Receptor-interacting Ser/Thr kinase 1 (RIPK1) and myosin IIA-dependent ceramidosomes form membrane pores that mediate blebbing and necroptosis.

Formation of membrane pores/channels regulates various cellular processes, such as necroptosis or stem cell niche signaling. However, the roles of membrane lipids in the formation of pores and their biological functions are largely unknown. Here, using the cellular stress model evoked by the sphingolipid analog drug FTY720, we show that formation of ceramide-enriched membrane pores, referred to here as ceramidosomes, is initiated by a receptor-interacting Ser/Thr kinase 1 (RIPK1)-ceramide complex transported to the plasma membrane by nonmuscle myosin IIA-dependent trafficking in human lung cancer cells. Molecular modeling/simulation coupled with site-directed mutagenesis revealed that Asp147 or Asn169 of RIPK1 are key for ceramide binding and that Arg258 or Leu293 residues are involved in the myosin IIA interaction, leading to ceramidosome formation and necroptosis. Moreover, generation of ceramidosomes independently of any external drug/stress stimuli was also detected in the plasma membrane of germ line stem cells in ovaries during the early stages of oogenesis in Drosophila melanogaster Inhibition of ceramidosome formation via myosin IIA silencing limited germ line stem cell signaling and abrogated oogenesis. In conclusion, our findings indicate that the RIPK1-ceramide complex forms large membrane pores we named ceramidosomes. They further suggest that, in addition to their roles in stress-mediated necroptosis, these ceramide-enriched pores also regulate membrane integrity and signaling and might also play a role in D. melanogaster ovary development.

A critical role for nucleoporin 358 (Nup358) in transposon silencing and piRNA biogenesis in Drosophila.

Piwi-interacting RNAs (piRNAs) are a class of small noncoding RNAs that bind Piwi proteins to silence transposons and to regulate gene expression. In Drosophila germ cells, the Aubergine (Aub)-Argonaute 3 (Ago3)-dependent ping-pong cycle generates most germline piRNAs. Loading of antisense piRNAs amplified by this cycle enables Piwi to enter the nucleus and silence transposons. Nuclear localization is crucial for Piwi function in transposon silencing, but how this process is regulated remains unknown. It is also not known whether any of the components of the nuclear pore complex (NPC) directly function in the piRNA pathway. Here, we show that nucleoporin 358 (Nup358) and Piwi interact with each other and that a germline knockdown (GLKD) of Nup358 with short hairpin RNA prevents Piwi entry into the nucleus. The Nup358 GLKD also activated transposons, increased genomic instability, and derailed piRNA biogenesis because of a combination of decreased piRNA precursor transcription and a collapse of the ping-pong cycle. Our results point to a critical role for Nup358 in the piRNA pathway, laying the foundation for future studies to fully elucidate the mechanisms by which Nup358 contributes to piRNA biogenesis and transposon silencing.

mRNA detection in budding yeast with single fluorophores.

Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells. Single-molecule mRNA Fluorescence In Situ Hybridization (FISH) has been established as the standard method for this purpose, but current protocols require a long region of mRNA to be targeted by multiple DNA probes. Here, we introduce a new single-probe FISH protocol termed sFISH for budding yeast, Saccharomyces cerevisiae using a single DNA probe labeled with a single fluorophore. In sFISH, we markedly improved probe specificity and signal-to-background ratio by using methanol fixation and inclined laser illumination. We show that sFISH reports mRNA changes that correspond to protein levels and gene copy number. Using this new FISH protocol, we can detect >50% of the total target mRNA. We also demonstrate the versatility of sFISH using FRET detection and mRNA isoform profiling as examples. Our FISH protocol with single-fluorophore sensitivity significantly reduces cost and time compared to the conventional FISH protocols and opens up new opportunities to investigate small changes in RNA at the single cell level.

Co-chaperone Hsp70/Hsp90-organizing protein (Hop) is required for transposon silencing and Piwi-interacting RNA (piRNA) biogenesis.

