In vitro transcription (IVT) using T7 RNA polymerase has become the most common method to synthesize RNAs for use in basic research and pharmaceutical applications. To solve the heterogeneity issue associated with the system, cis-acting ribozymes have been exploited to direct co-transcriptional processing to yield target RNAs with precisely defined ends. However, traditionally used ribozymes have many limitations, such as low efficiency and special sequence requirements of target RNAs. In addition, the introduction of ribozymes in IVT complicates the downstream purification of target RNAs. Here we describe a new cassette of engineered ribozymes (HHV-Kt and Twister-Kt) that can work in concert to produce RNA with defined 5’ and 3’ ends. The pair of ribozymes displayed reliably high activity when working with RNA of various lengths and structures. The engineered ribozymes also carry a K-turn RNA motif that enables fast post-IVT clearance of cleaved ribozymes and uncleaved precursors using K-turn affinity resins. Finally, we demonstrated the scalability of our system for the rapid production of large quantities of homogeneous RNA samples. RNA has attracted increasing research interest due to its variety of biological functions and the great potential for pharmaceutical use. Consequently, RNA production has become a routine and, in some applications, a critical task in many labs or industries. Most techniques are still in development and evolving. There are two ways to synthesize RNA molecules, chemically and enzymatically. Chemical synthesis is preferred for short RNA oligomers with less than 80 nucleotides, but efficiency drops considerably with increased length . EnzymatAlthough effective in generating homogeneous RNA, introducing ribozymes creates a situation where downstream purification of the target RNA becomes challenging. Two major side-products must be removed, the released ribozyme and the uncleaved precursor. In practice, IVT reactions are usually subject to denaturing gel electrophoresis to separate the target RNA from unwanted species based on their sizes . This tiHH and HDV ribozymes were first introduced in the application of homogeneous RNA production in the nineties of the last century when a very limited number of ribozymes were discovered ,11. ThisRecently, more ribozymes have been discovered and characterized ,13. We dMet (mt-tRNAMet) as a model system, we characterized the efficiency and sequence requirements of these ribozymes. We fused wild-type ribozymes with the mt-tRNAMet sequence and placed them under the T7 promoter for IVT. HHV, TS, and Pistol were placed upstream of the tRNA for producing a defined 5’ end, while Twister was installed downstream of the tRNA to make a defined 3’ end. A base-paring stem structure formed between the ribozyme and the target RNA is a conserved feature for most ribozymes and is required for maximum efficiency , Twister Sister (TS), Pistol, and Twister, appeared to have high activity ,13. We sficiency . For comMet. Nearly all mt-tRNAMet were released from the HHV-tRNA precursor. There is a persistence of minor uncleaved tRNA-Twister precursor throughout the reaction, which did not build up over time. For TS-tRNA, the uncleaved precursor is prominent. In the first three hours, about 60% of TS-tRNA was cleaved. Prolonged incubation yielded more of the total RNA and resulted in a higher level of cleavage. After 24 hours, TS-tRNA precursor accounted for ~22% of the total RNA. We only observed Pistol-tRNA precursor, suggesting Pistol could not fold into an active conformation when fused with mt-tRNAMet  to the CNBr-activated beads. For K-turn modified ribozyme, we conjugated the recombinant L7Ae protein from -through  A and C.Poliovirus genome (NC_002058), a highly structured 554-nt EMCV IRES from the encephalomyocarditis virus [Based on previous results, the HHV-Kt and Twister-Kt pair has emerged as a promising cassette of ribozymes that can be used in homogeneous RNA production. We next used this ribozyme cassette to synthesize more RNA species of diverse types. These include a 1000-nt RNA sequence taken from the 5’-end of the is virus , the 341is virus , and a 2ome NC_0058, a higMany applications, such as structural analysis and drug development, require large quantities of RNA molecules. Therefore, large-scale RNA production is often necessary. We started to scale up our system to make milligrams of EMCV and HCV IRES RNAs . A 500 μIn vitro transcription by T7 has become the most established method to manufacture RNA, despite synthesized RNA having heterogeneous terminal ends. However, there are many scenarios where RNA of high purity and homogeneity are strictly demanded, for example, in the development of RNA-based therapeutics, such as circular RNA, tRNA, sgRNA, etc. Here we have developed a system that comprises a cassette of engineered ribozymes (HHV-Kt and Twister-Kt) and a novel K-turn affinity resin. HHV-Kt and Twister-Kt working in concert, are capable of generating homogeneous terminal ends for a variety of RNAs, from short linear RNA to large complex RNA molecules. HHV-Kt and Twister-Kt deliver consistently high cleavage efficiency. In addition, HHV-Kt and Twister-Kt do not impose any sequence restrictions on RNA to be synthesized.Sulfolobus solfataricus or Archaeoglobus fulgidus can bind K-turn strongly with Kd in the range of picomoles [In this study, we screened three RNA motifs for an affinity tag and found that the K-turn motif allows effective clearance of ribozymes when IVT is done. The K-turn is a short RNA motif that comprises a three-nucleotide bulge flanked by A•G and G•A basepairs on one side, which can be recognized explicitly by many proteins in nature, including the ribosomal protein L7Ae . Archaeaicomoles . High afWe chose L7ae from a thermophilic archaeon because of its inherited thermal and chemical stability. The resultant K-turn affinity resin can be regenerated with a basic and high-salt solution without losing capacity. Furthermore, we demonstrated that the system could be scaled up to produce milligrams or more of RNA.Some target RNAs may contain a K-turn motif, so the K-turn purification system is unsuitable. In this case, the HHV-MS2/Twister-MS2 ribozyme cassette can be used with the MS2 affinity resin for RNA production.Taken together, we have provided a reliable solution for preparing high-quality RNA that can be adapted to different scales.All the ribozyme sequences (wildtype and engineered) used in this study were listed in Supplemental Table S1. All DNA sequences used as templates for RNA in-vitro transcription (IVT) were listed in Supplemental Table S2. All DNA was synthesized commercially and cloned into pUC57 vectors under the control of the T7 promoter. DNA templates for transcription were generated by PCR. RNA was prepared by a standard T7 RNAP run-off transcription reaction using the HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs). Typically, a 20 uL reaction was set up according to the instruction and incubated at 37°C for 3 to 24 hours. 2uL of the sample was taken from the reaction at various time points and was analysed by denaturing polyacrylamide gel electrophoresis. For statistical analysis of cleavage efficiency, three independent IVT reactions for each construct were carried out. The band intensities corresponding to precursors, cleaved ribozymes, and released RNA products in denaturing gels were quantified by ImageJ software. The cleavage efficiency was calculated as follows: cleavage efficiency = (cleaved ribozyme + RNA product)/(precursor + cleaved ribozyme + RNA product). The data were plotted as mean ± SD.Sulfolobus solfataricus was overexpressed in E.coli and purified as previously described [3, pH 8.4, 500 mM NaCl) was incubated with 2.3 mL CNBr-activated Sepharose media. The mixture was then rotated for 2 hours at room temperature for the conjugation to occur. The coupled resins were treated sequentially with the coupling buffer and the blocking buffer  and washed four times with high pH buffer  and low pH buffer . Finally, the resins were suspended in 50 mM Hepes-K, pH 7.5, and 200 mM KCl, and stored at 4°C.Recombinant L7Ae protein from escribed . Protein3, pH 8.4, 500 mM NaCl) and incubated with 3.2 mL CNBr-activated Sepharose medium. The rest of the steps were the same as for preparing the K-turn affinity resin. To purify RNA, 5 µL of IVT reaction was combined with 60 µL resins in a spin column. After centrifugation, the flow-through was collected and saved.Recombinant MS2 coat protein (MCP) carrying a 6xhis tag and MBP fusion protein at its N-terminal was prepared as previously described . To conj2) and loaded onto 200 µL Sephadex G-200 resin in a spin column. After 20-minute of incubation at room temperature, the resin was precipitated by centrifugation, and the flowthrough was collected. The resin was regenerated by washing twice with 300 µL of 8 M urea.To clear up D8-containing ribozymes, a 15 µL of IVT reaction was brought to 300 µL with the D8 binding buffer . After a quick spin, the flowthrough was collected and saved. Target RNA that binds to resin non-specifically can be eluted with additional washes with binding buffer. The K-turn affinity resins were regenerated by washing five times with stripping buffer containing 200 mM Tris base and 2 M KCl and resuspended in binding buffer.A prepacked column containing 1.2 mL of K-turn affinity resins was prepared to scale up the RNA production. The column was installed into a liquid chromatography system and equilibrated with the binding buffer . For RNA purification, a 500 µL of IVT reaction was diluted with 9 mL of binding buffer and applied to the column. Flowthrough was collected and saved. Additional RNA was eluted with a gradient to the stripping buffer . Fractions containing the target RNA were pooled and combined with the flowthrough. The final RNA product was buffer-exchanged to water and concentrated with Amicon® Ultra-15 Centrifugal Filter Devices (Merck Millipore).Click here for additional data file.


