Blocking CDK5/pho‐PPARγ significantly reduces CD44v+ BCSCs population in tumor tissues, thus abrogating metastatic progression in TNBC mouse model. Strikingly, diminishing stemness transformation reverses immunosuppressive microenvironment and enhances anti‐PD‐1 therapeutic efficacy on TNBC. Mechanistically, CDK5 switches the E3 ubiquitin ligase activity of PPARγ and directly protects ESRP1 from a ubiquitin‐dependent proteolysis. This finding firstly indicates that CDK5 blockade can be a potent strategy to diminish stemness transformation and increase the response to PD‐1 blockade in TNBC therapy.Triple‐negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, in which the higher frequency of cancer stem cells (CSCs) correlates with the poor clinical outcome. An aberrant activation of CDK5 is found to associate with TNBC progression closely. CDK5 mediates PPAR Cancer stem cells (CSCs) promote triple‐negative breast cancer (TNBC) metastasis and therapeutic resistance, including immunotherapy. Aberrant activation of CDK5 promotes TNBC progression by triggering stemness transformation. Mechanistically, CDK5 switches the non‐canonical E3 ubiquitin ligase activity of PPARγ and protects ESRP1 from a ubiquitin‐dependent proteolysis, which controls CD44v generation. Pharmacological interruption of CDK5 reverses immunosuppressive tumor microenvironment, and enhances anti‐PD‐1 curative effects. TNBC patients with CSCs markers mostly exhibit a worse prognosis. Eliminating CSCs is potentially beneficial to tumor therapy efficacy and durable tumor remission.Triple‐negative breast cancer (TNBC) is a highly malignant subtype of breast cancer (BC), which is defined by the lack of expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Up to 95% of TNBCs are classified as invasive mammary carcinomas, characterized by marked heterogeneity, aggressive distant metastasis, and relatively higher proportion of breast cancer stem cells (BCSCs) compared with other BC subtypes.1, 2 Its activation is primarily dependent on binding to the non‐cyclin activators CDK5R1 (p35) and CDK5R2 (p39), thereby phosphorylating a list of substrates, such as retinoblastoma (RB) protein, signal transducer and activator of transcription 3 (STAT3), and peroxisome proliferator‐activated receptor‐gamma (PPARγ). With these critical roles, CDK5 is intrinsically involved in many cellular events, including proliferation, adhesion, migration, and apoptosis, and has been revealed to serve as a key node in many physiological and pathological activities especially in cancer. A number of studies have shown that aberrant CDK5 activity contributes to tumor progression by promoting angiogenesis, metastasis, and immune evasion. In particular, as to the role of CDK5 in BC, it has been indicated that the inhibition and disruption of CDK5 attenuate BC growth via orchestrating a migration–proliferation dichotomy. CDK5 possesses the dominant effect in controlling the BC cell cycle in a CDK2‐independent manner. Additionally, inhibiting CDK5 can promote the sensitivities of BC cells to multi‐kinds of chemotherapeutic drugs, such as 5‐fluorouracil, 6‐thioguanine, and paclitaxel, significantly. These provocative findings imply that CDK5 might, therefore, be another key molecular target in the CDK family for cancer treatment after the identification of CDK4 and CDK6 as effective and stable drug targets. Indeed, a set of new drugs to inhibit CDK5 activity are being developed in several clinical trials . In this context, further deciphering of the mechanistic underpinning for CDK5 deregulation is urgently needed, and this may help develop new targeted molecular strategies and novel clinical combinations.Cyclin‐dependent kinase 5 (CDK5) is a non‐canonical cyclin‐dependent kinase that has been well‐characterized for the development of the central nervous system by regulating neuronal development and differentiation.7 Its  Indeed, the proof‐of‐principle studies with ICB in advanced‐stage TNBC have yielded promising results compared with the outcome from other BC subtypes. However, emerging data from preclinical and clinical trials suggest limited success with ICB monotherapy in TNBC. Thus, several investigations and clinical trials, evaluating the integration of ICB into the adjuvant combinatorial settings, are underway, and some of them have shown promising success in TNBC patients. Recently, increasing studies have revealed that the populations of tumor cells with CSC traits are elevated after immunotherapy, which is essential for adaptive resistance and tumor recurrence. Although, Mukherjee et al. have documented a novel link between CDK5 and glioma CSCs, the mechanism underlying CDK5‐mediated TNBC stemness remains unclear and it is still unknown whether CDK5 blockade efficiently enhances immunotherapy in TNBC. Here, we sought to investigate a rationale for combining CDK5 inhibition with ICB to achieve TNBC remission, while exploring the corresponding underlying mechanism by which CDK5 promotes stemness transformation in TNBC progression.Recently, the successful treatment of some patients with malignancies using immune checkpoint blockade (ICB) antibodies, such as those directed against the programmed cell death protein 1 and its ligand (PD‐1/PD‐L1) axis and cytotoxic T lymphocyte‐associated antigen 4, has demonstrated unprecedented clinical resolution, which has truly brought new hope to refractory TNBC.14 Ind22.1 This database is composed of 1699 BC cases including 679 Luminal A, 461 Luminal B, 220 HER2‐positive (HER2+), 299 TNBC, and 140 normal breast tissues. As shown in Figure S1A–C in the Supporting Information, CDK5 and CDK5R1, but not CDK5R2, were significantly elevated in TNBC at the mRNA level compared with other subtypes as well as normal breast tissues, indicating an essential role of CDK5 in TNBC. Consistently, the same results were seen in other cohorts of clinical samples . Kaplan–Meier curves were measured to analyze whether the survival time of each BC subtype was associated with the expression of CDK5. The results showed that among TNBC patients, those with higher expression of CDK5 showed a shorter overall survival time .18 Thie Figure ,D. Addit2.2 Additionally, the mice bearing the parental 4T1 cells were also challenged with roscovitine (Rosc), a potent CDK5 inhibitor (2 mg kg−1 5 days−1). The results showed that multiple tumor nodules that had metastasized to the lung surface were observed in the control group, but none was seen in the CDK5‐KO group . The efficient sgRNA was screened and selected to deplete endogenous CDK5 . Then, we established an orthotopic mouse model by using CDK5‐KO cells or parental 4T1 cells.19 Addp Figure . Using ehighCD24low BCSC population significantly . Moreover, the gene signature of CD44+CD24−/low BCSCs, proposed by Creighton et al., was governed by CDK5  population together with other subpopulations using flow cytometry  revealed that several stemness‐related pathways, such as Hedgehog, Hippo, JAK/STAT, PI3K/AKT/mTOR, and RAS/ERK/MAPK,4 were5 Figure . These ry Figure . As prevo Figure . Intereso Figure . Furthern Figure . Addition Figure . Collect2.3 As mentioned above, the aberrant expression of CDK5 was dominantly enriched in CD44‐positive 4T1 cells, indicating a close link between CDK5 and CD44. Therefore, we speculated that CD44 might be involved in CDK5‐induced TNBC stemness transformation. As expected, the cassette exon alternative splicing events in CD44 were impacted by CDK5 interruption according to the Sashimi plot  construction.23 As A Figure . To evalA Figure ,D. Toget spontaneously activated by CD44v overexpression in 4T1 cells , were markedly restrained subjecting them to CDK5 depletion  and CDK5 were closely co‐localized in the tumor tissues. Additionally, the pho‐PPARγ level was much higher in CDK5‐high specimens than that in CDK5‐low specimens. Pearson's correlation analysis showed a significantly positive correlation between pho‐PPARγ and CDK5 in the tumor tissues   Figure . Moreovee Figure , as welle Figure . Consistγ could control CD44v isoform switching. To do so, we produced 4T1 cells by overexpressing wild type or mutated PPARγ, as indicated in Figure S4D in the Supporting Information. The serine at site 273 of PPARγ was mutated into aspartic acid (PPARγ‐S273D) or alanine (PPARγ‐S273A) to mimic the hyper‐ or non‐phosphorylated form, respectively. Based on the CD44v‐specific PCR assay, which is similar to CDK5, PPARγ‐S273D enhanced CD44v isoform generation ; meanwhile, the self‐renewal capacity of 4T1 cells was parallelly analyzed using MFA . As shown in Figure S5D in the Supporting Information, the fact that PPARγ‐triggered self‐renewal capacity was closely associated with pho‐PPARγ level , rather than its transcriptional activity. These data implied that CDK5/PPARγ axis‐triggered TNBC cell stemness transformation was independent of PPARγ transcriptional activity.To elucidate the mechanism underlying the effect of CD44v isoform switching, we examined the mode of action of pho‐PPARγ‐interacting proteins to elucidate the rational mechanism by applying an anti‐Flag affinity purification mass spectrometry (MS) assay. To preliminarily evaluate the binding strength, the list of candidate proteins was subjected to online docking analysis tools, such as ZDOCK, PatchDOCK, and FireDOCK. Notably, epithelial splicing regulatory protein 1 (ESRP1) showed the strongest binding strength with PPARγ, as shown in Figure S5E in the Supporting Information. For further substantiation of the interaction between PPARγ and ESRP1, co‐immunoprecipitation assays were elaborately performed. As shown in Figure γ in PPARγ‐S273A cells. A similar result was observed in a reverse co‐immunoprecipitation assay . Especially, CD8+ cytotoxic T lymphocytes (CTLs) were notably infiltrated in tumors after CDK5 inhibition , which strongly predicted the response to ICB.Despite the importance of CSCs in tumor development,36 the Then, the mice were injected with Rosc (2 mg kg−1) or anti‐PD‐1 antibody (100 µg per mouse), individually or in combination, on days 7, 12, 17, and 22  increased significantly in tumors with combined treatment . High infiltration of TILs is a clinical biomarker, indicating the beneficial responses of ICB therapy. Furthermore, immunophenotyping tumors with combined treatment using flow cytometry showed, in addition to an increase in the overall percentage of CD3+ cells ‐expression CD8+ T‐cells, and M1‐prone macrophages,  and 28 (late‐stage) post‐transplantation. Prepared single‐cell suspensions were subsequently subjected to multicolor flow cytometric analysis. The gating strategy is shown in Figure S6A in the Supporting Information. In late‐stage tumor tissues, the ratio of anti‐tumoral immune cells, such as CTLs Figure  and M1‐p19 Not, Figure . By cont1 Figure ,K.