions around the duration of disability during the tuberculosis disease episode on values estimated by the WHO, stratified by treatment and HIV status . Mortality odds ratios for HIV and for receipt vs. non-receipt of tuberculosis treatment were based on mortality risks estimated by the WHO Global Tuberculosis Programme. We allowed for differences in survival between individuals with RS-TB receiving a first-line regimen , individuals with RR-TB receiving a second-line regimen, and individuals with RR-TB inappropriately receiving a first-line regimen, using country-reported data (details in Supplementary Information).There is little evidence of survival among individuals with RR-TB inappropriately receiving a first-line regimen, and we calculated this value as a weighted average of survival probabilities for individuals with RR-TB receiving a second-line regimen and untreated individuals, with a wide prior distribution specified for this weighting parameter  was based on a systematic review of observational cohort studies  mortality among individuals surviving tuberculosis disease, following a published approach (details in Supplementary Information)12. These methods were also used to estimate post-TB disability weights that were applied to tuberculosis survivors. We assumed elevated mortality rates and disability weights among individuals surviving RR-TB vs. RS-TB based on studies reporting a higher prevalence and severity of tuberculosis sequelae among individuals surviving RR-TB as compared to RS-TB13  YLLtb, representing DALYs lost to mortality during the tuberculosis disease episode; (3) YLDptb, representing DALYs lost to post-TB disability among TB survivors; and (4) YLLptb, representing DALYs lost due to post-TB mortality.Health outcomes were quantified as the years of life lost due to premature mortality (YLLs) and the years of healthy life lost due to non-fatal disability (YLDs). For each modeled stratum, we calculated total tuberculosis-attributable DALYs as the sum of: (1) YLD30. To do so, we re-estimated the model for each of the 1000 parameter sets created from a Latin hypercube sample from the parameter probability distributions and calculated intervals as the 2.5th and 97.5th percentiles of the resulting distribution. We also calculated partial-rank correlation coefficients (PRCCs), describing the sensitivity of the estimate for total global DALYs due to RR-TB to uncertainty in individual parameters31. For parameters that varied at the country level  post-TB mortality rates and disability weights for RR-TB assumed to be the same as for RS-TB. For each alternative specification, we estimated total global RR-TB DALYs, average DALYs per incident RR-TB case, and the fraction of global tuberculosis DALYs attributable to RR-TB.We recalculated results for several alternative analytic assumptions: (1) the duration of RR-TB disease assumed to be 50% greater than for RS-TB, (2) the case fatality for RR-TB inappropriately treated with a first-line regimen assumed to be the same as for untreated individuals, (3) case fatality for RR-TB inappropriately treated with a first-line regimen assumed to be the same as for individuals treated with a second-line regimen, (4) case fatality for untreated RR-TB assumed to be greater than for RS-TB (OR = 1.5), (5) post-TB mortality rates based on estimates reported by Lee Rodriguez et al.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


IL4R, IL12RB1, IL12RB2, IL6, TMPRSS2, and ACE2. Furthermore, a generalized linear mixed model identified statistically significant associations between prostate cancer risk and SNPs in IL12RB2, IL13, IL17A, IL4R, MAPT, and TFNRS1B. Finally, a statistically significant association was observed between IL2RA and TNFRSF1B and Gleason scores, and between SLC11A1, TNFRSF1B and PSA values. We identified SNPs in inflammation and two prostate cancer-associated genes. Our results provide new insights into the immunogenetic landscape of prostate cancer and the impact that SNPs on immune genes may have on affecting the susceptibility to prostate cancer.The immune system plays a critical role in modulating cancer development and progression. Polymorphisms in key genes involved in immune responses are known to affect susceptibility to cancer. Here, we analyzed 35 genes to evaluate the association between variants of genes involved in immune responses and prostate cancer risk. Thirty-five genes were analyzed in 47 patients with prostate cancer and 43 healthy controls using next-generation sequencing. Allelic and genotype frequencies were calculated in both cohorts, and a generalized linear mixed model was applied to test the relationship between prostate cancer risk and nucleotide substitution. Odds ratios were calculated to describe the association between each single nucleotide polymorphism (SNP) and prostate cancer risk. Significant changes in allelic and genotypic distributions were observed for  Prostate cancer (PCa) is one of the most widespread noncutaneous cancers in men. The incidence of PCa appears to be affected by several risk factors, among which inflammation plays a prominent role ,2. InflaACE, ACE2, ATG7, CD28, IFNG, IFNGR1, IFNGR2, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL17A, IL18, IL18R1, IL2, IL2RA, IL4, IL4R, IL5, IL6, IL7R, IRF5, LAG3, MAPT, NR1I2, PACGR, PARK2, PTPN22, SCLC11A1, TMPRSS2, TNFRS, TNFSF1B, TRAF6, and VDR). We then evaluated the possible association between the