High-grade serous ovarian carcinoma (HGSOC) represents the most common form of epithelial ovarian carcinoma. The absence of specific symptoms leads to late-stage diagnosis, making HGSOC one of the gynecological cancers with the worst prognosis. The cellular origin of HGSOC and the role of reproductive hormones, genetic traits , chromosomal instability, or dysregulation of crucial signaling pathways have been considered when evaluating prognosis and response to therapy in HGSOC patients. However, the detection of HGSOC is still based on traditional methods such as carbohydrate antigen 125 (CA125) detection and ultrasound, and the combined use of these methods has yet to support significant reductions in overall mortality rates. The current paradigm for HGSOC management has moved towards early diagnosis via the non-invasive detection of molecular markers through liquid biopsies. This review presents an integrated view of the relevant cellular and molecular aspects involved in the etiopathogenesis of HGSOC and brings together studies that consider new horizons for the possible early detection of this gynecological cancer. Ovarian cancer (OC), situated among the most aggressive and deadly gynecological malignancies, is the fifth leading cause of cancer-related death in females in developed countries, with a total of 313,959 new cases diagnosed in 2020 worldwide  and 12,810 predicted annual deaths and 19,880 predicted diagnoses in 2022 in the US ,5,6. TheCurrent clinical approaches that guide physicians in managing EOC remain primarily based on imaging, histological evaluation, serum markers such as CA125, or predictive models, which do not display sufficient sensitivity and sensibility ,19; howeDifficulties in the early detection of HGSOC, before the disease develops to advanced stages, can be attributed to the lack of specific symptoms, which are usually missed or attributed to other pathologies . In clinMUC16 gene—in HGSOC compared to healthy ovarian tissue [Initial studies found elevated levels of CA125—a transmembrane cell-surface protein encoded by the n tissue ,27. CA12n tissue ,30,31,32n tissue ,42,43,44n tissue .The Risk of Malignancy Index (RMI) algorithm combines CA125 levels, ultrasound results, and menopausal status ; howeverOther strategies based on the Risk of Ovarian Cancer Algorithm (ROCA)  aimed toAlternative strategies such as the Risk of Ovarian Malignancy Algorithm (ROMA), OVA1™, or OVERA™ deserve special consideration given their U.S. Food and Drug Administration (FDA) approval and their relevance to routine clinical practice. ROMA, which displays high performance in menopausal patients, evaluates HE4 and CA125 levels and stratifies patients with adnexal masses into low or high risk of malignancy ,64. HE4 WT1, TP53, and P16 expression profile that distinguishes HGSCO from LGSOC [While ongoing gene expression profiling studies have suggested that HGSOC represents a highly heterogeneous pathology , expressom LGSOC , while Lom LGSOC .HOX, FAP (myofibroblast markers), and ANGPTL1/2 (markers of microvascular pericytes) or high expression of HMGA2, SOX11 (transcription factors), MCM2, and PCNA (proliferation markers) is correlated with worse prognosis [MUC gene (MUC16) expression or CXCL11, CXCL10 (chemokine ligands), and CXCR3 (receptor) expression [Different gene expression profiles reported by studies such as the Cancer Genome Atlas (TCGA) project ,83 can brognosis ,86,87. Apression ,86,87.NR5A1, GATA4, FOXL2, TP53, and BMP7 possess different profiles when comparing primary OC tumors and their metastases, and could eventually be used to predict patient survival [BRCA1, ATR, FANCD2, BRIP1, BARD1, and RAD51) is associated with a better prognosis in HGSOC cases, whereas the expression of epithelial-to-mesenchymal genes  is associated with a worse prognosis [Gene expression panels such as survival . Additiorognosis .HIF1α and TP53 in therapy-unresponsive patients as an indicator of a worse overall prognosis [CTNNBL1, CKB, GNG11, IGFBP7, and PLCG2) for classifying wild-type BRCA HGSOC patients into sensitive- and resistant-to-therapy groups [Studies have also assessed differential gene expression in HGSOC to guide the optimal therapeutic choice and measure expected responses . A meta-rognosis . Lee et rognosis . Most rey groups .UBE2Q1 as a prognostic biomarker associated with poor relapse-free overall survival in HGSOC patients [Novel bioinformatic approaches are currently attempting to refine gene expression signatures that predict therapeutic responses  and diffpatients .Overall, using DEG analysis in early diagnosis has the potential to improve the management and survival of HGSOC patients.Scientists and physicians have attempted to elucidate the possible cellular origin of HGSOC for over a century; unfortunately, a clear origin has yet to be established, since no specific precancerous lesion has been recognized, which represents a significant obstacle to early diagnosis approaches ,96,97.The first theory for HGSOC’s cell of origin points to the ovarian surface epithelium (OSE) . FathallBRCA1 or BRCA2 mutations undergoing salpingectomies [BRCA gene mutations [P53 gene mutations, and a strong correlation in CCNE1 copy number amplification [The observation of genetically and phenotypically altered cells  in the ciliated end of the fallopian tube in female patients carrying ectomies ,106 provectomies ,110,111 ectomies ,112,113.ectomies , includiutations ,115, idefication ,117. STIfication ,118,119.fication  and telofication ,121; thefication ,123. Latfication , which wAlthough recent evidence suggests the likelihood of a tubular origin of HGSOC precursor lesions, a possible dual origin (OSE or FTE) for this tumor is also currently accepted ,127,128.Gonadotropin hypothesis: The risk of serous EOC increases due to the excessive ovarian tissue uptake of FSH and LH [(a) H and LH ,137. TheH and LH  and the H and LH ,140 or EH and LH  support Androgen/progestin hypothesis: This dual hypothesis acknowledges high androgen levels  or obesity) as an EOC risk factor [(b) k factor ,143,144 k factor ,133. Higk factor ,146 in Hk factor ,148 and k factor . Conversk factor ,150.The heterogeneous expression of estrogen and the estrogen receptor (ER) alters depending on tumor stage and subcellular location and even changes with different prognoses in HGSOC patients ,129,130;In line with these two hypotheses, differing physiological conditions could be considered to be risk factors  or protective factors . During ovulation and menopause, elevated FSH and LH flux from the pituitary gland to the ovarian epithelium increases the risk of tumor development . Indeed,Based on this knowledge, strategies that consider the expression of AR, ER, and PR have been explored to predict survival rates and responses to therapy in HGSOC ,162.The complex genomic landscape of HGSOC, which arises from a combination of inherited and/or somatic mutations and chromosomal abnormalities, epigenetic alterations, and signaling pathway dysregulation , has beeBRCA1 and BRCA2 genes, present in 13–22% of HGSOC cases [RAD51, BRIP1, PALB2, CHEK2, MRE11A, RAD50, and ATM, which belong to the Fanconi anemia–BRCA pathway and play crucial roles in the HRR system for double-stranded DNA breaks [RAD51 [BRCA genes [HGSOC possesses a robust heritable component involving germline mutations that contribute to the development of disease in 25% of cases . GermlinOC cases ,185,186,OC cases . Other rA breaks ,189,190.A breaks ,191,192.A breaks ,194. News [RAD51  or calcuCA genes .Genome-wide association studies (GWASs) have revealed associations between single-nucleotide polymorphisms (SNPs) and pathogenic phenotypes within the population  or the iHOXD9, CDC42, and CDA8 [CCNE1, CPNE1, HEATR3, and STRCP1 [As most SNPs occur in non-coding regulatory regions, transcriptome-wide association studies (TWASs) and expression quantitative trait locus (eQTL) analyses have sought to overcome this drawback when studying disease predisposition. Lawrenson et al. described three statistically significant eQTL associations for and CDA8 , while Gand CDA8 , indicatd STRCP1 . Novel ad STRCP1 . These dTP53, the most frequently mutated gene associated with HGSOC, functions as “the guardian of the genome” by regulating upstream or downstream genes to control tumor suppression and maintain cellular homeostasis [TP53 mutations in around 93–96% of HGSOC patients [TP53 mutation rates of 100% in late-stage cases and 82% in early-stage cases [TP53 mutational load in HGSOC patients [TP53 mutations are mostly missense (60.52%) but can also be frameshift (15.24%), splice-site (10.53%), nonsense (10.73%), and in-frame (3.22%) mutations [BRCA1/2 and RAD51D may also undergo somatic mutations [CSMD3 and FAT3 (tumor-suppressor genes), MLH3 (with known roles in DNA mismatch repair), and CDK12 (RNA splicing regulation) [eostasis ,202. Thepatients ,201,203.ge cases , consistpatients . TP53 muutations . R273, Rutations ,206,207 utations ,211,212.utations ,213,214.ulation) ,171.Corona et al. described several point mutations that converge on TEAD4/PAX8-binding sites during OC’s progression , highligTP53 mutations [CNNE1, MYC, and MECOM) and focal deletion areas  [CNNE1 amplification [PTEN loss [Chromosomal instability (CIN) leads to aberrant gene expression patterns and protein functions related to primary tumor growth/development and metastatic burden in several cancers . Specifiutations , cell-cyutations . CIN affand NF1) ,172; confication  or PTEN TEN loss . InteresTEN loss .MLF1, BRCA1, CCNE1, TP53, ARID1A, MYC, and PIK3CA [TP53 mutations, CNNE1 amplification, or RB1 loss [Chromothripsis, a well-reported form of CIN with a critical role in cancer , is corrd PIK3CA . FinallyRB1 loss ,225.Epigenetic mechanisms involving DNA methylation , histone modifications, or non-coding RNAs (ncRNAs) can regulate gene expression, participate in the onset of HGSOC, and influence therapeutic outcomes ,226,227.BRCA1 promoter hypermethylation in families affected by HGSOC without BRCA1/2 germline mutations is correlated with allelic BRCA1 loss [DNA methylation is generally associated with decreased gene expression and can affect the promoters of tumor-suppressor genes. For instance, CA1 loss . HypometCA1 loss ,177. MorCA1 loss .NOXA [Histone modifications carried out by methyltransferases (HMTs) or deacetylases (HDACs) have also been implicated in the development of HGSOC . SpecifiNOXA . FurtherNOXA ,231.CTBP1-DT lncRNA and its link to the malignant behavior of HGSOC cells and disease progression, suggesting this ncRNA as a possible biomarker for early diagnosis or a therapeutic target [The expression of ncRNAs—specifically, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs)—represents an additional epigenetic mechanism with importance in the pathophysiology of cancer ,232. A sc target .Relevant signaling pathways involved in HGSOC include RB (67% of cases), PI3K/RAS (45% of cases), NOTCH (22% of cases), and FOXM1 (85% of cases) ,172,237 RB1 loss combined with HRD are associated with unusually long survival rates after chemotherapy, providing opportunities for more precise stratification of HGSOC [The RB pathway controls the G1-to-S phase transition in mammalian cells ,239. Oveof HGSOC .PIK3CA (20% of cases) or AKT isoforms (15–20% of cases) [KRAS oncogene amplifications (11% of cases), MAPK amplification (20% of cases), NF1 loss (8% of cases), and other less frequent changes in NRAS or BRAF [Alterations in the PI3K/Akt/mTOR pathway in HGSOC mainly derive from genomic