γ axis causes an obvious immunophenotyping shift, thus augmenting the efficacy of anti‐PD‐1 therapy in TNBC.Conclusively, pharmacologically diminishing tumor cells stemness transformation by targeting the CDK5/pho‐PPAR3Figure  Notably, a class of selective CDK4/6 inhibitors, including palbociclib, ribociclib, and abemaciclib, has shown significant clinical activities across a range of malignancies. Unlike CDK4/6, CDK5 is a non‐stereotypical CDK member and its activation is majorly dependent on binding to non‐cyclin activators CDK5R1 and CDK5R2. The multifunctional properties of CDK5, specifically in regulating carcinogenesis and cancer progression have been overlooked for many years. Until recently, several studies have provided increasing evidence at the gene, mRNA, and protein levels unveiling the role of CDK5 in human cancers, including BC, non‐small cell lung cancers (NSCLCs), and brain cancers. It has been proven that CDK5 inhibition contributes to eliminate the progression of certain cancers and overcome the resistance to anticancer therapies efficiently. These investigations indicated the possibility that CDK5 could be an intriguing drug target. Indeed, several pharmacological CDK5 inhibitors have been undergoing preclinical/clinical trials for possible anticancer applications and are yielding promising responses. In the current study, we first found the existence of CDK5 activation in tumor tissues of TNBC patients at different stages and then established the primary positive correlation between aberrant CDK5 activation and TNBC progression. By applying multiple experimental approaches, we demonstrated that CDK5 is indeed critically required for TNBC cell stemness transformation, which has a strong association with enhanced metastasis and the constitution of an immunosuppressive environment. CDK5 blockade not only results in profound metastasis abrogation but also promote anti‐tumor immunity.In the current study, we provided novel insights into the mechanisms underlying TNBC cell stemness transformation and demonstrated that CDK5 plays an essential role during TNBC cell stemness transformation in vitro and in vivo Figure 7. In th Actually, the knowledge gained about the exact interpretation of the linkage between CDK5 and its substrates in BC cells has helped in understanding the neoplastic roles of CDK5 and further broadened the spectrum for CDK5‐targeted treatment in BC. Here, on account of the detailed context‐based quantitative analysis of specimens from TNBC patients, we observed a close correlation between CDK5 activation and PPARγ phosphorylation at the Ser273 site. Thus, we postulated that TNBC‐related CDK5 activation might lead to PPARγ phosphorylation. Indeed, further comprehensive analysis suggested that there was an eventual considerable kinase‐substrate relationship between CDK5 and PPARγ in the context of TNBC. Retrospectively, Bruce's group first found in adipocytes an event where CDK5 induces PPARγ phosphorylation at the Ser273 site. This finding indicates a novel modality of post‐translational modification of PPARγ creating a new setting to further explore the biological actions of PPARγ. Enlightened by this idea, we performed a series of tests, including focused genetic screening and subsequent mechanistic studies, to elucidate the precise significance of PPARγ phosphorylation mediated by CDK5 in TNBC progression. First, unraveling the interaction between PPARγ and ESRP1 revealed that PPARγ exhibited E3 ligase activity. Strikingly, CDK5 switched this non‐canonical biological activity of PPARγ and directly protected ESRP1 from ubiquitin‐dependent proteolysis. All these molecular events occurred during BC cell stemness transformation inducing CD44v generation, which is the canonical biomarker of CSCs and a valuable indicator of poor prognosis. In summary, these data implied that CDK5 surely control TNBC cell stemness transformation. With this, we tuned the action of the CDK5/pho‐PPARγ axis to analyze the exact role of this node in programming stemness. The results demonstrated that CDK5/pho‐PPARγ axis activation is positively responsible for the malignant progression of TNBC in both human specimens and experimental models. The pharmacological inhibition or genetic interruption of CDK5/pho‐PPARγ truly diminished tumor development in the TNBC mouse model. Collectively, these results strongly suggest that the CDK5/pho‐PPARγ axis exists in the context of TNBC stemness. This molecular axis is a pivotal driver of the aggressive development of TNBC.There is a consensus recognition that CDK5 triggers a cascade of events to promote tumor progression by phosphorylating its specific substrates on serine and/or threonine residues.8 Actu According to the restricted CD44v expression in cancer cells and the conclusive association between CD44v isoform and poor prognosis, CD44v has been regarded as a valuable diagnostic and therapeutic target in certain human cancers, including gastric, colorectal, hepatocellular, and breast carcinoma On this basis, a number of humanized antibodies against CD44v are undergoing clinical trials, and have shown promising curative effects (NCT01358903 and NCT01641250). However, there remains the challenge of the clinical application of antibody‐based treatments. For example, the immunogenic accumulation of antibodies in nontumor areas limited the clinical use of bivatuzumab . Additionally, the highly complicated and context‐dependent expression of CD44v isoforms substantiates the necessity and urgency to generate adequate new antibodies with high affinity against each isoform. Although some other approaches appear to interrupt CD44v in addition to a humanized antibody, the fundamental questions about CD44v‐targeted treatment are still unsolved. Therefore, the good knowledge of the upstream regulatory mechanism of CD44 alternative splicing is helpful for us to understand how CD44v promotes malignant transformation. From a broader perspective, the pivotal molecular nodes identified in the network responsible for CD44v generation prefer to provide potential druggable targets for further pharmaceutical development. Of note, the novel regulatory pathway centering on CDK5 for controlling CD44v generation was originally shown in this study. More importantly, using the small molecules, including interrupting CDK5 activity and inhibiting PPARγ phosphorylation at 273, have proven to efficiently block CD44v isoform switching, which exhibits promotive antitumoral effects. The regulatory mechanism developed by us opens a new avenue to device a novel strategy against CD44v by targeting CDK5 for TNBC therapy.Developmental preclinical studies have demonstrated the tremendously promotive anti‐tumoral efficacy of antibody‐based strategies against CD44v in multiple human cancers.42 Acc Although the mechanism that regulates the interaction of CSCs with the immune system has not yet been well defined, we speculated that successfully targeting CSCs proactively remodel their microenvironment, thus enhancing immunotherapeutic efficacy. To confirm this hypothesis, we established and comprehensively verified the therapeutic effects of combinatorial treatments with CDK5 inhibitor and anti‐PD‐1 antibody in the TNBC mouse model. As expected, diminishing stemness transformation by targeting CDK5 profoundly promoted anti‐PD‐1 curative effects. Moreover, we observed the underappreciated function of CDK5 in enhancing TILs infiltration, indicating a narrow association between CSCs and T‐cell infiltration in tumor tissues. In conclusion, these results bring us new hope that targeting CDK5 may sensitize TNBC and other immunologically “cold” cancer types to immunotherapy.CSC has been reported to create and maintain populations of tumor‐associated immune cells, such as TAMs and Tregs, to build up an immunosuppressive niche for immune evasion.46 Alt4γ E3 ubiquitin ligase activity but also opened new avenues for devising a novel strategy against CD44v by targeting CDK5 for TNBC therapy. Additionally, CDK5 interruption with anti‐PD‐1 therapy displayed a striking therapeutic efficacy in TNBC. With this pilot study, more efforts are needed to further address CDK5‐targeted pharmaceutical development and translational issues in the future.This study unearthed the vital function of CDK5 in inducing the stemness transformation of TNBC cells and promoting malignant progression. Concerning the molecular mechanism, CDK5 was uncovered to enhance CD44 alternative splicing from CD44s to CD44v isoform in driving TNBC cell stemness transformation. On one hand, enhanced CD44v generation was proven to provoke the self‐renewal capacity of TNBC cells. On the other hand, CD44v‐positive CSCs would also facilitate immunosuppressive microenvironment construction. The latter mechanism was verified in BC cells during stemness transformation. This novel mechanism not only highlighted the essential role of CDK5 in regulating PPAR52.The cell lines were mycoplasma tested.The 4T1 (GDC0294) murine mammary cell lines were obtained from China Center for Type Culture Collection , and maintained in RPMI 1640 media supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum (Gibco). All cells were cultured at 37 °C with 5% COhttp://crispr.mit.edu/, donated by Zhang Feng) to target the common exons for all mice, CDK5 isoforms were synthesized as follows: 5′‐ ACAGCCGCAACGTGCTACAT ‐3′, and cloned to lentiCRISPRv2 (one vector system) plasmids from Zhang lab. Scramble sequence: 5′‐ GGCTCCAACCGTCCAGAATA ‐3′.Online sgRNA designer were used . The primers are listed as follows: PPARγ‐S273A forward:5′‐CAGGAAAGACAACGGACAAAGCACCATTTGTCATCTACGAC‐3′, PPARγ‐S273A reverse: 5′‐GTCGTAGATGACAAATGGTGCTTTGTCCGTTGTCTT TCCTG‐3′; PPARγ‐S273D forward: 5′‐CAGGAAAGACAACGGACAAAGAACCA TTTGTCATCTACGAC‐3′, PPARγ‐S273D reverse: 5′‐GTCGTAGATGACAAATGG TTCTTTGTCCGTTGTCTTTCCTG‐3′. The changes in the nucleotide bases are underlined. The site‐specific mutations and absence of any spurious mutations were confirmed by DNA sequencing at Genscript Company.The PPAR Briefly, the splenic B cells were purified and fused with mouse myeloma Sp2/0 cells by PEG. Positive hybridomas were tested by ELISA and Western blot analysis, and were cloned by limiting dilution process. The hybridoma cell lines that secreted antibody with high titers were massively cultured for hybridoma injection. Seven days later, ascites were collected and centrifuged at 12 000 r min−1 for 30 min to obtain the supernatant. The anti‐CD44v monoclonal antibody was purified by using protein A/G affinity chromatography. Specificity and titration were examined by Western blot, IHC, and immunofluorescence microscopy in cells or tissues.The purified His‐CD44v (V2‐V10) fusion protein was used to immunize female BALB/c mice according to the conventional procedure.48 Bri5 cells suspended in RPMI, 1640 were injected into the inguinal mammary fat pads of syngeneic, immunocompetent BALB/c mice. Treatments were given as single agents or in combinations with the following regimen for each drug. The CDK5 inhibitor Roscovitine was administered by intraperitoneal (i.p.) injection every 5 days at 2 mg kg−1. Anti‐PD‐1 antibody  was intravenously (i.v.) injected every 5 days. Treatment was initiated when tumors became palpable on day 7 ending on day 27 post tumor implant. Tumors were measured every second day with the vernier caliper, and the volume (0.5 × length × width × width) was calculated.Female BALB/c mice (6–8 weeks old) were purchased from the Model Animal Research Center of the Nanjing University, Nanjing, China and bred in the animal facilities under specific pathogen‐free conditions. All mouse procedures and experiments for this study were approved by Institutional Animal Care and Use Committee, Nanjing University (Accreditation No. 2017‐S09‐72). For 4T1 model, 2 × 10This study was approved by the Institutional Review Board at The Nanjing University. All human tissues were obtained from Chinese PLA General Hospital, Beijing. All patients provided written informed consent in accordance with the declaration of Helsinki before enrolling in the study. The protocol in this study was approved by the institutional review board at the Chinese PLA General Hospital, Beijing. No commercial sponsor was involved in this study. Human tissue microarrays from 70 cases of human TNBC (including 26 cases in stage 1 and 44 cases in stage 2 according to the TNM staging system) were analyzed by immunohistochemistry staining using CDK5 (1:50) and CDK5R1 (1:50) antibodies. The immunostained slides were quantified by Histoscore and TissueGnostics software.https://cancergenome.nih.gov/) was used to analyze selected genes expression during BC progression in different subtypes of BC. Only patients who had been classified into different BC subtypes  according to PAM50 were taken into account . Normalized expression data and z‐scores for mRNA expression data were downloaded from cBioPortal .Gene expression data of human BC from The Cancer Genome Atlas (TCGA) database  was then calculated to reflect the degree to which a set gene was overrepresented. The ES was then normalized for each gene set to account for the size of the set, as normalized enrichment score (NES). False discovery rate was used to generate the p values for significant enrichment.GSEA was performed by using preranked tool as described previously.49 For Briefly, proteins were extracted from each cell lines. Then protein extracts were incubated with PTMScan Motif specific antibody following manufacturer's recommendations. The capture proteins were then eluted with dilute acid and analyzed by LC–MS/MS.Phosphoproteome was performed as previously described.50 Bri−3m tris, pH 8.5, and were subjected to tryptic digestion. 