Post-COVID-19 condition in pregnant and postpartum women: a long-term follow-up, observational prospective study.

Background: Post-COVID-19 condition has recently been defined as new or persistent common COVID-19 symptoms occurring three months after disease onset. The pathology of the disease is unclear, but immune and vascular factors seem to play a significant role. The incidence, severity, and implications of the disease after COVID-19 infection in pregnancy have not been established. We aimed to study the incidence and main risk factors for post-COVID-19 condition in an obstetric population and their implications for maternal and perinatal morbimortality. Methods: This is a prospective observational cohort study undertaken including women during pregnancy or at admission for labour with acute COVID-19 infection from March 9th, 2020 to June 11th, 2022. The inclusion criteria were confirmed acute COVID-19 infection during the recruitment period, a lack of significant language barrier and consent for follow-up. Patients were clinically followed-up by telephone via semi structured questionnaires. The exclusion criteria were loss to follow-up, spontaneous miscarriage, and legal termination of pregnancy. Patients were classified into groups according to the severity of symptoms at onset. We included patients from the first six first waves of the pandemic according to national epidemiological data in Spain. We studied the incidence of post-COVID-19 condition and their main demographic, clinical and obstetric risk factors. Findings: A total of 409 pregnant women were recruited at acute diagnosis, and 286 were followed-up. The mean time to follow-up was 92 weeks (standard deviation ± 28 weeks; median 100 weeks (Interquartile range: 76; 112)). A total of 140 patients had at least one post-COVID-19 symptom at least three months after acute infection. Neurological (60%) and cutaneous (55%) manifestations were the most frequent findings. The following profiles were identified as presenting a higher risk of post-COVID-19 condition: migrant women born in countries with lower Human Development Index; multiparous women; women with COVID-19 during pregnancy, mainly during the first and third trimesters, and in the first and second waves of the pandemic; women who had a higher number of symptoms; women who had a higher incidence of moderate and severe symptoms; women who required hospitalisation due to COVID-19 complications; and women who were not vaccinated before disease onset. We did not find any significant difference in perinatal results, such as gestational week at delivery, birthweight, the need for neonatal care or 5-min Apgar score, and newborns benefited from a high rate of breastfeeding at discharge. Women who were infected during successive waves of the pandemic had a significant and constant decrease in the risk of post-COVID-19 condition comparing to estimated risk in the first wave (OR: 0.70; 95% CI: 0.62, 0.92). Symptoms tended to resolve over time heterogeneously. Symptoms of myalgia and arthralgia took longer to resolve (mean of 60 weeks and 54 weeks, respectively). In a small but significant proportion of patients, neurological and psycho-emotional symptoms tended to become chronic after 90 weeks. Interpretation: At least 34.2% of obstetric patients from our cohort with acute COVID-19 infection presented post-COVID-19 condition symptoms. Demographic and acute disease characteristics as well as specific pregnancy-related risk factors were identified. This is the first study to assess post-COVID-19 condition in pregnant women. Further analysis on the biological pathophysiology of post-COVID-19 is needed to explain the characteristics of the disease. Funding: This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project "PI21/01244" and co-funded by the European Union, as well as P2022/BMD-7321 (Comunidad de Madrid) and ProACapital, Halekulani S.L. and MJR.

Small Ruminant Lentivirus Infection in Sheep and Goats in North Portugal: Seroprevalence and Risk Factors.

Small ruminant lentiviruses (SRLVs) are transmitted among ovine and caprine species. This disease is a severe problem for small ruminant production, not only for animals' well-being but also for flocks' efficiency. The main aim of this research was to quantify the seroprevalence and associated risk factors for SRLV infection in the northern region of Portugal. Samples were collected from a total of 150 flocks, of which 129 (86.0%; 95% CI: 80.67%-91.33%) had at least one seropositive animal. Out of 2607 individual blood samples, 1074 (41.2%) were positive for SRLVs. Risk factors associated with SRLV infection were species (caprine), age (>2 years old), flock size (>100 animals), production system (intensive), food production system (milk), type of activity (professional), participation in livestock competitions (yes), replacement young ewe bought (yes), and natural feeding management (yes). This knowledge empowers the implementation of effective preventive measures. Overall, biosecurity measures should be promoted and implemented with the main aim of reducing viral transmission and reducing the prevalence of this disease. We recognise that government authorities should promote and audit voluntary control and eradication programs in small ruminant flocks in the region studied.

