TAC1 Research Articles

Mu-opioid receptors in tachykinin-1-positive cells mediate the respiratory and antinociceptive effects of the opioid fentanyl.

Opioid drugs are potent analgesics that carry the risk of respiratory side effects due to actions on μ-opioid receptors (MORs) in brainstem regions that control respiration. Substance P is encoded by the Tac1 gene and is expressed in neurons regulating breathing, nociception, and locomotion. Tac1-positive cells also express MORs in brainstem regions mediating opioid-induced respiratory depression. We determined the role of Tac1-positive cells in mediating the respiratory effects of opioid drugs. In situ hybridization was used to determine Oprm1 mRNA expression (gene encoding MORs) in Tac1-positive cells in regions regulating respiratory depression by opioid drugs. Conditional knockout mice lacking functional MORs in Tac1-positive cells were produced and the respiratory and locomotor responses to the opioid analgesic fentanyl were assessed using whole-body plethysmography. A tail immersion assay was used to assess the antinociceptive response to fentanyl. Oprm1 mRNA was highly expressed (>80%) in subpopulations of Tac1-positive cells in the preBötzinger Complex, nucleus tractus solitarius, and Kölliker-Fuse/lateral parabrachial region. Conditionally knocking out MORs in Tac1-positive cells abolished the effects of fentanyl on respiratory rate, relative tidal volume, and relative minute ventilation compared with control mice. Importantly, the antinociceptive response of fentanyl was eliminated in mice lacking functional MORs in Tac1-positive cells, whereas locomotor effects induced by fentanyl were preserved. Our findings suggest that Tac1-positive cells mediate the respiratory depressive and antinociceptive effects of the opioid fentanyl, providing important insights for the development of pain therapies with reduced risk of respiratory side effects.

Examination of the Effect of Dimethyl Trisulfide in Acute Stress Mouse Model with the Potential Involvement of the TRPA1 Ion Channel.

Polysulfides are endogenously produced in mammals and generally associated with protective functions. Our aim was to investigate the effect of dimethyl trisulfide (DMTS) in a mouse model of acute stress. DMTS activates transient receptor potential ankyrin 1 (TRPA1) channels and leads to neuropeptide release, potentially that of substance P (SP). We hypothesize that DMTS might inhibit the degrading enzymes of endocannabinoids, so this system was also investigated as another possible pathway for mediating the effects of DMTS. Trpa1 gene wild-type (WT) and knockout (KO) mice were used to confirm the role of the TRPA1 ion channel in mediating the effects of DMTS. C57BL/6J, NK1 gene KO, and Tac1 gene KO mice were used to evaluate the effect of DMTS on the release and expression of SP. Some C57BL/6J animals were treated with AM251, an inhibitor of the cannabinoid CB1 receptor, to elucidate the role of the endocannabinoid system in these processes. Open field test (OFT) and forced swim test (FST) were performed in each mouse strain. A tail suspension test (TST) was performed in Trpa1 WT and KO animals. C-FOS immunohistochemistry was carried out on Trpa1 WT and KO animals. The DMTS treatment increased the number of highly active periods and decreased immobility time in the FST in WT animals, but had no effect on the Trpa1 KO mice. The DMTS administration induced neuronal activation in the Trpa1 WT mice in the stress-related brain areas, such as the locus coeruleus, dorsal raphe nucleus, lateral septum, paraventricular nucleus of the thalamus, and paraventricular nucleus of the hypothalamus. DMTS may have a potential role in the regulation of stress-related processes, and the TRPA1 ion channel may also be involved in mediating the effects of DMTS. DMTS can be an ideal candidate for further study as a potential remedy for stress-related disorders.

Substance P Promotes Leukocyte Infiltration in the Liver and Lungs of Mice with Sepsis: A Key Role for Adhesion Molecules on Vascular Endothelial Cells.