100 µg digested peptides were labeled with TMT isobaric labeling reagents (ThermoFisher Scientific) in buffer with a hydrous acetonitrile for 1 h at room temperature, and 8 µL of 5% hydroxylamine was added to quench the reaction. Equivalent labeled peptides were combined and desalted on a C18 Extraction Disk Cartridge . The labeled peptides were subjected to online strong anion exchange (SCX)‐reversed phase liquid chromatography‐tandem MS.The whole proteins were dissolved in 2% sodium dodecyl sulfate (SDS), 100 × 104 per well in 2 mL DMEM/F12 media supplemented with insulin, transferrin, selenium (ITS) and 20 ng mL−1 EGF (Sigma Aldrich). Cells were cultured at 37 °C with 5% CO2. Spheres greater than 50 µm were counted 12–14 days later.6‐Well plates were pre‐coated with 1 mL poly (2‐hydroxyethyl methacrylate) (pHEMA) . The pre‐coated plates were then placed at 37 °C for 48–72 h. Cultured cells were trypsinized and resuspended in DMEM/F12 media (Gibco). The number of viable cells were calculated, and then the cells were seeded as 1 × 10 Primers are listed in Table S1 in the Supporting Information. All antibody information is listed in Table S2 in the Supporting Information.Immunohistochemistry, immunofluorescence, RT‐qPCR and Western blot were performed according to standard protocols as previously described.51 PriFor the immunoprecipitation assays, whole‐cell extracts were prepared after transfection, incubated overnight with the appropriate antibodies, and subsequently incubated with protein A/G beads (Beyotime) for 2 h. The beads were then washed five times with low‐salt lysis buffer, and the immunoprecipitates were eluted from beads in 1× SDS loading buffer (Beyotime) and boiled for 10 min, and then resolved by SDS‐PAGE. Proteins were transferred to PVDF membranes (Bio‐Rad) and incubated with the appropriate antibodies. The LumiGlo Chemiluminescent Substrate System (Thermo Fisher Scientific) was used for protein detection.6 cells in 100 µL PBS. Fluorescent antibodies and isotype antibodies were consequently added at the indicated concentration, and cells were incubated for a further 30 min at 4 °C. Then cells were washed twice with ice‐cold PBS. Flow cytometry analysis was performed with the BD FACS calibur and data were analyzed with FlowJo software.All cells were pre‐incubated with FcR blocking antibody  for 15 min at 4 °C at concentration of 1 µg per 1 × 10t‐test. Multiple comparisons between two populations were conducted by multiple t‐tests with type 1 error correction. Differences among multiple groups were calculated by one‐ or two‐way ANOVA. Differences in survival were calculated by Log‐rank Mantel‐Cox test. Differences between tumor growth curve were determined by repeated measures two‐way ANOVA. Significance was set at p values of or below 0.05. For all figures, *p < 0.05, **p < 0.01, ***p < 0.001. Unless noted in the figure legend, all data are shown as mean ± SEM. Generally, all experiments were carried out with n ≥ 3 biological replicates.All statistical analyses were performed by GraphPad Prism 5 (GraphPad). TCGA RNAseq_V2 gene expression data was downloaded from cBioPortal website. Differences between two groups were calculated by Student's The authors declare no conflict of interest.P.S. supervised the project and wrote the manuscript; Y.B. designed the study, performed most experiments, and bioinformatics analysis; N.C., T.C., and Y.S. designed the study and performed experiments; X.Z. conducted the MS experiments; B.L., J.W., and Q.L. provided clinical TNBC samples and supervised the in vivo assays performed by Y.Y., N.Y., Wei.Z., Wenlong.Z. and H.S.; W.G.Zhu, and J.J. designed the experiments and modified the manuscript. All authors critically reviewed the article and approved the final manuscript.Supporting InformationClick here for additional data file.


Understanding the different neural networks that support human language is an ongoing challenge for cognitive neuroscience. Which divisions are capable of distinguishing the functional significance of regions across the language network? A key separation between semantic cognition and phonological processing was highlighted in early meta-analyses, yet these seminal works did not formally test this proposition. Moreover, organization by domain is not the only possibility. Regions may be organized by the type of process performed, as in the separation between representation and control processes proposed within the Controlled Semantic Cognition framework. The importance of these factors was assessed in a series of activation likelihood estimation meta-analyses that investigated which regions of the language network are consistently recruited for semantic and phonological domains, and for representation and control processes. Whilst semantic and phonological processing consistently recruit many overlapping regions, they can be dissociated  only when using both formal analysis methods and sufficient data. Both semantic and phonological regions are further dissociable into control and representation regions, highlighting this as an additional, distinct dimension on which the language network is functionally organized. Furthermore, some of these control regions overlap with multiple-demand network regions critical for control beyond the language domain, suggesting the relative level of domain-specificity is also informative. Multiple, distinct dimensions are critical to understand the role of language regions. Here we present a proposal as to the core principles underpinning the functional organization of the language network.  Though it remains unclear how precisely language is organized in the brain, different facets of language can be dissociated from one another at the behavioral and neuropsychological levels; in particular, a key division between semantics and phonology . Yet queThe neural correlates of language may differ based on the importance of semantic versus phonological demands. Phonological processing is the perception, analysis and use of language sounds to understand and produce spoken and written language . The proThe neural correlates of phonology and semantics were the subject of a seminal review of organization across the language network by In the years since Like semantic cognition, phonology is supported by control processes, though these have not been emphasized to the same extent, nor have their neural correlates been clearly isolated to date. However, some functional neuroimaging work has found activation in left IFG, precentral gyrus, SMG and dmPFC for controlled, effortful phonological tasks  . To deten-back working memory task ; and the multiple demand network map from There were three steps to the present study. Firstly, formal ALE analyses compared the regions implicated in the semantic and phonological subdomains, testing the supposition that these subdomains rely on distinct neural correlates. To determine the relative impact of changing method of analysis versus dataset, this was first performed using 2n-back working memory task.Activation Likelihood Estimation (ALE) meta-analyses were conducted for the semantic and phonology domains, independently for Vigneau et al.’s data and the full datasets, which were compared to one another using formal contrast analyses. Subsequently, formal ALE analyses were conducted for semantic control, phonological control, and the 2.1All meta-analyses included only peer-reviewed English language articles, describing task-based fMRI and PET studies that reported peak coordinates of a univariate contrast in standard space  and focused on a young healthy adult sample (below 40 years old). Contrasts were excluded if they focused on patients, clinical trials or individual differences . Finally, any contrasts that overlapped with the other domains – e.g., phonological working memory tasks – were excluded, to allow for meaningful comparisons to be made between domains. Within each paper, wherever multiple task contrasts were reported for the same participant sample, all the peak activation coordinates were analyzed as a single contrast, following the recommendation from For each domain, studies were sourced from one or more existing published meta-analyses, providing appropriate inclusion and exclusion criteria in keeping with the accepted definition of that particular domain. As the phonology meta-analyses alone did not bring the literature included up-to-date, the same criteria were applied to a literature search. This gained more data and allowed fair comparison with the semantics domain. In addition, each contrast had to meet the general inclusion criteria provided above. For semantics and phonology, formal analyses were conducted employing the data from The studies for the semantics domain (Supplementary Table 2) were taken from a meta-analysis by To determine which semantic regions are involved in control, an additional semantic control assessment was included from https://clarivate.com/products/web-of-science/) search to extend the timeframe of inclusion. n-back working memory ALE analysis. Coverage from these two meta-analyses ended in 2009, therefore the Web of Science search was conducted between 2010-2021. Search terms were ‘phonology’ or ‘phonological’ in conjunction with ‘MRI’ or ‘PET’, resulting in 316 results that were assessed for their fit to the inclusion criteria. This yielded an additional 306 foci from 18 experiments that met the inclusion criteria. The final dataset included 82 experiments with a total of 1176 foci (Supplementary Table 3). It was necessary to assess phonological control differently to semantic control, due to the dearth of studies directly focusing on controlled phonological processing. In lieu of a dedicated ALE analysis, the dataset for the phonological domain was divided into hard versus easy tasks, for subsequent formal ALE contrast. Tasks that were passive or simply required a straightforward stimulus-response mapping, e.g., repetition or listening, were classed as easier, while those that involved decision-making, such as judgement of rhyme or syllable number, were classified as harder. In total, 26 experiments with 376 foci in total were included for easy phonology, and 57 experiments with 800 foci in total for hard phonology.For the phonological