Epidemiological and molecular evaluation of BRAF, KRAS, NRAS genes and MSI in the development of colorectal cancer.

BACKGROUND AND OBJECTIVES: KRAS, NRAS, BRAF mutations and microsatellite instability (MSI) can be associated with Colorectal Cancer (CRC) development. MATERIAL AND METHODS: We evaluated 828 medical records of CRC patients from a school hospital from January/2016 to December/2020. Variables such as age, gender, ethnicity, literacy level, smoking, alcoholism, primary anatomical site, tumor staging, presence of BRAFV600E, KRAS, NRAS mutations and MSI , survival and metastasis were identified. The statistical analyses were performed (p < 0.05 was considered significant). RESULTS: There was a predominance of males (51.93%), whites (90.70%), low education (72.34%), smokers (73.79%), and non-alcoholics (79.10%). Rectum was the most affected site (42.14%), advanced tumor stage was most prevalent (62.07%), and metastasis occurred in (64.61%). Of the enrolled patients; 204 were investigated for BRAF mutation and detected in (2.94%); 216 for KRAS gene and detected in (26.08%); 210 for NRAS gene, and detected in (25.36%); 370 for MSI and detected in (44.68%). A significant association of CRC with NRAS mutation and alcohol habit (p = 0.043) was observed. The presence of MSI was associated with primary site proximal colon (p < 0.000), distal colon (p = 0.001) and rectum (p = 0.010). CONCLUSION: Patients with CRC are male, over 64 years old, white, with low education, smokers and non-alcoholics. The most affected primary site is rectum in advanced stage with metastasis. CRC is associated with NRAS mutation and alcohol habit, there is increased risk for primary site of proximal colon and MSI; decreased risk for distal colon and rectum in the presence of MSI.

A new order, Entrophosporales, and three new Entrophospora species in Glomeromycota.

As a result of phylogenomic, phylogenetic, and morphological analyses of members of the genus Claroideoglomus, four potential new glomoid spore-producing species and Entrophospora infrequens, a new order, Entrophosporales, with one family, Entrophosporaceae (=Claroideoglomeraceae), was erected in the phylum Glomeromycota. The phylogenomic analyses recovered the Entrophosporales as sister to a clade formed by Diversisporales and Glomeraceae. The strongly conserved entrophosporoid morph of E. infrequens, provided with a newly designated epitype, was shown to represent a group of cryptic species with the potential to produce different glomoid morphs. Of the four potential new species, three enriched the Entrophosporales as new Entrophospora species, E. argentinensis, E. glacialis, and E. furrazolae, which originated from Argentina, Sweden, Oman, and Poland. The fourth fungus appeared to be a glomoid morph of the E. infrequens epitype. The physical association of the E. infrequens entrophosporoid and glomoid morphs was reported and illustrated here for the first time. The phylogenetic analyses, using nuc rDNA and rpb1 concatenated sequences, confirmed the previous conclusion that the genus Albahypha in the family Entrophosporaceae sensu Oehl et al. is an unsupported taxon. Finally, the descriptions of the Glomerales, Entrophosporaceae, and Entrophospora were emended and new nomenclatural combinations were introduced.

A serosurvey of Mycobacterium avium subsp. paratuberculosis infection of goats in the North of Portugal.

