[Biological threats to global malaria elimination II Deletion in the malaria rapid diagnostic test target Plasmodium falciparum histidine-rich protein 2/3 genes].

The global malaria epidemic is still severe. Because of simple procedures, rapid detection and accuracy results, rapid diagnostic test (RDT) has become the most important and the most widely used diagnostic tool for malaria prevention and control. However, deletions in the RDT target Plasmodium falciparum histidine-rich protein 2/3 (Pfhrp2/3) genes may cause false-negative results of RDT, which has been included as one of the four biological threats to global malaria elimination. This article reviews the applications of RDT in the global malaria diagnosis, analyzes the threats and challenges caused by Pfhrp2/3 gene deletion, proposes methods for monitoring Pfhrp2/3 gene deletion, and summarizes the causes and countermeasures of negative RDT detections, so as to provide insights into consolidation of malaria elimination achievements in China and contributions to global malaria elimination.

The peptidoglycan-associated lipoprotein gene mutant elicits robust immunological defense in mice against Salmonella enteritidis.

Background: Salmonella enteritidis (S. enteritidis), a zoonotic pathogen with a broad host range, presents a substantial threat to global public health safety. Vaccination stands as an effective strategy for the prevention and control of S. enteritidis infection, highlighting an immediate clinical need for the creation of safe and efficient attenuated live vaccines. Methods: In this study, a S. enteritidis peptidoglycan-associated lipoprotein (pal) gene deletion strain (Δpal), was constructed. To assess its virulence, we conducted experiments on biofilm formation capability, motility, as well as cell and mouse infection. Subsequently, we evaluated the immune-protective effect of Δpal. Results: It was discovered that deletion of the pal gene reduced the biofilm formation capability and motility of S. enteritidis. Cell infection experiments revealed that the Δpal strain exhibited significantly decreased abilities in invasion, adhesion, and intracellular survival, with downregulation of virulence gene expression, including mgtC, invH, spvB, sipA, sipB, ssaV, csgA, and pipB. Mouse infection experiments showed that the LD50 of Δpal increased by 104 times, and its colonization ability in mouse tissue organs was significantly reduced. The results indicated that the pal gene severely affected the virulence of S. enteritidis. Further, immunogenicity evaluation of Δpal showed a significant enhancement in the lymphocyte transformation proliferation capability of immunized mice, producing high titers of specific IgG and IgA, suggesting that Δpal possesses good immunogenicity. Challenge protection tests demonstrated that the strain could provide 100% immune protection against wild-type strains in mice. Discussion: This study proves that the pal gene influences the virulence of S. enteritidis, and Δpal could serve as a candidate strain for attenuated live vaccines, laying the foundation for the development of attenuated live vaccines against Salmonella.

Expanding the genetic toolbox for the obligate human pathogen Streptococcus pyogenes.

Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. To overcome this limitation and enable the straightforward investigation of gene functions in S. pyogenes, we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for S. pyogenes. The new replicative plasmids feature low- and high-copy replicons combined with different resistance cassettes and a standardized multiple cloning site for rapid cloning procedures. We designed site-specific integrative plasmids and verified their integration by nanopore sequencing. To minimize the effect of plasmid integration on bacterial physiology, we screened publicly available RNA-sequencing datasets for transcriptionally silent sites. We validated this approach by designing the integrative plasmid pSpy0K6 targeting the transcriptionally silent gene SPy_1078. Analysis of the activity of different constitutive promoters indicated a wide variety of strengths, with the lactococcal promoter P 23 showing the strongest activity and the synthetic promoter P xylS2 showing the weakest activity. Further, we assessed the functionality of three inducible regulatory elements including a zinc- and an IPTG-inducible promoter as well as an erythromycin-inducible riboswitch that showed low-to-no background expression and high inducibility. Additionally, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. We therefore adapted the chemically defined medium called RPMI4Spy that showed reduced autofluorescence and enabled efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS*, which enabled the scarless deletion of the sagB gene. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.

