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This article reviews state-of-the-art microdroplet technological solutions for screening microorganisms mutant and GMO strains. Microorganisms used in the production of various products – single-cell protein, single-cell oil, enzymes, pigments and other bioactive compounds – can always be improved and their properties enhanced to increase the production of products of interest, to simplify microbial cultivation process, improve efficiency or adapted strains to use cheaper raw materials such as agroindustrial by-products. Microorganisms can be improved using either classical mutagenesis techniques or genetic engineering methods. Regardless of the selected method for mutant or GMO creation, during the process most promising microorganism strains must be selected, which is usually a slow and labour-intensive process. The use of microdroplets is a promising technological solution to speed up strain selection. This review looks at the latest developments in microdroplet technology, compares their variations, and identifies future prospects.

Thyroid hormone (TH) and thyroid hormone receptor (THR) regulate stem cell proliferation and differentiation during development, as well as during tissue renewal and repair in the adult. THR undergoes posttranslational modification by small ubiquitin-like modifier (SUMO). We generated the THRA (K283Q/K288R)−/− mouse model for in vivo studies and used human primary preadipocytes expressing the THRA sumoylation mutant (K283R/K288R) and isolated preadipocytes from mutant mice for in vitro studies. THRA mutant mice had reduced white adipose stores and reduced adipocyte cell diameter on a chow diet, compared to wild-type, and these differences were further enhanced after a high fat diet. Reduced preadipocyte proliferation in mutant mice, compared to wt, was shown after in vivo labeling of preadipocytes with EdU and in preadipocytes isolated from mice fat stores and studied in vitro. Mice with the desumoylated THRA had disruptions in cell cycle G1/S transition and this was associated with a reduction in the availability of cyclin D2 and cyclin-dependent kinase 2. The genes coding for cyclin D1, cyclin D2, cyclin-dependent kinase 2 and Culin3 are stimulated by cAMP Response Element Binding Protein (CREB) and contain CREB Response Elements (CREs) in their regulatory regions. We demonstrate, by Chromatin Immunoprecipitation (ChIP) assay, that in mice with the THRA K283Q/K288R mutant there was reduced CREB binding to the CRE. Mice with a THRA sumoylation mutant had reduced fat stores on chow and high fat diets and reduced adipocyte diameter.

Črevesne bakterije Campylobacter jejuni so od leta 2005 najpogostejši povzročitelj gastrointestinalnih okužb ljudi v Evropski Uniji. Glavni vir okužb z bakterijami C. jejuni pri ljudeh so uživanje nezadostno obdelanega perutninskega mesa, nepasteriziranega mleka in kontaminirane vode. Tu nastane vprašanje, kako bakterije C. jejuni, ki so poznane kot zelo občutljive na okoljske razmere, vseeno preživijo v okolju in pozvročijo nadaljne kontaminacije hrane in okužbe ljudi. Raziskave v zadnjih letih nakazujejo na sposobnost preživetja bakterij C. jejuni v biofilmih, ki jim omogočijo zaščito pred neugodnimi razmerami v okolju. Predvidevamo, da bi preprečitev pritrditve bakterij C. jejuni na površine, ki je prva stopnja tvorbe biofilma, lahko negativno vplivala na vse nadaljne stopnje tvorbe biofilma. V magistrski nalogi smo ovrednotili vpliv mutacij v genih, vključenih v različne stopnje nastanka biofilma.Vkjlučili smo: (i) šest sevov mutantov bakterij C. jejuni z enojno mutacijo v genih vključenih v različne stopnje nastanka biofilma (sevi mutantov v genih luxS, kpsM, omp50, csrA, rrpA in rrpB) ter (ii) tri seve mutantov bakterij C. jejuni z dvojno mutacijo v genih vključenih v različne stopnje nastanka biofilma (gena luxS in kpsM, gena luxS in omp50, ter gena rrpA in rrpB). Rezultati so pokazali pomembno vlogo posameznih genov, prevsem genov luxS, csrA in omp50, ki torej predstavljajo potencialni tarčni mehanizem alternativnih strategij nazdora nad patogenimi bakterijami C. jejuni. Intestinal bacteria Campylobacter jejuni have been the most common cause of gastrointestinal infections in humans in the European Union since 2005. The main source of C. jejuni infections in humans is the consumption of insufficiently processed poultry meat, unpasteurized milk and contaminated water. This raises the question of how C. jejuni bacteria, which are known to be very sensitive to external environmental conditions, still survive in the environment and cause further food contamination and human infections. Research in recent years indicates the ability of C. jejuni bacteria to survive in biofilms, which enable them to protect themselves from adverse environmental conditions. We hypothesize that preventing the adhesion of C. jejuni bacteria to surfaces, which is the first stage of biofilm formation, could negatively affect all subsequent stages of biofilm formation. In this master's thesis, we evaluated the impact of mutations in genes involved in different stages of biofilm formation. We included: (i) six mutants of bacteria C. jejuni with single mutations in genes involved in different stages of biofilm formation (mutants in genes luxS, kpsM, omp50, csrA, rrpA and rrpB) and (ii) three mutants of bacterial C. jejuni with a double mutation in genes involved in different stages of biofilm formation (luxS and kpsM genes, luxS and omp50 genes, and rrpA and rrpB genes). The results showed an important role of individual genes, especially, genes luxS, csrA and omp50, which therefore represent a potential target mechanism of alternative control strategies over the pathogenic bacteria C. jejuni.