amplification of f cases) ,243. Nonf cases) ,245. Ras or BRAF .BRCA1/2 promoters represent downstream targets of the RB, PI3K, and RAS signaling pathways, offering opportunities for therapeutic interventions using CDK, RTK, and MAPK inhibitors such as cediranib, nindetanib or pazopanib  [Interestingly, survival ,247), or tumors) ,248. Oth tumors) ,250.E2F copy number gain and amplification or the overexpression/inactivation of FOXM1 upstream transcription factors such as ERBB2 (tyrosine kinase receptor) or TP53, might cause overexpression of FOXM1 in HGSOC. Abnormal FOXM1 overactivation, in addition to copy number gains on this gene, coincides with abnormal cell-cycle processes that promote cell proliferation, cancer stemness, genomic instability, and poor prognosis [Different molecular traits, such as rognosis ,252.CCND, p21CIP1, NF-κβ, and c-MYC [CXCR4/SDF1α signaling dysregulation [DK1 overexpression  [Notch family members are transmembrane receptors that, upon binding and cleavage, migrate to the nucleus and target the promoters of genes such as nd c-MYC . Other fgulation  or DK1 o ligand) , with boThe upward trend in the number of clinical trials focused on OC—and on HGSOC in particular—has translated into an increase in survival and positive outcomes when comparing the last five years with the early 2000s .Up to 949 clinical trials have been registered in the clinicaltrials.gov portal of the U.S. National Institutes of Health (NIH) within the last five years , with 19It should be noted that while much existing research has focused on early and non-invasive detection, the need for a very large study population for general screening represents an obstacle to the design of such studies ,258,259.Liquid biopsies, based on the non-invasive detection of molecular biomarkers (mainly through NGS) released from tumor cells into the bloodstream, represent an alternative approach to the early detection of HGSOC . Here, wCTCs are released from a primary or metastatic tumor and shed into the peripheral blood, where they can be isolated by size selection  or by tactDNA is the fraction of total cfDNA released from tumor cells, mainly through necrosis and/or apoptosis . ctDNA lOPCML, RASSF1A, RUNX3, and APC [COL23A1, C2CD4D, and WNT6 [HOXA9 and HIC1 [TP53 mutations and other somatic variants have also been detected in ctDNA from stage I and II HGSOC cases [Early detection strategies using ctDNA rely on evaluating methylation patterns and detecting somatic mutations or aberrant fragmentation patterns ,279,280. and APC ,282,283,and WNT6 , or HOXAand HIC1  have beeOC cases ,287,288.OC cases ,290, as OC cases . FinallyOC cases .miR-1246, miR-595, and miR-2278) [CASC11 [Detection of ctRNA, as a fraction of the total cfRNA released from tumor cells, represents a promising approach in liquid biopsies, since this strategy offers a snapshot of the transcriptional landscape and mirrors potential pathogenic processes . ctRNA hiR-2278) ,297 overiR-2278)  or lncRNiR-2278)  and CASC [CASC11 ) upregul [CASC11 .Exosomes are a subtype of EVs that are released from tumors and extravasate into the bloodstream, where they can avoid the immune system , promoteRanked as one of the most aggressive and deadly forms of gynecological cancer, HGSOC is currently considered to be a public healthcare issue that significantly impacts female patients’ quality of life. Current challenges arise from the difficulty in establishing an early and effective diagnosis in patients with adnexal masses. HGSOC is mostly diagnosed in advanced FIGO stages (III-IV) due to the lack of symptoms and the use of traditional diagnostic methods. The performance of CA125—the only available molecular serum biomarker—alone or in combination with other biomarkers or TVS, has yet to support significant reductions in mortality rates.TP53 gene, genes belonging to the HRR system, and CIN. Furthermore, alterations in multiple signaling pathways and epigenomic mechanisms have also been described.Other strategies have focused on the possible dual origin of HGSOC (the FTE or OSE) and the influence of hormones in promoting or protecting against the development of disease. Furthermore, HGSOC is characterized by extensive genomic instability, promoted by almost universal mutations in the Research in this field is rapidly moving forward, allowing the molecular management of HGSOC by encompassing multiple disease-associated features in high-throughput, personalized approaches. Proof of such advances includes the putative gene panels and expression analyses intended for the stratification of HGSOC, the establishment of treatment-guided decisions, and the monitoring of disease progression, which can be extended to early detection.Most recently, liquid biopsies have gained momentum as an emerging strategy in the field of gynecological oncology, providing a non-invasive approach with promising applications from several clinical standpoints, including early diagnosis of adnexal masses or monitoring of HGSOC patients’ prognosis and treatment effectiveness.This review sought to provide an update of those studies concerning the molecular and cellular characterization of HGSOC tumors, which may serve as a starting point for the design of new strategies aimed at improving management of this subtype of cancer.


Anterior cingulate cortex mediates the flexible updating of an animal’s choice responses upon rule changes in the environment. However, how anterior cingulate cortex entrains motor cortex to reorganize rule representations and generate required motor outputs remains unclear. Here, we demonstrate that chemogenetic silencing of the terminal projections of cingulate cortical neurons in secondary motor cortex in the rat disrupts choice performance in trials immediately following rule switches, suggesting that these inputs are necessary to update rule representations for choice decisions stored in the motor cortex. Indeed, the silencing of cingulate cortex decreases rule selectivity of secondary motor cortical neurons. Furthermore, optogenetic silencing of cingulate cortical neurons that is temporally targeted to error trials immediately after rule switches exacerbates errors in the following trials. These results suggest that cingulate cortex monitors behavioral errors and updates rule representations in motor cortex, revealing a critical role for cingulate-motor circuits in adaptive choice behaviors. The anterior cingulate cortex allows an animal to update its behaviour when the environment changes. In this work, the authors identify a pathway from cingulate to secondary motor cortex, critical for updating motor rules following behavioural errors. Previous studies in primates suggested that premotor cortex and supplementary motor areas underlie the planning and execution of sequential movements7. More recently, it has been demonstrated that neurons in the rodent secondary motor cortex (M2) similarly code initiation of sequential movements, motor planning, memory, and values for upcoming choice actions, suggesting that they maintain representations of sensorimotor associations for adaptive choice behavior14. These findings raise a question of what circuit mechanisms enable M2 neurons to process context-dependent information such as task rules. More specifically, how is this information provided from input brain regions and how are they processed by M2 circuits?A central feature of animal intelligence is the hierarchical organization of behaviors and the capacity to adopt flexible strategies that allow complex sequential actions20. However, despite abundant anatomical evidence of cingulate projections to motor cortices24, there are several fundamental issues that remain poorly understood. (1) Dimensions of task rule representations in ACC: what aspects of task rules ACC circuits represent? We addressed this issue by using a behavioral task in which rats were required to adapt their sequential choice behaviors between single-step and two-step choice responses. The rule switches in this task paradigm are asymmetric in their design  and this feature allowed us to distinguish whether ACC circuits are recruited in rule switches that demand an increment of response steps , a decrement of response steps  or in both types of rule switches. (2) Relevance of ACC to motor output pathways: how does ACC entrain motor cortex to update neural representations for rules upon sudden task rule changes? To address this issue, we perturbed the projections from ACC to M2 using chemogenetic and optogenetic methods and examined what aspects of choice behaviors were mediated by the ACC→M2 circuit and how ACC circuits modulate neural activity in M2 after rule switches in which mapping of choice actions to rewards are suddenly changed. Combining these pathway-specific and task period-specific manipulations with animals’ behaviors in a choice decision task and with neural activity measurements from M2, this study revealed a specific functional role of ACC→M2 pathway in flexible updating of rule representations upon rule switches during animals’ sequential choice responses.Studies in humans and other animals have shown that medial prefrontal areas including anterior cingulate cortex (ACC) mediate flexible decisions in the face of rule changes  or under uncertain conditionsWe devised a behavioral task in which an animal updates its sequential choice response for rewards upon abrupt rule changes . Briefly, in the CAS task, animals are required to choose left or right ports based on an auditory tone cue stimulus. Under the 1 step rule condition, animals received a reward after correctly poking a left or right port instructed by one of two tone cues, and could start the next trial after an inter-trial interval (ITI) 26 received projections from ACC (area 24a’/24b’)  Fig. 27,28. Weses Fig. .Fig. 3AnWe then asked what aspects of choice behaviors are mediated by the ACC→M2 circuit that we identified. More specifically, we wanted to examine the potential roles of projections from ACC to M2 in the animal’s ability to update sequential choices upon rule switches. Using bilateral infusion cannulae targeting M2 in rats that had been injected with the inhibitory DREADD virus in ACC, we infused CNO solution in M2 and examined its effect on task performance  Fig. . We obta29, which measures the degree of overlap between two response distributions31. For each M2 single-unit the preferred and non-preferred rule conditions were compared, given two distributions of neuronal activity (see Methods). An ROC curve was then generated by taking the observed firing rate of a neuron and then the area under the ROC curve was calculated. A value of 0.5 indicates that the two distributions were completely overlapped, and thus the neuron is not selective to the rules. A value of 1.0, on the other hand, indicates that the two distributions were completely separated and so the neuron is very selective. Time course of rule selectivity of the representative M2 single-unit showed an increase in rule selectivity in the pre-choice period in the ipsilateral choice condition but not in the contralateral condition  analysision Fig. . We repeion Fig. . Such anion Fig. . We spliTo test if a perturbation of ACC inputs to M2 neurons during pre-choice period could affect an animal’s sequential choice performance in trials following rule switches, we optogenetically excited ACC terminals in M2 specifically during pre-choice period. Indeed, such perturbation affected 2nd choice performance  . Therefore, animals were expected to adjust their 