domain, studies were sourced from two existing meta-analyses, n-back domain were sourced from n between 0 and 3. For the purposes of the present study, only those contrasts that reported a higher > lower n-value and met the general inclusion criteria were included. A total of 11 contrasts that contained meaningful  or phonological stimuli were also excluded due to the potential overlap with semantic and phonological processing. The final dataset for this domain included 66 experiments with a total of 1216 foci (Supplementary Table 5).The studies for the 2.2https://brainmap.org/ale/; n-back domains, for semantic control and for the Vigneau semantics and phonology datasets. The activation maps for the semantics and phonology domains and for hard and easy phonology were also directly contrasted using GingerALE, creating pairwise thresholded conjunction and subtraction ALE images using a p-value of .001 with 10000 permutations and a minimum cluster volume of 20 mm3.Meta-analyses were performed using activation likelihood estimation (ALE) performed in GingerALE version 3.0.2  which brain regions are consistently activated across studies employing semantic and phonology tasks, 2) how distinct are the networks for semantic and phonological processing, 3) does control versus representation provide an additional informative way to separate the functions of regions in the language network and 4) how domain-specific or domain-general are the control areas implicated in semantic and phonological tasks? In considering the division between the neural correlates of semantic cognition and phonology, we first consider whether a formal ALE meta-analysis would reveal this division in the datasets employed by 3.1The formal meta-analysis of the 3. Whilst these clusters may reflect true differences between semantics and phonology, these small differences do not provide strong evidence of distinct networks for phonological and semantic cognition.Directly contrasting Vigneau et al.’s semantic and phonology datasets  revealedper se; however, when all contrasts featuring sentences or phrases are removed, the resulting ALE map still implicates the same regions with the exception of the right ATL . This region may be involved in syntactic processing or may simply fail to reach sufficient power with fewer studies despite a semantic role , three additional clusters are revealed in the right fusiform gyrus extending into the cerebellum, the left fusiform gyrus and the right IFG (pars triangularis), and the left frontal cluster extends into the precentral gyrus , indicating that these regions may also reflect control demands in the phonology domain. Like the semantic network, the regions implicated in phonology may be divided on the basis of performing control versus representation processes. Specifically, the bilateral STG may underpin phonological representation, whilst IFG, inferior parietal cortex, precentral gyrus and posterior ITG may all contribute to phonological control.Phonological control, represented by the formal contrast of hard > easy phonology, is displayed in 3.3n-back task and a mask of the multiple demand network , dorsal IFG (pars opercularis and triangularis), precentral gyrus, dmPFC and posterior ITG were implicated in both semantic and phonological control. These regions may reflect language-general or domain-general control regions. To help to distinguish these two possibilities, we examined the overlap with two measures of domain-general control: an ALE analysis of the working memory ork from . A forman-back working memory ALE analysis yielded a distributed bilateral network for domain-general control , and some regions responsible for domain-general control .The  control , highly 4. Phonological representation is supported by bilateral STG with control dependent on the left supramarginal and superior angular gyri, dorsal IFG/dlPFC and posterior lateral temporal cortex, as well as the dmPFC. The left precentral gyrus is identified in the phonological control contrast at lower thresholds , and thus may also be involved in phonological control, perhaps specifically of complex motor sequences including those needed for articulation (A multidimensional approach is necessary to describe the underlying functional organization of the language network. Formal analyses confirmed prior hypotheses that distinguishing semantic and phonological subdomains provides one key organizational principle. Yet this domain-based separation alone is not sufficient. Distinguishing the processes of representation and control provides additional, distinct indications of the functional roles of regions throughout the language network. Consistent with prior assessments, the semantic network comprises the bilateral ATL for representation, alongside control regions in the left IFG, bilateral dmPFC and left posterior lateral temporal lobe . The AG culation , or in wculation . The finculation , yet proculation . SemantiActivation likelihood estimation is a powerful meta-analytic tool. Visual analysis of the distribution of peaks is clearly insufficient to determine the regions involved in a task or compare two distributed networks. However, it is the difference between the two ALE analyses that may be more surprising. Despite claims of distinct distributions, the Many models of the language network focus on the separation of subdomains as the single organizing principle . For exaLanguage and executive function are often considered behaviorally and neurally independent, due to the ‘special’ nature of language . Yet areUnlike phonological control, not all of the areas implicated in semantic control are responsible for domain-general control. Ventral inferior frontal and posterior middle temporal regions were not identified as part of the MDN, in keeping with prior research . InsteadThe authors declare no competing interest.


Tenebrio molitor larvae flour was recently approved by the European Food Safety Authority (EFSA) as a novel food. The aim of the present work was to create protein-rich healthy cracker from insect flour, achieving the claim “source of protein” with a target market focused on the healthy products for consumption on the go. Contents of T. molitor flour from 2 to 20% (%w/w) were tested, using a previously optimized formulation and the comparison in terms of nutritional, physical, and sensory properties with a standard formulation was performed. T. molitor incorporation allowed an improvement in the nutritional profile of snacks, through an increase of 15% in protein content and an enrichment in minerals . The crackers containing a 6% of insect flour were the most appreciated by the panelists. The incorporation of T. molitor induced a reduction in firmness and an increase in crispness, resulting from the impact of the protein on the structure. This aspect has a positive impact with respect of the acceptance of snacks—70% of the panelists consider the possibility to buy the crackers with 6% enrichment. A darkening of the samples with the increase in the incorporation of T. molitor flour was also observed, accompanied by a reduction of about 20% of the L* values. Globally, insect protein can play an important role in redesigning food diets, making them more sustainable, with less environmental impact and equally balanced.Entomophagy is still a widespread practice in Africa and Asia, although it is declining due to the westernization of diets. Today, the issue of its rehabilitation is underway; indeed, the nutritional economic and ecological stakes of this consumption are strategic. It can be considered an important way to face the scarcity of natural resources, environmental pressures due to the increasing world population, and demand for protein.  Entomophagy, which refers to the consumption of insects by humans, is still a widespread practice in Africa and Asia, although it is declining due to the westernization of diets . In WestThe consumption of insects by humans as an alternative to more conventional animal proteins, can present significant nutritional and environmental benefits. Insects contain an equivalent level of protein, and a high level of nutrients and unsaturated fats. In addition, insect production has less environmental impact and requires less land and water compared to other animal protein sources . On the Tenebrio molitor , more commonly known as “mealworms”, were used. T. molitor larvae have been recently approved by EFSA 1/2.The color of the crackers was instrumentally measured using a colorimeter (Minolta CR-400) with a D65 colorimetric standard and a visual angle of 2°. The results were expressed with the values of: L*, lightness including positive values from 0 to 100; a*, greenness (60 to −60 positive to negative); and b*, yellowness (60 to −60 positive to negative), according to the CIELab standard. The saturation, C*All color measurements were also performed 24 h after baking, under the same lighting conditions and with a standard white with the following parameters: L* = 94.61, a* = 0.53 and b* = 3.62. The measurements were repeated 10 times for each formulation (one measurement per cracker).−1, with a 5 kg load cell at room temperature (20 ± 2 °C). Hardness was calculated as the peak force (N) in the force versus time texturogram. This peak corresponds to the maximum force required to break the cracker. Crispiness was also determined from the same graph, and it is considered as the time needed to reach the maximum peak(s). The shorter the time in which the break occurs, the crispier the material. So, crispiness can be obtained from the time needed to break the cracker, which is inversely related to crispiness; the faster the breakage occurs, the crispier the cracker will be [The texture of the crackers was characterized, 24 h after baking, with a TA.XTplus texturometer , by a penetration test (5 mm distance), on a perforated base, using a stainless probe of 2 mm diameter with a speed of 3 mm·sperature  ± 2 °C. n = 56, age = 12–62, male = 18, female = 38) to assess which concentration of insect flour was most appreciated. To do so, the crackers containing 6% and 15% T. molitor flour were presented randomly, together with a control cracker. All tasters were previously informed that the crackers contained edible insects, approved by the EFSA. The cracker samples were evaluated in terms of color, smell, taste, texture, and overall liking . Purchase intention was also assessed, with five levels ranging from “I would definitely buy” to “I would definitely not buy”. The tests were carried out in a standardized sensory analysis room, according to EN ISO 8589 [Crackers were evaluated by un untrained sensory panel (ISO 8589 .w) was determined using a thermo-hygrometer  at 20 ± 1 °C. The measurements were performed in triplicates from powdered samples.The water content and water activity of the crackers were determined. Water content was measured by the loss of weight of a sample when heated to 100–105 °C, until a constant weight was obtained. Water activity (aT. molitor flour and crackers were performed accordingly with AOAC procedures [3 and HCl (3:1) at 105 °C (with staged heating) in a DigiPrep MS digester . The determination of mineral elements was performed by atomic absorption spectrophotometry and ICP-OES  [The nutritional composition of ocedures . The proocedures ,24. The MA, USA) . All anaMA, USA) .For total phenolic content and antioxidant capacity quantification, extracts were prepared according to the procedure used by Barreira et al.  and Reis−1) and are expressed in mg of equivalent gallic acid per gram of sample (mg