Cell state dependent effects of Bmal1 on melanoma immunity and tumorigenicity.

The circadian clock regulator Bmal1 modulates tumorigenesis, but its reported effects are inconsistent. Here, we show that Bmal1 has a context-dependent role in mouse melanoma tumor growth. Loss of Bmal1 in YUMM2.1 or B16-F10 melanoma cells eliminates clock function and diminishes hypoxic gene expression and tumorigenesis, which could be rescued by ectopic expression of HIF1α in YUMM2.1 cells. By contrast, over-expressed wild-type or a transcriptionally inactive mutant Bmal1 non-canonically sequester myosin heavy chain 9 (Myh9) to increase MRTF-SRF activity and AP-1 transcriptional signature, and shift YUMM2.1 cells from a Sox10high to a Sox9high immune resistant, mesenchymal cell state that is found in human melanomas. Our work describes a link between Bmal1, Myh9, mouse melanoma cell plasticity, and tumor immunity. This connection may underlie cancer therapeutic resistance and underpin the link between the circadian clock, MRTF-SRF and the cytoskeleton.

Na+/H+-exchanger 1 enhances antitumor activity of engineered NK-92 natural killer cells.

Adoptive cell transfer (ACT) immunotherapy has remarkable efficacy against some hematological malignancies. However, its efficacy in solid tumors is limited by the adverse tumor microenvironment (TME) conditions, most notably that acidity inhibits T and natural killer (NK) cell mTOR complex 1 (mTORC1) activity and impairs cytotoxicity. In several reported studies, systemic buffering of tumor acidity enhanced the efficacy of immune checkpoint inhibitors. Paradoxically, we found in a c-Myc-driven hepatocellular carcinoma model that systemic buffering increased tumor mTORC1 activity, negating inhibition of tumor growth by anti-PD1 treatment. Therefore, in this proof-of-concept study, we tested the metabolic engineering of immune effector cells to mitigate the inhibitory effect of tumor acidity while avoiding side effects associated with systemic buffering. We first overexpressed an activated RHEB in the human NK cell line NK-92, thereby rescuing acid-blunted mTORC1 activity and enhancing cytolytic activity. Then, to directly mitigate the effect of acidity, we ectopically expressed acid extruder proteins. Whereas ectopic expression of carbonic anhydrase IX (CA9) moderately increased mTORC1 activity, it did not enhance effector function. In contrast, overexpressing a constitutively active Na+/H+-exchanger 1 (NHE1; SLC9A1) in NK-92 did not elevate mTORC1 but enhanced degranulation, target engagement, in vitro cytotoxicity, and in vivo antitumor activity. Our findings suggest the feasibility of overcoming the inhibitory effect of the TME by metabolically engineering immune effector cells, which can enhance ACT for better efficacy against solid tumors.

First international external quality assessment scheme of nucleic acid amplification tests for the detection of Schistosoma and soil-transmitted helminths, including Strongyloides: A pilot study.

BACKGROUND: Nucleic acid amplification tests (NAATs) are increasingly being used as diagnostic tools for soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Ancylostoma duodenale and A. ceylanicum), Strongyloides stercoralis and Schistosoma in human stool. Currently, there is a large diversity of NAATs being applied, but an external quality assessment scheme (EQAS) for these diagnostics is lacking. An EQAS involves a blinded process where test results reported by a laboratory are compared to those reported by reference or expert laboratories, allowing for an objective assessment of the diagnostic performance of a laboratory. In the current study, we piloted an international EQAS for these helminths (i) to investigate the feasibility of designing and delivering an EQAS; (ii) to assess the diagnostic performance of laboratories; and (iii) to gain insights into the different NAAT protocols used. METHODS AND PRINCIPAL FINDINGS: A panel of twelve stool samples and eight DNA samples was validated by six expert laboratories for the presence of six helminths (Ascaris, Trichuris, N. americanus, Ancylostoma, Strongyloides and Schistosoma). Subsequently this panel was sent to 15 globally dispersed laboratories. We found a high degree of diversity among the different DNA extraction and NAAT protocols. Although most laboratories performed well, we could clearly identify the laboratories that were poorly performing. CONCLUSIONS/SIGNIFICANCE: We showed the technical feasibility of an international EQAS for the NAAT of STHs, Strongyloides and Schistosoma. In addition, we documented that there are clear benefits for participating laboratories, as they can confirm and/or improve the diagnostic performance of their NAATs. Further research should aim to identify factors that explain poor performance of NAATs.