2nd choice responses based on the outcome feedback information. To address whether and how ACC circuits provide M2 neurons with outcome feedback information upon rule switches, we examined M2 activity during the outcome feedback period  (see Methods). We first tested if an M2 neuron showed a greater or smaller mean firing rate during the outcome feedback period than that during the baseline period and conducted several analyses using the database of such neurons. A chemogenetic silencing of ACC neurons decreased the proportions of neurons that were activated either during the positive outcome feedback period  or during the negative outcome feedback period   and 2nd/3rd epochs Fig. . We founTo test if suppression of ACC circuits during the outcome period could affect animals’ sequential choice performance as was suggested by the chemogenetic modulation of activities of outcome-related neurons in M2 Fig. , we optoWe delivered the light for 4 s immediately following the animals’ incorrect 2nd choices Fig.  or the aWe repeated similar experiments with 4 s light applied immediately after the animals’ correct 2nd choices Fig. . Light dFinally, we tested if an optogenetic excitation of ACC inputs to M2 neurons during the outcome period could affect an animal’s sequential choice performance , suggesting that the role of ACC-M2 projection is not limited to performing an extra step but rather is linked to flexibly adjusting the performance of an additional step in an action sequence upon rule switches.What is the functional role of ACC-M2 pathway in an animal’s sequential decision making and rule switching? Do ACC inputs just increase the excitability/activity of M2 neurons in a way that enables them to function normally or do they actually convey task-related information? If the latter is the case, what information do ACC-M2 projections convey? A previous rat study showed that inhibiting M2 neurons caused decrease of left/right binary and single-step choice performance, suggesting that modulating M2 activity per se affects single-step choice performance5. Given that a chemogenetic silencing of ACC-affected rule selectivity of M2 neurons during pre-choice period in trials that immediately followed rule switches  might be necessary to extinguish such non-rewarded movements upon rule switches.We observed an effect of chemogenetic silencing of ACC circuits in updating rules from 1 step to 2 steps but not in the opposite direction  as measured by non-rewarded second actions. This asymmetric effect of chemogenetic silencing may indicate that ACC circuits are recruited in rule switches that demands an increment but not a decrement of response actions. Given that making a 2 steps choice would demand a greater cost on the part of the animals compared to making a single-step response, the asymmetric effect  raises a possibility that ACC circuits are recruited specifically in task settings that demand an increased effort/cost for making choice responsesChemogenetic silencing of ACC neurons increased 2nd choice omission errors in the first epoch of 2 steps condition and, on average, this effect emerged toward the end of the first epoch Figs.  and 4f. 39. It is an open question whether and how such processes could be recruited in distinct ways for flexibly updating sequential choice responses upon changes of rules in the environment.A chemogenetic silencing of ACC neurons or their terminals in M2 decreased 2nd choice performance by around 20% on average but did not completely abolish animals’ ability to update their choice responses upon rule switches Figs. . It migh40. In this study, we minimized the within-trial working memory load while requiring animals to hold rule representations  across trials in specific rule blocks. The cognitive loads of different time scales  might affect how an update of behavioral response would rely on DLPFC vs. ACC circuits32. Alternatively, the reason might be due to the difference in the characters of the task rule that we focused on in this study: in previous rodent studies that showed the involvement of prelimbic/infralimbic cortices in rule switching, these were related to choices in space  while, in this study, we tested animals’ ability of updating internal representations of rules related to response sequences 42. It is an open question what distinct roles ACC and prelimbic/infralimbic circuits play in flexibly adjusting responses upon rule switches.Interestingly, we found no effect of chemogenetic silencing of prelimbic/infralimbic cortex  while previous studies have shown that DLPFC also plays a critical role in updating rule representations in the brain43. Given these considerations, while we found no behavioral effect in ChR2 excitation experiments during the outcome period, a ChR2 excitation with a higher light pulse frequency can possibly affect animals’ task performance. Including this issue, precise circuit mechanisms regarding how ACC-M2 circuits work in rule switching remain unexplored and it is an open question whether and how ACC neurons could modulate downstream M2 neurons possibly in cooperation with local inhibitory neuronal networks.An optogenetic excitation of ACC-M2 projections during the outcome period did not affect animals’ choice performance while an optogenetic suppression of ACC neurons during the same task period could affect it Fig. . This diWe confirm that our research complies with U.S. National Institutes of Health guidelines and the Massachusetts Institute of Technology Department of Comparative Medicine and Committee of Animal Care .Wild-type male Long-Evans rats (>300 g) were used (Charles River). Rats were pair-housed during initial behavioral training and then single-housed after being injected with viruses or being implanted with electrodes, fiberoptic implants, or cannulae. Rats were kept on a reverse 12 h light/dark cycle, and trained and tested in their dark cycle. Food was available ad libitum, and rats had scheduled access to water for motivating them to work for water reward while monitoring their body weight to ensure they were over 85% of their initial weight.Behavior took place in a custom-made chamber  inside a sound-attenuating cubicle (MED Associates). The cubicle was electromagnetically shielded by a copper mesh sheet or nickel/silver fabric in electrophysiology recording experiments. The behavioral setup consisted of a stainless-steel lever at the center (MED Associates) and two ports equipped with infrared photodiodes on the left and right sides of the lever, arranged side-by-side with a center-to-center distance of 65 mm on a stainless-steel wall. An interruption of the infrared beam signaled port entry. A sipper tube was installed on the front wall 25 mm above the center lever and was connected to a water supply that was controlled by a computer-controlled solenoid. In addition, there were two speakers mounted on the side walls (about 150 mm away from the center lever). Timing of presentations of sounds from the speakers  and delivery of water rewards were controlled using a multifunction digital input/output board  with custom programs written in C++  and LabVIEW  on a computer running a Windows 10 operating system. Behavioral events were timestamped with a precision of <1 ms.Rats self-initiated each trial with a push on the center lever to receive the tone cue stimulus. After a delay of 10 ms, a tone of either 8 or 12 kHz with a sound pressure level of 75 dB was presented in pseudorandom order in each session. The tone was kept on until an animal entered one of the side ports (left or right) as the 1st choice. Choices were rewarded with ~25 μl water if they poked the correct side port. An error feedback buzzer sound was delivered as a penalty if they poked the incorrect side port followed by an elongated period of ITI. A feedback buzzer sound accompanied by an elongated ITI was also delivered if animals made a 2nd choice commission error, that is, entered the opposite side port after making a correct 1st choice and before initiating the next trial (pushing the center lever after an ITI). This completed a trial and, after an ITI period , an LED turned on, signaling the animals to initiate the next trial by pushing the center lever.In the 2 steps condition, in addition to making the first-choice response to one of the side ports (depending on the tone cue), animals were required to make a 2nd response by entering the side port opposite the one that animals chose as the 1st response, instead of pushing the center lever. A correct entry to the side port as a 2nd response was rewarded with ~25 μl water. If animals pushed the center lever before making a correct 2nd response , an error feedback buzzer sound was delivered. In this way, switches from 1 step to 2 steps rules were directly signaled to the rats: when the rule was switched from 1 step to 2 steps conditions and if the rats completes the 1st response , rats would push the center lever to initiate the next trial, causing an error buzzer feedback as an explicit feedback signal.1 step and 2 steps conditions were switched in every 55 trials  in a block-wise manner. In some sessions, behavior experiments started from the 2 steps condition (1st block) and then switched to the 1 step condition (2nd block) and so forth , rats were first trained to either push the lever or enter a side port (left or right) to receive a water reward. Next, rats were trained to perform a contingency task in which a tone sound (4 kHz) was delivered when the animal pushed the center lever, and then the animal was required to poke either the left- or right-side port to receive a reward . Then rats were trained on auditory discrimination in which they were required to choose left- or right-side ports depending on the tone cue stimulus (either 8 or 12 kHz). Once they reached over 70% correct performance for two consecutive days, they were trained with the CAS task without a rule change between the 1 step and 2 steps conditions, requiring animals to make 2 step responses throughout the session. Once they reached over 70% correct performance for 2 consecutive days, they were trained on the CAS task with rule changes between the 1 step and 2 steps conditions once every 70–100 trials per block. The length of a block was gradually shortened to 55 trials to complete the task training phase. In many training sessions and in some testing sessions, we adjusted the proportion of two trial types  to abate animals’ choice bias to specific side ports. Such adjustments were typically limited to a ratio of less than 2.