EAG·g−1).Polyphenols were quantified using the Folin−Ciocalteu reagent according with the procedure described by Mohankumar et al. . This me−1).To evaluate the antioxidant activity, two methods were used. The DPPH method was performed according to the method described by Brand-Williams et al. , with so3+ from potassium ferricyanide in the TPTZ -1,3,5-triazine) reagent to ferrous iron Fe2+ in the presence of antioxidant, whereby 90 µL of the sample was mixed with 270 µL of distilled water and 2.7 mL of FRAP reagent. After mixing, the solution was placed in a water bath at 37 °C for 30 min. The absorbance was measured at 595 nm and the results calculated from a Trolox standard range (0 to 700 µmol·L−1).FRAP method described by Benzie and Strain  was alsoFor both methods, the results are expressed as mg of Trolox per mg of dry extract. All analysis were repeated in triplicate and performed on powdered cracker samples and Tenebrio flour.T. molitor flour and on the pure flour. Results were obtained from a range of dilutions in liquid medium, from 10−1 to 10−3 [To control the quality of the finished product, an enumeration on GYP medium  was carried out for the cracker with 6%  to 10−3 . Enumerap < 0.05). All results were presented as mean ± standard deviation.All results were statistically analyzed using Prism 5 software and subjected to analysis of variance (one-way ANOVA) and Tukey’s test with a margin of error of 5% (The appearance of the crackers is shown in p < 0.05) from those containing 4%, 6%, 10%, 15%, and 20% of T. molitor flour; a reduction of about 20% of L* value was observed for 15 and 20% of Tenebrio flour incorporation. On the contrary, the incorporation of Tenebrio flour induces an increase in the color intensity level from red to green (a*). The control cracker a* value (2.64) is significantly lower than the crackers with incorporations of 4%, 6%, 10%, 15%, and 20%. Numerous studies have shown similar results for the enrichment of bread [T. molitor flour. These results can be explained on the one hand by the formation of Maillard reaction compounds, favored by the presence of insect flour with high protein content (Regarding the brightness (L*) of the product, a decrease was observed inversely to the amount of insect flour incorporated. Indeed, the L* value of the control cracker (67.18) is significantly higher p < 0.0 from thoof bread , cookiesof bread , and shoof bread  with T.  content  and, on p > 0.05) influence the color of the finished product.For all parameters, the incorporation of a small amount of insect flour (2%) does not significantly (p < 0.05) and a similar effect was observed for spread parameter. It was noticed that the spread is higher when there was more insect flour is incorporated. However, regarding the density of the crackers, no significant difference was observed up to 15% incorporation. This is a relevant quality parameter, as consumers generally want less heaviness and more density [T. molitor does not alter the gas retention in the crackers, which is an important phenomenon related to the cracker’s texture and sensory attributes.The characteristic dimensions of the crackers are shown in  density . This suWhen developing cracker-type snacks, one of the most important parameters is the texture of the final product. When consuming this type of product, texture that crunches under the tooth are generally appreciated . In ordeT. molitor content flour present, the less force required to cause the breakage, i.e., meaning that the crackers become softer. This decrease in hardness is accompanied by a reduction in the breakage time of the structure, i.e., the breakage of the structure occurs earlier, which is associated with greater crispiness. Indeed, the addition of insect flour disturbs the formation of the gluten network responsible for the homogeneity of the dough. This was prominent during the realization of the dough, which was more difficult to mix and to handle. Similar results were observed, especially in the study of Zielińska et al. [T. molitor and cricket flour. This study showed that mealworm enrichment resulted in much softer crackers, but also in elasticity, resilience, cohesion, and chewiness of muffins, except for 6% and 10% flour incorporation (as in our study). However, this study shows that mealworm flour has a more significant impact on the overall texture of the product.In general, a decrease in hardness was observed with the incorporation of insect flour—a reduction around 30% in the firmness values was observed, comparing the control and the crackers with 15% incorporation. In fact, the more a et al.  on muffiT. molitor flour and the control was conducted. According to the type of sensory analysis performed (untrained panel), more than three samples lead to results that are difficult to compare. Thus, according to the texture results, it is clear that incorporations of more than 6% result in much softer crackers. On the other hand, the crackers with 20% of Tenebrio flour were too dark and had a bitter taste; thus, its sensory appreciation was not considered acceptable. So, sensory evaluation on the snack crackers containing 6% and 15% Tenebrio flour . Moreover, 70% of the panelists assumed that they would be willing to buy the crackers with 6% io flour . HoweverT. molitor flour, in comparison with the control.Considering the results obtained for the sensory analysis, all the experiments that followed were performed on the cracker most appreciated by the panel, i.e., the cracker enriched with 6% T. molitor flour. These values are quality parameters. Indeed, for this type of food, the water content and water activity strongly influence the crispness of the product, its sensory acceptance, and its shelf life. A study conducted by Arimi et al. [i et al.  shows thT. molitor flour, which has a lower water content than the other ingredients. As mentioned before, it is possible that the addition of high concentrations of insect flour leads to a weaker gluten network, unable to effectively trap gas bubbles and water molecules, resulting in a decrease in product moisture. Similar results were observed in studies conducted by Min et al. [T. molitor flour. Likewise, there is a consistent reduction in water activity with the addition of Tenebrio flour. Likewise, there is a consistent reduction in water activity with the addition of Tenebrio flour. Crackers showed aw values around 1.6, meaning that, in general, the addition of insect flour imparts a positive impact on the shelf life of the crackers (and the consequent loss of crispness). These results may suggest that wheat flour has a greater water holding capacity than T. molitor flour. The aw values obtained are low (<0.17), which is associated with a longer shelf life, since spoiling bacteria do not have optimal conditions to grow [A significant decrease in water content was observed in the cracker enriched with insect flour, compared to the control (from 0.162 to 0.132). The water present in the dough comes only from the water added to the system (according to the formulations presented) and from the water content of the raw materials. The proportional change in water content was, therefore, caused by the replacement of wheat flour with n et al.  and Zieln et al.  on the e to grow .T. molitor meal was determined to be 51% protein of dry weight, which is relatively close to the values found in the literature [T. molitor flour, can use the claim “source of protein”, since its protein content represents more than 12% of the total energy. However, the amount of protein does not allow the mention “rich in protein”. There was also a significant increase (p < 0.05) in the amount of total ash (2.18%), which reflects the increase in mineral content.The biochemical and mineral composition of the crackers was reported in terature ,18,41. Tterature , the finp > 0.05) were observed between the control and enriched crackers, with crude fat contents ranging from 11.1% to 12.7%  and ash (from 1.90 to 2.18 g/100 g) content, and a decrease in carbohydrate content (from 72.7 to 71.0 g/100 g) as the concentration of worm flour increased, which is associated with a reduction in the calorie intake.T. molitor flour is a source of potassium, magnesium, phosphorus, and iron. This induces similar results in crackers containing it. According to the recommended daily values (RDI) established by the European Regulation N°1924/2006; Directive N 90/494 (EC) [T. molitor flour can be a “source of potassium, magnesium, phosphorus, and iron”. In terms of sodium, a non-significant (p > 0.05) increase was observed. In comparison with literature, similar results were observed for pure Tenebrio molitor flour. In fact, the study carried out by Costa et al. [Tenebrio flour presents results with the same order of magnitude, especially in potassium (800 mg/100 g), magnesium (282.3 g/100 g), phosphorus (797.0 mg/100 g), copper (0.78 mg/100 g), zinc (9.65 mg/100 g), and manganese (1.10 mg/100 g). Similar values are stated in the supported document published by EFSA [T. molitor as a novel food [Regarding the mineral profile, the results are listed in 494 (EC) , the craa et al.  about thTenebrio may present a nutritional claim “source of” in the case of potassium, phosphorus, iron, copper, zinc, and manganese, since they have contents higher than 15% of DVR (highlighted in the table), according with the Regulation (CE) No 1924/2006 [Considering the values of 15% of RDV summarized in the table, of each one of the minerals, it is possible to verify that the crackers enriched with 924/2006 .p > 0.05) in the total phenolic compounds (more than double compared to the control—from 5.02 mg GAE·g−1 to 12.49 mg GAE.g−1). However, since Tenebrio flour has a content of total phenolic compounds of 15 mg GAE·g−1, it can be expected that the value of phenolic compounds may be overestimated in crackers with 6% of Tenebrio flour. This fact may result from the interference of the protein in the quantification of phenolic compounds, by the Folin−Ciocalteu reagent. It should also be noted that the control sample (9.65 g/100 g protein) already has a considerable content of phenolic compounds (5.02 mg GAE·g−1).These phenolic compounds make a decisive contribution to the antioxidant capacity; from It is important to evaluate the antioxidant capacity of our final product, since the presence of antioxidants plays an important role in our diet. These compounds play a role in the prevention of diseases related to oxidative stress, in cardiovascular diseases, diabetes, or the aging process, which are all among the major public health problems nowadays . In addiThe antioxidant properties of the crackers and flour were evaluated in accordance with the ability to neutralize the DPPH radical and the measurement of reducing power (FRAP). The overall results are presented in p < 0.05) was observed when the insect flour was incorporated. Similar results were observed by ferric ion reduction (FRAP method).Regarding the antioxidant activity accessed by DPPH radical capture, a significant increase (p < 0.05) increased the free radical scavenging capacity, as indicated by the DPPH scavenging activity. In addition, numerous studies have identified bioactive compounds in insects, such as polyphenols, chitins, peptides, and proteins. Since insects are known to be high in protein and are potential sources of bioactive proteins and peptides responsible for antioxidant activity [Navarro del Hierro et al.  studied activity ,46,47.Tenebrio flour. This fact can also be explained by the existence of the Maillard reactions already