To better understand the epizootiology of caprine paratuberculosis in the North of Portugal, a cross-sectional study was conducted from 2014 to 2015. The seroprevalence and risk factors for Mycobacterium avium subsp. paratuberculosis (Map) seropositivity were evaluated. Antibodies against Map were determined by a commercial ELISA. In 936 sera tested from 56 goat herds, 120 (12.8%, 95% CI: 10.8-15.1%) goats and 34 (60.7%, 95% CI: 47.6-72.4%) herds were positive. Risk factors for seropositivity were investigated by logistic regression models. The odds of Map seropositivity were found to be higher for animals with clinical signs, OR = 5.1 (95% CI: 2.7-9.6%), animals belonging to herds with previous wasting disease, OR = 2.3 (95% CI: 1.1-4.8%), and accumulation of manure in the herd, OR = 3.1 (95% CI: 1.7-5.7%). The potential risk factors identified in this study support the current recommendations for the control of paratuberculosis in these and other animals.

A New Multiplatform Model for Outpatient Prenatal and Postpartum Care in a Cohort of COVID-19-Affected Obstetric Patients.

Spain was one of the epicenters of the first wave of the COVID-19 pandemic. We describe in this article the design and results of a new telephone-and-telematic multiplatform model of systematic prenatal and postpartum follow-up for COVID-19-affected women implemented in a tertiary reference hospital in Madrid. We included patients with RT-PCR-confirmed COVID-19 during pregnancy or delivery from 10 March 2020 to 15 December 2020. We had a total of 211 obstetric patients: 148 (70.1%) were tested at the onset of suspicious clinical manifestations and 62 (29.4%) were tested in the context of routine screening. Of all the patients, 60 women (28.4%) were asymptomatic and 97 (46%) presented mild symptoms. Fifty-one women (24.2%) were admitted to our hospital for specific treatment because of moderate or severe symptoms. We had no missed cases and a good adherence. The mean number of calls per patient was 2.3. We performed 55 in-person visits. We analyzed the complexity of our program over time, showing a two-wave-like pattern. One patient was identified as needing hospitalization and we did not record major morbidity. Telemedicine programs are a strong and reproducible tool to reach to pregnant population affected by COVID-19, to assess its symptoms and severity, and to record for pregnancy-related symptoms both in an outpatient regime and after discharge from hospital.

Establishment of Colorectal Cancer Organoids in Microfluidic-Based System.

Colorectal cancer is the second leading cause of cancer death worldwide. Significant advances in the molecular mechanisms underlying colorectal cancer have been made; however, the clinical approval of new drugs faces many challenges. Drug discovery is a lengthy process causing a rapid increase in global health care costs. Patient-derived tumour organoids are considered preclinical models with the potential for preclinical drug screening, prediction of patient outcomes, and guiding optimized therapy strategies at an individual level. Combining microfluidic technology with 3D tumour organoid models to recapitulate tumour organization and in vivo functions led to the development of an appropriate preclinical tumour model, organoid-on-a-chip, paving the way for personalized cancer medicine. Herein, a low-cost microfluidic device suitable for culturing and expanding organoids, OrganoidChip, was developed. Patient-derived colorectal cancer organoids were cultured within OrganoidChip, and their viability and proliferative activity increased significantly. No significant differences were verified in the organoids' response to 5-fluorouracil (5-FU) treatment on-chip and on-plate. However, the culture within the OrganoidChip led to a significant increase in colorectal cancer organoid-forming efficiency and overall size compared with conventional culture on a 24-well plate. Interestingly, early-stage and late-stage organoids were predominantly observed on-plate and within the OrganoidChip, respectively. The OrganoidChip thus has the potential to generate in vivo-like organotypic structures for disease modelling and drug screening applications.

Characterization and strong risk association of TLR2 del -196 to -174 polymorphism and Helicobacter pylori and their influence on mRNA expression in gastric cancer.