Piwi-interacting RNAs (piRNAs) are 26-30-nucleotide germ line-specific small non-coding RNAs that have evolutionarily conserved function in mobile genetic element (transposons) silencing and maintenance of genome integrity. Drosophila Hsp70/90-organizing protein homolog (Hop), a co-chaperone, interacts with piRNA-binding protein Piwi and mediates silencing of phenotypic variations. However, it is not known whether Hop has a direct role in piRNA biogenesis and transposon silencing. Here, we show that knockdown of Hop in the germ line nurse cells (GLKD) of Drosophila ovaries leads to activation of transposons. Hop GLKD females can lay eggs at the same rate as wild-type counterparts, but the eggs do not hatch into larvae. Hop GLKD leads to the accumulation of γ-H2Av foci in the germ line, indicating increased DNA damage in the ovary. We also show that Hop GLKD-induced transposon up-regulation is due to inefficient piRNA biogenesis. Based on these results, we conclude that Hop is a critical component of the piRNA pathway and that it maintains genome integrity by silencing transposons.

Isolation and characterization of Vagococcus sp from midgut of Culex quinquefasciatus (Say) mosquito.

BACKGROUND & OBJECTIVES: Mosquito gut is a rich source of microorganisms. These microorganisms exhibit close association and contribute various physiological processes taking place in mosquito gut. The present study is aimed to characterize two bacterial isolates M19 and GB11 recovered from the gut of Culex quinquefasciatus mosquito collected from Bhuj and Jamnagar districts of Gujarat, India. METHODS: Both the strains were characterized using polyphasic approach including, phenotypic characterization, whole cell protein profiling and sequencing of 16S rRNA gene and groESL region. RESULTS: Sequences of 16S rRNA gene of M19 and GB11 were 99% similar to Vagococcus carniphilus and Vagococcus fluvialis. But phenotypic profile, whole cell protein profile and sequence of groESL region of both isolates were found to be similar to V. fluvialis. CONCLUSION: Based on phenotypic, genotypic and protein profiling, both the strains were identified as V. fluvialis. So far this species was known from domestic animals and human sources only. This is the first report of V. fluvialis inhabiting midgut of Cx. quinquefasciatus mosquito collected from Arabian sea coastal of India.

Hexavalent chromium-induced epigenetic instability and transposon activation lead to phenotypic variations and tumors in Drosophila.

Developmental robustness represents the ability of an organism to resist phenotypic variations despite environmental insults and inherent genetic variations. Derailment of developmental robustness leads to phenotypic variations that can get fixed in a population for many generations. Environmental pollution is a significant worldwide problem with detrimental consequences of human development. Understanding the genetic basis for how pollutants affect human development is critical for developing interventional therapies. Here, we report that environmental stress induced by hexavalent chromium, Cr(VI), a potent industrial pollutant, compromises developmental robustness, leading to phenotypic variations in the progeny. These phenotypic variations arise due to epigenetic instability and transposon activation in the somatic tissues of the progeny rather than novel genetic mutations and can be reduced by increasing the dosage of Piwi - a Piwi-interacting RNA-binding protein, in the ovary of the exposed mother. Significantly, the derailment of developmental robustness by Cr(VI) exposure leads to tumors in the progeny, and the predisposition to develop tumors is fixed in the population for at least three generations. Thus, we show for the first time that environmental pollution can derail developmental robustness and predispose the progeny of the exposed population to develop phenotypic variations and tumors.

Direct NP- A cost-effective extraction-free RT-qPCR based test for SARS-CoV-2.

Over 2.4 million daily total tests are currently being performed for SARS-CoV-2, in the United States. The most common SARS-CoV-2 tests require RNA extraction and purification. Extraction of RNA is a time-consuming and costly step that requires a constant supply of reagents and accessories. With the current testing demand, the supply chain remains the bottleneck for RNA extraction. Here, we report Direct NP- a cost-effective extraction-free RT-qPCR based dualplex test for SARS-CoV-2 from Nasopharyngeal (NP) swab specimens. Direct NP detects SARS-CoV-2 viral RNA from heat-denatured patient specimens using a dualplex RT-qPCR assay. Direct NP showed 92.5% positive percentage agreement (PPA) (95% Confidence Interval (CI) = 79.61%-98.43%) and 97% negative percent agreement (NPA) (95% CI = 89.11-100%) with the CDC assay. Direct NP reduces the cost per test to $2, making it suitable for broad-scale testing while lowering the cost burden on the healthcare system.