Candida albicans biofilms are a complex multilayer community of cells that are resistant to almost all classes of antifungal drugs. The bottommost layers of biofilms experience nutrient limitation where C. albicans cells are required to respire. We previously reported that a protein Ndu1 is essential for Candida mitochondrial respiration; loss of NDU1 causes inability of C. albicans to grow on alternative carbon sources and triggers early biofilm detachment. Here, we screened a repurposed library of FDA-approved small molecule inhibitors to identify those that prevent NDU1-associated functions. We identified an antihelminthic drug, Niclosamide (NCL), which not only prevented growth on acetate, C. albicans hyphenation and early biofilm growth, but also completely disengaged fully grown biofilms of drug-resistant C. albicans and Candida auris from their growth surface. To overcome the suboptimal solubility and permeability of NCL that is well known to affect its in vivo efficacy, we developed NCL-encapsulated Eudragit EPO (an FDA-approved polymer) nanoparticles (NCL-EPO-NPs) with high niclosamide loading, which also provided long-term stability. The developed NCL-EPO-NPs completely penetrated mature biofilms and attained anti-biofilm activity at low microgram concentrations. NCL-EPO-NPs induced ROS activity in C. albicans and drastically reduced oxygen consumption rate in the fungus, similar to that seen in an NDU1 mutant. NCL-EPO-NPs also significantly abrogated mucocutaneous candidiasis by fluconazole-resistant strains of C. albicans, in mice models of oropharyngeal and vulvovaginal candidiasis. To our knowledge, this is the first study that targets biofilm detachment as a target to get rid of drug-resistant Candida biofilms and uses NPs of an FDA-approved nontoxic drug to improve biofilm penetrability and microbial killing.

Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. While its ability to replicate within cells has been studied in much detail, the bacterium also encodes a less characterised type 4 pili (T4P) system. T4Ps are dynamic adhesive organelles identified as major virulence determinants in many human pathogens. In F. tularensis, the T4P is required for adherence to the host cell, as well as for protein secretion. Several components, including pilins, a pili peptidase, a secretin pore and two ATPases, are required to assemble a functional T4P, and these are encoded within distinct clusters on the Francisella chromosome. While some of these components have been functionally characterised, the role of PilO, if any, still is unknown. Here, we examined the role of PilO in the pathogenesis of F. novicida. Our results show that the PilO is essential for pilus assembly on the bacterial surface. In addition, PilO is important for adherence of F. novicida to human monocyte-derived macrophages, secretion of effector proteins and intracellular replication. Importantly, the pilO mutant is attenuated for virulence in BALB/c mice regardless of the route of infection. Following intratracheal and intradermal infection, the mutant caused no histopathology changes, and demonstrated impaired phagosomal escape and replication within lung liver as well as spleen. Thus, PilO is an essential virulence determinant of F. novicida.