BACKGROUND: Toll-like receptor-2 (TLR2) is responsible for recognizing Helicobacter pylori (H. pylori) and activating the immune response. Polymorphisms in TLR2 may modulate gastric carcinogenesis. AIM: To evaluate whether the TLR2 19216T/C (rs3804099) and TLR2 -196 to -174 ins/del (rs111200466) polymorphisms contribute to gastric carcinogenesis in the Brazilian population, and to determine the influence of both polymorphisms and H. pylori infection on TLR2 mRNA expression. METHODS: DNA was extracted from 854 peripheral blood leukocyte or gastric tissue samples [202 gastric cancer (GC), 269 chronic gastritis (CG), and 383 control/healthy (C)] and genotyped by allele-specific PCR or restriction fragment length polymorphism (RFLP)-PCR. Quantitative polymerase chain reaction by TaqMan® assay was used to quantify TLR2 mRNA levels in fresh gastric tissues (48 GC, 36 CG, and 14 C). RESULTS: Regarding the TLR2 -196 to -174 polymorphism, the ins/del and del/del genotypes were associated with a higher risk of GC by comparison with the C in all of the analyzed inheritance models (codominant, dominant, recessive, overdominant and log-additive; P < 0.0001). Similarly, an increased risk was observed when comparing the GC and CG groups [codominant (P < 0.0001), dominant (P < 0.0001), recessive (P = 0.0260), overdominant (P < 0.0001) and log-additive (P < 0.0001)]. In contrast, TLR2 19216T/C was associated with a protective effect in the GC group compared to the C group [dominant (P = 0.0420) and log-additive (P = 0.0300)]. Regarding the association of polymorphisms with H. pylori infection, individuals infected with H. pylori and harboring the TLR2 -196 to -174 ins/del polymorphism had an increased risk of gastric carcinogenesis [codominant (P = 0.0120), dominant (P = 0.0051), overdominant (P = 0.0240) and log-additive (P = 0.0030)], while TLR2 19216T/C was associated with a protective effect [codominant (P = 0.0039), dominant (P < 0.0001), overdominant (P = 0.0097) and log-additive (P = 0.0021)]. TLR2 mRNA levels were significantly increased in the GC group (median RQ = 6.95) compared to the CG group (RQ = 0.84, P < 0.0001) and to the normal mucosa group (RQ = 1.0). In addition, both H. pylori infection (P < 0.0001) and the presence of the polymorphic TLR2 -196 to -174del (P = 0.0010) and TLR2 19216 C (P = 0.0004) alleles influenced TLR2 mRNA expression. CONCLUSION: The TLR2 -196 to -174 ins/del and TLR2 19216 T/C polymorphisms are strongly associated with GC. TLR2 mRNA expression levels are upregulated in neoplastic tissues and influenced by both the presence of H. pylori and variant genotypes.

A non-canonical RNA degradation pathway suppresses RNAi-dependent epimutations in the human fungal pathogen Mucor circinelloides.

Mucorales are a group of basal fungi that includes the casual agents of the human emerging disease mucormycosis. Recent studies revealed that these pathogens activate an RNAi-based pathway to rapidly generate drug-resistant epimutant strains when exposed to stressful compounds such as the antifungal drug FK506. To elucidate the molecular mechanism of this epimutation pathway, we performed a genetic analysis in Mucor circinelloides that revealed an inhibitory role for the non-canonical RdRP-dependent Dicer-independent silencing pathway, which is an RNAi-based mechanism involved in mRNA degradation that was recently identified. Thus, mutations that specifically block the mRNA degradation pathway, such as those in the genes r3b2 and rdrp3, enhance the production of drug resistant epimutants, similar to the phenotype previously described for mutation of the gene rdrp1. Our genetic analysis also revealed two new specific components of the epimutation pathway related to the quelling induced protein (qip) and a Sad-3-like helicase (rnhA), as mutations in these genes prevented formation of drug-resistant epimutants. Remarkably, drug-resistant epimutant production was notably increased in M. circinelloides f. circinelloides isolates from humans or other animal hosts. The host-pathogen interaction could be a stressful environment in which the phenotypic plasticity provided by the epimutant pathway might provide an advantage for these strains. These results evoke a model whereby balanced regulation of two different RNAi pathways is determined by the activation of the RNAi-dependent epimutant pathway under stress conditions, or its repression when the regular maintenance of the mRNA degradation pathway operates under non-stress conditions.

EGFR-Based Immunoisolation as a Recovery Target for Low-EpCAM CTC Subpopulation.