Probing gene function in Candida albicans wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus.

The adaptation of gene deletion methods based on the CRISPR-Cas9 system has facilitated the genetic manipulation of the pathogenic yeast Candida albicans, because homozygous mutants of this diploid fungus can now be generated in a single step, allowing the rapid screening of candidate genes for their involvement in a phenotype of interest. However, the Cas9-mediated double-strand breaks at the target site may result in an undesired loss of heterozygosity (LOH) on the affected chromosome and cause phenotypic alterations that are not related to the function of the investigated gene. In our present study, we harnessed Cas9-facilitated gene deletion to probe a set of genes that are constitutively overexpressed in strains containing hyperactive forms of the transcription factor Mrr1 for a possible contribution to the fluconazole resistance of such strains. To this aim, we used gene deletion cassettes containing two different dominant selection markers, caSAT1 and HygB, which confer resistance to nourseothricin and hygromycin, respectively, for simultaneous genomic integration in a single step, hypothesizing that this would minimize undesired LOH events at the target locus. We found that selection for resistance to both nourseothricin and hygromycin strongly increased the proportion of homozygous deletion mutants among the transformants compared with selection on media containing only one of the antibiotics, but it did not avoid undesired LOH events. Our results demonstrate that LOH on the target chromosome is a significant problem when using Cas9 for the generation of C. albicans gene deletion mutants, which demands a thorough examination of recombination events at the target site. IMPORTANCE: Candida albicans is one of the medically most important fungi and a model organism to study fungal pathogenicity. Investigating gene function in this diploid yeast has been facilitated by the adaptation of gene deletion methods based on the bacterial CRISPR-Cas9 system, because they enable the generation of homozygous mutants in a single step. We found that, in addition to increasing the efficiency of gene replacement by selection markers, the Cas9-mediated double-strand breaks also result in frequent loss of heterozygosity on the same chromosome, even when two different selection markers were independently integrated into the two alleles of the target gene. Since loss of heterozygosity for other genes can result in phenotypic alterations that are not caused by the absence of the target gene, these findings show that it is important to thoroughly analyze recombination events at the target locus when using Cas9 to generate gene deletion mutants in C. albicans.

Smad4 is essential for epiblast scaling and morphogenesis after implantation, but nonessential before implantation.

Bone morphogenic protein (BMP) signaling plays an essential and highly conserved role in embryo axial patterning in animal species. However, in mammalian embryos, which develop inside the mother, early development includes a preimplantation stage, which does not occur in externally developing embryos. During preimplantation, the epiblast is segregated from extra-embryonic lineages that enable implantation and development in utero. Yet, the requirement for BMP signaling is imprecisely defined in mouse early embryos. Here, we show that, in contrast to previous reports, BMP signaling (SMAD1/5/9 phosphorylation) is not detectable until implantation when it is detected in the primitive endoderm - an extra-embryonic lineage. Moreover, preimplantation development appears to be normal following deletion of maternal and zygotic Smad4, an essential effector of canonical BMP signaling. In fact, mice lacking maternal Smad4 are viable. Finally, we uncover a new requirement for zygotic Smad4 in epiblast scaling and cavitation immediately after implantation, via a mechanism involving FGFR/ERK attenuation. Altogether, our results demonstrate no role for BMP4/SMAD4 in the first lineage decisions during mouse development. Rather, multi-pathway signaling among embryonic and extra-embryonic cell types drives epiblast morphogenesis postimplantation.

A simplified and efficient method for single-step, targeted gene deletion in mycobacteria.

Targeted gene deletion in mycobacteria remain complicated, requiring expertise and multiple steps. Here we present a single-step, easy to understand and perform method for targeted gene deletion. Using this method, we successfully deleted several genes in both M. smegmatis and M. abscessus. We believe this method will facilitate molecular research of mycobacteria and make it accessible to a greater number of researchers throughout the world.

Development of a telomere vector-based approach to overcome limitations caused by lethal phenotypes in the study of essential genes in Magnaporthe oryzae.