Liposome-based transfection enhances RNAi and CRISPR-mediated mutagenesis in non-model nematode systems.

Nematodes belong to one of the most diverse animal phyla. However, functional genomic studies in nematodes, other than in a few species, have often been limited in their reliability and success. Here we report that by combining liposome-based technology with microinjection, we were able to establish a wide range of genomic techniques in the newly described nematode genus Auanema. The method also allowed heritable changes in dauer larvae of Auanema, despite the immaturity of the gonad at the time of the microinjection. As proof of concept for potential functional studies in other nematode species, we also induced RNAi in the free-living nematode Pristionchus pacificus and targeted the human parasite Strongyloides stercoralis.

Heritable genetic transformation of Strongyloides stercoralis by microinjection of plasmid DNA constructs into the male germline.

Heretofore, transgenesis in the parasitic nematode genus Strongyloides has relied on microinjecting transgene constructs into gonadal syncytia of free-living females. We now report transgenesis in Strongyloides stercoralis by microinjecting constructs into the syncytial testes of free-living males. Crosses of individual males microinjected with a construct encoding GFP with cohorts of 12 non-injected females produced a mean of 7.28±2.09 transgenic progeny. Progeny of males and females microinjected with distinct reporter constructs comprised 2.6%±0.7% of individuals expressing both paternal and maternal transgenes. Implications of this finding for deployment of CRISPR/Cas9 mutagenesis in Strongyloides spp. are discussed.

Detection of piggyBac-mediated Transposition by Splinkerette PCR in Transgenic Lines of Strongyloides ratti.

Splinkerette PCR (spPCR) is a newly developed and efficient method to ascertain and characterize genomic insertion sites of transgenes. The method described in this protocol was successfully applied to confirm piggyBac transposon-mediated integration of transgenes into chromosomes of the parasitic nematode Strongyloides ratti. This work is described in detail in Shao et al. (2012) and presented here in a simplified diagram (Figure 1). Using this method, chromosomal loci of integration were determined based on target site and 5'- and 3' flanking sequences. Therefore, spPCR can be a useful method to confirm integrative transgenesis in functional genomic studies of parasitic nematodes. Potter and Luo (2010) contains a protocol for use of spPCR to detect and map piggyBac transposon-mediated chromosomal integrations in Drosophila, and was the source of our method for Strongyloides. The splinkerette- and piggyBac-specific oligos described in that reference could be used without modification in Strongyloides. For interested readers, a general review of the biology of parasitic nematodes in the genus Strongyloides may be found in Viney and Lok (2007), and a methods-based article on S. stercoralis as an experimental model, with information on transgenesis, may be found in Lok (2007).

Transposon-mediated chromosomal integration of transgenes in the parasitic nematode Strongyloides ratti and establishment of stable transgenic lines.

Genetic transformation is a potential tool for analyzing gene function and thereby identifying new drug and vaccine targets in parasitic nematodes, which adversely affect more than one billion people. We have previously developed a robust system for transgenesis in Strongyloides spp. using gonadal microinjection for gene transfer. In this system, transgenes are expressed in promoter-regulated fashion in the F1 but are silenced in subsequent generations, presumably because of their location in repetitive episomal arrays. To counteract this silencing, we explored transposon-mediated chromosomal integration of transgenes in S. ratti. To this end, we constructed a donor vector encoding green fluorescent protein (GFP) under the control of the Ss-act-2 promoter with flanking inverted tandem repeats specific for the piggyBac transposon. In three experiments, free-living Strongyloides ratti females were transformed with this donor vector and a helper plasmid encoding the piggyBac transposase. A mean of 7.9% of F1 larvae were GFP-positive. We inoculated rats with GFP-positive F1 infective larvae, and 0.5% of 6014 F2 individuals resulting from this host passage were GFP-positive. We cultured GFP-positive F2 individuals to produce GFP-positive F3 L3i for additional rounds of host and culture passage. Mean GFP expression frequencies in subsequent generations were 15.6% in the F3, 99.0% in the F4, 82.4% in the F5 and 98.7% in the F6. The resulting transgenic lines now have virtually uniform GFP expression among all progeny after at least 10 generations of passage. Chromosomal integration of the reporter transgenes was confirmed by Southern blotting and splinkerette PCR, which revealed the transgene flanked by S. ratti genomic sequences corresponding to five discrete integration sites. BLAST searches of flanking sequences against the S. ratti genome revealed integrations in five contigs. This result provides the basis for two powerful functional genomic tools in S. ratti: heritable transgenesis and insertional mutagenesis.