−1) and xylazine (8 mg kg−1), rats were placed in an isoflurane induction chamber for 5–10 min. Then rats were moved to a stereotactic frame and their nose was placed in a cone, which provided 1.5–2% continuous isoflurane flow. After verifying surgical levels of anesthesia with pinch tests and eye blink tests, rats were secured in non-rupture ear bars (Kopf Instruments). The concentration of isoflurane was maintained at 0.75–1.5% throughout the surgery. Slow release buprenorphine (1 mg kg−1) and Ringer’s solution were administered in the middle and at the end of the surgery, respectively. Rats remained on a heating pad until they made a full recovery from anesthesia after surgery. Supplemental nutrient gels were supplied to rats after they were returned to their home cages. Rats were monitored during their recovery from surgery for at least 4 days before restarting experiments.All surgeries were performed under isoflurane anesthesia (1.0–2.0%) using the standard stereotactic technique. Following an IP administration of a cocktail solution of ketamine . For optogenetic silencing of ACC neurons  ml−1 for AAV5-CamKIIa-hM4Di-mCherry, 1.9 × 1013 GC ml−1 for AAV5-CamKIIa-mCherry, 1.3 × 1013 GC ml−1 for AAV9-CamKIIa-eNpHR3.0-eYFP, and 1.1 × 1013 GC ml−1 for AAV9-hSyn-hChR2(H134R)-eYFP viruses. For viruses used in viral tracing experiments . The micropipette was slowly lowered to the target site and remained for 5 min before starting injections . After injections, the micropipette stayed for 10 min before it was withdrawn.Each animal underwent bilateral craniotomies using a 1/4 size drill bit. The virus solutions with a volume of 1 μl were injected using a mineral oil-filled glass micropipette joined by a microelectrode holder to a 10 µl Hamilton micro syringe. A micro syringe pump was used to control the speed of virus injections  and AAVrh8-synP-DIO-sTpEpB-WPRE-bGH with a mixed ration of 1:1 at M2 44. One to two weeks later, RVΔG-4mCherry (EnvA) (1 μl) was injected at the same coordinates45. Viral titers for the injected solution were 1.05 × 1013 GC ml−1 and 1.15 × 1012 GC ml−1 for pENN.AAV.CaMKII.0.4.Cre.SV40 and AAVrh8-synP-DIO-sTpEpB-WPRE-bGH, respectively, and 1.7 × 1010 GC ml−1 for RVΔG-4mCherry (EnvA). One to two weeks  after the injection of the rabies virus, the animal was perfused, brain extracted, sectioned, immunostained, and imaged.For exploring brain regions projecting to M2 in the rat, we used a genetically engineered rabies virus13 GC ml−1 for AAVretro-pmSyn1-EBFP-Cre and 4.7 × 1012 GC ml−1 for AAV5-hSyn-DIO-hM4Di-mCherry.To validate projections from ACC to M2, we injected AAVretro-pmSyn1-EBFP-Cre (Addgene#51507-AAVrg) at M2  and AAV5-hSyn-DIO-hM4Di-mCherry (Addgene#44362) at ACC . The viral titers were 1.1 × 10−1 in PBS), incubated for 30 min at room temperature, and then mounted on microscope slides. Antibodies used for staining were as follows: to stain for hM4Di-mCherry or mCherry alone, slices were incubated with primary rabbit anti-RFP  and visualized using anti-rabbit Alexa 555 or Alexa 568 (1:200). To stain for eNpHR3.0-eYFP and hChR2-eYFP, slices were incubated with primary chicken anti-GFP  and visualized using anti-rabbit Alexa 488 (1:200). Immuno-stained slices were imaged using an epifluorescence (Zeiss Imager.Z2) or a confocal microscope (Zeiss LSM700) with ×5 or ×10 objective lenses. Intensity of each fluorescence channel in imaging data was adjusted using ZEN software .Rats were deeply anesthetized using sodium pentobarbital and then transcardially perfused with saline and 4% paraformaldehyde (PFA). Brains were extracted and incubated in 4% PFA at room temperature overnight. Brains were transferred to PBS, and 50 μm coronal slices were prepared using a vibratome. For immunostaining, each slice was placed in PBS + 0.1% Triton X-100 (PBS-T), with 10% normal goat serum for 1 h and then incubated with primary antibody at 4 °C for 12 h. Slices then underwent three wash steps for 10 min each in PBS-T, followed by 2 h incubation with secondary antibody. After three more wash steps of 10 min each in PBS-T, slices were transferred to DAPI solution  was prepared by first dissolving CNO in dimethyl sulfoxide  followed by adding saline solution . CNO solutions were intraperitoneally injected 35–40 min before starting behavioral testing. In some sessions, we started behavior experiments 60 min after the IP injection of CNO solution to examine whether the duration from the CNO administration to the start time of behavioral testing could affect the animal’s performance. In saline control conditions, a vehicle solution  was intraperitoneally injected 35–40 min before behavioral sessions.Rats were bilaterally injected with AAV5-CaMKIIa-hM4Di-mCherry virus in ACC (area 24a’/24b’) Fig. 27,28. At−1) was bilaterally infused through a 33-gauge dual internal cannula (Plastics One) at a speed of 0.2 μl min−1. After waiting for 4 min after infusions, the infusion cannula was removed. Rats were kept in their home cage for 30 min until we started behavioral experiments.For locally infusing CNO solution or its control solution in M2 in rats infected with inhibitory DREADD virus in ACC, we implanted 26-gauge dual guide cannula (Plastics One) targeting bilateral M2   under light anesthesia, we resumed neural activity measurements. Continuous voltage signals were recorded in the hard disc for offline data analysis. To obtain multiunit spike timestamps in offline analysis, the continuous voltage signals were high-pass filtered (400 Hz) digitally, and multiunit spikes were detected by thresholding the continuous signals at 4 SDs above the baseline level46.For testing if an IP administration of CNO could suppress neural activity in ACC, we measured multiunit spiking activity in ACC in two rats infected with the inhibitory DREADD virus and implanted with silicon probes (Neuronexus)  at around this coordinate elicited contractions of the shoulders or limbs of the rats.The neural activity data were obtained from six rats in which Utah array electrodes with 4 × 8 matrix shanks  (Blackrock Microsystems) were implanted in M2 targeting an area covering the Bregma coordinate of ML 1.3 mm, AP + 2.0 mm in either left or right hemispheres . This location was chosen because it was the center of the distribution of stimulation sites that resulted in contralateral orienting movement and neurons related to orienting responses were recorded in previous studiesFor implant surgery, animals were anesthetized in the isoflurane chamber and placed in the stereotaxic frame. After applying eye ointment and washing the incision sites with betadine and ethanol, an incision was created over the scalp and connective tissue was removed. The skull was drilled for attaching bone screws and for implanting the array electrodes in M2. After installing bone screws, we performed a durotomy and slowly inserted the array electrodes at M2 using a manipulator and a mounting probe by applying a vacuum to steady the array. Dental cement was applied to secure the cable to the screws and the craniotomy was filled with a surgical silicone adhesive (Kwik-sil). The vacuum was turned off and the array was further secured using dental cement.All recordings were conducted after the rats were fully recovered (at least seven days after surgery). The ground was taken from one of the skull screws typically above the cerebellum. The reference channel was chosen from one of the skull screws or one of the recording channels in which no clear spiking activity was observed. Data were recorded using a unity gain amplifier forwarded, filtered , and stored in the Digital Lynx SX System  for offline data analysis using Cheetah 5 software .Dual fiberoptic cannulae  were implanted in the ACC in task-trained rats under isoflurane anesthesia . A yellow-green laser  with a fiberoptic patch cable  was installed in the behavioral chamber. A TTL pulse was delivered from the behavior system through an interface board  that determined the timing of laser light delivery for optogenetic intervention. Light was continuously delivered for 4 s during the outcome feedback period following the animals’ 2nd choices  were implanted in M2 cortex in task-trained rats under isoflurane anesthesia . A blue laser  with a fiberoptic patch cable  was installed in the behavioral chamber. A TTL pulse was delivered from the behavior system through an interface board  that determined the laser timing for optogenetic intervention. 473 nm light with 5 ms pulses was delivered at 10 or 20 Hz for 1 s during the pre-choice period  attached to an optical fiber  with the tip of the fiber extending beyond the tip of the electrode by 200–300 μm were used for simultaneous optical stimulation and extracellular recordings in anesthetized condition. The optrode was slowly lowered to cingulate cortex where the eNpHR3.0 virus or hChR2 virus was injected. The optical fiber was connected to a yellow-green (561 nm) laser (for eNpHR3.0 virus) or to a blue (473 nm) laser (for hChR2 virus) and was controlled by a beam shutter and a shutter controller. The power intensity of light emitted from the optrode was calibrated to 13–16 mW (for yellow-green laser) and 5–6 mW (for blue laser), respectively, as measured with an optical power/energy meter, which is consistent with the power intensity used in behavior experiments. Then, 561 nm light pulses were delivered at each depth of the optrode (pulses with 1 s duration at 5 Hz for eNpHR3.0 virus and with 5 ms duration at 10 or 20 Hz for hChR2 virus) while neuronal activity in ACC was collected for 10–20 sweeps. Continuous voltage data were monitored online using an oscilloscope and a sound speaker, fed into a preamplifier, transferred to an interface board, and saved in the hard disc. Continuous voltage traces were high-pass filtered with a Butterworth filter and then thresholded typically at around −50 to −80 μV using Offline Sorter software (Plexon). Spike rasters and peri-event time histograms (PETHs) of spiking activities of isolated single-units were plotted using Neuro Explorer software . Rats were sacrificed, and brains were collected and sectioned for histological confirmation of recorded sites.Spike sorting was conducted using Offline Sorter software (Plexon). Spike rastergram Fig.  and someAnimals’ task performance of the first choices in 1 step rule or 2 steps rule blocks  was quantified using a percent error rate in their 1st choice responses . Note that, in 1 step condition, animals were required to make a single choice response  and thus there is no correct 2nd choice although, for convenience, we referred to the correct choice responses in 1 step condition as correct 1st choice responses; indeed, in 1 step condition, committing a 2nd response  was not rewarded and, instead, an error feedback tone was provided. In group analysis, the percent 1st choice error rate for two trial types  were separately calculated and then averaged.In addition, in 2 steps rule blocks, after an animal makes a correct 1st choice response, animals can make two kinds of choice responses. (1) Choose a side port opposite to the one that the animal chose in its 1st response (correct 2nd choice response). (2) Alternatively, animals may omit poking to the side port and, instead, may push the center lever for initiating the next trial (incorrect 2nd choice response). Such a response was classified as an incorrect 2nd choice (or a 2nd choice omission error) and was used as a behavioral measure to quantify the animal’s ability to adapt to switches from 1 step to 2 steps conditions  was calculated as the difference in the timing of center lever entry and first-choice port entry (Choice 1). Similarly, response time for the 2nd choice response (RT2) was calculated as the difference in the timing of second choice port entry (Choice 2) and first-choice port entry (Choice 1).To compare animals’ task performance between the 1st block in the session and the following Rule Switch-blocks (either 1 step or 2 steps rule block), sessions were not included in the group analysis if they do not have Rule Switch-blocks . Also, to compare animals’ task performance across three epochs within a block (either 1 step or 2 steps rule -block), sessions were not included in the group analysis if they have less than 10 trials in the 3rd epoch of at least one Rule Switch-block. To compare the task performance in the first, middle, and third sections in a block, a block was subdivided into three epochs: trial no. 1–18 (1st), 19–36 (2nd), and 37–55 (3rd) for sessions