Reverse genetic approaches are common tools in genomics for elucidating gene functions, involving techniques such as gene deletion followed by screening for aberrant phenotypes. If the generation of gene deletion mutants fails, the question arises whether the failure stems from technical issues or because the gene of interest (GOI) is essential, meaning that the deletion causes lethality. In this report, we introduce a novel method for assessing gene essentiality using the phytopathogenic ascomycete Magnaporthe oryzae. The method is based on the observation that telomere vectors are lost in transformants during cultivation without selection pressure. We tested the hypothesis that essential genes can be identified in deletion mutants co-transformed with a telomere vector. The M. oryzae gene MoPKC, described in literature as essential, was chosen as GOI. Using CRISPR/Cas9 technology transformants with deleted GOI were generated and backed up by a telomere vector carrying a copy of the GOI and conferring fenhexamid resistance. Transformants in which the GOI deletion in the genome was not successful lost the telomere vector on media without fenhexamid. In contrast, transformants with confirmed GOI deletion retained the telomere vector even in absence of fenhexamid selection. In the latter case, the maintenance of the telomere indicates that the GOI is essential for the surveillance of the fungi, as it would have been lost otherwise. The method presented here allows to test for essentiality of genes when no mutants can be obtained from gene deletion approaches, thereby expanding the toolbox for studying gene function in ascomycetes.

Biolistic Transformation of Cryptococcus neoformans.

Biolistic transformation of Cryptococcus neoformans is used as a molecular tool to genetically alter or delete targeted genes. The DNA is introduced into the yeast on DNA-coated gold beads by a helium shock wave produced using a biolistic particle system. The procedure often involves insertion of a dominant selectable marker into the desired site by homologous recombination. To increase the likelihood of homologous recombination, large fragments of overlapping DNA are used. The two most used dominant selectable markers are nourseothricin and Geneticin. With the need to generate multiple gene deletions in the same strain, there are recyclable marker systems, such as the bacteriophage P1 Cre-loxP system or CRISPR that provide additional useful molecular tools. While newer strategies exist to generate deletions and introduce markers and other gene modifications, biolistic transformation has remained a viable tool to facilitate the construction of genetically modified yeast strains. This chapter provides a working protocol on how to delete and restore a gene in C. neoformans.

Identification and Diagnosis of Complete Haptoglobin Gene Deletion, One of the Genes Responsible for Adverse Posttransfusion Reactions.

Allergic reactions are the most frequent adverse events in blood transfusion, and anaphylactic shock, although less frequent, is systemic and serious. The cause of allergic reactions to blood transfusions are largely unknown, but deficiencies in serum proteins such as haptoglobin (Hp) can lead to anaphylactic shock. A complete deletion of the haptoglobin gene (HPdel) was first identified in families with anomalous inheritance and then verified as a genetic variant that can cause anaphylactic shock because homozygotes for HPdel have complete Hp deficiency. Thereby, they may produce antibodies against Hp from blood transfusions. HPdel is found in East and Southeast Asian populations, with a frequency of approximately 0.9% to 4%, but not in other populations. Diagnosis of Hp deficiency due to HPdel prior to transfusion is advisable because severe adverse reactions can be prevented by washing the red blood cells and/or platelets with saline or by administering plasma products obtained from an Hp-deficient donor pool. This review outlines the background of the identification of HPdel and several genetic and immunological methods developed for diagnosing Hp deficiency caused by HPdel.

A Novel Method for Floxed Gene Manipulation Using TAT-Cre Recombinase in Ex Vivo Precision-Cut Lung Slices (PCLS).