Substance P (SP), encoded by the Tac1 gene, has been shown to promote leukocyte infiltration and organ impairment in mice with sepsis. Neurokinin-1 receptor (NK1R) is the major receptor that mediates the detrimental impact of SP on sepsis. This investigation studied whether SP affects the expression of adhesion molecules, including intercellular cell adhesion molecule-1 (ICAM1) and vascular cell adhesion molecule-1 (VCAM1) on vascular endothelial cells in the liver and lungs, contributing to leukocyte infiltration in these tissues of mice with sepsis. Sepsis was induced by caecal ligation and puncture (CLP) surgery in mice. The actions of SP were inhibited by deleting the Tac1 gene, blocking NK1R, or combining these two methods. The activity of myeloperoxidase and the concentrations of ICAM1 and VCAM1 in the liver and lungs, as well as the expression of ICAM1 and VCAM1 on vascular endothelial cells in these tissues, were measured. The activity of myeloperoxidase and the concentration of ICAM1 and VCAM1 in the liver and lungs, as well as the expression of ICAM1 and VCAM1 on vascular endothelial cells in these tissues, increased in mice with CLP surgery-induced sepsis. Suppressing the biosynthesis of SP and its interactions with NK1R attenuated CLP surgery-induced alterations in the liver and lungs of mice. Our findings indicate that SP upregulates the expression of ICAM1 and VCAM1 on vascular endothelial cells in the liver and lungs, thereby increasing leukocyte infiltration in these tissues of mice with CLP surgery-induced sepsis by activating NK1R.

Precise genome editing underlines the distinct contributions of mutations in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in Candida parapsilosis.

Candida parapsilosis has recently emerged as a major threat due to the worldwide emergence of fluconazole-resistant strains causing clonal outbreaks in hospitals and poses a therapeutic challenge due to the limited antifungal armamentarium. Here, we used precise genome editing using CRISPR-Cas9 to gain further insights into the contribution of mutations in ERG11, ERG3, MRR1, and TAC1 genes and the influence of allelic dosage to antifungal resistance in C. parapsilosis. Seven of the most common amino acid substitutions previously reported in fluconazole-resistant clinical isolates (including Y132F in ERG11) were engineered in two fluconazole-susceptible C. parapsilosis lineages (ATCC 22019 and STZ5). Each mutant was then challenged in vitro against a large array of antifungals, with a focus on azoles. Any possible change in virulence was also assessed in a Galleria mellonella model. We successfully generated a total of 19 different mutants, using CRISPR-Cas9. Except for R398I (ERG11), all remaining amino acid substitutions conferred reduced susceptibility to fluconazole. However, the impact on fluconazole in vitro susceptibility varied greatly according to the engineered mutation, the stronger impact being noted for G583R acting as a gain-of-function mutation in MRR1. Cross-resistance with newer azoles, non-medical azoles, but also non-azole antifungals such as flucytosine, was occasionally noted. Posaconazole and isavuconazole remained the most active in vitro. Except for G583R, no fitness cost was associated with the acquisition of fluconazole resistance. We highlight the distinct contributions of amino acid substitutions in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in C. parapsilosis.

Suppressing the Substance P-NK1R Signalling Protects Mice against Sepsis-Associated Acute Inflammatory Injury and Ferroptosis in the Liver and Lungs.