Pathogenic Leptospira are the causative agents of leptospirosis, the most widespread zoonotic infectious disease. Leptospirosis is a potentially severe and life-threatening emerging disease with highest burden in sub-tropical areas and impoverished populations. Mechanisms allowing pathogenic Leptospira to survive inside a host and induce acute leptospirosis are not fully understood. The ability to resist deadly oxidants produced by the host during infection is pivotal for Leptospira virulence. We have previously shown that genes encoding defenses against oxidants in L. interrogans are repressed by PerRA (encoded by LIMLP_10155), a peroxide stress regulator of the Fur family. In this study, we describe the identification and characterization of another putative PerR-like regulator (LIMLP_05620) in L. interrogans. Protein sequence and phylogenetic analyses indicated that LIMLP_05620 displayed all the canonical PerR amino acid residues and is restricted to pathogenic Leptospira clades. We therefore named this PerR-like regulator PerRB. In L. interrogans, the PerRB regulon is distinct from that of PerRA. While a perRA mutant had a greater tolerance to peroxide, inactivating perRB led to a higher tolerance to superoxide, suggesting that these two regulators have a distinct function in the adaptation of L. interrogans to oxidative stress. The concomitant inactivation of perRA and perRB resulted in a higher tolerance to both peroxide and superoxide and, unlike the single mutants, a double perRAperRB mutant was avirulent. Interestingly, this correlated with major changes in gene and non-coding RNA expression. Notably, several virulence-associated genes (clpB, ligA/B, and lvrAB) were repressed. By obtaining a double mutant in a pathogenic Leptospira strain, our study has uncovered an interplay of two PerRs in the adaptation of Leptospira to oxidative stress with a putative role in virulence and pathogenicity, most likely through the transcriptional control of a complex regulatory network., Author summary Leptospirosis is a widespread infectious disease responsible for over one million of severe cases and 60 000 fatalities annually worldwide. This neglected and emerging disease has a worldwide distribution, but it mostly affects populations from developing countries in sub-tropical areas. The causative agents of leptospirosis are pathogenic bacterial Leptospira spp. There is a considerable deficit in our knowledge of these atypical bacteria, including their virulence mechanisms. In addition to the Leptospira PerRA regulator that represses defenses against peroxide, we have identified and characterized a second PerR regulator in pathogenic Leptospira species (PerRB) that participates in Leptospira tolerance to superoxide. Phenotypic and transcriptomic analyses of single PerRA and PerRB mutants suggest that the two PerRs fulfill distinct functions in the adaptation to oxidative stress. Concomitant inactivation of PerRA and PerRB resulted in a higher tolerance to both peroxide and superoxide. Moreover, the perRAperRB mutant lost its virulence. Major changes in gene expression, including a decreased expression of several virulence factors, were observed in the double perRAperRB mutant. Our study suggests that PerRA and PerRB cooperate to orchestrate a complex regulatory network involved in Leptospira virulence.

Background and Objectives Remarkable infectivity of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV2) is due to the rapid emergence of various strains which enable the virus to ruling the world. Over the course of SARS-CoV2 pandemic, the scientific communities worldwide are responding to newly emerging genetic variants. However, mechanism behind the persistent infection of these variants is still not known due to the paucity of study of these variants at molecular level. In this scenario, computational methods have immense utility in understanding the molecular and functional properties of different variants. Methods The various mutants (MTs) of SpikeS1 receptor binding domain (RBD) of highly infectious SARS-CoV2 strains were manifested and elucidated the protein structure and binding strength using molecular dynamics (MD) simulation and protein-protein docking approaches. Results MD simulation study showed that all MTs exhibited stable structures with altered functional properties. Furthermore, the binding strength of different MTs along with WT (wildtype) was revealed that MTs showed differential binding affinities to host protein with high binding strength exhibited by V367F and V483A MTs. Conclusion Hence, this study shed light on the molecular basis of infection caused by different variants of SARS-CoV2, which might play an important role in to cease the transmission and pathogenesis of virus and also implicate in rational designing of a specific drug.