Precision-cut lung slices (PCLS), ex vivo 3D lung tissue models, have been widely used for various applications in lung research. PCLS serve as an excellent intermediary between in vitro and in vivo models because they retain all resident cell types within their natural niche while preserving the extracellular matrix environment. This protocol describes the TReATS (TAT-Cre recombinase-mediated floxed allele modification in tissue slices) method that enables rapid and efficient gene modification in PCLS derived from adult floxed animals. Here, we present detailed protocols for the TReATS method, consisting of two simple steps: PCLS generation and incubation in a TAT-Cre recombinase solution. Subsequent validation of gene modification involves live staining and imaging of PCLS, quantitative real-time PCR, and cell viability assessment. This four-day protocol eliminates the need for complex Cre-breeding, circumvents issues with premature lethality related to gene mutation, and significantly reduces the use of animals. The TReATS method offers a simple and reproducible solution for gene modification in complex ex vivo tissue-based models, accelerating the study of gene function, disease mechanisms, and the discovery of drug targets. Key features • Achieve permanent ex vivo gene modifications in complex tissue-based models within four days. • Highly adaptable gene modification method that can be applied to induce gene deletion or activation. • Allows simple Cre dosage testing in a controlled ex vivo setting with the advantage of using PCLS generated from the same animal as true controls. • With optimisation, this method can be applied to precision-cut tissue slices of other organs.

CURE on yeast genes of unknown function increases students' bioinformatics proficiency and research confidence.

Course-based undergraduate research experiences (CUREs) can reduce barriers to research opportunities while increasing student knowledge and confidence. However, the number of widely adopted, easily transferable CUREs is relatively small. Here, we describe a CURE aimed at determining the function of poorly characterized Saccharomyces cerevisiae genes. More than 20 years after sequencing of the yeast genome, nearly 10% of open reading frames (ORFs) still have at least one uncharacterized Gene Ontology (GO) term. We refer to these genes as "ORFans" and formed a consortium aimed at assigning functions to them. Specifically, over 70 faculty members attended summer workshops to learn the bioinformatics workflow and basic laboratory techniques described herein. Ultimately, this CURE was adapted for implementation at 34 institutions, resulting in over 1,300 students conducting course-based research on ORFans. Pre-/post-tests confirmed that students gained both (i) an understanding of gene ontology and (ii) knowledge regarding the use of bioinformatics to assign gene function. After using these data to craft their own hypotheses, then testing their predictions by constructing and phenotyping deletion strains, students self-reported significant gains in several areas, including computer modeling and exposure to a project where no one knows the outcome. Interestingly, most net gains self-reported by ORFan Gene Project participants were greater than published findings for CUREs assessed with the same survey instrument. The surprisingly strong impact of this CURE may be due to the incoming lack of experience of ORFan Project participants and/or the independent thought required to develop testable hypotheses from complex data sets.

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients enrolled at 100 health facilities throughout Tanzania: February to July 2021.

Plasmodium falciparum with the histidine rich protein 2 gene (pfhrp2) deleted from its genome can escape diagnosis by HRP2-based rapid diagnostic tests (HRP2-RDTs). The World Health Organization (WHO) recommends switching to a non-HRP2 RDT for P. falciparum clinical case diagnosis when pfhrp2 deletion prevalence causes ≥ 5% of RDTs to return false negative results. Tanzania is a country of heterogenous P. falciparum transmission, with some regions approaching elimination and others at varying levels of control. In concordance with the current recommended WHO pfhrp2 deletion surveillance strategy, 100 health facilities encompassing 10 regions of Tanzania enrolled malaria-suspected patients between February and July 2021. Of 7863 persons of all ages enrolled and providing RDT result and blood sample, 3777 (48.0%) were positive by the national RDT testing for Plasmodium lactate dehydrogenase (pLDH) and/or HRP2. A second RDT testing specifically for the P. falciparum LDH (Pf-pLDH) antigen found 95 persons (2.5% of all RDT positives) were positive, though negative by the national RDT for HRP2, and were selected for pfhrp2 and pfhrp3 (pfhrp2/3) genotyping. Multiplex antigen detection by laboratory bead assay found 135/7847 (1.7%) of all blood samples positive for Plasmodium antigens but very low or no HRP2, and these were selected for genotyping as well. Of the samples selected for genotyping based on RDT or laboratory multiplex result, 158 were P. falciparum DNA positive, and 140 had sufficient DNA to be genotyped for pfhrp2/3. Most of these (125/140) were found to be pfhrp2+/pfhrp3+, with smaller numbers deleted for only pfhrp2 (n = 9) or only pfhrp3 (n = 6). No dual pfhrp2/3 deleted parasites were observed. This survey found that parasites with these gene deletions are rare in Tanzania, and estimated that 0.24% (95% confidence interval: 0.08% to 0.39%) of false-negative HRP2-RDTs for symptomatic persons were due to pfhrp2 deletions in this 2021 Tanzania survey. These data provide evidence for HRP2-based diagnostics as currently accurate for P. falciparum diagnosis in Tanzania.