Substance P (SP), encoded by the TAC1/Tac1 gene, acts as a significant mediator in dysregulated systemic inflammatory response and associated organ injury in sepsis by activating the neurokinin-1 receptor (NK1R). This study investigated the impact of SP-NK1R signaling on ferroptosis in the liver and lungs of mice with sepsis. Sepsis was induced by caecal ligation puncture (CLP) surgery in mice. The SP-NK1R signaling was suppressed by Tac1 gene deletion, NK1R blockade, and a combination of these two approaches. The physiological conditions of mice were recorded. The profile of the SP-NK1R cascade, inflammatory response, ferroptosis, and tissue histology were investigated in the liver and lungs. Several manifestations of sepsis occurred in Tac1+/+ mice during the development of sepsis. Notably, hypothermia became significant four hours after the induction of sepsis. In the liver and lungs of mice subjected to CLP surgery, the concentrations of SP and NK1R were upregulated. Additionally, the concentrations of pro-inflammatory mediators, including cytokines (IL-1β, IL-6, and TNF-α) and chemokines (MCP-1 and MIP-2), were increased. Moreover, ferroptosis was elevated, as evidenced by increased concentrations of iron and MDA and reduced concentrations of GSH, Nrf2, and Gpx4. Suppressing the SP-NK1R cascade significantly mitigated CLP-surgery-induced alterations in mice. Importantly, these three approaches used to suppress SP-NK1R signaling showed similar effects on protecting mice against sepsis. In conclusion, increased SP-mediated acute inflammatory response and injury in the liver and lungs in mice with CLP-surgery-induced sepsis was associated with elevated ferroptosis. The detrimental effect of SP on sepsis was predominantly mediated by NK1R. Therefore, the suppression of increased SP-NK1R signaling and ferroptosis may be a promising adjuvant therapeutic candidate for sepsis and associated acute liver and lung injury.

Phylogenetic and functional analysis of tiller angle control homeologs in allotetraploid cotton.

Plants can adapt their growth to optimize light capture in competitive environments, with branch angle being a crucial factor influencing plant phenotype and physiology. Decreased branch angles in cereal crops have been shown to enhance productivity in high-density plantings. The Tiller Angle Control (TAC1) gene, known for regulating tiller inclination in rice and corn, has been found to control branch angle in eudicots. Manipulating TAC1 in field crops like cotton offers the potential for improving crop productivity. Using a homolog-based methodology, we examined the distribution of TAC1-related genes in cotton compared to other angiosperms. Furthermore, tissue-specific qPCR analysis unveiled distinct expression patterns of TAC1 genes in various cotton tissues. To silence highly expressed specific TAC1 homeologs in the stem, we applied CRISPR-Cas9 gene editing and Agrobacterium-mediated transformation, followed by genotyping and subsequent phenotypic validation of the mutants. Gene duplication events of TAC1 specific to the Gossypium lineage were identified, with 3 copies in diploid progenitors and 6 copies in allotetraploid cottons. Sequence analysis of the TAC1 homeologs in Gossypium hirsutum revealed divergence from other angiosperms with 1-2 copies, suggesting possible neo- or sub-functionalization for the duplicated copies. These TAC1 homeologs exhibited distinct gene expression patterns in various tissues over developmental time, with elevated expression of A11G109300 and D11G112200, specifically in flowers and stems, respectively. CRISPR-mediated loss of these TAC1 homeologous genes resulted in a reduction in branch angle and altered petiole angles, and a 5 to 10-fold reduction in TAC1 expression in the mutants, confirming their role in controlling branch and petiole angles. This research provides a promising strategy for genetically engineering branch and petiole angles in commercial cotton varieties, potentially leading to increased productivity.

The Effects of Tachykinin1 Gene Products on Prepubertal Dabry's Sturgeon (Acipenser dabrynus) Pituitary Hormone Secretion and Gene Expression.

As an ancient and endangered species unique to the Yangtze River in China, the wild population of the Dabry's sturgeon has become scarce. Due to the long time till the first sexual maturity of Dabry's sturgeon, the population of artificially bred Dabry's sturgeon recovered slowly. As a member of the tachykinin family, TAC1 has been reported to have a variety of functions in mammals such as pain control, smooth muscle contraction and reproductive cycle regulation, but the function of Tac1 in fish has been rarely reported. In this study, we synthesized two tac1 gene products, Substance P (SP) and neurokinin A (NKA), and further verified the effect of two tac1 gene products on the secretion of related hormones in the pituitary of Dabry's Sturgeon by intraperitoneal injection and co-culture of primary cells. Expression studies revealed that the newly cloned tac1 were mainly distributed in the hypothalamus and pituitary tissue of the brain. In prepubertal Dabry's sturgeon, this study showed that the two gonadotropins' mRNA levels in pituitary tissue can be significantly increased by SP and NKA through intraperitoneal injection, and the LH protein level in serum was also increased. Further study showed that both NKA and SP could promote the two gonadotropins' mRNA expression in pituitary cells of Dabry's sturgeon. In addition, we explored the optimal dose and time of SP and NKA on pituitary cells is 24 h and over 10 nM. These results, as a whole, suggested that tac1 gene products play an important role in gonadotropin release and gonadal development in prepubertal Dabry's sturgeon.