Carbon Monoxide Activates PERK-Regulated Autophagy to Induce Immunometabolic Reprogramming and Boost Antitumor T-cell Function.

Mitochondria and endoplasmic reticulum (ER) share structural and functional networks and activate well-orchestrated signaling processes to shape cells' fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS promotes mitochondrial function. Strategies to boost antitumor T-cell function by targeting ER-mitochondria cross-talk have not yet been exploited. Here, we used carbon monoxide (CO), a short-lived gaseous molecule, to test whether engaging moderate ERS conditions can improve mitochondrial and antitumor functions in T cells. In melanoma antigen-specific T cells, CO-induced transient activation of ERS sensor protein kinase R-like endoplasmic reticulum kinase (PERK) significantly increased antitumor T-cell function. Furthermore, CO-induced PERK activation temporarily halted protein translation and induced protective autophagy, including mitophagy. The use of LC3-GFP enabled differentiation between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter the process (LC3-GFPneg). LC3-GFPpos T cells showed strong antitumor potential, whereas LC3-GFPneg cells exhibited a T regulatory-like phenotype, harbored dysfunctional mitochondria, and accumulated abnormal metabolite content. These anomalous ratios of metabolites rendered the cells with a hypermethylated state and distinct epigenetic profile, limiting their antitumor activity. Overall, this study shows that ERS-activated autophagy pathways modify the mitochondrial function and epigenetically reprogram T cells toward a superior antitumor phenotype to achieve robust tumor control. SIGNIFICANCE: Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.

Bacterial kinetics-controlled shape-directed biosynthesis of silver nanoplates using Morganella psychrotolerans.

We show for the first time that by controlling the growth kinetics of Morganella psychrotolerans, a silver-resistant psychrophilic bacterium, the shape anisotropy of silver nanoparticles can be achieved. This is particularly important considering that there has been no report that demonstrates a control over shape of Ag nanoparticles by controlling the growth kinetics of bacteria during biological synthesis. Additionally, we have for the first time performed electrochemistry experiments on bacterial cells after exposing them to Ag(+) ions, which provide significant new insights about mechanistic aspects of Ag reduction by bacteria. The possibility to achieve nanoparticle shape control by using a "green" biosynthesis approach is expected to open up new exciting avenues for eco-friendly, large-scale, and economically viable shape-controlled synthesis of nanomaterials.

Biological synthesis of metallic nanoparticles.

The synthesis of metallic nanoparticles is an active area of academic and, more importantly, "application research" in nanotechnology. A variety of chemical and physical procedures could be used for synthesis of metallic nanoparticles. However, these methods are fraught with many problems including use of toxic solvents, generation of hazardous by-products, and high energy consumption. Accordingly, there is an essential need to develop environmentally benign procedures for synthesis of metallic nanoparticles. A promising approach to achieve this objective is to exploit the array of biological resources in nature. Indeed, over the past several years, plants, algae, fungi, bacteria, and viruses have been used for production of low-cost, energy-efficient, and nontoxic metallic nanoparticles. In this review, we provide an overview of various reports of synthesis of metallic nanoparticles by biological means. FROM THE CLINICAL EDITOR: This review provides an overview of various methods of synthesis of metallic nanoparticles by biological means. Many chemical and physical procedures used for synthesis of metallic nanoparticles are fraught with major problems: toxic solvents, hazardous by-products, high energy consumption. Over the past several years, plants, algae, fungi, bacteria, and viruses have been used for production of low-cost, energy-efficient, and nontoxic metallic nanoparticles.

Tracking the influence of long-term chromium pollution on soil bacterial community structures by comparative analyses of 16S rRNA gene phylotypes.