identified significant genetic polymorphisms and the clinical parameters of patients with PCa.Advances in next-generation sequencing (NGS) technologies have enabled the simultaneous sequencing of millions of nucleotides and investigation of unique sets of genes for diagnostic, prognostic, therapeutic, and research purposes. Many studies have investigated the association between polymorphisms in immune system-related genes and cancer risk ,7,8,9,10IL4R, IL12RB1, IL12RB2, IL6, TMPRSS2, and ACE2 were found associated with PCa risk. Both the C allele and the CC genotype of IL4R rs2301807 were inversely associated with PCa risk . The AA genotype in the 3′ UTR of IL12RB1 rs3746190, along with the C allele of IL12RB2 rs2307145, were found associated with a lower risk of PCa . The G allele of the intronic variant IL6 rs2069832 was positively associated with a higher risk of PCa . In addition, both the A allele and CA heterozygous genotype of the intronic variant TMPRSS2 rs140141551 were both associated with a higher risk of PCa . Conversely, the G allele and the AG genotype of TMPRSS2 rs11701576 were found to be associated with a decreased risk of PCa . Finally, a synonymous variant was identified within the ACE2 gene, and the T allele of rs35803318 was found to be associated with a higher risk of PCa .The present study analyzed a total of 47 patients with PCa and 43 HCs using NGS, and comprehensive gene profiling was conducted on a panel of 35 genes. p < 0.05, with a few exceptions for borderline values, which were retained to avoid premature selection in intermediate models  was implemented. The first GLMM runs were applied individually to each gene to identify loci statistically associated with the occurrence of PCa. Statistical significance was set at e models . Odds rae models . StatistIL12RB2 rs2228420  = 0.000347), IL13 rs20541  = 0.000138), IL17A rs7747909  = 0.015979), MAPT rs2258689  = 0.012260), TNFRSF1B rs2275416  = 0.027425). A negative association with PCa risk was instead observed for IL12RB2 rs2229546  = 0.001876), IL4R rs2301807  = 1.87 × 10−6), and TNFRSF1B rs636964  = 0.034034). Additionally, no statistically significant association between PCa risk and the nucleotide substitutions found within ACE, ATG7, CD28, IFNG, IFNGR1, IFNGR2, IL12A, IL12B, IL18, IL18R1, IL2, IL2RA, IL4R, IL5, IL7R, IRF5, LAG3, R1I2, PACGR, PTPN22, TNFRS, TRAF6, and VDR.Using the predictors identified in Finally, GLMM analysis was performed to explore the relationship between statistically relevant variants identified as useful predictors in the initial model  and the IL2RA rs7093069 and TNFRSF1B rs2275416) were significantly associated with GS. Accordingly, the variables rs7093069 and rs2275416  represent good predictors for the PCa severity. Indeed, with GS with values of around 6, rs7093069 and rs2275416 do not appear as informative variables, while with values of around 9, they appear strongly associated with the severity of the pathology. In addition, SLC11A1 rs17221959 and both TNFRSF1B rs636964 and rs2275416 were found to be significantly associated with serum PSA levels. Overall, only GS and PSA have been positively selected by the model, while other variables  were removed during the backward selection. Indeed, in the dataset here, analyzed age, cTNM, and BMI demonstrated to have no association with PCa, nor alone or taken together.Two polymorphisms (ACE2 and TMPRSS2) in a cohort of patients with PCa and a cohort of HCs. We identified several novel polymorphisms that contributed to PCa susceptibility. In particular, we found that six SNPs in several key genes, namely IL4R, IL12RB1, IL12RB2, TMPRSS2, and TNFRSF1B, were associated with a lower risk of PCa. In contrast, the eight SNPs identified in IL6, TMPRSS2, ACE2, IL12RB2, IL13, IL17A, MAPT, and TNFRSF1B were associated with a higher risk of PCa. PCa patients with specific polymorphisms in T-helper cytokine genes (IL13 rs20541 and IL4R rs2301807) may have an altered T-helper response and consequently, a potential imbalance in their immune system. Altered expression and increased circulating levels of both pro- and anti-inflammatory cytokines have been reported in several types of cancer [IL6 rs2069832 was positively associated with a higher risk of PCa is consistent with that of Slattery et al. [IL12RB1 rs3746190, IL12RB2 rs2307145, and rs2229546 were associated with decreased PCa risk. Interleukin 12 (IL-12) plays a pivotal role in stimulating natural killer (NK) cell cytotoxicity, inflammation, and boosting T-helper type I cells [IL12RB2 rs2229546, our results are in line with another study that found an association between rs2229546 and a reduced risk of cervical squamous cell carcinoma and HPV-18-positive cervical cancer [IL17A rs7747909 belongs to the 3′-UTR region of interleukin 17A (IL-17A) and was found associated with a higher risk of PCa. Interestingly, Bedoui et al. reported a strong association between IL17A polymorphisms, including rs7747909, and the development of tolerance to colon rectal cancer therapy [IL17 has been reported more frequently in CRPC and neuroendocrine prostate cancer than in hormone-naïve PCa [IL13 rs20541 polymorphism, located in exon 4, appears to be strongly associated with increased serum IgE levels in children [IL13 rs20541 was positively associated with the risk of PCa, whereas it was previously reported to be associated with a decreased risk of glioma [TMPRSS2 rs140141551 and ACE2 rs35803318 