FtsH belongs to the AAA+ ATP-dependent family of proteases, which participate in diverse cellular processes and are ubiquitous among bacteria, chloroplasts, and mitochondria. FtsH is poorly characterized in most organisms, especially compared to other major housekeeping proteases. In the current study, we examined the source of FtsH essentiality in the human oral microbiome species Streptococcus mutans, one of the primary etiological agents of dental caries. By creating a conditionally lethal ftsH mutant, we were able to identify a secondary suppressor missense mutation in the vicR gene, encoding the response regulator of the essential VicRK two-component system (TCS). Transcriptomic analysis of the vicR (G195R) mutant revealed significantly reduced expression of 46 genes, many of which were located within the genomic island Tnsmu2, which harbors the mutanobactin biosynthetic gene cluster. In agreement with the transcriptomic data, deletion of the mutanobactin biosynthetic gene cluster suppressed ftsH essentiality in S. mutans. We also explored the role of FtsH in S. mutans physiology and demonstrated its critical role in stress tolerance, especially acid stress. The presented results reveal the first insights within S. mutans for the pleiotropic regulatory function of this poorly understood global regulator.

Engineered Genetic Incompatibility (EGI) is a method to create species-like barriers to sexual reproduction. It has applications in pest control that mimic Sterile Insect Technique when only EGI males are released. This can be facilitated by introducing conditional female-lethality to EGI strains to generate a sex-sorting incompatible male system (SSIMS). Here, we demonstrate a proof of concept by combining tetracycline-controlled female lethality constructs with a pyramus-targeting EGI line in the model insect Drosophila melanogaster. We show that both functions (incompatibility and sex-sorting) are robustly maintained in the SSIMS line and that this approach is effective for population suppression in cage experiments. Further we show that SSIMS males remain competitive with wild-type males for reproduction with wild-type females, including at the level of sperm competition.

Although depression is the leading cause of disability worldwide, its pathophysiology is poorly understood. Our previous study showed that hippocampal peroxisome proliferator-activated receptor δ (PPARδ) overexpression displays antidepressive effect and enhances hippocampal neurogenesis during chronic stress. Herein, we further extended our curiosity to investigate whether downregulating PPARδ could cause depressive-like behaviors through downregulation of neurogenesis.

No abstract available

Recent investigations into mechanisms behind the development of osteoporosis suggest that suppressing PPARγ-mediated adipogenesis can improve bone formation and bone mineral density. In this study, we investigated a co-treatment strategy to enhance bone formation by combining NELL-1, an osteogenic molecule that has been extensively studied for its potential use as a therapeutic for osteoporosis, with two methods of PPARγ suppression. First, we suppressed PPARγ genetically using lentiviral PPARγ-shRNA in immunocompromised mice for a proof of concept. Second, we used a PPARγ antagonist to suppress PPARγ pharmacologically in immunocompetent senile osteopenic mice for clinical transability. We found that the co-treatment strategy significantly increased bone formation, increased the proliferation stage cell population, decreased late apoptosis of primary mouse BMSCs, and increased osteogenic marker mRNA levels in comparison to the single agent treatment groups. The addition of PPARγ suppression to NELL-1 therapy enhanced NELL-1's effects on bone formation by upregulating anabolic processes without altering NELL-1's inhibitory effects on osteoclastic and adipogenic activities. Our findings suggest that combining PPARγ suppression with therapeutic NELL-1 may be a viable method that can be further developed as a novel strategy to reverse bone loss and decrease marrow adiposity in age-related osteoporosis.

Aedes aegypti is the primary vector of arthropod-borne viruses including dengue, chikungunya and Zika. Vector population control methods are reviving to impede disease transmission. An efficient sex separation for male-only releases is crucial for area-wide mosquito population suppression strategies. Here, we report on the construction of two genetic sexing strains using red- and white-eye colour mutations as selectable markers. Quality control analysis showed that the Red-eye genetic sexing strains (GSS) is better and more genetically stable than the White-eye GSS. The introduction of an irradiation-induced inversion (Inv35) increases genetic stability and reduces the probability of female contamination of the male release batches. Bi-weekly releases of irradiated males of both the Red-eye GSS and the Red-eye GSS/Inv35 fully suppressed target laboratory cage populations within six and nine weeks, respectively. An image analysis algorithm allowing sex determination based on eye colour identification at the pupal stage was developed. The next step is to automate the Red-eye-based genetic sexing and validate it in pilot trials prior to its integration in large-scale population suppression programmes. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.

Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells.