with rule switches in every 55 trials. Similarly, in two sessions in which the rule was switched in every 40 trials, a block was subdivided into three epochs: trial no. 1–13 (1st), 14–26 (2nd), and 27–40 (3rd).In some sessions, behavior experiments started from the 2 steps condition (1st block) and then switched to the 1 step condition (2nd block) and so forth  while 5 rats were used for experiments locally infusing CNO solutions with a dose of 1 μg μl−1 (one rat was used for both IP injection experiment and cannula infusion experiment). Two animals (three sessions) were also tested with an IP injection of CNO with a dose of 40 mg kg−1 but the data were not included in the group analysis. Also, one rat was pilot-tested with a local infusion of CNO solutions with a dose of 0.4 and 4 μg μl−1 but these data were not included in the group analysis.We used a total of 31 rats for chemogenetics experiments; 15 rats were injected with the inhibitory DREADD virus (AAV5-CaMKIIa-hM4Di-mCherry) in ACC, 5 rats were injected with the mCherry control virus (AAV5-CaMKIIa-mCherry) in ACC, 6 and 5 rats were injected with the inhibitory DREADD virus in prelimbic/infralimbic cortex and ventral thalamic nuclei, respectively. Among the 15 rats that were injected with the inhibitory DREADD virus in ACC, 11 rats were used for experiments intraperitoneally administering CNO solutions  were removed from electrophysiology data analysis .48. We analyzed neural data collected in 43 sessions from 5 rats: 29 sessions with saline control , 14 sessions with an IP injection of CNO solution with a dose of 20 mg kg−1 . From these session data, a total of 900 single-units were isolated: 594 units and 306 units for saline and CNO conditions, respectively. All these units were included in the group analysis of mean firing rate . Only a unit with a refractory period (>2 ms) in the auto-correlogram was accepted as single-units−1 in the 1 s window immediately before the animals made the choice in either cue stimulus condition in 1 step condition or before animal’s making the 1st choice in either cue stimulus condition in 2 steps condition .A single-unit was included in the group analysis of rule selectivity Fig.  if it sh31. An ROC analysis measures the degree of overlap between two response distributions. For each M2 single-unit the preferred and non-preferred rule conditions were compared, given two distributions, P and N respectively, of neuronal activity. For example, for some neurons . We then calculated the area under ROC curve (auROC) using these firing rates. To examine how rule selectivity changes throughout the Rule Switch-blocks, a block was subdivided into three epochs: trial nos. 1–18 (1st epoch), 19–36 (2nd epoch), and 37–55 (3rd epoch) for sessions with rule switches in every 55 trials. Similarly, in two sessions in which the rule was switched in every 40 trials, a block was subdivided into three epochs: trial nos. 1–13 (1st epoch), 14–26 (2nd epoch), and 27–40 (3rd epoch).Then ROC analysis was conducted for each epoch separately. For example, to calculate rule selectivity in the 1st epoch of a block following rule switches from 1→2 steps conditions, ROC curves were calculated using distributions of mean firing rates during the pre-choice period of trials in the 2nd and 3rd epochs in the preceding 1 step rule block and of mean firing rates during the pre-choice period of trials in the 1st epoch of subsequent 2 steps rule block. Similarly, rule selectivity in the 2nd (or 3rd) epoch of a block following rule switches from 1→2 steps conditions were calculated using distributions of mean firing rates during the pre-choice period of trials in the 2nd and 3rd epochs in the preceding 1 step rule block and of mean firing rates during pre-choice period of trials in the 2nd (or 3rd) epoch of subsequent 2 steps rule block. Similarly, rule selectivity in each of three epochs of a block following rule switches from 2→1 steps conditions was calculated using distributions of mean firing rates during the pre-choice period of trials in the 2nd and 3rd epochs in the preceding 2 steps rule block and of mean firing rates during the pre-choice period of trials in each of three epochs of subsequent 1 step rule block.30. To test the statistical significance of rule selectivity, we used a bootstrap analysis and repeated the ROC analysis 200 times to obtain 95% percentile threshold in which we assigned the rule condition  at random to each trial and calculated the auROC. In group analysis, trial conditions were grouped in ipsilateral and contralateral -conditions depending on the hemisphere implanted with the electrode array in each animal: in ipsilateral trial conditions, animals were required to first choose the ipsilateral side port and then the contralateral side port as its 2nd choice  while, in “contralateral” trial conditions, they were required to select the contralateral side port as its 1st choice and then to the ipsilateral side port as its 2nd choice   all the single-units included in the single-unit database were sorted in descending order according to their mean firing rates. (2) The firing rates of single-units measured in the same percentiles were paired between CNO and saline conditions. (3) Then, the spikes of single-units in saline condition were randomly removed so that the mean firing rates across trials were matched between the two single-units in the same percentiles of the sorted lists of neurons measured in CNO and saline conditions. (4) Once the firing rates were matched between CNO and saline conditions, an auROC was estimated in the same manner as in Fig. t-test, one-sided, P = 0.025). Positive outcome-activated neurons were defined as single-units that showed significantly greater firing rates in the positive outcome feedback period  than those in the baseline period. Similarly, positive outcome-suppressed neurons were defined as single-units that showed significantly smaller firing rates in the positive outcome feedback period  than those in the baseline period. Negative outcome-activated neurons and negative outcome-suppressed neurons were defined in similar manners except that, for these neurons, the outcome feedback period was a 3 s period following an error feedback tone instead of a reward delivery (negative outcome feedback period). In Fig. All the isolated M2 single-units were included in the group analysis . They were classified according to their activity in the outcome feedback period. Each single-unit was tested if the mean firing rates in the outcome feedback period were significantly greater or smaller than those in the baseline period   and R (R Studio) were used for data analysis. No statistical analysis was conducted to pre-determine the sample sizes of each experiment. Statistics were run two-sided except mentioned otherwise. In testing the statistical significance of the effect of CNO dose or epoch in Rule Switch-blocks or their interaction in task performance Figs.  and 4d–gFurther information on research design is available in the Supplementary InformationPeer Review FileDescription to Additional Supplementary InformationReporting Summary


Host-induced gene silencing (HIGS) refers to the silencing of genes in pathogens and pests by expressing homologous double-stranded RNAs (dsRNA) or artificial microRNAs (amiRNAs) in the host plant. The discovery of such trans-kingdom RNA silencing has enabled the development of RNA interference-based approaches for controlling diverse crop pathogens and pests. Although HIGS is a promising strategy, the mechanisms by which these regulatory RNAs translocate from plants to pathogens, and how they induce gene silencing in pathogens, are poorly understood. This lack of understanding has led to large variability in the efficacy of various HIGS treatments. This variability is likely due to multiple factors, such as the ability of the target pathogen or pest to take up and/or process RNA from the host, the specific genes and target sequences selected in the pathogen or pest for silencing, and where, when, and how the dsRNAs or amiRNAs are produced and translocated. In this review, we summarize what is currently known about the molecular mechanisms underlying HIGS, identify key unanswered questions, and explore strategies for improving the efficacy and reproducibility of HIGS treatments in the control of crop diseases. A review of what is known and unknown about the molecular mechanisms underlying the silencing of pathogen and pest genes via the expression of complementary RNAs in the host plant. CHALCONE SYNTHASE1), but instead silenced this gene through a posttranscriptional mechanism  mutants showed that the corresponding genes encoded core proteins/enzymes that are now known to mediate RNAi across eukaryotes: RNA-dependent RNA polymerase  was first described in plants over 30 years ago, when plant scientists attempted to overexpress a gene involved in purple pigment production in petunia  that associate with AGO proteins and then base-pair with complementary mRNAs. The AGO proteins can then slice the mRNA, inhibit translation of the mRNA, or recruit protein complexes that mediate RNA-directed DNA methylation of complementary DNA  is associated with the DNA methylation of the target gene, which inhibits the binding of RNA polymerase II Pol II; . Post-TGCaenorhabditis elegans (Nicotiana benthamiana plants expressing a gene encoding green fluorescent protein (GFP) that had been silenced. RNA gel-blot analyses of this total RNA showed that it included siRNAs complementary to both the sense and anti-sense strands of GFP. Injection of this total RNA into transgenic C. elegans resulted in silencing of GFP, showing that the silencing activity produced by the plant was functional in the nematode. Notably, size fractionation of the RNA showed that the most active fraction was 80-90 nt in length, which suggested that the silencing activity was conferred by longer dsRNAs rather than mature siRNAs.The first evidence that silencing-related RNAs produced in plants might be able to silence genes in another organism came from work done with the free-living nematode  elegans . In thatHeterodera glycines) in a buffered solution containing dsRNA, which was found to be taken up by an oral route  stage soybean cyst nematodes  of a nematode gene . The autnt crops . Numerount crops , oomycetnt crops , and insnt crops . For exaAlthough HIGS represents a promising approach for limiting crop losses caused by pathogens and pests, many questions remain, including how silencing RNAs are secreted from plant cells, and how cells in the pathogen or pest take up these RNAs. It is also not clear which proteins from the plant and pathogen are required for successful HIGS. In this review, we focus on the mechanisms underlying HIGS. We also discuss recent findings highlighting how plants use endogenous RNAi to overcome pathogen infection, how plant cells recognize long dsRNA and produce small interfering RNAs (siRNAs), and how we can improve the efficacy of siRNA production using various dsRNA constructs. We describe possible mechanisms of RNA delivery, including whether extracellular vesicles (EVs) play a role in this process. Lastly, we suggest future directions aimed at addressing the major unanswered questions about how HIGS functions and how it can be improved.The molecular mechanisms underlying HIGS may differ between insects and nematodes versus fungi and oomycetes (filamentous pathogens). For the herbivorous insects, it appears that long dsRNAs (including hpRNAs) are taken