mentioned, which also originated compounds with high antioxidant power. These reactions occur during food processing at high temperatures (as baking), between amine groups (from proteins) and carbonyl compounds (from reducing sugars), inducing the formation of compounds with antioxidant activity, such as reactive oxygen species scavenging activity or metal-chelating activity. Although some of the compounds formed in Maillard reactions have a negative impact on health, when consumed regularly and in large amounts, their contribution to an increase in antioxidant capacity has been duly studied by several authors, such as Shen et al. [Depending on the type of edible insects, these proteins may change the DPPH radical scavenging activity and the reducing power of ferric ion. These changes may depend on the molecular weight of the protein or peptide in question, as well as the amino acid composition . Considen et al.  who studT. molitor incorporation and in the raw flour were evaluated. The total mold and yeast contents are relatively close for the 6% cracker and the raw flour (4.26 and 4.91 log cfu·mL−1 respectively). Similar results were found for T. molitor flour by Vandeweyer et al. [−1) and in the study by Costa et al. [T. molitor as a new food source (4.4 log cfu·mL−1). In order to have more in-depth results on the microbiological aspect of T. molitor, it would have been judicious to also count bacterial colonies , as well as to search for certain microorganism-types responsible for food infection . Indeed, several studies have demonstrated the presence of Enterobacteriaceae, i.e., 6.3 log cfu·mL−1 and 7.6 log cfu·mL−1 by Costa et al. [T. molitor larvae. These values are higher than the legal limit set for minced meat or raw material used in the preparation of meat [−1 for enterobacteria) is, according to some authors, applicable to insects because there is no specific regulation for their consumption. However, only the Belgian Higher Health Council (HSC)  and the Federal Agency for the Safety of the Food Chain (FASFC) , as well as the Dutch Food and Consumer Product Safety Authority (NVWA) , have written an opinion on the food safety aspects of edible insects [Salmonella, Listeria monocytogenes, and Escherichia coli in meat and shellfish dishes. Thus, the detection of Salmonella spp. and L. monocytogenes must lead to an undetectable amount in 25 g of sample. These results were notably found by Vandeweyer et al. [The counts of molds and yeasts in the crackers with 6% r et al.   incorporation was accepted in terms of sensory evaluation. However, for this incorporation level, the nutritional qualities of the cracker, especially in terms of proteins and minerals, was achieved, with a slight impact on textural properties of the product. As T. molitor flour is rich in proteins and certain minerals, its incorporation in the appetizer crackers has allowed the claim “source of protein” and “source of potassium, magnesium, phosphorus iron and zinc”. The use of this flour is also interesting in terms of the antioxidant capacity of the product.The present work highlighted the interest of incorporating T. molitor flour also affected the color of the crackers ; however, for low incorporation levels , the resulting browning was well accepted, without altering the overall appreciation by the panelists. In fact, the crackers with 6% (w/w) of insect flour were the most appreciated by the panelists, as a large part of them (70%) even said that they were ready to buy them. These results are relatively satisfactory and suggest that insect-based crackers could become widely appreciated and consumed as functional foods in the near future in Western countries, where insect consumption is still low.The enrichment of T. molitor flour as an ingredient with the potential to promote the nutritional profile of a food. This sustainable alternative source of food can be considered a positive impact in nutritional terms and can represent an alternative to the protein shortage in future decades all over the world.The obtained results support the idea of using


Cyclic hydroxylamines, which upon oxidation yield their respective stable nitroxide radicals, have been suggested as spin probes of peroxynitrite and superoxide. The present study investigated this approach by following the kinetics of peroxynitrite decay in the absence and presence of various 5-membered and 6-membered ring hydroxylamines, and comparing the yield of their respective nitroxides using electron paramagnetic spectroscopy. The results demonstrate that hydroxylamines do not react directly with peroxynitrite, but are oxidized to their respective nitroxides by the radicals formed during peroxynitrite self-decomposition, namely •OH and •NO2. The accumulated nitroxides are far below their expected yield, had the hydroxylamines fully scavenged all these radicals, due to multiple competing reactions of the oxidized forms of the hydroxylamines with •NO2 and ONOO−. Therefore, cyclic hydroxylamines cannot be used for quantitative assay of peroxynitrite in vitro. The situation is even more complex in vivo where •OH and •NO2 are formed also via other oxidizing reactions systems. The present study also compared the yield of accumulated nitroxides under constant flux of superoxide in the presence of various cyclic hydroxylamines. It is demonstrated that certain 5-membered ring hydroxylamines, which their respective nitroxides are poor SOD-mimics, might be considered as stoichiometric monitors of superoxide in vitro at highest possible concentrations and pH.There is a considerable need for methods that allow quantitative determination in vitro and in vivo of transient oxidative species such as peroxynitrite (ONOOH/ONOO This approach assumes efficient oxidation of cyclic hydroxylamines by superoxide and peroxynitrite to produce stable nitroxide radicals as end-products, which can be quantitatively determined by electron paramagnetic resonance (EPR) spectroscopy 0 denotes the accumulated concentration of nitroxide in the absence of SOD.Superoxide was radiolytically generated in aerated or oxygenated solutions containing formate, phosphate buffer and 50 μM DTPA to bind all traces of redox-active transition metals thus avoiding metal-catalyzed dismutation of superoxide. Under such experimental conditions all radicals produced by the radiation are converted into superoxide . The rat•]0/[RNO•]-1) vs. [SOD] yields a straight line and one has to determine kcat to calculate kRNO-H. As previously described, kcat = (2.8 ± 0.2) × 109 M−1 s−1 was determined using CytIII as a competing reagent in aerated solutions containing 20 μM CytIII, 5 mM formate and 1 mM phosphate buffer at pH 7.8 where k(CytIII + O2•−) = 1.1 × 106 M−1 s−1 [kcat is highly affected by ionic strength and in the presence of 0.1 M formate it decreases to kcat = (1.3 ± 0.1) × 109 M−1 s−1 [A plot of  = 4.3 [K1(3-CP-H) = 5.85 [K1(TEMPOL-H) = 7.1 [K1(TEMPO-H) = 7.5 [K2 is significantly higher, e.g., 13.7 has been estimated for TEMPOL-H and TEMPO-H [+HOH.The value of pH) = 4.3 , pK1(3-C) = 5.85 , pK1(TEMH) = 7.1  and pK1(H) = 7.5 , 7.96 [2H) = 7.5 . The val TEMPO-H . Therefo− and 10 mM NaOH. The final pH as measured at the outlet of the stopped-flow apparatus was 4.3–4.4. Therefore, the predominant form of the hydroxylamine under such experimental conditions is RN+HOH. Although the maximum absorption of ONOOH is around 250 nm [+HOH absorption in this spectral region. In the absence of RN+HOH the decay of ONOOH followed first-order kinetics where kd = 1.32 ± 0.03 s−1, in agreement with literature data [2+, 1–5 mM TEMPO-H2+, or 2.5–10 mM 3-CP-H2+ . The rate of peroxynitrite decay monitored at 302 nm was unaffected upon increasing the concentration of TEMPONE-H/TEMPON-H2+ from 0.625 mM to 2.5 mM or TEMPOL-H/TEMPOL-H2+ from 4 mM to 40 mM.The reaction of ONOOH/ONOO+HOH at pH 4.3–4.6 (50 mM acetate buffer and 50 µM DTPA) yields RNO•, but its yield was significantly lower than ca. 60% expected if all •OH and •NO2 radicals , the yield of RNO2•/O2•−, pKa = 4.8) [The present study compared the efficacies of superoxide scavenging by 5-membered and 6-membered ring hydroxylamines, where their respective nitroxides have different SOD-mimic activities. Superoxide was radiolitically generated in oxygenated solutions containing 0.1 M formate, 10 mM phosphate buffer and 50 μM DTPA. Under such experimental conditions all radicals produced by the radiation are converted into superoxide  using SOD as a competing agent as described in the experimental section (The rate constant of 3-CTPO-H reaction with superoxide has been determined to be (4.6 ± 0.2) × 10 section .k = (4.7 ± 0.2) × 103 M−1 s1 for 3-CP-H and (1.8 ± 0.1) × 103 M−1 s−1 for TEMPOL-H  = 6 × 109 M−1 s−1 [2, peroxynitrite decomposes to yield ·OH and ·NO2 radicals  [+HOH is expected to be less reactive than RNO-H towards •OH and •NO2 radicals, and the values of k5 and k6 reflect the apparent rate constants at the pH studied.The results demonstrate that RNe of CO2 , which i of DMSO ,12. In fposition , where k M−1 s−1 . In the radicals , which o1 = 7.5) ,15,25,26•OH and •NO2 radicals, but due to multiple competing reactions 4–8, the determination of RNO• by EPR spectroscopy cannot be used for quantitative assay of peroxynitrite in vitro. The situation is even more complicated under in vivo conditions where •OH and •NO2 are formed by other oxidizing reactions systems, such as Fenton-like reactions and peroxidase/H2O2/nitrite, respectively.Peroxynitrite self-decomposition yields ca. 30% Eo(RN+=O/RNO•) [3–104 M−1 s−1 [Eo(RN+=O/RNO•) = 1.0 V and 0.87 V, respectively [Similar argumentation holds for the proposed use of hydroxylamines as monitors of superoxide, particularly in cases where the accumulated nitroxides are effective SOD-mimics. Seemingly, the accumulation of nitroxides, as end-products of hydroxylamines oxidation might serve for monitoring the superoxide flux. However, effective catalytic removal of superoxide radicals by nitroxides, particularly 6-membered ring nitroxides, totally pre-empts such an approach. It has been demonstrated that 6-membered ring nitroxides are better SOD-mimics compared to 5-membered ones, and that the SOD-activity decreases with the increase in the reduction potential of the nitroxide, =O/RNO•) ,15. Thus M−1 s−1 ,10,11), ectively ,15, i.e.2, and obviously in its presence, which is the predominant path of peroxynitrite decomposition in vivo. Peroxynitrite induces hydroxylamines oxidation to their respective nitroxides indirectly via reactions with the transient radical products of peroxynitrite self-decomposition, i.e., •OH and •NO2. However, the accumulated nitroxides are far below their expected yield, had the hydroxylamines fully scavenged all these radicals, due to multiple competing reactions of the oxidized forms of the hydroxylamines with •NO2 and ONOO−. Hence, cyclic hydroxylamines cannot be used for quantitative assay of peroxynitrite in vitro. The situation is even more complex in vivo where •OH/CO3•− and •NO2 are formed also by other oxidizing reactions systems, such as Fenton-like reactions and peroxidase/H2O2/nitrite, respectively.The present study demonstrates that peroxynitrite does not react directly with cyclic hydroxylamines in the absence of COThe oxidation of cyclic hydroxylamines by superoxide yields their respective nitroxides, which are known as SOD-mimic. Therefore, the 5-membered ring hydroxylamines, which their respective nitroxides are poor SOD-mimics, might be considered as stoichiometric monitors of superoxide in vitro at highest possible concentrations and pH.