In the malaria parasite Plasmodium falciparum, synthesis of isoprenoids from glycolytic intermediates is essential for survival. The antimalarial fosmidomycin (FSM) inhibits isoprenoid synthesis. In P. falciparum, we identified a loss-of-function mutation in HAD2 (P. falciparum 3D7_1226300 [PF3D7_1226300]) as necessary for FSM resistance. Enzymatic characterization revealed that HAD2, a member of the haloacid dehalogenase-like hydrolase (HAD) superfamily, is a phosphatase. Harnessing a growth defect in resistant parasites, we selected for suppression of HAD2-mediated FSM resistance and uncovered hypomorphic suppressor mutations in the locus encoding the glycolytic enzyme phosphofructokinase 9 (PFK9). Metabolic profiling demonstrated that FSM resistance is achieved via increased steady-state levels of methylerythritol phosphate (MEP) pathway and glycolytic intermediates and confirmed reduced PFK9 function in the suppressed strains. We identified HAD2 as a novel regulator of malaria parasite metabolism and drug sensitivity and uncovered PFK9 as a novel site of genetic metabolic plasticity in the parasite. Our report informs the biological functions of an evolutionarily conserved family of metabolic regulators and reveals a previously undescribed strategy by which malaria parasites adapt to cellular metabolic dysregulation.IMPORTANCE Unique and essential aspects of parasite metabolism are excellent targets for development of new antimalarials. An improved understanding of parasite metabolism and drug resistance mechanisms is urgently needed. The antibiotic fosmidomycin targets the synthesis of essential isoprenoid compounds from glucose and is a candidate for antimalarial development. Our report identifies a novel mechanism of drug resistance and further describes a family of metabolic regulators in the parasite. Using a novel forward genetic approach, we also uncovered mutations that suppress drug resistance in the glycolytic enzyme PFK9. Thus, we identify an unexpected genetic mechanism of adaptation to metabolic insult that influences parasite fitness and tolerance of antimalarials.

To clarify the detailed molecular mechanisms underlying the development of asthma, we assessed the potential immune effects of prenatal osteoprotegerin (OPG) inhibition in the pathogenesis of asthma. The effects of OPG deficiency on the development of asthma were evaluated using an ovalbumin-induced asthma model in OPG knockout mice. Histological analysis demonstrated that OPG was mainly detected in airway epithelial cells in wild type mice. After ovalbumin sensitization and challenge, accumulation of inflammatory cells, gene expression of T helper 2-related cytokines and mucus hypersecretion in lung tissues were inhibited by OPG deficiency. Importantly, the serum level of IgE was not increased in OPG KO mice after ovalbumin sensitization and challenge. Based on these findings, OPG knockout mice were protected against methacholine-induced airway hyperresponsiveness. OPG expression is thought to be essential for induction of the allergic immune response in asthma.

To maximize crop growth, crops need to capture sunlight efficiently. This property is primarily influenced by the shape of the crops such as the angle, area, and arrangement of leaves. We constructed a rice (Oryza sativa L.) inbred line that displayed an ideal transition of plant shapes in terms of sunlight receiving efficiency. During vegetative growth, this line exhibited tiller spreading with increased tiller number, which formed a parabolic antenna-like structure. The architecture probably improved light reception efficiency of individuals compared with the recurrent parent. The line achieved not only acceleration of the vegetative growth, but also significant suppression of weed growth under the canopy. The increased light reception efficiency of the line has consequently reduced the amount of incident light to the ground and supplied significant competitiveness against weeds. The spread tillers became erect from the entry of the reproductive growth phase, adaptively sustaining light reception efficiency in thicker stands. The line carries a small chromosomal segment from Oryza rufipogon Griff., a putative progenitor of Asian cultivated rice. The introduced chromosome segment had little effect on grain yield and quality. Our results shed light on potentials hidden in the wild rice chromosome segment to achieve the valuable traits.

Cellular gene expression machinery has coevolved with molecular parasites, such as viruses and transposons, which rely on host cells for their expression and reproduction. We previously reported that a wild-derived allele of mouse Nxf1 (Tap), a key component of the host mRNA nuclear export machinery, suppresses two endogenous retrovirus-induced mutations and shows suggestive evidence of positive selection. Here we show that Nxf1(CAST) suppresses a specific and frequent class of intracisternal A particle (IAP)-induced mutations, including Ap3d1(mh2J), a model for Hermansky-Pudlak syndrome, and Atcay(hes), an orthologous gene model for Cayman ataxia, among others. The molecular phenotype of suppression includes approximately two-fold increase in the level of correctly-spliced mRNA and a decrease in mutant-specific, alternatively-processed RNA accumulating from the inserted allele. Insertional mutations involving ETn and LINE elements are not suppressed, demonstrating a high degree of specificity to this suppression mechanism. These results implicate Nxf1 in some instances of pre-mRNA processing, demonstrate the useful range of Nxf1(CAST) alleles for manipulating existing mouse models of disease, and specifically imply a low functional threshold for therapeutic benefit in Cayman ataxia.