Deciphering the Interrelationship of arnT Involved in Lipid-A Alteration with the Virulence of Salmonella Typhimurium.

The lipopolysaccharide (LPS) that resides on the outermost surface and protects Gram-negative bacteria from host defenses is one of the key components leading to Salmonella infection, particularly the endotoxic lipid A domain of LPS. Lipid A modifications have been associated with several genes such as the arnT that encodes 4-amino-4-deoxy-L-arabinose transferase, which can be critical for bacteria to resist cationic antimicrobial peptides and interfere with host immune recognition. However, the association of arnT with virulence is not completely understood. Thus, this study aimed to elucidate the interrelationship of the major lipid A modification gene arnT with Salmonella Typhimurium virulence. We observed that the arnT-deficient S. Typhimurium (JOL2943), compared to the wild type (JOL401), displayed a significant decrease in several virulence phenotypes such as polymyxin B resistance, intracellular survival, swarming, and biofilm and extracellular polymeric substance (EPS) production. Interestingly, the cell-surface hydrophobicity, adhesion, and invasion characteristics remained unaffected. Additionally, LPS isolated from the mutant induced notably lower levels of endotoxicity-related cytokines in RAW and Hela cells and mice, particularly IL-1ß with a nine-fold decrease, than WT. In terms of in vivo colonization, JOL2943 showed diminished presence in internal organs such as the spleen and liver by more than 60%, while ileal infectivity remained similar to JOL401. Overall, the arnT deletion rendered the strain less virulent, with low endotoxicity, maintained gut infectivity, and reduced colonization in internal organs. With these ideal characteristics, it can be further explored as a potential attenuated Salmonella strain for therapeutics or vaccine delivery systems.

CheV enhances the virulence of Salmonella Enteritidis, and the Chev-deleted Salmonella vaccine provides immunity in mice.

BACKGROUND: Salmonella enteritidis (SE) is a major zoonotic pathogen and causes infections in a variety of hosts. The development of novel vaccines for SE is necessary to eradicate this pathogen. Genetically engineered attenuated live vaccines are more immunogenic and safer. Thus, to develop a live attenuated Salmonella vaccine, we constructed a cheV gene deletion strain of SE (named ΔcheV) and investigated the role of cheV in the virulence of SE. First, the ability to resist environmental stress in vitro, biofilm formation capacity, drug resistance and motility of ΔcheV were analyzed. Secondly, the bacterial adhesion, invasion, intracellular survival assays were performed by cell model. Using a mouse infection model, an in vivo virulence assessment was conducted. To further evaluate the mechanisms implicated by the reduced virulence, qPCR analysis was utilized to examine the expression of the strain's major virulence genes. Finally, the immune protection rate of ΔcheV was evaluated using a mouse model. RESULTS: Compared to C50336, the ΔcheV had significantly reduced survival ability under acidic, alkaline and thermal stress conditions, but there was no significant difference in survival under oxidative stress conditions. There was also no significant change in biofilm formation ability, drug resistance and motility. It was found that the adhesion ability of ΔcheV to Caco-2 cells remained unchanged, but the invasion ability and survival rate in RAW264.7 cells were significantly reduced. The challenge assay results showed that the LD50 values of C50336 and ΔcheV were 6.3 × 105 CFU and 1.25 × 107 CFU, respectively. After the deletion of the cheV gene, the expression levels of fimD, flgG, csgA, csgD, hflK, lrp, sipA, sipB, pipB, invH, mgtC, sodC, rfbH, xthA and mrr1 genes were significantly reduced. The live attenuated ΔcheV provided 100% protection in mice against SE infection. CONCLUSION: All the results confirmed that the deletion of the cheV gene reduces the virulence of SE and provides significant immune protection in mice, indicating that ΔcheV could be potential candidates to be explored as live-attenuated vaccines.