The study of the microstructure of dough and bread also confirmed the established dependencies. This percentage of replacement of refined flour with whole wheat flour from sprouted wheat grain can be recommended as the best for obtaining bread of good quality with high rheological characteristics.Whole wheat flour from sprouted wheat grain is a full-fledged raw ingredient containing essential amino acids, easily digestible sugars, and dietary fiber, with increased digestibility and enzymatic activity. The use of this raw material in the production of food products will contribute to the creation of products for a healthy diet of the population. This study is aimed at studying the possibility of using whole grain flour from sprouted wheat in the production of bread and its effect on the rheological and microstructural properties of dough and finished products. It was found that whole wheat flour from sprouted wheat grain had an even particle size and was characterized by a uniform distribution of particles over the size range (from 53 to 209 microns—61 ± 3%); large particles from 297 to 497 microns were present in an amount of no more than 10 ± 3%. The replacement of 20% refined flour with whole wheat flour from sprouted wheat grain resulted in better values of the farinograph quality index (200 ± 3 mm). The bread obtained according to this recipe had a high specific volume (4.21 ± 0.62 mL.g In recent years, sprouting of grain crops has been actively used to obtain new raw materials used in the manufacture of food products. This is due to an increase in their nutritional value and improved nutrient absorption . A numbeThe increase in enzymatic activity caused by germination can negatively affect the rheological properties of the dough and the quality of the finished bread, since the germination process reduces the baking properties of the flour. At the same time, this process is considered as a tool for improving the quality of food and increasing the functional potential of health promotion . This isWhole grain flour is rich in vitamins, minerals, and dietary fiber, as all the components of the wheat grain are completely absorbed into its composition, making it a raw ingredient of high nutritional value. However, the effect of wheat germination on the physico-chemical, rheological, and baking properties of flour and its microstructural changes has not been properly studied in the previous works, and it is of scientific interest.The aim of the study was to evaluate the possibility of using whole grain flour from sprouted wheat in the production of bread and its effect on the rheological and microstructural properties of dough and finished products.Triticum aestivum L.), a variety of Lubava, harvested in 2014-2018, grown in the Ural region, Russia (grains nongerminated). The protein content was 12.5 ± 0.3 g/100 g in terms of humidity.This study used a grain of soft spring white-grain wheat (The ingredients used to make the bread were purchased at a market in the city of Chelyabinsk. Refined wheat flour (RF)  was provided by the manufacturer OOO Soyuzpishcheprom, Chelyabinsk, Russia, and was used as the main raw material in the manufacture of bakery products.Lux extra, manufacturer of OOO SAF-NEVA, food salt of the highest grade, manufacturer of OOO Russol).Yeast and salt were purchased in the retail network of the city of Chelyabinsk  was soaked in water 22 ± 2°C for 6 hours until a humidity of 35% was reached. The samples were then placed in germination trays, which were located in a temperature- and humidity-controlled chamber . Germination was carried out at a temperature of 22 ± 2°C and a relative humidity of 95 ± 3%. The sprouted wheat was removed from the chamber after reaching the sprout size of 1.5-2.0 mm in more than 90% of the grains; the germination time was 14.6 ± 0.8 hours [The sprouted wheat was dried  at a temperature of 35–40°C for 10 hours until the final moisture content of 8–14% was reached.Whole wheat flour was obtained by milling the grain using a Perten 3100 laboratory mill, with a fixed speed of 20,000 revolutions per minute , equipped with a 0.8 mm metal mesh; then, the flour was sifted through a 0.6 mm sieve . The milThe value of the drop number (FN) was determined using the international method AACC 56-81.03 with the FN 1500 system  with a flour sample size of 7 g (14% of humidity) in 25 ml of water.(1) Physico-Chemical Analysis of Whole Wheat Flour. The moisture content was determined using the international method AACC 44-15.02. The protein content in whole wheat flour was calculated using the Kjeldahl micromethod [romethod . The masromethod ; the douFor the use of whole wheat flour from sprouted wheat grain in the production of bread, mixtures of RF and WWF were prepared in the following proportions: (a) 90 : 10, (b) 90 : 20, and (c) 70 : 30.The rheological properties of the RF and WWF dough were determined in accordance with the AACC 54-21.02 (2010) method on a Farinograph-AT .The dough was prepared according to the method of Kim et al. , with soThe test laboratory baking was carried out using the international method AACC 10-10.03. The recipe of the obtained samples is presented in The amount of water was calculated based on the water absorption capacity of the flour at the rate of 650 E.F. (± 20) on the Brabender Farinograph equipment. The water temperature was 22 ± 2°C.The dough was divided by hand into portions of 450 g, and fermentation was carried out in a controlled fermentation chamber , at 30 ± 1°C and a relative humidity of 80% for 90 minutes. Baking was carried out in a laboratory oven  at 220°C for 20 minutes. The bread was cooled at room temperature and tested after 3 hours.−1, test of 1.7 mm s−1, reverse stroke of 10.0 mm s−1, and compressive force of 40%.The volume of bread was determined by the seed displacement according to the method AACC 10-05.01, 2010. The bread was weighed, and its specific volume was calculated. The moisture content of the bread crumb was determined according to AACCI 44-15.02, 2010. The deformation of the bread crumb was determined in accordance with the AACC 74-09.01, 2010 method using a texturometer . From the bread samples (4 hours after baking), slices up to 25 mm thick were cut, reducing the size of the slices to 25 mm long by 25 mm wide, and the crust layer was removed. For the analysis, an aluminum cylindrical probe P/20 (radius 20 mm) was used, and the following experimental parameters were used: preliminary testing of 1.0 mm sThe microstructure of dough and bakery products was studied using transmission electron microscopy (JSM-7001F (JEOL), magnification 1000x). The samples were predried on freeze-drying equipment , then sputtered with platinum.The studies were conducted in a fivefold repetition. Grain germination, whole wheat flour production, and bread samples were carried out under the same conditions to ensure the accuracy of the results. The experimental data were processed on the basis of mathematical statistics methods using Microsoft Excel and MathCad. The obtained data are presented with a confidence factor of 0.95.α-amylase is considered one of the main limiting factors for use in the production of bread flour from sprouted wheat grain; it is able to hydrolyze starch molecules, which leads to a sharp decrease in the viscosity of the dough, loss of shape stability of the products, and the production of sticky crumbs. In the process of making bread, different batches of flour with different FN values can be mixed to produce high-quality bakery products.Previously, Olaerts et al. described that the germination process leads to a significant decrease in FN, which is associated with increased activity of the alpha-amylase enzyme in wheat grain . Based oIn this study, the change in the FN value was determined during the germination of wheat grain in order to control this process . It was According to , millingBased on the literature , the proThe particle size distribution is a very important factor from a technological point of view, as it affects the properties of the flour and the subsequent processing steps . The resA strong influence of the germination method on the particle size distribution was noted; the WWF sample was characterized by a uniform distribution of particles over the size range; large particles from 296 to 498 microns were not more than 10 ± 3%.p ≤ 0.05) from WGWF.In all flour samples, the moisture content showed values close to 13.5%; the ash content did not change during germination. The slight increase in flour lipids during germination may be due to an increase in free lipids provided by the germination process . As for WGWF flour has the lowest gluten index and the lowest wet gluten content, which can be attributed to its low gluten quality and low protein content in nonsprouted wheat. The WWF sample has large gluten values. These findings are consistent with studies by Hadnadev et al., which reported similar results, where wheat most exposed to high temperatures and precipitation prior to harvest presented the highest values of wet gluten content . The gluThe changes observed in the formation of the gluten structure after germination may be due to the formation of phenolic compounds capable of binding sulfides available for the formation of disulfide bridges. This is important for the formation of the structure of gluten, and protein oxidation and strengthening of gluten can also occur , 24.α-amylase tablets, in order to increase the enzymatic activity and increase the volume of bread. Therefore, in further studies, the addition of part of the flour from the sprouted wheat grain was carried out by partially replacing the refined flour. Mixtures of RF and WWF were prepared in the following proportions: (a) 90 : 10, (b) 90 : 20, and (c) 70 : 30. A dough sample obtained from 100% RF was used as a control. The effect of replacing refined flour with whole wheat flour from sprouted wheat grain on the rheological characteristics of the dough is presented in Bread manufacturers do not want to get flour from wheat with a low FN, because products made from wheat flour with a low FN value have a sticky, crumbly crumb, and a low specific volume . While wThe control sample of the refined flour dough was characterized by high water absorption (61%) and fairly good stability (8.2 min), which is typical for wheat flour with a sufficiently strong gluten.p ≤ 0.05) decrease in water absorption. Pasqualone et al. in their studies [Replacing RF with WWF resulted in a significant ; the dough stability increases before replacing RF with WWF in the amount of 20%, and when replacing 30%, it drops sharply to 4.4 minutes, indicating a weakening of the dough as a result of the increased action of portions . This phportions , 33. Thi-amylase . The micThe structure of both the control and experimental dough samples  is characterized by the presence of a large number of oval-shaped particles with a size of 5 to 30 microns, which in their characteristics correspond to starch grains. The grain surface is smooth, without cracks, grooves, and pores . It is pIn the dough samples (control and a), more fine- and medium-sized starch granules are visualized. Starch is present in the form of round or elliptical grains. Individual grains are slightly deformed; more often, this is observed in large starch grains. Individual starch grains have attached protein particles, which gives them an angularity. In samples (b) and (c), swollen, significantly increased in size large starch grains with a size of 20 to 30 microns predominate.The protein matrix is developed in all samples somewhat differently. In the control sample, it is not sufficiently developed; only in rare cases, several grains of medium and fine starch are covered with a layer of protein , in someIn samples (b) and (c), the protein matrix is more clearly distinguishable, evenly distributed, and surrounds most of the starch grains, combining them. Numerous holes from the formed air bubbles are visualized on the surface of the protein matrix, which indirectly confirms a more intensive fermentation process of the dough.The influence of WWF on the quality of bread was evaluated by the specific volume, humidity, and deformation characteristics of the crumb . The useThe addition of WWF also had a positive effect on the deformation characteristics of the bread crumb. In samples (a) and (b), a slight increase in the total deformation index can be noted, while the elastic deformation also has high values, which indicates a high recovery capacity and a well-developed porous structure.α-amylase reduces the retrogradation of amylopectin and makes the crumb structure more elastic [Several studies have shown , 38 that elastic .At the same time, an excessive amount of WWF, which is observed in the sample (c), leads to a significant increase in the plastic deformation index, which indicates the formation of a sticky, jammed crumb, and a deterioration in the quality of bread. This fact may be due to the weakening of gluten, which makes it difficult to retain gas in the dough and contributes to a denser crumb.The crumb of bread after baking can be represented as a single structural system of starch, protein, and moisture molecules. The crumb structure of the control bread sample  is charaThe control sample is characterized by the presence of a single swollen structureless elastic jelly. Individual starch grains or the protein matrix are not visualized.In the crumb samples (a) and (b), the presence of small pores is visualized, bounded by the interstitial walls that make up the spongy skeleton. The interstitial walls consist of a solid mass of protein coagulated during baking, which is interspersed with swollen gelatinized starch grains, which are almost impossible to visually determine, since they are closely adjacent to the mass of coagulated protein with their entire surface, and therefore, there is no sharp, clearly visible border between them. Also, on the surface, there is a developed network of thin protein fibrils. It should be noted that the protein matrix in samples (a), (b), and (c) is more developed, which is due to the intensification of the fermentation process due to the introduction of WWF.As a result of the study of the rheological properties of the dough, the quality of bread, and its rheological characteristics, as well as microscopic analysis of the samples, the optimal size of the replacement of refined flour with whole wheat flour from sprouted wheat grain was determined, which was 20%. This percentage of replacement led to better values of the farinograph quality index (200 ± 3 mm).−1) and optimal rheological characteristics: total deformation13.7 ± 0.3 mm, plastic4.3 ± 0.3 mm, and elastic9.4 ± 0.3 mm. The study of the microstructure of dough and bread also confirmed the established dependencies.The bread obtained according to this recipe had a high specific volume (4.21 ± 0.62 mL.gIt is recommended to conduct further studies to study the nutritional value and antioxidant activity of whole wheat flour from sprouted wheat and the dynamics of changes during the baking process, as well as to evaluate the effectiveness of sprouted WWF in various final products of cereals to improve their functionality or increase their nutritional value.