The breast cancer susceptibility gene BRCA1 is well known for its function in double-strand break (DSB) DNA repair. While BRCA1 is also implicated in transcriptional regulation, the physiological significance remains unclear. COBRA1 (also known as NELF-B) is a BRCA1-binding protein that regulates RNA polymerase II (RNAPII) pausing and transcription elongation. Here we interrogate functional interaction between BRCA1 and COBRA1 during mouse mammary gland development. Tissue-specific deletion of Cobra1 reduces mammary epithelial compartments and blocks ductal morphogenesis, alveologenesis and lactogenesis, demonstrating a pivotal role of COBRA1 in adult tissue development. Remarkably, these developmental deficiencies due to Cobra1 knockout are largely rescued by additional loss of full-length Brca1. Furthermore, Brca1/Cobra1 double knockout restores developmental transcription at puberty, alters luminal epithelial homoeostasis, yet remains deficient in homologous recombination-based DSB repair. Thus our genetic suppression analysis uncovers a previously unappreciated, DNA repair-independent function of BRCA1 in antagonizing COBRA1-dependent transcription programme during mammary gland development.

PTGS (post-transcriptional gene silencing) is used to counter pathogenic invasions, particularly viruses. In return, many plant viruses produce proteins which suppress silencing directed against their RNA. The diversity of silencing suppression at the species level in natural hosts is unknown.

The contributions of TGF-β signaling to cancer are complex but involve the inflammatory microenvironment as well as cancer cells themselves. In mice encoding a TGF-β mutant that precludes its binding to the latent TGF-β binding protein (Tgfb1(-/C33S)), we observed multiorgan inflammation and an elevated incidence of various types of gastrointestinal solid tumors due to impaired conversion of latent to active TGF-β1. By genetically eliminating activators of latent TGF-β1, we further lowered the amount of TGF-β, which enhanced tumor frequency and multiorgan inflammation. This model system was used to further investigate the relative contribution of TGF-β1 to lymphocyte-mediated inflammation in gastrointestinal tumorigenesis. Toward this end, we generated Tgfb1(-/C33S);Rag2(-/-) mice that lacked adaptive immune function, which eliminated tumor production. Analysis of tissue from Tgfb1(-/C33S) mice indicated decreased levels of P-Smad3 compared with wild-type animals, whereas tissue from Tgfb1(-/C33S);Rag2(-/-) mice had normal P-Smad3 levels. Inhibiting the inflammatory response normalized levels of interleukin (IL)-1β and IL-6 and reduced tumor cell proliferation. In addition, Tgfb1(-/C33S);Rag2(-/-) mice exhibited reduced paracrine signaling in the epithelia, mediated by hepatocyte growth factor produced by gastric stroma. Together, our results indicate that many of the responses of the gastric tissue associated with decreased TGF-β1 may be directly or indirectly affected by inflammatory processes, which accompany loss of TGF-β1, rather than a direct effect of loss of the cytokine.

The biosynthesis and incorporation of polyunsaturated fatty acids into phospholipid membranes are unique features of certain marine Gammaproteobacteria inhabiting high-pressure and/or low-temperature environments. In these bacteria, monounsaturated and saturated fatty acids are produced via the classical dissociated type II fatty acid synthase mechanism, while omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) are produced by a hybrid polyketide/fatty acid synthase-encoded by the pfa genes-also referred to as the secondary lipid synthase mechanism. In this work, phenotypes associated with partial or complete loss of monounsaturated biosynthesis are shown to be compensated for by severalfold increased production of polyunsaturated fatty acids in the model marine bacterium Photobacterium profundum SS9. One route to suppression of these phenotypes could be achieved by transposition of insertion sequences within or upstream of the fabD coding sequence, which encodes malonyl coenzyme A (malonyl-CoA) acyl carrier protein transacylase. Genetic experiments in this strain indicated that fabD is not an essential gene, yet mutations in fabD and pfaA are synthetically lethal. Based on these results, we speculated that the malonyl-CoA transacylase domain within PfaA compensates for loss of FabD activity. Heterologous expression of either pfaABCD from P. profundum SS9 or pfaABCDE from Shewanella pealeana in Escherichia coli complemented the loss of the chromosomal copy of fabD in vivo. The co-occurrence of independent, yet compensatory, fatty acid biosynthetic pathways in selected marine bacteria may provide genetic redundancy to optimize fitness under extreme conditions. IMPORTANCE A defining trait among many cultured piezophilic and/or psychrophilic marine Gammaproteobacteria is the incorporation of both monounsaturated and polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of these different classes of fatty acid molecules is linked to two genetically distinct co-occurring pathways that utilize the same pool of intracellular precursors. Using a genetic approach, new insights into the interactions between these two biosynthetic pathways have been gained. Specifically, core fatty acid biosynthesis genes previously thought to be essential were found to be nonessential in strains harboring both pathways due to functional overlap between the two pathways. These results provide new routes to genetically optimize long-chain omega-3 polyunsaturated fatty acid biosynthesis in bacteria and reveal a possible ecological role for maintaining multiple pathways for lipid synthesis in a single bacterium.