Genetic variants in the TAC1 transcriptional regulatory region affect on trainability and excitability levels in Belgian Malinois dogs.

Trainability in dogs is affected by learning aptitude and memory capacity. While this trait has a heritable basis in canines, the specific genetic loci responsible for it remain unknown. Our previous results suggested that the BDNF, CCK and TAC1 genes are associated with learning and memory in canines. Experimental validation is crucial to confirm the effects of these candidate genes on trainability. Understanding the genetic foundation of this trait would offer insight into the inheritance pattern of complex behavioural characteristics. This study aimed to assess the genetic variations within candidate genes and explore their potential associations with behavioural phenotypes in dogs. The behavioural characteristics of 123 male Belgian Malinois dogs were assessed using a customised questionnaire. Target regions of candidate genes were screened for genetic variation by single-strand conformational polymorphism (SSCP). Following that, SSCP banding patterns were sequenced, and putative transcription factor binding sites were predicted using bioinformatics tools. Quantitative association analysis between identified genetic variants and behavioural trait scores was performed using the general linear model (GLM). Sequencing the coding and flanking regions revealed three mutations (c.-89C>T, c.-162G>C and c.*33T>A) in the dog TAC1 gene. Bioinformatics analysis predicted two single nucleotide polymorphisms (SNPs) were located within the putative TAC1 promoter and could disrupt transcription factor binding sites. Statistical tests revealed that the c.-89C>T was significantly associated with excitability (p < 0.01), while the c.-162G>C was significantly associated with trainability level (p < 0.05). In summary, we identified two regulatory SNPs in the 5'-UTR promoter region of the TAC1 gene that are associated with excitability and trainability in Belgian Malinois dogs. These genetic variations have the potential to alter the binding sites of transcription factors NRF1 and OTX1, consequently influencing TAC1 expression and related behavioural characteristics. Our findings implicate TAC1 polymorphisms as candidates influencing breed-specific behavioural characteristics in canines. Further studies on diverse breeds of dogs are necessary to validate these SNPs' effects.

Molecular Identification of Fungal Species through Multiplex-qPCR to Determine Candidal Vulvovaginitis and Antifungal Susceptibility.

Vulvovaginal candidiasis (VVC) is a prevalent condition affecting women worldwide. This study aimed to develop a rapid qPCR assay for the accurate identification of VVC etiological agents and reduced azole susceptibility. One hundred and twenty nine vaginal samples from an outpatient clinic (Bilbao, Spain) were analyzed using culture-based methods and a multiplex qPCR targeting fungal species, which identified Candida albicans as the predominant species (94.2%). Antifungal susceptibility tests revealed reduced azole susceptibility in three (3.48%) isolates. Molecular analysis identified several mutations in genes associated with azole resistance as well as novel mutations in TAC1 and MRR1 genes. In conclusion, we developed a rapid multiplex qPCR assay that detects C. albicans in vulvovaginal specimens and reported new mutations in resistance-related genes that could contribute to azole resistance.