Circulating tumour cells (CTCs) play a key role in the metastasis process, as they are responsible for micrometastasis and are a valuable tool for monitoring patients in real-time. Moreover, efforts to develop new strategies for CTCs isolation and characterisation, and the translation of CTCs into clinical practice needs to overcome the limitation associated with the sole use of Epithelial Cell Adhesion Molecule (EpCAM) expression to purify this tumour cell subpopulation. CTCs are rare events in the blood of patients and are believed to represent the epithelial population from a primary tumour of epithelial origin, thus EpCAM immunoisolation is considered an appropriate strategy. The controversy stems from the impact that the more aggressive mesenchymal tumour phenotypes might have on the whole CTC population. In this work, we first characterised a panel of cell lines representative of tumour heterogeneity, confirming the existence of tumour cell subpopulations with restricted epithelial features and supporting the limitations of EpCAM-based technologies. We next developed customised polystyrene magnetic beads coated with antibodies to efficiently isolate the phenotypically different subpopulations of CTCs from the peripheral blood mononuclear cells (PBMCs) of patients with metastatic cancer. Besides EpCAM, we propose Epidermal Growth Factor Receptor (EGFR) as an additional isolation marker for efficient CTCs detection.

A non-canonical RNA silencing pathway promotes mRNA degradation in basal Fungi.

The increasing knowledge on the functional relevance of endogenous small RNAs (esRNAs) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. In the basal fungus Mucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share common machinery for their biogenesis consisting of an RNase III endonuclease Dicer, a single Argonaute protein and two RNA-dependent RNA polymerases. We show in this study that, besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the rdrp genes are involved in a novel dicer-independent degradation process of endogenous mRNAs. The analysis of esRNAs accumulated in wild type and silencing mutants demonstrates that this new rdrp-dependent dicer-independent regulatory pathway, which does not produce sRNA molecules of discrete sizes, controls the expression of target genes promoting the specific degradation of mRNAs by a previously unknown RNase. This pathway mainly regulates conserved genes involved in metabolism and cellular processes and signaling, such as those required for heme biosynthesis, and controls responses to specific environmental signals. Searching the Mucor genome for candidate RNases to participate in this pathway, and functional analysis of the corresponding knockout mutants, identified a new protein, R3B2. This RNase III-like protein presents unique domain architecture, it is specifically found in basal fungi and, besides its relevant role in the rdrp-dependent dicer-independent pathway, it is also involved in the canonical dicer-dependent RNAi pathway, highlighting its crucial role in the biogenesis and function of regulatory esRNAs. The involvement of RdRPs in RNA degradation could represent the first evolutionary step towards the development of an RNAi mechanism and constitutes a genetic link between mRNA degradation and post-transcriptional gene silencing.

The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides.

BACKGROUND: RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which they derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants. RESULTS: Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway. CONCLUSION: This work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential and stationary growth phases and opens up an important avenue for in-depth study of genes involved in the regulation of physiological and developmental processes in this fungal model.

Nano-droplet systems by surfactant self-assembly and applications in the pharmaceutical industry.

Liquid systems containing droplets with size in the nanoscale range are attractive from both scientific and technological points of view as they have many current and potential applications in several industries and products. The formation and stabilization of nano-droplet systems are mostly based on the self-assembly of surfactant (amphiphilic) molecules at interfaces, driven by the solvophobic effect. Surfactants are involved in both top-bottom (high energy) and bottom- up (low energy) methods. Several devices have also been developed to aid in liquid fragmentation down to the nanometer scale. Nano-droplet systems can be both thermodynamically stable (microemulsions) or metastable (nanoemulsions), and appropriate formulation is a key for optimum product design in terms of droplet size, maximum solubilization, colloidal stability, and optical and rheological properties, among others. Such characteristics are determined by molecular packing, interfacial curvature, droplet-droplet interactions, film elasticity and nature of the dispersed and continuous phase. These properties can be engineered by proper understanding of the molecular structure and phase behavior of the multicomponent systems involved and by a range of experimental characterization techniques. Nano-droplet systems can help to solve specific issues in pharmaceutical products such as processing, limitations in drug solubility or stability, control on drug release, drug targeting and absorption; there are many examples to prove that. However, several practical aspects should be considered for preclinical and clinical tests and product development.