Gene editing in allergic diseases: Identification of novel pathways and impact of deleting allergen genes.

Gene editing technology has emerged as a powerful tool in all aspects of health research and continues to advance our understanding of critical and essential elements in disease pathophysiology. The clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology has been used with precision to generate gene knockouts, alter genes, and identify genes that cause disease. The full spectrum of allergic/atopic diseases, in part because of shared pathophysiology, is ripe for studies with this technology. In this way, novel culprit genes are being identified and allow for manipulation of triggering allergens to reduce allergenicity and disease. Notwithstanding current limitations on precision and potential off-target effects, newer approaches are rapidly being introduced to more fully understand specific gene functions as well as the consequences of genetic manipulation. In this review, we examine the impact of editing technologies of novel genes relevant to peanut allergy and asthma as well as how gene modification of common allergens may lead to the deletion of allergenic proteins.

A child with TSC2/PKD1 contiguous gene deletion syndrome successfully treated with tolvaptan for rapidly enlarging renal cysts.

Tolvaptan, a vasopressin receptor antagonist, has been shown to be effective in the treatment of renal cysts in ADPKD. However, tolvaptan is not indicated for pediatric patients, and reports of its use are rare, making its efficacy and adverse reactions unclear. Herein, we present the case of an 11-year-old girl who had vitiligo from birth. She was diagnosed with West syndrome at 6 months of age and tuberous sclerosis at 2 years of age. At the age of 6 years, an abdominal magnetic resonance imaging (MRI) revealed multiple bilateral renal cysts, and she was diagnosed with ADPKD. Abdominal MRI scans performed at 10 years and 11 years showed rapid renal cyst enlargement, and the renal prognosis was judged to be poor. The patient was treated with tolvaptan to delay cyst exacerbation. There were no apparent adverse events after the initiation of treatment, and the MRI performed 12 months after treatment initiation showed that renal cyst enlargement was suppressed. The results suggest that tolvaptan may be effective in pediatric patients with severe ADPKD who have rapidly enlarging renal cysts, although evaluation of renal cyst enlargement and side effects should be continued.

[Production of adeno-associated virus in insect cells using multiple gene deleted baculovirus].

Adeno-associated virus (AAV) is one of the most frequently used viral vectors in the field of gene therapy. However, the industrial production of AAV is facing key bottlenecks such as low yield and high-cost. The aim of this study was to establish a technology system for production of AAV in the double virus infected insects by using multiple-gene deleted baculovirus. First, a multiple gene deleted baculovirus for AAV production was constructed, and the baculovirus titer and its effect on infected cells was examined. Subsequently, the insect cells were co-infected with the double baculovirus and the infection conditions were optimized. At the final stage, we performed AAV production based on optimized conditions, and evaluated relevant parameters including production titer and quality. The results showed that the titer of AAV produced in the multiple gene deleted baculovirus was not different from that of the wild type, but the rate of cell death was significantly slower upon infection. Using the double virus route for optimized production of AAV, the genome titers were 1.63×1011 VG/mL for Bac4.0-1 and 1.02×1011 VG/mL for Bac5.0-2, which were elevated 240% and 110%, respectively, compared with that of the wild-type. Electron microscopy observations revealed that all three groups exhibited normal AAV viral morphology and they showed similar transduction activity. Taken together, we developed an AAV production system based on the infection of insect cells using multiple-gene deleted baculovirus, which significantly improved the virus yield and showed application potential.