Leptospira interrogans, the causative agent of most cases of human leptospirosis, must respond to myriad environmental signals during its free-living and pathogenic lifestyles. Previously, we compared L. interrogans cultivated in vitro and in vivo using a dialysis membrane chamber (DMC) peritoneal implant model. From these studies emerged the importance of genes encoding the Peroxide responsive regulators PerRA and PerRB. First described in in Bacillus subtilis, PerRs are widespread in Gram-negative and -positive bacteria, where regulate the expression of gene products involved in detoxification of reactive oxygen species and virulence. Using perRA and perRB single and double mutants, we establish that L. interrogans requires at least one functional PerR for infectivity and renal colonization in a reservoir host. Our finding that the perRA/B double mutant survives at wild-type levels in DMCs is noteworthy as it demonstrates that the loss of virulence is not due to a metabolic lesion (i.e., metal starvation) but instead reflects dysregulation of virulence-related gene products. Comparative RNA-Seq analyses of perRA, perRB and perRA/B mutants cultivated within DMCs identified 106 genes that are dysregulated in the double mutant, including ligA, ligB and lvrA/B sensory histidine kinases. Decreased expression of LigA and LigB in the perRA/B mutant was not due to loss of LvrAB signaling. The majority of genes in the perRA and perRB single and double mutant DMC regulons were differentially expressed only in vivo, highlighting the importance of host signals for regulating gene expression in L. interrogans. Importantly, the PerRA, PerRB and PerRA/B DMC regulons each contain multiple genes related to environmental sensing and/or transcriptional regulation. Collectively, our data suggest that PerRA and PerRB are part of a complex regulatory network that promotes host adaptation by L. interrogans within mammals., Author summary Leptospirosis is a neglected tropical disease with a worldwide distribution. Globally, ~1 million cases and ~60,000 deaths are reported each year. The majority of cases of human leptospirosis are associated with Leptospira interrogans. Infection begins when a naïve reservoir (or incidental) host comes into direct or indirect contact with urine from an infected reservoir host. While infection in reservoir hosts, including rats and mice, is generally asymptomatic, incidental hosts, including humans, may develop clinical symptoms ranging from mild flu-like illness to fulminant disease. The gene products required by leptospires for infection remain poorly understood. Herein, we establish that the FUR family regulators PerRA and PerRB function in parallel, contributing to infectivity and renal colonization in mice. By comparative transcriptomics, we identified >100 genes that were dysregulated in the perRA/B double mutant cultivated in rat peritoneal cavities, including the virulence determinants LigA and LigB. Importantly, the PerRA, PerRB and PerRA/B DMC regulons contain multiple genes related to environmental sensing and/or transcriptional regulation. Our data suggest that PerRA and PerRB are part of a complex regulatory network that promotes host adaptation by L. interrogans within mammals.

SF3B1 mutations occur in many cancers, and the highly conserved His662 residue is one of the hotspot mutation sites. To address effects on splicing and development, we constructed strains carrying point mutations at the corresponding residue His698 in Drosophila using the CRISPR-Cas9 technique. Two mutations, H698D and H698R, were selected due to their frequent presence in patients and notable opposite charges. Both the sf3b1-H698D and–H698R mutant flies exhibit developmental defects, including less egg-laying, decreased hatching rates, delayed morphogenesis and shorter lifespans. Interestingly, the H698D mutant has decreased resistance to fungal infection, while the H698R mutant shows impaired climbing ability. Consistent with these phenotypes, further analysis of RNA-seq data finds altered expression of immune response genes and changed alternative splicing of muscle and neural-related genes in the two mutants, respectively. Expression of Mef2-RB, an isoform of Mef2 gene that was downregulated due to splicing changes caused by H698R, partly rescues the climbing defects of the sf3b1-H698R mutant. Lariat sequencing reveals that the two sf3b1-H698 mutations cause aberrant selection of multiple intronic branch sites, with the H698R mutant using far upstream branch sites in the changed alternative splicing events. This study provides in vivo evidence from Drosophila that elucidates how these SF3B1 hotspot mutations alter splicing and their consequences in development and in the immune system., Author summary In the past decade, one of the important findings in the RNA splicing field has been that somatic SF3B1 mutations widely occur in many cancers. Including R625, H662, K666, K700 and E902, there are five hotspot mutation sites in the highly conserved HEAT repeats of SF3B1. Several kinds of H662 mutations have been found widely in MDS, AML, CLL and breast cancers; however, it remains unclear how these H662 mutations alter splicing and whether they have in vivo effects on development. To address these questions, in this manuscript, we first summarized the H662 mutations in human diseases and constructed two corresponding Drosophila mutant strains, sf3b1-H698D and -H698R using CRISPR-Cas9. Analyses of these two fly strains find that the two oppositely charged Sf3b1-H698 mutants are defective in development. In addition, one mutant has decreased climbing ability, whereas the other mutant has impaired immune response. Further RNA-seq allows us to find responsible genes in each mutant strain, and lariat sequencing reveals that both mutations cause aberrant selection of the intronic branch sites. Our findings provide the first in vivo evidence that Sf3b1 mutations result in defective development, and also reveal a molecular mechanism of these hotspot histidine mutations that enhance the use of cryptic branch sites to alter splicing. Importantly, we demonstrate that the H698R mutant prefers to use far upstream branch sites.