up directly from the host and then processed using the RNAi machinery within the insect or nematode to induce gene silencing. For filamentous pathogens, the available evidence suggests that siRNAs and microRNAs (miRNAs) produced within the host plant are taken up by the pathogen, which then induce gene silencing, although in most systems, translocation of long dsRNA has not been ruled out. Here we discuss key experiments addressing these issues within each pest and pathogen group.Leptinotarsa decemlineata) and cotton bollworm (Helicoverpa armigera), was much higher when dsRNA was expressed inside chloroplasts, indicating that long dsRNA is the key translocated RNA, rather than siRNAs. Such a plastid-based system is unlikely to work against phloem-feeding insects, however, as these insects are unlikely to ingest whole chloroplasts.In herbivorous insects, the translocation of dsRNA appears to be more effective at inducing gene silencing than the translocation of siRNAs. This was demonstrated by comparing the HIGS efficacy of dsRNA expression inside chloroplasts versus the nucleus. Chloroplast-expressed dsRNAs are protected against host cell DCL enzymes and thus can accumulate to much higher levels than dsRNAs expressed from nucleus-encoded genes, which are rapidly processed into siRNAs . ImportaH. armigera by expressing an artificial miRNA (amiRNA) construct in N. benthamiana that used the backbone of an insect miRNA precursor gene. This modified amiRNA precursor remained largely unprocessed in N. benthamiana due to the lack of recognition by plant DCLs. However, feeding on transgenic leaves expressing this construct led to efficient silencing of H. armigera genes and high levels of mortality and growth abnormalities. These results indicate that the unmodified precursor of the amiRNA was taken up by insect cells and processed by the insect’s RNAi machinery.The conclusion that insect’s RNAi machinery contributes to efficient HIGS in insects is further supported by a recent study from Sitobion avenae) were switched back to feeding on nontransgenic plants . Consistent with this finding, silencing of the gene encoding the clathrin heavy chain in Colorado potato beetles reduced target gene silencing, as did pharmacological inhibition of endocytosis (Systemic Interference Defective 1 (sid-1)-like A and C (SilA and SilC)) also reduced target gene silencing, suggesting that dsRNA may also be taken up via a channel-based mechanism. The sid-1 gene was first identified in the nematode Caenorhabditis elegans when screening for mutants lacking the systemic RNAi phenotype  and filamentous pathogens. Optimizing the expression of such amiRNAs is thus one avenue toward improving the efficacy of HIGS. Standard amiRNA constructs are based on endogenous primary miRNA (pri-miRNA) genes, which encode RNAs that form complex hairpin structures that are processed by DCL1 in plants to produce 21- or 22-nt mature miRNAs. amiRNAs are generated by replacing just the helical region of the pri-miRNA corresponding to the mature miRNA. Optimizing amiRNA production thus involves optimizing the expression and processing of the pri-amiRNA. For example, the loop region in pri-miRNAs plays an important role in controlling the maturation of pri-miRNAs and can directly contribute to the repression of target mRNAs, at least in human cells . In addiNicotiana tabacum) plants under the control of the CaMV 35S promoter, showed similar efficacies in reducing the fecundity of the aphid Myzus persicae  targeting two different genes, the phytoene desaturase gene and the ribosomal protein L10 (RPL10) gene. RNA-seq analysis of total transcripts in N. benthamiana following infection with VIGS constructs designed to either maximize or minimize off-target silencing confirmed that the tool was effective at minimizing off-target effects while maximizing target gene silencing  of a target gene and spread to the coding regions . As a clt 3′-UTR . As anotIn addition to selecting the appropriate pathogen gene and target site sequence within that gene, HIGS efficacy is likely dependent on obtaining high levels of hpRNA and/or siRNAs in the tissue/cells colonized by the pathogen or pest. Most HIGS experiments have employed strong constitutive promoters to accomplish this goal, such as the 35S promoter of cauliflower mosaic virus, which is transcribed by RNA Pol II. An RNA Pol III promoter could also be used to drive high expression of hpRNAs, but such promoters have proven to be less effective in HIGS . This isF. graminearum in both barley and Arabidopsis . Most parasitic plants establish vascular connections with their host plants via structures called haustoria. SUC2 in Arabidopsis to drive expression of a hpRNA targeting the STM gene of dodder, which is required for haustoria formation. Phloem-specific expression of this hpRNA was highly effective at reducing dodder growth on tobacco. The use of a tissue-specific promoter in this example localizes expression of the hpRNA to areas of likely infection while limiting expression in other parts of the plant, which should reduce potential off-target effects on host genes. Given that HIGS appears to act very early during the fungal infection process, prior to the formation of haustoria  contains 10 AGO genes (Brachypodium genome (a monocot) contains 16  also preferentially binds 24-nt RNAs but appears to function by inhibiting the translation of targeted mRNAs, as it associates with polysomes in the cytoplasm  or through TGS (e.g. DNA methylation).Mucor circinelloides, two classes of 21 and ∼25-nt siRNAs are produced from the expression of extra-chromosomal DNA. These two classes of siRNAs cause PTGS of target genes in M. circinelloides, indicating that this fungus possesses all the machinery required to carry out RNAi  leaves rather than the host AGO1 protein. Among the co-precipitated sRNAs was a miRNA-like RNA named miR8788-3p. This sRNA was predicted to target the potato gene StABH1, which encodes an alpha/beta hydrolase of unknown function. Evidence that miR8788-3p is translocated is indirect: StABH1 transcript levels decreased during infection by P. infestans, but not when miR8788 levels were reduced in P. infestans using a target site mimic. Furthermore, these knockdown strains displayed reduced virulence on potato. It remains unclear, however, whether miR8788-3p is translocated as a complex with P. infestans AGO1 or if it also associates with the host’s AGO1.A second example of an oomycete sRNA being translocated to host cells comes from work on nfestans . The autF.  graminearum infections on barley  and restrictions on releasing GMOs into the environment have encouraged plant scientists to develop new approaches for delivering RNAs into pathogens. In a pioneering study, n barley . In thisDICER-LIKE 1 gene, which strongly indicates that the fungus was taking up long dsRNA from the plant and that this dsRNA was then triggering the RNAi machinery within the fungus  demonstrated that the delivery of siRNA bound to gold nanoparticles (AuNSs) does not require the internalization of gold nanoparticles. The infiltration of 10-nm spherical AuNSs loaded with siRNAs into the leaves of S.  avenae) Shp gene  when applying dsRNA to detached barley leaves. However, MIF1, MIF2, and MIF3) in the same aphid species, even though nymphs fed an artificial diet containing this dsRNA showed efficient silencing. Furthermore, the authors were unable to detect transport of fluorescently labeled dsRNA into phloem cells, contradicting the findings of Although SIGS appears to be a promising strategy, questions remain about its underlying mode of action, overall efficacy, and reproducibility . For insVerticillium dahliae, miR166 and miR159. These miRNAs are taken up by the fungus, resulting in the silencing of two endogenous genes required for virulence. Verticillium  dahliae strains expressing versions of these genes in which the miRNA target sites were modified to no longer base-pair with the miRNAs exhibited enhanced virulence, demonstrating that these miRNAs contribute directly to plant immunity by targeting fungal genes. Although an exciting finding, this work raises questions as to why mutations in these target sites in the fungus have not been selected over the course of evolution given that miR166 and miR159 both function in plant development and are highly conserved across angiosperms  increased susceptibility to B. cinerea, suggesting that siRNAs play a direct role in immunity. In support of this conclusion, predicted target genes of these siRNAs in B. cinerea were downregulated during infection, but not in B. cinerea collected from infected rdr6 mutant plants , the RNAs in these pellets were found to be located outside EVs  and identified several species of RNA, including siRNAs, microRNAs, spliceosomal RNAs, and tRNA fragments. The latter were shown to interfere with translation. Similar results were reported by In addition to the apoplast, another likely location for RNA transfer from plants to pathogens and pests is the phloem. Long-distance RNA translocation through the phloem was confirmed by assessing sRNA/mRNA movement across graft junctions . SimilarAlthough diverse RNA species have been detected in both the phloem  and apopVitis vinifera) and introduced through the base of cut petioles into N. benthamiana , which have been shown to target viral RNAs . Some ofSome fungal pathogens also secrete ribonuclease-like effectors, presumably to target plant-derived RNAs .Proteomic analyses of phloem sap, xylem sap, and AWFs have shown that these three fluids share overlapping sets of proteins, including multiple RNA-binding proteins . For exaCmPSRP1 as an RNA-binding protein that binds to siRNAs in the phloem of pumpkin and facilitates intercellular movement. Interestingly, a recent study on siRNA movement in Arabidopsis revealed that siRNAs move long distance and cell-to-cell as double-stranded rather than single-stranded siRNAs and are not bound to AGO proteins (Similarly, several RNA-binding proteins in phloem sap have been reported to play critical roles in long-distance RNA translocation . Yoo et proteins , suggestC.  elegans, suggesting that one of its functions is to enhance the transmission of RNAs from worker bees to larvae (The above examples mostly focus on the intra-organismal movement of RNAs. However, RNA-binding proteins likely play important roles in inter-organismal movement as well. An interesting example of this has been described in honeybee colonies, where diverse protein-coding and noncoding RNAs are secreted into royal jelly by worker bees and fed to developing larvae in the hive (Additional investigations are required to understand the mechanisms of RNA uptake by both plants and pathogens, as these insights should lead to improvements in the efficacy of both HIGS and SIGS. Basic questions remain to be answered, such as how specific RNAs and RNA-binding proteins are selected for export, and how they are exported. The roles of pathogen RNAi machineries in HIGS and SIGS also need to be determined. Does SIGS require specific AGO proteins from plants or pathogens? What is the mechanism of RNA uptake and translocation by pathogens? Once RNA is taken up by a pathogen, how is it released into the cytoplasm to engage with pathogen RNAs? Addressing this long list of questions will require a robust HIGS system in which candidate genes can be disrupted in both the host and pathogen, allowing individual genes and gene families to be assessed for their contributions to HIGS and SIGS.