Bacterial community structures of highly chromium-polluted industrial landfill sites (G1 and G2) and a nearby control site (G3) were assessed using cultivation-dependent and cultivation-independent analyses. Sequencing of 16S rRNA genes discerned a total of 141 distinct operational taxonomic units (OTUs). Twelve different bacterial phyla were represented amongst 35, 34 and 72 different bacterial genera retrieved from sites G1, G2 and G3, respectively. The bacterial community of site G1 consisted of Firmicutes (52.75%), Gammaproteobacteria (18%), Actinobacteria (14.5%), Bacteriodetes (9.5%) and Deinococcus-Thermus (5.25%) and that of site G2 consisted of Firmicutes (31.25%), Alphaproteobacteria (7%), Betaproteobacteria (8%), Gammaproteobacteria (19%), Deltaproteobacteria (9.5%), Epsilonproteobacteria (3%), Actinobacteria (13%), Bacteriodetes (7.75%) and Deinococcus-Thermus (1.5%). The bacterial community of site G3 consisted of Firmicutes (6.25%), Alphaproteobacteria (7.5%), Betaproteobacteria (17.25%), Gammaproteobacteria (29.75%), Deltaproteobacteria (7.5%), Epsilonproteobacteria (4%), Actinobacteria (9.5%), Bacteriodetes (11.25%), Gemmatimonadetes (2.5%), Deinococcus-Thermus (1.8%), Chloroflexi (1.5%) and Planctomycetes (1.2%). The phyla of Gemmatimonadetes, Chloroflexi and Planctomycetes were not detected in sites G1 and G2; likewise, Alpha, Beta, Delta and Epsilon subdivisions of Proteobacteria were not recovered from site G1. These findings reveal that long-term chromium-induced perturbation results in community shifts towards a dominance of Firmicutes from Proteobacteria in the soil environment.

Extracellular synthesis of crystalline silver nanoparticles and molecular evidence of silver resistance from Morganella sp.: towards understanding biochemical synthesis mechanism.

There has been significant progress in the biological synthesis of nanomaterials. However, the molecular mechanism of synthesis of such bio-nanomaterials remains largely unknown. Here, we report the extracellular synthesis of crystalline silver nanoparticles (AgNPs) by using Morganella sp., and show molecular evidence of silver resistance by elucidating the synthesis mechanism. The AgNPs were 20+/-5 nm in diameter and were highly stable at room temperature. The kinetics of AgNPs formation was investigated. Detectable particles were formed after an hour of reaction, and their production remained exponential up to 18 h, and saturated at 24 h. Morganella sp. was found to be highly resistant to silver cations and was able to grow in the presence of more than 0.5 mM AgNO(3). Three gene homologues viz. silE, silP and silS were identified in silver-resistant Morganella sp. The homologue of silE from Morganella sp. showed 99 % nucleotide sequence similarity with the previously reported gene, silE, which encodes a periplasmic silver-binding protein. The homologues of silP and silS were also highly similar to previously reported sequences. Similar activity was totally absent in closely related Escherichia coli; this suggests that a unique mechanism of extracellular AgNPs synthesis is associated with silver-resistant Morganella sp. The molecular mechanism of silver resistance and its gene products might have a key role to play in the overall synthesis process of AgNPs by Morganella sp. An understanding of such biochemical mechanisms at the molecular level might help in developing an ecologically friendly and cost-effective protocol for microbial AgNPs synthesis.

Bacterial synthesis of copper/copper oxide nanoparticles.

A bacterial mediated synthesis of copper/copper oxide nanoparticle composite is reported. A Gram-negative bacterium belonging to the genus Serratia was isolated from the midgut of Stibara sp., an insect of the Cerambycidae family of beetles found in the Northwestern Ghats of India. This is a unique bacterium that is quite specific for the synthesis of copper oxide nanoparticles as several other strains isolated from the same insect and common Indian mosquitoes did not result in nanoparticle formation. By following the reaction systematically, we could delineate that the nanoparticle formation occurs intracellularly. However, the process results in the killing of bacterial cells. Subsequently the nanoparticles leak out as the cell wall disintegrates. The nanoparticles formed are thoroughly characterized by UV-Vis, TEM, XRD, XPS and FTIR studies.