were associated with a higher risk of PCa than TMPRSS2 rs11701576, which was negatively associated with PCa risk. TMPRSS2 encodes a transmembrane serine protease 2 (TMPRSS2), which is expressed by prostate epithelial cells. To date, TMPRSS2 physiological function remains unclear, albeit its expression appears to be regulated by androgens in PCa cells [ACE2 encodes the angiotensin-converting enzyme (ACE2), which is aberrantly expressed in several cancers [The importance of the immune system in cancer onset and progression has been well established in cancer research. In the last decade, the notion of engaging the immune system as a patient-personalized weapon against cancer cells has undoubtedly revolutionized cancer therapy. A growing body of evidence has investigated the presence of variants in genes implicated in anti-tumor immune responses and cancer risk. Therefore, we investigated the presence of polymorphisms in a panel of 35 genes involved in pro- and anti-inflammatory responses, autophagy-lysosome pathway and two PCa-associated genes  whose expression is not restricted to neurons. In truth, MAPT overexpression was previously associated with lower GS, suggesting its potential as marker of PCa prognosis [IL2RA rs7093069 and TNFRSF1B rs2275416, implying the strong impact of genetic variability within immune genes on cancer development, progression, and staging. Alterations in IL2RA relative expression have been reported in groups of patients with different GS [TNFRSF1B rs2275416 was associated with serum PSA levels, GS, and PCa risk. TNFRSF1B encodes the receptor for tumor necrosis factor-α (TNFR2), which plays a crucial role in promoting tumor growth, invasion, and metastasis. TNFR2 is highly expressed in T regulatory cells (Tregs) and its presence is crucial in the TME [TNFRSF1B rs2275416 may also affect PCa prognosis, given its association with both GS and PSA levels. SLC11A1 rs17221959 was also associated with serum PSA levels. This finding is also consistent with the results recently reported by Zhu et al. in which SLC11A1 rs7573065 C > T was associated with increased risk of PCa and low overall survival; meanwhile, higher mRNA expression was found in PCa tissues compared to normal tissues [tability ,42,43,44rognosis  and resprognosis . In our erent GS . On the  the TME . Hence,  the TME ,50,51. A the TME , it is p tissues . Despite+-EDTA test tubes. PCa assessment was based on the clinical examination and histopathological analysis of isolated biopsies performed in all patients. The ethical committee of the AOU, Sassari, approved this study. Two cohorts, comprising 47 patients with PCa and 43 HCs, were recruited. The demographic and clinical characteristics of the patients with PCa and HCs are summarized in PBMCs were isolated by Ficoll-Histopaque gradient centrifugation . PBMCs were washed twice using phosphate-buffered saline (PBS 1X) and stored at −80 °C in fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) . Genomic DNA extraction from PCa and HCs samples was performed using the DNeasy Blood and Tissue Kit . After thawing, cells were washed twice in PBS 1X to remove DMSO and FBS traces and then resuspended in 200 μL PBS 1X before proceeding according to the manufacturer’s instructions. The final DNA concentration of each sample was measured using Nanodrop One , and DNA quality was assessed by calculating the ratios of absorbance at A260/A280 and A260/A230.Library preparation and DNA sequencing were performed at the CRS4 Research Center . Thirty-five genes involved in both innate and adaptive immune responses as well as PCa-associated genes were sequenced. Two AmplySeq primer pools were designed and purchased from Illumina for the gene panel analysis. The PCR protocol and reagents were adapted from QIAGEN Application Note (Qiagen 1104745 10/2016) to amplify the entire length of each gene. The PCR products were quantified using Qubit, and equimolar amounts were pooled. Gene-panel PCR pools were grouped to achieve the appropriate sequencing coverage. Libraries were obtained using Nextera DNA Flex with 100 ng of DNA and indexed with IDT for the Illumina Nextera DNA UD Indexes Primer Set . PCR products were purified with 1X AMPure XP beads , and the libraries were quantified using Qubit. A loading pool consisting of 90 samples (47 PCa and 43 HCs) was diluted to 9 pM before sequencing using the MiSeq Reagent Kit v3 600-cycle (Illumina). The BaseSpace Sequence Hub (Illumina) was used to conduct demultiplexing and fastq file generation, and two distinct analyses were performed for each sample. Gene panel sequences were analyzed using an in-house pipeline. http://ihg2.helmholtzmuenchen.de/cgi-bin/hw/hwa1.pl, accessed on 1 January 2022). Fisher exact test, chi-square test, and both allelic and genotypic odds ratios (ORs) were used to compare genetic variants between PCa and HC cohorts. To test the relationship between cancer onset and nucleotide substitution for a given locus, multivariate regression analysis was carried out by applying backward selection, in which non-significant covariates were removed step by step until the final model was obtained. A GLMM implemented in the R package lme4 [https://www.r-project.org/, accessed on 1 January 2022), was used to perform the analysis. Odds ratios with 97.5% confidence interval were calculated using the R-package “oddsratio” .The allele and genotype frequency estimates were calculated using (age lme4  in the R