Leishmania parasites. The aims of this study were to investigate the effect of NAIs therapy on Leishmania major (L. major) the causative agent of CL in in vitro and in a murine model.Cutaneous leishmaniasis (CL) is a Neglected Tropical Disease (NTD) that causes high morbidity in the tropics and sub-tropics. Despite the remarkable advancements in the treatment of CL, the available therapeutics are far from ideal and also cause serious adverse side effects. Negative air ions (NAIs) generators are widely available for domestic and industrial uses. Several studies have reported on positive effects of NAIs therapy on human health as a non-pharmaceutical treatment for respiratory disease, allergy, or stress-related health conditions, including infectious diseases. To our knowledge, no studies have examined the effectiveness of the NAIs therapy against In vitro anti-leishmanial effects of NAIs therapy were measured by parasitological methods. NAIs therapy was assessed in vivo in L. major infected BALB/c mice by measuring the footpad (FP) lesion size and parasite load using metric caliper tool and qPCR, respectively. Immune responses in treated and non-treated mice were assessed by measuring the levels of IFN-γ, IL-4, NO and arginase activity. In vitro NAIs therapy significantly decreased the viability of Leishmania promastigotes and of amastigotes cultured in macrophages, but did not affect the host cells. NAIs therapy of L. major infected BALB/c mice resulted in reduced FP lesion size, diminished parasite burden, and importantly decreased induction of IL-4 and arginase activity in the presence of NAIs. In contrast IFN-γ and NO levels were significantly enhanced. NAIs therapy significantly diminished the progression of disease compared to the control group, but was less effective than amphotericin B treatment.in vitro and in Leishmania-infected mice, elicited a T-helper 1 (Th1) response and increased efficient cellular immunity, resulting in a diminished parasite load. Therefore, NAIs therapy can be considered as a useful and safe tool that can contribute to clearing L. major infections without inducing toxicity in host cells. The applications and mechanisms of NAIs therapy warrant further investigation especially in humans suffering from CL.Our study shows that NAIs treatment was effective  Leishmania (L) species, which are intracellular protozoan parasites  was directly mixed with 100 μl of culture supernatant of stimulated splenocytes. After a10 min incubation at room temperature, the colored (azo dye) complex absorbance was measured at an OD = 570 nm. The NO absorbance values of each sample were quantified in terms of standard curve of nitrate [Arginase activity were carried out in the FPs of those mice used for splenocyte culture preparation. The arginase activity test quantifies the conversion of L-arginine to L-ornithine, as described elsewhere , 30. NO  nitrate , 30, 31.Leishmania parasite load were calculated by applying Spearman correlation method. P values <0.05 were considered as statistically significant. The results are shown as the mean ± SD of three consecutive tests with identical results that were carried out in triplicate.All statistical analyses were performed using Prism 8.0 software . One-way ANOVA and Student’s t-test were used for performing the comparison between groups. The association between the cytokines (IFN- γ /IL-4) induction and differences in L. major promastigote forms after exposure to 106 NAI/cm3 was determined and expressed in %-age to the non-treated controls .The viability of controls . NAIs reL. major amastigote-infected macrophages (P<0.001), while no loss of viability was seen in non-infected J774A.1 macrophages (P<0.001).The effects of NAI exposure on the macrophage cell line J774A.1  and on Lrophages  were alsL. major promastigotes into the FP. Following infection CL lesion development and swelling were monitored weekly for about 6 weeks (P<0.001).To evaluate the effect of NAIs therapy on the CL lesion in experimentally infected BALB/c mice, animals were infected subcutaneously by inoculation of  6 weeks . NAIs th 6 weeks . Note thLeishmania parasites in the LNs and FP of infected BALB/c mice was assessed using quantitative real-time PCR (qPCR) (L. major parasite load was remarkably reduced and positively correlated to the duration of treatments (G1 to G4) compared to negative control groups G5 (no exposure). Groups G4 (16 h) and G6 (Amphotericin B as positive reference drug), had the lowest parasite loads in comparison with other groups (P<0.001). No differences were seen in the number of viable Leishmania parasites in the LN and FP of the infected BALB/c mice   and convR (qPCR) . Assessmice Figs  and 4.(P<0.001). Conversely, significantly lower levels of IL-4 were measured in culture supernatants of splenocytes of the amphotericin B group (G6) and all groups receiving NAI therapy compared to the non-treated control groups (G5 and G7). Increase of IFN- ɣ and decrease of IL-4 levels occurred in a time-dependent manner  and arginase activities. In mice treated with NAIs therapy during 6 and 16 h per day , arginase activity was significantly decreased, while no effects were evident in the 2 and 4 h per day treatment groups  and carbon dioxide (CO2) to generate distinct anions such as O3-, O2-and CO3- [2-and CO3- ions. Importantly, the generation of ozone, nitric oxide and nitrous oxide [Pseudomonas aeruginosa the main principal cause of NAIs-induced bacterial inactivation was exposure to ozone but in Mycobacterium parafortuitum electroporation ending from exposure to the electric field was postulated to be the major principal cause of cell death [Leishmania promastigotes as well as intracellular amastigotes.NAIs have been previously demonstrated to have a lethal effect on several other microorganisms  especialreatment . A studyod size. . Howeverant role . Severalant role , 19. Thend CO3- , 35. Theus oxide  is also ll death , 33, 36.L. major resulted in diminished CL lesion size and in reduced parasite load in the FP and LNs of treated versus non-treated mice. The treatment was most efficient when applied for 6 and 16 h per day . In addition, the continuous NAIDs treatment applied in this study was safe, as no detrimental effects were noted in any of the animals exposed to treatment. In vivo efficacy of any treatment is often associated with immunity, and we demonstrate an increased production of IFN-γ and iNOS in NAIs-treated mouse splenocytes, which indicates that a T-helper-1 (TH1)-biased immune response is induced. Conversely, production of IL-4 and arginase, two indicators of T-helper-2 (TH2) activation were decreased. Under normal circumstances, Leishmania parasites residing within infected murine macrophages inhibit the induction of TH1-inducing cytokines such as IFN-γ and cell mediated responses and NO production, but modulate the host cell to produce IL-4 and arginase, in order to promote parasite survival. One of the important points in the current study relates to the effects of NAIs therapy on parasite reduction. According to our data, NAIs therapy can reduce parasite replication and spread. Thus, the effects on the TH1 responses can be due to reduction in the parasite suppression, rather than TH1-inducing cytokines induction.NAIs therapy in BALB/c mice infected in the FP with Leishmania infection. They are potent producers of IFN-γ, and depletion of mouse NK) cells dramatically enhances susceptibility against infection of normally resistant mice [In vitro studies have shown that promastigotes are directly lysed by NK cells, but NK cell activity is also modulated by promastigotes. In human patients suffering from acute cutaneous leishmaniasis, impaired NK responses and reduced NK cell numbers have been observed, and studies in other animal models have shown that NK cells play a key role in the induction and direction of the immune response. Thus, the beneficial effects of NAI therapy observed herein could also be based on effects on NK cells [Other effects of NAIs therapy on immune cells have been described. For instance, NAIs therapy applied after methylcholan-threne carcinogenesis in mice increased the activity of natural killer cell (NK) cell activity and led to tumor regression and prolonged survival , 37. NAIant mice . In vitrNK cells .L. major infection in BALB/c mice and delayed CL lesion development, NAI therapy did not lead to the complete clearance and destruction of the parasite in the lesion site. According to studies in humans, ulcerative CL lesions are usually related to low Leishmania parasite burden and impaired parasite clearance. In BALB/c mice tissue damage due to CL lesions is extensive and Leishmania replication is unrestricted. Thus, high burden of parasites trigger Th2-biased immune responses and production of distinct cytokines such as IL-10 which results in necrosis and fibrosis due to chronic tissue remodeling [Although this study demonstrated that NAI therapy reduced modeling , 40. TheL. major promastigotes and intracellular amastigotes in vitro, and in vivo studies showed that NAIs therapy cures CL lesions and reduces the parasite burden in L. major infected mice. NAIs therapy also impacts on the immune response, by inducing a TH1-biased cellular response. These results underline the potential of NAI therapy as a promising alternative treatment for CL caused by L. major. NAIs treatment is non-invasive, inexpensive, safe and easy to use. However, more critical studies need to be carried out on the direct and indirect effects on NAIs on humans and other animal models, the mechanisms by which NAIs therapy increase cellular immune responses, and how NAIs directly act on these parasites. Ongoing research will provide insights into these aspects.In conclusion, this study demonstrates that NAIs exposure directly affect