Dual-probe RNA FRET-FISH in Yeast.

mRNA Fluorescence In Situ Hybridization (FISH) is a technique commonly used to profile the distribution of transcripts in cells. When combined with the common single molecule technique Fluorescence Resonance Energy Transfer (FRET), FISH can also be used to profile the co-expression of nearby sequences in the transcript to measure processes such as alternate initiation or splicing variation of the transcript. Unlike in a conventional FISH method using multiple probes to target a single transcript, FRET is limited to the use of two probes labeled with matched dyes and requires the use of sensitized emission. Any widefield microscope capable of sensitive single molecule detection of Cy3 and Cy5 should be able to measure FRET in yeast cells. Alternatively, a FRET-FISH method can be used to unambiguously ascertain identity of the transcript without the use of a guide probe set used in other FISH techniques.

Single-probe RNA FISH in Yeast.

Quantitative profiling of mRNA expression is an important part of understanding the state of a cell. The technique of RNA Fluorescence In Situ Hybridization (FISH) involves targeting an RNA transcript with a set of 40 complementary fluorescently labeled DNA oligonucleotide probes. However, there are many circumstances such as transcripts shorter than 200 nt, splicing variations, or alternate initiation sites that create transcripts that would be indistinguishable to a set of multiple probes. To this end we adapted the standard FISH protocol to allow the use of a single probe with a single fluorophore to quantify the amount of transcripts inside budding yeast cells. In addition to allowing the quantification of short transcripts or short features of transcripts, this technique reduces the cost of performing FISH.

Suppression of expression between adjacent genes within heterologous modules in yeast.

Recent studies have shown that proximal arrangement of multiple genes can have complex effects on gene expression. For example, in the case of heterologous gene expression modules, certain arrangements of the selection marker and the gene expression cassette may have unintended consequences that limit the predictability and interpretability of module behaviors. The relationship between arrangement and expression has not been systematically characterized within heterologous modules to date. In this study, we quantitatively measured gene expression patterns of the selection marker (KlURA3 driven by the promoter, pKlURA) and the gene expression cassette (GFP driven by the galactose-inducible GAL1 promoter, pGAL1) in all their possible relative arrangements in Saccharomyces cerevisiae. First, we observed that pKlURA activity depends strongly on the relative arrangement and the activity of pGAL1. Most notably, we observed transcriptional suppression in the case of divergent arrangements: pKlURA activity was reduced when pGAL1 was inactive. Based on our nucleosome occupancy data, we attribute the observed transcriptional reduction to nucleosome repositioning. Second, we observed that pGAL1 activity also depends on the relative arrangement of pKlURA. In particular, strains with divergent promoters showed significantly different pGAL1 activation patterns from other strains, but only when their growth was compromised by lack of uracil. We reasoned that this difference in pGAL1 activation patterns arises from arrangement-dependent pKlURA activity that can affect the overall cell physiology (i.e., cell growth and survival in the uracil-depleted condition). Our results underscore the necessity to consider ramifications of promoter arrangement when using synthetic gene expression modules.

The effect of an intervening promoter nucleosome on gene expression.

Nucleosomes, which are the basic packaging units of chromatin, are stably positioned in promoters upstream of most stress-inducible genes. These promoter nucleosomes are generally thought to repress gene expression due to exclusion; they prevent transcription factors from accessing their target sites on the DNA. However, the role of promoter nucleosomes that do not directly occlude transcription factor binding sites is not obvious. Here, we varied the stability of a non-occluding nucleosome positioned between a transcription factor binding site and the TATA box region in an inducible yeast promoter and measured downstream gene expression level. We found that gene expression level depends on the occupancy of the non-occluding nucleosome in a non-monotonic manner. We postulated that a non-occluding nucleosome can serve both as a vehicle of and a barrier to chromatin remodeling activity and built a quantitative, nonequilibrium model to explain the observed nontrivial effect of the intervening nucleosome. Our work sheds light on the dual role of nucleosome as a repressor and an activator and expands the standard model of gene expression to include irreversible promoter chromatin transitions.