Transgenesis in the parasitic nematode Strongyloides ratti.

Strongyloides and related genera are advantageous subjects for transgenesis in parasitic nematodes, primarily by gonadal microinjection as has been used with Caenorhabditis elegans. Transgenesis has been achieved in Strongyloides stercoralis and in Parastrongyloides trichosuri, but both of these lack well-adapted, conventional laboratory hosts in which to derive transgenic lines. By contrast, Strongyloides ratti develops in laboratory rats with high efficiency and offers the added advantages of robust genomic and transcriptomic databases and substantial volumes of genetic, developmental and immunological data. Therefore, we evaluated methodology for transgenesis in S. stercoralis as a means of transforming S. ratti. S. stercoralis-based GFP reporter constructs were expressed in a proportion of F1 transgenic S. ratti following gonadal microinjection into parental free-living females. Frequencies of transgene expression in S. ratti, ranged from 3.7% for pAJ09 to 6.8% for pAJ20; respective frequencies for these constructs in S. stercoralis were 5.6% and 33.5%. Anatomical patterns of transgene expression were virtually identical in S. ratti and S. stercoralis. This is the first report of transgenesis in S. ratti, an important model organism for biological investigations of parasitic nematodes. Availability of the rat as a well-adapted laboratory host will facilitate derivation of transgenic lines of this parasite.

Involvement of T1R3 in calcium-magnesium taste.

Calcium and magnesium are essential for survival but it is unknown how animals detect and consume enough of these minerals to meet their needs. To investigate this, we exploited the PWK/PhJ (PWK) strain of mice, which, in contrast to the C57BL/6J (B6) and other inbred strains, displays strong preferences for calcium solutions. We found that the PWK strain also has strong preferences for MgCl2 and saccharin solutions but not representative salty, sour, bitter, or umami taste compounds. A genome scan of B6 x PWK F2 mice linked a component of the strain difference in calcium and magnesium preference to distal chromosome 4. The taste receptor gene, Tas1r3, was implicated by studies with 129.B6ByJ-Tas1r3 congenic and Tas1r3 knockout mice. Most notably, calcium and magnesium solutions that were avoided by wild-type B6 mice were preferred (relative to water) by B6 mice null for the Tas1r3 gene. Oral calcium elicited less electrophysiological activity in the chorda tympani nerve of Tas1r3 knockout than wild-type mice. Comparison of the sequence of Tas1r3 with calcium and saccharin preferences in inbred mouse strains found 1) an inverse correlation between calcium and saccharin preference scores across primarily domesticus strains, which was associated with an I60T substitution in T1R3, and 2) a V689A substitution in T1R3 that was unique to the PWK strain and thus may be responsible for its strong calcium and magnesium preference. Our results imply that, in addition to its established roles in the detection of sweet and umami compounds, T1R3 functions as a gustatory calcium-magnesium receptor.

Genetic loci affecting body weight and fatness in a C57BL/6J x PWK/PhJ mouse intercross.

To determine the genetic variation that contributes to body composition in the mouse, we interbred a wild-derived strain (PWK/PhJ; PWK) with a common laboratory strain (C57BL/6J; B6). The parental, F(1), and F(2) mice were phenotyped at 18 weeks old for body weight and composition using dual-energy X-ray absorptiometry (DEXA). A total of 479 (244 male and 235 female) F(2) mice were genotyped for 117 polymorphic markers spanning the autosomes. Twenty-eight suggestive or significant linkages for four traits (body weight, adjusted lean and fat weight, and percent fat) were detected. Of these, three QTLs were novel: one on the proximal portion of Chr 5 for body weight (Bwq8; LOD = 4.7), one on Chr 3 for lean weight (Bwtq13; LOD = 3.6), and one on Chr 11 for percent fat (Adip19; LOD = 5.8). The remaining QTLs overlapped previously identified linkages, e.g., Adip5 on Chr 9. One QTL was sex-specific (present in males only) and seven were sex-biased (more prominent in one sex than the other). Most alleles that increased body weight were contributed by the B6 strain, and most alleles that increased percent fat were contributed by the PWK strain. Eight pairs of interacting loci were identified, none of which exactly overlapped the main-effect QTLs. Many of the QTLs found in the B6 x PWK cross map to the location of previously reported linkages, suggesting that some QTLs are common to many strains (consensus QTLs), but three new QTLs appear to be particular to the PWK strain. The location and type of QTLs detected in this new cross will assist in future efforts to identify the genetic variation that determines the ratio of lean to fat weight as well as body size in mice.