International Labour Organization (ILO) report indicates more than 2.4 million workers die from work-related diseases and accidents each year. Work-related respiratory ailments related to airborne particulate matter such as flour dust are responsible for about 386,000 deaths and 6.6 million illness-adjusted life years. Even though exposure to flour dust together with the extreme expansions of flour mill sectors is a priority health concern, extent of the problem is little investigated in Ethiopia. The aim of this study was to evaluate the magnitude and risk factors of work-related respiratory symptoms among flour mill workers in Bahir Dar City, Ethiopia.< 0.05 p value. Adjusted odds ratio (AOR) with a confidence interval (CI) of 95% was calculated to determine a strength of association.This study employed a comparative cross-sectional survey of 560 samples (280 exposed group from flour mill workers and 280 unexposed group from office workers) with a stratified random sampling technique. The study was conducted from March to April 2019 in Bahir Dar City, Northwest Ethiopia. We used the British Medical Research Council (BMRC) questionnaire to assess work-related respiratory symptoms. The questionnaire was pretested and interview administered to collect data. Binary logistic regression analysis was fitted to evaluate significant factors of respiratory symptoms at a Χ2 = 107.11; p < 0.0001). Chest illness among flour mill workers was higher, 43.6% (N = 122) compared to that of among control group, 7.9% (N = 22) followed by dyspnea, 33.6% (N = 94) versus 2.5% (N = 7) among flour mill workers and control group, respectively. Age > 35 years , work set up with inadequate ventilation , work experience > 5 years  and having no training in safety  significantly affected the experience of respiratory symptoms among flour mill workers whereas age , monthly salary  and exposure status  were detected to be significant factors of respiratory symptoms in the exposed and the unexposed combined model.All the sampled participants had fully responded to the interview. The median age of exposed and unexposed groups was 28.5 interquartile range  and 31  years, respectively. The prevalence of work-related respiratory symptoms among flour mill workers was substantially higher than that of among controls, 63.9% and 20.7%, respectively , use of administrative measures  and provision of a suitable personal protective equipment (PPE). Furthermore, it is vital to integrate workplace health and safety programs to the wider public health policies and strategies to effectively mitigate the burden of work-related respiratory conditions. We also encourage future studies to evaluate concentration of flour dusts combined with physical examinations to establish plausible associations of respiratory symptoms with dusts of flour mill-related origin. Recent International Labor Organization (ILO) survey demonstrates more than 2.4 million workers die from work-related illnesses and accidents annually . World H3 per day) for a prolonged time also leads to the development of asthma symptoms . The prevalence of respiratory symptoms was significantly higher among flour mill workers than among office workers . The perceived symptoms reported among flour mill workers were cough (29.3%), productive cough (26.1%), phlegm (30.4%), chest illness (43.6%), and dyspnea (33.6%)  and unexposed (office workers) was 63.9% [95% CI ] and 20.7% [95% CI ], respectively. Whereas the overall prevalence rate in both exposed and unexposed groups was 42.3%  were available, the wind stream in various working units was obstructed because of poor workstations, poor plan, and design of the machines and working units. We also noticed that each working department did not have adequate illuminations. There has been no safety communication in the majority of department in the premises. None of the plants rehearsed sodden wiping to restrict the introduction of flour dusts. There were no basic facilities such as washing facilities at many of the observed plants. The majority of workers are not provided with appropriate PPE and those few workers who were provided with PPE did not wear it, handled it unsafely, and placed it somewhere at their worksite. On the other hand, where PPE is available, it is not a standard; it has been teared and placed by the workers and taken to their homes as there is no supervisory mechanism for its use. In general, the observation confirmed that the working environment of the flour mills might justify the higher proportion of the respiratory symptoms reported in the study.Two models were fitted to determine the associated factors of respiratory symptoms; one large model for the entire data set, and the second for the flour mill workers. In the former model, the effect of exposure status on respiratory symptoms was determined after adjusting confounding factors, like sex, age, educational status, monthly income, physical exercise, and BMI. The result indicated that the odds of respiratory symptoms were 5.18 times higher among flour mill workers than among the control . Age and monthly income were also associated with respiratory symptoms in the larger model (combination of exposed and unexposed group) (Table In the second model (the model consisting only flour mill workers), age, educational status, status of working unit ventilation, year of service, and health and safety trainings were significantly associated with respiratory symptoms Table .Table 5FThe current study showed that age was significantly associated with respiratory symptoms among flour mill employees. Workers with > 35 years of age were 2.03 more likely to develop respiratory symptoms . The chance of developing respiratory symptoms among flour mill workers who had no education were 54% higher than those of the literate workers . Workers who worked in an inadequately ventilated unit had 2.05 times higher at risk to experience respiratory symptoms than those who operate in well-ventilated working units . Workers with more than 3 years of services/work experience had 89% increased chance of developing respiratory symptoms than those with less than 3 years of services . The probability of developing respiratory symptoms is increased by a factor of 2.45 among workers who did not receive trainings in health and safety than those who received the training  . Workers in flour mill factories are more likely subject to exposure to a high level of flour dusts for a prolonged period. The report by scholars in Sudan  and ItalLower prevalence rate of respiratory symptoms has been documented among flour mill workers in studies in Ethiopia (Hawassa) (56.6%) , Iran  has also detected that age is a significant predictor of respiratory symptoms but not the model in unexposed group. This finding is consistent with other reports , 37. AgiIn the current survey, flour mill workers with no education reported higher respiratory impairments than educated participants. This report replicates findings of numerous epidemiological studies , 38, 39.Similar to our finding, multiple studies have proven that the length of employment substantially affects the likely occurrence of respiratory symptom , 40–42. The lack of adequately ventilated working units was the other important factors influencing the incidence of respiratory conditions among flour mill workers. Previous evidence has registered a reliable finding . The levThe lack of ventilation also may lead to the accumulation of flour dust in the indoor environment and decreases the mixing and dilution of those dusts , 28, 44.Our analysis revealed that the lack of trainings in safety and health of the workplaces considerably affected the occurrence of work-related respiratory problems. Evidence in the literature has confirmed our result , 26. SafSimilar to every other developing country, workers in small- and medium-size industries are usually least considered in health and safety regulations in Ethiopia. The health conditions that the workers often encounter have been little investigated in such industries. Therefore, there has been a shortage of reliable data that help develop preventive measures, which commensurate with the risks emerging alongside with the extensive growth of the industries. We believe that the findings of this survey cannot be overlooked as it minimizes those gaps. We also employed a comparative cross-sectional design to support the hypothesis that exposure to the inherent nature of hazards in flour mill industries is substantially linked to the experience of work-related respiratory conditions. Besides, the interview data collection technique used in the current investigation has also increased the response rate, which in turn ensures that the findings are representative.However, there are some limitations which cannot be revoked in the current investigation. First, because of the feasibility concern, measurements for the lung function and other examinations such as prick tests were not carried out. Second, the study failed to determine the levels and characteristics of flour dust exposures. Third, there was a lack of information on the types of energy the participants used at their homes. Fourth, the study was carried out in specific types of workplaces (flour mill factories), which precludes the findings from being expanded to other economic sectors. Moreover, because the unexposed group participants were selected from public workers, they might differ from the flour mill workers in many characteristics. Finally, because of a recall bias, underreporting may be anticipated, as the study relied on self-reporting data. We fitted separate models to minimize the effects of some confounders, and used a validated assessment tool to evaluate the symptoms.Respiratory symptoms related to occupational exposure to flour dusts were significantly higher among flour mill workers than those of control group. Older age, lower level of education, higher length of employment, lack of trainings in safety and health, and inadequate ventilation in the workplaces were important factors of work-related respiratory impairments. We recommend that employers need to effectively implement health and safety programs that account for the reduction of dusts at source, the use of engineering controls , the use of administrative measures , and provision of suitable personal protective equipment (PPE) in the workplace. Moreover, aligning workplace health and safety programs with wider public health policies and strategies to effectively mitigate the burden of respiratory conditions is recommended. We also encourage future studies to evaluate concentration of flour dusts combined with physical examinations to establish plausible associations of respiratory symptoms with dusts of flour mill-related origin.