A single argonaute gene participates in exogenous and endogenous RNAi and controls cellular functions in the basal fungus Mucor circinelloides.

The mechanism of RNAi is well described in metazoans where it plays a role in diverse cellular functions. However, although different classes of endogenous small RNAs (esRNAs) have been identified in fungi, their biological roles are poorly described due, in part, to the lack of phenotype of mutants affected in the biogenesis of these esRNAs. Argonaute proteins are one of the key components of the RNAi pathways, in which different members of this protein family participate in the biogenesis of a wide repertoire of esRNAs molecules. Here we identified three argonaute genes of the fungus Mucor circinelloides and investigated their participation in exogenous and endogenous RNAi. We found that only one of the ago genes, ago-1, is involved in RNAi during vegetative growth and is required for both transgene-induced RNA silencing and the accumulation of distinct classes of esRNAs derived from exons (ex-siRNAs). Classes I and II ex-siRNAs bind to Ago-1 to control mRNA accumulation of the target protein coding genes. Class III ex-siRNAs do not specifically bind to Ago-1, but requires this protein for their production, revealing the complexity of the biogenesis pathways of ex-siRNAs. We also show that ago-1 is involved in the response to environmental signals, since vegetative development and autolysis induced by nutritional stress are affected in ago-1(-) M. circinelloides mutants. Our results demonstrate that a single Ago protein participates in the production of different classes of esRNAs that are generated through different pathways. They also highlight the role of ex-siRNAs in the regulation of endogenous genes in fungi and expand the range of biological functions modulated by RNAi.

[Hyperdense carotid T in stroke: 'tree sign']. / Bifurcacion carotidea (T) hiperdensa en el ictus: el 'signo del arbol'

TITLE: Bifurcacion carotidea (T) hiperdensa en el ictus: el 'signo del arbol'

Experimental evidences in favour of the hydroxylamine→nitrene-water tautomerization on the coordination sphere of Ir(III) centres.

And, to round off … A series of Ir(III) 5-membered metallacycles with an Ir-CH2 bond, react with aq. NH2OH with formation of hydride 6-membered iridacyclic complexes, which contain an Ir-NH=CH- imine functionality (see scheme).

Catalytic amine-borane dehydrogenation by a PCP-pincer palladium complex: a combined experimental and DFT analysis of the reaction mechanism.

Catalytic dehydrogenation of ammonia-borane (NH(3)·BH(3), AB) and dimethylamine borane (NHMe(2)·BH(3), DMAB) by the Pd(II) complex [((tBu)PCP)Pd(H(2)O)]PF(6) [(tBu)PCP = 2,6-C(6)H(3)(CH(2)P(t)Bu(2))(2)] leads to oligomerization and formation of spent fuels of general formula cyclo-[BH(2)-NR(2)](n) (n = 2,3; R = H, Me) as reaction byproducts, while one equivalent of H(2) is released per amine-borane equivalent. The processes were followed through multinuclear ((31)P, (1)H, (11)B) variable temperature NMR spectroscopy; kinetic measurements on the hydrogen production rate and the relative rate constants were also carried out. One non-hydridic intermediate could be detected at low temperature, whose chemical nature was explored through a DFT modeling of the reaction mechanism, at the M06//6-31+G(d,p) computational level. The computational output was of help to propose a reliable mechanistic picture of the process.

Molecular tools for carotenogenesis analysis in the zygomycete Mucor circinelloides.

The carotene producer fungus Mucor circinelloides is the zygomycete more amenable to genetic manipulations by using molecular tools. Since the initial development of an effective procedure of genetic transformation, more than two decades ago, the availability of new molecular approaches such as gene replacement techniques and gene expression inactivation by RNA silencing, in addition to the sequencing of its genome, has made Mucor a valuable organism for the study of a number of processes. Here we describe in detail the main techniques and methods currently used to manipulate M. circinelloides, including transformation, gene replacement, gene silencing, RNAi, and immunoprecipitation.