Gene expression changes of angiogenesis factors during basal skin cancer laser treatment

Background: Basal cell carcinoma is the most widespread malignant skin neoplasm. Angiogenesis is critical for the growth and metastasis of malignant tumors.&#x0D; Aims: To study the levels of representation of transcripts in the foci of basal cell skin cancer before and after the therapy of genes for angiogenesis proteins and their receptors.&#x0D; Materials and methods: The study included 31 patients with confirmed basal cell skin cancer who received treatment, using a pulsed dye laser and long-pulsed neodymium laser. The patients provided skin punch biopsies from BCC lesions and after therapy from the same localization. The gene expression was analyzed with real-time reverse transcription PCR using endogeneous control, and the gene expression ration changes during the therapy were calculated according to Livaks double delta formulae.&#x0D; Results:&#x0D; An increased expression of the matrix metalloproteinase MMP9 and the tachykinin precursor TAC1 genes were revealed in skin biopsy samples of the superficial and nodular form of basal cell skin cancer during laser pulsed therapy. The expression of tumor necrosis factor TNF, epidermal growth factor receptor EGFR, fibroblast growth factor FGF2 genes increases to a lesser extent.&#x0D; It was shown that the expression of the calcitonin-related polypeptide alpha CALCA gene in the skin of patients is at basal level, which makes it possible to exclude the influence of the neuropeptide on the basal cell skin cancer pathogenesis.&#x0D; Conclusions:&#x0D; Among the factors of neoangiogenesis potentially influencing the development of basal cell skin cancer, the leading role of expression of the MMP9 matrix metalloproteinase and TAC1 precursor protein of tachykinin has been shown. Simultaneous changes in the level of these proteins may be due to neuroimmune interactions in the epidermis, which is probably realized by mast cells as the microenvironment of the basal cell carcinoma.

TAC1 Gene Therapy in the Gut to Reduce Long-Term Memory Loss in Tg4-42 Alzheimer Diseased Mice: A Research Proposal

Alzheimer’s disease (AD) is a neurodegenerative disorder that mainly affects a large percentage of the older adult population. AD can cause many problems, most notably long-term memory (LTM) loss. Many studies have observed a decreased level of substance P (SP) in the hippocampus of individuals with AD. In this paper, we propose a novel strategy to limit AD-related decline of LTM using probiotics transformed with SP. These transformed bacteria will contain varying concentrations of SP and will be injected into three different segments of the proximal colon in AD mouse models. LTM will be measured through Morris Water Maze (MWM) and Barnes Maze (BM) tests over three months to examine improvements in spatial memory. It is anticipated that this experiment will demonstrate that increased concentrations of SP in the proximal colon will result in the greatest reduction in LTM loss in AD individuals. This experiment will establish a new therapeutic option for AD individuals to slow the progression of LTM loss.

Molecular dissection studies of TAC1, a transcription activator of Candida drug resistance genes of the human pathogenic fungus Candida albicans.

The up-regulation of ABC transporters Cdr1p and Cdr2p that efflux antifungal azole drugs are a leading cause of Multi-Drug Resistance (MDR) in the white fungus Candida albicans. C. albicans was reported to infect patients following the recent Covid-19 pandemic after they were given steroids for recovery. Previously, the TAC1 gene was identified as the transcriptional activator of Candida drug resistance genes (CDR1 and CDR2) and has no known human homologs. This makes it a good target for the development of novel antifungals. We, therefore, carried out the molecular dissection study of TAC1 to understand the functional regulation of the ABC transporter genes (CDR1 and CDR2) under its control. The N-terminal DNA Binding Domain (DBD) of Tac1p interacts with the Drug Responsive Element (DRE) present in the upstream promoter region of CDR1 and CDR2 genes of C. albicans. The interaction between DBD and DRE recruits Tac1p to the promoter of CDR genes. The C-terminal Acidic Activation Domain (AAD) of Tac1p interacts with the TATA box Binding Protein (TBP) and thus recruits TBP to the TATA box of CDR1 and CDR2 genes. Taking a cue from a previous study involving a TAC1 deletion strain that suggested that Tac1p acts as a xenobiotic receptor, in this study, we identified that the Middle Homology Region (MHR) of Tac1p acts as a probable xenobiotic binding domain (XBD) which plays an important role in Candida drug resistance. In addition, we studied the role of Tac1p in the regulation of some lipid profiling genes and stress response genes since they also contain the DRE consensus sequence and found that some of them can respond to xenobiotic stimuli.