Midgut microbial community of Culex quinquefasciatus mosquito populations from India.

The mosquito Culex quinquefasciatus is a ubiquitous species that serves as a major vector for west nile virus and lymphatic filariasis. Ingestion of bloodmeal by females triggers a series of physiological processes in the midgut and also exposes them to infection by these pathogens. The bacteria normally harbored in the midgut are known to influence physiology and can also alter the response to various pathogens. The midgut bacteria in female Cx. quinquefasciatus mosquitoes collected over a large geographical area from India was studied. Examination of 16S ribosomal DNA amplicons from culturable microflora revealed the presence of 83 bacterial species belonging to 31 bacterial genera. All of these species belong to three phyla i.e. Proteobacteria, Firmicutes and Actinobacteria. Phylum Proteobacteria was the most dominant phylum (37 species), followed by Firmicutes (33 species) and Actinobacteria (13 species). Phylum Proteobacteria, was dominated by members of γ-proteobacteria class. The genus Staphylococcus was the largest genus represented by 11 species whereas Enterobacter was the most prevalent genus and recovered from all the field stations except Leh. Highest bacterial prevalence was observed from Bhuj (22 species) followed by Nagrota (18 species), Masimpur (18 species) and Hathigarh (16 species). Whereas, least species were observed from Leh (8 species). It has been observed that individual mosquito harbor extremely diverse gut bacteria and have very small overlap bacterial taxa in their gut. This variation in midgut microbiota may be one of the factors responsible for variation in disease transmission rates or vector competence within mosquito population. The present data strongly encourage further investigations to verify the potential role of the detected bacteria in mosquito for the transmission of lymphatic filariasis and west nile virus. To the best of our knowledge this is the first study on midgut microbiota of wild Cx. quinquefasciatus from over a large geographical area.

Genus-wide physicochemical evidence of extracellular crystalline silver nanoparticles biosynthesis by Morganella spp.

This study was performed to determine whether extracellular silver nanoparticles (AgNPs) production is a genus-wide phenotype associated with all the members of genus Morganella, or only Morganella morganii RP-42 isolate is able to synthesize extracellular Ag nanoparticles. To undertake this study, all the available Morganella isolates were exposed to Ag+ ions, and the obtained nanoproducts were thoroughly analyzed using physico-chemical characterization tools such as transmission electron microscopy (TEM), UV-visible spectrophotometry (UV-vis), and X-ray diffraction (XRD) analysis. It was identified that extracellular biosynthesis of crystalline silver nanoparticles is a unique biochemical character of all the members of genus Morganella, which was found independent of environmental changes. Significantly, the inability of other closely related members of the family Enterobacteriaceae towards AgNPs synthesis strongly suggests that AgNPs synthesis in the presence of Ag+ ions is a phenotypic character that is uniquely associated with genus Morganella.

Bacteria-mediated precursor-dependent biosynthesis of superparamagnetic iron oxide and iron sulfide nanoparticles.

The bacterium Actinobacter sp. has been shown to be capable of extracellularly synthesizing iron based magnetic nanoparticles, namely maghemite (gamma-Fe2O3) and greigite (Fe3S4) under ambient conditions depending on the nature of precursors used. More precisely, the bacterium synthesized maghemite when reacted with ferric chloride and iron sulfide when exposed to the aqueous solution of ferric chloride-ferrous sulfate. Challenging the bacterium with different metal ions resulted in induction of different proteins, which bring about the specific biochemical transformations in each case leading to the observed products. Maghemite and iron sulfide nanoparticles show superparamagnetic characteristics as expected. Compared to the earlier reports of magnetite and greigite synthesis by magnetotactic bacteria and iron reducing bacteria, which take place strictly under anaerobic conditions, the present procedure offers significant advancement since the reaction occurs under aerobic condition. Moreover, reaction end products can be tuned by the choice of precursors used.