The matrix (M) protein of vesicular stomatitis virus (VSV) has a complex role in infection and immune evasion, particularly with respect to suppression of Type I interferon (IFN). Viral strains bearing the wild-type (wt) M protein are able to suppress Type I IFN responses. We recently reported that the 22–25 strain of VSV encodes a wt M protein, however its sister plaque isolate, strain 22–20, carries a M[MD52G] mutation that perturbs the ability of the M protein to block NFκB, but not M-mediated inhibition of host transcription. Therefore, although NFκB is activated in 22–20 infected murine L929 cells infected, no IFN mRNA or protein is produced. To investigate the impact of the M[D52G] mutation on immune evasion by VSV, we used transcriptomic data from L929 cells infected with wt, 22–25, or 22–20 to define parameters in a family of executable logical models with the aim of discovering direct targets of viruses encoding a wt or mutant M protein. After several generations of pruning or fixing hypothetical regulatory interactions, we identified specific predicted targets of each strain. We predict that wt and 22–25 VSV both have direct inhibitory actions on key elements of the NFκB signaling pathway, while 22–20 fails to inhibit this pathway.

To study viral evolutionary processes within patients, mathematical models have been instrumental. Yet, the need for stochastic simulations of minority mutant dynamics can pose computational challenges, especially in heterogeneous systems where very large and very small sub-populations coexist. Here, we describe a hybrid stochastic-deterministic algorithm to simulate mutant evolution in large viral populations, such as acute HIV-1 infection, and further include the multiple infection of cells. We demonstrate that the hybrid method can approximate the fully stochastic dynamics with sufficient accuracy at a fraction of the computational time, and quantify evolutionary end points that cannot be expressed by deterministic models, such as the mutant distribution or the probability of mutant existence at a given infected cell population size. We apply this method to study the role of multiple infection and intracellular interactions among different virus strains (such as complementation and interference) for mutant evolution. Multiple infection is predicted to increase the number of mutants at a given infected cell population size, due to a larger number of infection events. We further find that viral complementation can significantly enhance the spread of disadvantageous mutants, but only in select circumstances: it requires the occurrence of direct cell-to-cell transmission through virological synapses, as well as a substantial fitness disadvantage of the mutant, most likely corresponding to defective virus particles. This, however, likely has strong biological consequences because defective viruses can carry genetic diversity that can be incorporated into functional virus genomes via recombination. Through this mechanism, synaptic transmission in HIV might promote virus evolvability., Author summary The evolution of human immunodeficiency virus within patients is an important part of the disease process. In particular, the presence of mutants that are resistant against anti-viral drugs can result in challenges to the long-term control of the infection. To study disease progression, computer simulations have been useful. However, in some cases these simulations can be difficult because of the complexity of the model. Here, we use a computational complexity reducing algorithm to simulate mutant dynamics in large populations, which can approximate the full model at a fraction of the time. The use of this algorithm allows us to study different transmission methods, viral processes that occur between virus strains within individual cells, and important quantities such as the mutant distribution or the probability of mutant existence at a given infected cell population size. We find that the direct synaptic cell-to-cell transmission of the virus through virological synapses can have strong biological consequences because it can promote potentially defective viruses that carry genetic diversity which can be incorporated into functional virus genomes during infection. Through this process, synaptic transmission in human immunodeficiency virus might promote virus evolvability.

Helicase regulation involves modulation of unwinding speed to maintain coordination of DNA replication fork activities and is vital for replisome progression. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB. We discovered that these four SEW mutations stabilize constricted states, with more fully constricted mutants having a generally greater impact on genomic stress, suggesting a dynamic model for helicase regulation that involves both excluded strand interactions and conformational states. These dnaB mutations result in increased chromosome complexities, less stable genomes, and ultimately less viable and fit strains. Specifically, dnaB:mut strains present with increased mutational frequencies without significantly inducing SOS, consistent with leaving single-strand gaps in the genome during replication that are subsequently filled with lower fidelity. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving a spectrum of DnaB conformational changes and relates current mechanistic understanding to functional helicase behavior at the replication fork., Author summary DNA replication is a vital biological process, and the proteins involved are structurally and functionally conserved across all domains of life. As our fundamental knowledge of genes and genetics grows, so does our awareness of links between acquired genetic mutations and disease. Understanding how genetic material is replicated accurately and efficiently and with high fidelity is the foundation to identifying and solving genome-based diseases. E. coli are model organisms, containing core replisome proteins, but lack the complexity of the human replication system, making them ideal for investigating conserved replisome behaviors. The helicase enzyme acts at the forefront of the replication fork to unwind the DNA helix and has also been shown to help coordinate other replisome functions. In this study, we examined specific mutations in the helicase that have been shown to regulate its conformation and speed of unwinding. We investigate how these mutations impact the growth, fitness, and cellular morphology of bacteria with the goal of understanding how helicase regulation mechanisms affect an organism’s ability to survive and maintain a stable genome.