Gene Expression Behavior of a Set of Genes in Platelet and Tissue Samples from Patients with Breast Cancer.

The tumor microenvironment (TME) is constituted by a great diversity of highly dynamic cell populations, each of which contributes ligands, receptors, soluble proteins, mRNAs, and miRNAs, in order to regulate cellular activities within the TME and even promote processes such as angiogenesis or metastasis. Intravasated platelets (PLT) undergo changes in the TME that convert them into tumor-educated platelets (TEP), which supports the development of cancer, angiogenesis, and metastasis through the degranulation and release of biomolecules. Several authors have reported that the deregulation of PF4, VEGF, PDGF, ANG-1, WASF3, LAPTM4B, TPM3, and TAC1 genes participates in breast cancer progression, angiogenesis, and metastasis. The present work aimed to analyze the expression levels of this set of genes in tumor tissues and platelets derived from breast cancer patients by reverse transcription-quantitative polymerase chain reaction (RTqPCR) assays, in order to determine if there was an expression correlation between these sources and to take advantage of the new information to be used in possible diagnosis by liquid biopsy. Data from these assays showed that platelets and breast cancer tumors present similar expression levels of a subset of these genes' mRNAs, depending on the molecular subtype, comorbidities, and metastasis presence.

ODP330 Long-term Effects of Prenatal Undernutrition on Hypothalamic KNDy Gene Expression in Female Rats

Abstract Accumulating evidence suggested that the nutritional environment during development periods induces metabolic programming, leading to metabolic disorders and detrimental influences on human reproductive health. The purpose of this study was to determine whether intrauterine malnutrition has a long-term adverse effect on a reproductive center, kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus (ARC) of the female offspring. In female offspring from 50% undernutrition (UN) dams, first virginal estrus tended to be advanced, and hypothalamic neurokinin B gene (Tac3) expression was increased in adolescents. Conversely, the UN caused the significant suppression of ARC kisspeptin (Kiss1) and Tac3 gene expression after 29 weeks of age. These results suggested that intrauterine undernutrition stress caused developmental programming in KNDy neurons, which changed in their gene expression depending on life stages. Furthermore, our data showed obesogenic effects of prenatal undernutrition in the female rats; UN offspring showed low birth weight, rapid catch-up growth at four weeks of age, visceral fat accumulation, and an increase in calorie intake and plasma triglyceride levels at adulthood. The current study demonstrated that intrauterine undernutrition affected hypothalamic developmental programming in female rats, which induced long-term effects on hypothalamic KNDy gene expression and could influence metabolism and reproductive function later in life. Presentation: No date and time listed

OR20-4 Expression of the Neuropeptide Substance P and the NK1 Receptor in Aldosterone-Producing Adenomas

Abstract Background Aldosterone-producing adenoma (APA) is a major cause of primary aldosteronism (PA) which is the most frequent form of secondary hypertension (1-2). The pathophysiology of PA is quite complex and still incompletely understood. Substance P (SP) encoded by the TAC1 gene belongs to a family of bioactive peptides named tachykinins. A recently published study indicated that SP, released by subcapsular nerve fibres, stimulates aldosterone production through activation of the neurokinin type 1 receptor (NK1R) in the human adrenal gland (3). The aim of our work was to investigate the presence of SP fibres and the NK1 receptor in a large series of APA in order to assess the potential role of this peptide in the pathophysiology of PA. Methods APA tissues were studied by molecular and histological approaches. Expression of SP and NK1 receptor was searched for in a series of 51 APA by RT-Q-PCR and immunohistochemistry. Results Quantitative RT-PCR data indicated that adenomas strongly express TAC1 mRNA. Immunohistochemistry showed the presence of SP-positive nerve fibres in APAs tissues. SP was also detected in a subpopulation of adenomatous cells. Adenomas strongly express mRNA encoding the NK1 receptor. The distribution of the NK1 receptor within APAs is similar to that of aldosterone synthase (CYP11B2), suggesting that SP may regulate aldosterone secretion by APAs. Conclusion These results suggest that SP and its NK1 receptor may be involved in the pathophysiology of aldosterone hypersecretion by APAs.