Elevated heart rate represents an independent risk factor for cardiovascular outcome in patients with heart disease. In the sinoatrial node, rate increase is mediated by β(1) adrenoceptor mediated activation of the Gα(s) pathway. We hypothesized that genetic inactivation of the stimulatory Gα(s) protein in the sinoatrial node would provide sinus rate control and would prevent inappropriate heart rate acceleration during β-adrenergic activation.

Early-onset epileptic encephalopathies with burst suppression (EOEE-BS) are a group of neonatal epileptic syndromes characterized by intractable epilepsy and severe psychomotor delay with structural and metabolic factors accounting for major etiologies. However, recent advances in gene sequencing have identified that genetic factors might also play a significant role in the development of EOEE-BS. Herein, we used various genetic tests to identify pathogenic genetic variants in EOEE-BS irrespective of structural malformations and analyzed the clinical features associated with each different etiology.

Transplanted mesenchymal stem cells (MSCs) have been shown to contribute to myocardial repair after myocardial infarction (MI), primarily through production and secretion some growth factors and cytokines related to cell survival and regeneration. Further improvement of the therapeutic potential of MSCs appears to be an attractive strategy for MI treatment. CXC chemokine receptor (CXCR) 7 is the receptor for stromal cell-derived factor-1 (SDF-1), an important chemokine that is essential for tissue repair and angiogenesis. SDF-1/CXCR7 axis plays a critical role in the mobilization, recruitment and function of MSCs during tissue regeneration. Here, we depleted miR-142 that targets CXCR7 in MSCs cells through expression of antisense of miR-142, resulting in enhanced expression of CXCR7 in these miR-142-depleted MSCs (md-MSCs). In vitro, presence of md-MSCs reduced hypoxia-induced cardiac muscle cell apoptosis in a more pronounced manner than MSCs. In vivo, compared to transplantation of MSCs, transplantation of md-MSCs further enhanced cardiac re-vascularization and further improved cardiac functions after MI in mice. Together, our data suggest that depletion of miR-142 in MSCs may improve their therapeutic effects on MI.

How myosin II localizes to the cleavage furrow in Dictyostelium and metazoan cells remains largely unknown despite significant advances in understanding its regulation. We designed a genetic selection using cDNA library suppression of 3xAsp myosin II to identify factors involved in myosin cleavage furrow accumulation. The 3xAsp mutant is deficient in bipolar thick filament assembly, fails to accumulate at the cleavage furrow, cannot rescue myoII-null cytokinesis, and has impaired mechanosensitive accumulation. Eleven genes suppressed this dominant cytokinesis deficiency when 3xAsp was expressed in wild-type cells. 3xAsp myosin II's localization to the cleavage furrow was rescued by constructs encoding rcdBB, mmsdh, RMD1, actin, one novel protein, and a 14-3-3 hairpin. Further characterization showed that RMD1 is required for myosin II cleavage furrow accumulation, acting in parallel with mechanical stress. Analysis of several mutant strains revealed that different thresholds of myosin II activity are required for daughter cell symmetry than for furrow ingression dynamics. Finally, an engineered myosin II with a longer lever arm (2xELC), producing a highly mechanosensitive motor, could also partially suppress the intragenic 3xAsp. Overall, myosin II accumulation is the result of multiple parallel and partially redundant pathways that comprise a cellular contractility control system.

Chronic estrogen treatment results in elevated levels of the gelatinase pro-MMP-9 in the pituitary of tumor-susceptible Fischer 344 rats. In contrast, pituitary pro-MMP-9 level is not increased by estrogen treatment in rats of the tumor-resistant BN strain nor in rats of an F(1) hybrid of these strains. The gelatinase MMP-2 is also detected in rat pituitary, but its level is not affected by either estrogen or rat genotype. In 124 estrogen-treated rats from a backcross of the F(1) hybrid to the F344 strain, the levels of both monomer and dimer forms of pro-MMP-9 correlate with the tumor phenotypes mass, total DNA, and hemoglobin content of the pituitary. In this backcross, the QTL Edpm5 (_e_strogen-_d_ependent _p_ituitary _m_ass on Chromosome 5) has a significant effect on MMP-9 levels, with inheritance of the BN allele of Edpm5 correlating with suppression of estrogen-dependent MMP-9 expression.