Evolution and horizontal transfer of a DD37E DNA transposon in mosquitoes.

ITmD37E, a unique class II transposable element (TE) with an ancient origin, appears to have been involved in multiple horizontal transfers in mosquitoes as ITmD37E sequences from 10 mosquito species of five genera share high nucleotide (nt) identities. For example, ITmD37E sequences from Aedes aegypti and Anopheles gambiae, which have an estimated common ancestor of 145-200 million years ago, display 92% nt identity. The comparison of ITmD37E and host mosquito phylogenies shows a lack of congruence. The wide distribution of conserved ITmD37Es in mosquitoes and the presence of intact copies suggest that this element may have been recently active.

Random mutagenesis of proximal mouse chromosome 5 uncovers predominantly embryonic lethal mutations.

A region-specific ENU mutagenesis screen was conducted to elucidate the functional content of proximal mouse Chr 5. We used the visibly marked, recessive, lethal inversion Rump White (Rw) as a balancer in a three-generation breeding scheme to identify recessive mutations within the approximately 50 megabases spanned by Rw. A total of 1003 pedigrees were produced, representing the largest inversion screen performed in mice. Test-class animals, homozygous for the ENU-mutagenized proximal Chr 5 and visibly distinguishable from nonhomozygous littermates, were screened for fertility, hearing, vestibular function, DNA repair, behavior, and dysmorphology. Lethals were identifiable by failure to derive test-class animals within a pedigree. Embryonic lethal mutations (total of 34) were overwhelmingly the largest class of mutants recovered. We characterized them with respect to the time of embryonic death, revealing that most act at midgestation (8.5-10.5) or sooner. To position the mutations within the Rw region and to guide allelism tests, we performed complementation analyses with a set of new and existing chromosomal deletions, as well as standard recombinational mapping on a subset of the mutations. By pooling the data from this and other region-specific mutagenesis projects, we calculate that the mouse genome contains approximately 3479-4825 embryonic lethal genes, or about 13.7%-19% of all genes.

Intra- and inter-specific diversity of Tc3-like transposons in nematodes and insects and implications for their evolution and transposition.

Tc3 of Caenorhabditis elegans is one of the founding members of the Tc1 family which includes DNA transposons in vertebrates, insects, nematodes and fungi. It is one of the best characterized eukaryotic transposons in terms of structure and transposition mechanism. A Tc3-like transposon MsqTc3 has been recently described in a mosquito. Here we present the characterization of a number of Tc3-like transposons in C. elegans, Caenorhabditis briggsae, and Drosophila melanogaster, which has revealed high levels of inter- and intra-specific diversity and further suggests a broad distribution of the Tc3-like transposons. These newly defined transposons and the previously described Tc3 and MsqTc3 form a highly divergent yet distinct clade in the Tc1 family. The above phylogenetic analysis of the Tc3-like transposons and their high levels of intra-specific diversity underscore interesting questions of their evolutionary dynamics in their respective hosts. The majority of the Tc3-like transposons contain two putative binding sites for their transposases. The first is near the terminus and the second is approximately 164-184 bp from the first site. Comparative analysis suggests that the second binding site may have been maintained for an important function in vivo. There is a large amount of variation in the length (27-566 bp) and structure of the terminal inverted repeats (TIRs) of Tc3-like transposons. Long (318-566 bp) TIRs that extend significantly beyond the second binding site are only found in the first described Tc3 and its close relatives, whose transposases form a recently derived clade among the Tc3-like transposons. Thus, these unique TIRs may have evolved recently together with their corresponding transposases.