Expression pattern of drug-resistance genes ERG11 and TAC1 in Candida albicans Clinical isolates.

Candida albicans (C. albicans) is an opportunistic fungus and the most common cause of vulvovaginal candidiasis (VVC). In recent years, the use of antifungal drugs has led to the incidence of drug-resistant C. albicans strains. The purpose of this study is twofold: to determine the pattern of drug susceptibility and the relationship between demographic factors and the incidence of drug resistance among C. albicans isolates and to investigate the expression pattern of drug-resistance genes ERG11 and TAC1 in C. albicans isolates. This descriptive cross-sectional study was conducted on 50C. albicans isolates from women with VVC. Antifungal susceptibility of the isolates was tested by M27-A3/S4 broth micro dilution method following the Clinical and Laboratory Standards Institute (CLSI) guidelines. High susceptibility rates were recorded for itraconazole and voriconazole (68%), followed by ketoconazole (46%). Fluconazole had the lowest susceptibility to C. albicans with susceptibility of 36%. The change in ERG11 and TAC1 genes expression was determined by qPCR. The mean ∆Ct values of ERG11 and TAC1genes were significantly different between fluconazole-resistant and susceptible groups (p < 0.001). Interestingly, we found that 77% of fluconazole-susceptible isolates had significantly upregulated ERG11 gene (2.9-99.0 fold). In addition, the expression of TAC1 was upregulated in 44% of fluconazole-susceptible isolates (3.86-89.8 fold). Our finding revealed that incidence of drug resistance in C. albicans is not simply controlled by genes but is a multi-factorial phenomenon, where several factors and mechanisms are involved in the process of drug resistance.

Single nucleotide polymorphisms and chromosomal copy number variation may impact the Sporothrix brasiliensis antifungal susceptibility and sporotrichosis clinical outcomes

Feline-transmitted sporotrichosis has garnered attention due to the recent high incidence and the lack of efficient control in the epicenter of the epidemic, Rio de Janeiro, Brazil. Sporothrix brasiliensis is the major pathogen involved in feline-to-human sporotrichosis in Brazil and displays more virulent genotypes than the closely related species S. schenckii. Over the last two decades, several reports of antifungal-resistant strains have emerged. Sequencing and comparison analysis of the outbreak strains allowed us to observe that the azole non-wild-type S. brasiliensis strain CFP 1054 had significant chromosomal variations compared to wild-type strains. One of these variants includes a region of 231 Kb containing 75 duplicated genes, which were overrepresented for lipid and isoprenoid metabolism. We also identified an additional strain (CFP 1055) that was resistant to itraconazole and amphotericin B, which had a single nucleotide polymorphism in the tac1 gene. The patients infected with these two strains showed protracted clinical course and sequelae. Even though our sample size is modest, these results suggest the possibility of identifying specific point mutations and large chromosomal duplications potentially associated with antifungal resistance and clinical outcomes of sporotrichosis.