Isolation and draft genome sequence of Enterobacter asburiae strain i6 amenable to genetic manipulation.

Enterobacter asburiae is a species of Gram-negative bacteria that is found in soil, water, and sewage. E. asburiae is generally considered to be an opportunistic pathogen, but has also been reported as a plant growth-promoting bacterium (PGPB), which may have beneficial effects on plant growth and development. However, genetic analysis of E. asburiae has been limited, possibly due to its redundant enzymes that digest exogenous DNA in the cell. Here, an E. asburiae strain i6 was isolated from soil in Nara, Japan. This strain was amenable to transformation and the one-step gene inactivation method based on λ Red recombinase. The transformation efficiency of the i6 strain with the 10 kb plasmid DNA pCF430 was at least four orders of magnitude higher than that of the previously sequenced E. asburiae strain ATCC 35953, which could not be transformed with the same plasmid DNA. A draft genome sequence of the i6 strain was determined and deposited into the database, allowing several factors that may determine transformation efficiency to be perturbed and tested. Together with the amenability of the i6 strain to genetic manipulation, the information from the i6 genome will facilitate characterization and fine-tuning of the beneficial and detrimental traits of this species.

Smad4 is essential for epiblast scaling and morphogenesis after implantation, but nonessential prior to implantation in the mouse.

Bone Morphogenic Protein (BMP) signaling plays an essential and highly conserved role in axial patterning in embryos of many externally developing animal species. However, in mammalian embryos, which develop inside the mother, early development includes an additional stage known as preimplantation. During preimplantation, the epiblast lineage is segregated from the extraembryonic lineages that enable implantation and development in utero. Yet, the requirement for BMP signaling in mouse preimplantation is imprecisely defined. We show that, in contrast to prior reports, BMP signaling (as reported by SMAD1/5/9 phosphorylation) is not detectable until implantation, when it is detected in the primitive endoderm - an extraembryonic lineage. Moreover, preimplantation development appears normal following deletion of maternal and zygotic Smad4, an essential effector of BMP signaling. In fact, mice lacking maternal Smad4 are viable. Finally, we uncover a new requirement for zygotic Smad4 in epiblast scaling and cavitation immediately after implantation, via a mechanism involving FGFR/ERK attenuation. Altogether, our results demonstrate no role for BMP4/SMAD4 in the first lineage decisions during mouse development. Rather, multi-pathway signaling among embryonic and extraembryonic cell types drives epiblast morphogenesis post-implantation.

Two-step regulation by matrix Gla protein in brown adipose cell differentiation.

OBJECTIVE: Bone morphogenetic protein (BMP) signaling is intricately involved in adipose tissue development. BMP7 together with BMP4 have been implicated in brown adipocyte differentiation but their roles during development remains poorly specified. Matrix Gla protein (MGP) inhibits BMP4 and BMP7 and is expressed in endothelial and progenitor cells. The objective was to determine the role of MGP in brown adipose tissue (BAT) development. METHODS: The approach included global and cell-specific Mgp gene deletion in combination with RNA analysis, immunostaining, thermogenic activity, and in vitro studies. RESULTS: The results revealed that MGP directs brown adipogenesis at two essential steps. Endothelial-derived MGP limits triggering of white adipogenic differentiation in the perivascular region, whereas MGP derived from adipose cells supports the transition of CD142-expressing progenitor cells to brown adipogenic maturity. Both steps were important to optimize the thermogenic function of BAT. Furthermore, MGP derived from both sources impacted vascular growth. Reduction of MGP in either endothelial or adipose cells expanded the endothelial cell population, suggesting that MGP is a factor in overall plasticity of adipose tissue. CONCLUSION: MGP displays a dual and cell-specific function in BAT, essentially creating a "cellular shuttle" that coordinates brown adipogenic differentiation with vascular growth during development.