Tachykinins amplify the action of capsaicin on central histaminergic neurons

Substance P (SP), a product of the tachykinin 1 (Tac1) gene, is expressed in many hypothalamic neurons. Its wake-promoting potential could be mediated through histaminergic (HA) neurons of the tuberomamillary nucleus (TMN), where functional expression of neurokinin receptors (NKRs) waits to be characterized. As in the process of nociception in the peripheral nervous system (PNS) capsaicin-receptor (transient potential vanilloid 1: TRPV1) signalling is amplified by local release of histamine and SP, we tested the involvement of tachykinins in the capsaicin-induced long-lasting enhancement (LLEcaps) of HA neurons firing by investigating selective neurokinin receptor ligands in the hypothalamic mouse brain slice preparation using patch-clamp recordings in cell-attached mode combined with single-cell RT-PCR. We report that the majority of HA neurons respond to SP (EC50 3 nM), express the SP precursor tachykinin 1 (Tac1) gene and at least one of the neurokinin receptors. Responses to selective agonists of three known neurokinin receptors were sensitive to corresponding antagonists. LLEcaps was significantly impaired by the neurokinin receptor antagonists, indicating that in hypothalamus, as in the PNS, release of tachykinins downstream to TRPV1 activation is able to boost the release of histamine. The excitatory action of SP on histaminergic neurons adds another pathway to the noradrenergic and orexinergic ones to synergistically enhance cortical arousal. We show NK1R to play a prominent role on HA neurons and thus the control of wakefulness.

Knockout of tac3 genes in zebrafish shows no impairment of reproduction

Neurokinin B (NKB) plays a pivotal role in the regulation of reproduction in vertebrates. However, whether this neuropeptide is dispensable for reproduction in teleosts remains unknown. In order to reveal its authentic functions in fish, in this study, two tachykinin 3 (tac3) genes encoding Nkbs were functional mutated in zebrafish using the Transcription Activator-like Effector Nucleases (TALEN) technology. We established tac3a−/−, tac3b−/− and tac3a−/−;tac3b−/− mutant lines, and investigated their reproductive performance and ontogeny. According to our study, spermatogenesis and folliculogenesis were not impaired in tac3a−/−, tac3b−/− and tac3a−/−;tac3b−/− mutant lines, but changes in the expression of genes related to reproductive axis were observed after loss of Tac3, suggesting that possible compensatory response was activated to maintained the reproductive function in zebrafish. In summary, our results indicate that mutation of tac3 genes do not disrupt the reproduction in zebrafish unlike in mammals, revealing the plasticity of reproductive neuroendocrine system in the brain of zebrafish.

Different pituitary action of NK3Ra and NK3Rb in grass carp

In mammals, NK3R is the specific receptor for NKB, which played an important role in reproduction. Recently, two NK3R isoforms, namely NK3Ra and NK3Rb, have been identified in fish. However, little is known about the pituitary actions of the two NK3R isoforms in fish. In this study, both NK3Ra and NK3Rb were isolated from grass carp pituitary. Although their sequence similarity was only 61.6%, the two NK3R isoforms displayed similar ligand selectivity and binding affinity to TAC3 gene products (NKBa, NKBRPa and NKBRPb). In addition, both NK3Ra and NK3Rb displayed similar signaling pathways, including PKA, PKC, MAPK and Ca2+ cascades. Tissue distribution indicated that both NK3Ra and NK3Rb were highly detected in grass carp pituitary. Further study found that NK3Ra was mainly located in pituitary LHβ cells, while NK3Rb was only detected in pituitary SLα cells. Furthermore, NK3Ra and NK3Rb activation could induce LHβ and SLα promoter activity, respectively. These results suggested that the two NK3R isoforms displayed different pituitary actions in fish. Using grass carp pituitary cells as model, we found that PACAP could significantly reduce NK3Ra, but induce NK3Rb mRNA expression coupled with cAMP/PKA and PLC/PKC pathways. Interestingly, PACAP could also significantly inhibit LHβ, but stimulate SLα mRNA expression in grass carp pituitary cells. Furthermore, NK3R antagonist could not only inhibit LHβ mRNA expression, but also block PACAP-induced SLα mRNA expression in grass carp pituitary cells. These results suggested that NK3Ra and NK3Rb could mediate PACAP-reduced LHβ and -induced SLα mRNA expression in grass carp pituitary, respectively.