TMEM175 Is an Organelle K(+) Channel Regulating Lysosomal Function.

Potassium is the most abundant ion to face both plasma and organelle membranes. Extensive research over the past seven decades has characterized how K(+) permeates the plasma membrane to control fundamental processes such as secretion, neuronal communication, and heartbeat. However, how K(+) permeates organelles such as lysosomes and endosomes is unknown. Here, we directly recorded organelle K(+) conductance and discovered a major K(+)-selective channel KEL on endosomes and lysosomes. KEL is formed by TMEM175, a protein with unknown function. Unlike any of the ∼80 plasma membrane K(+) channels, TMEM175 has two repeats of 6-transmembrane-spanning segments and has no GYG K(+) channel sequence signature-containing, pore-forming P loop. Lysosomes lacking TMEM175 exhibit no K(+) conductance, have a markedly depolarized ΔΨ and little sensitivity to changes in [K(+)], and have compromised luminal pH stability and abnormal fusion with autophagosomes during autophagy. Thus, TMEM175 comprises a K(+) channel that underlies the molecular mechanism of lysosomal K(+) permeability.

mTOR regulates lysosomal ATP-sensitive two-pore Na(+) channels to adapt to metabolic state.

Survival in the wild requires organismal adaptations to the availability of nutrients. Endosomes and lysosomes are key intracellular organelles that couple nutrition and metabolic status to cellular responses, but how they detect cytosolic ATP levels is not well understood. Here, we identify an endolysosomal ATP-sensitive Na(+) channel (lysoNa(ATP)). The channel is a complex formed by two-pore channels (TPC1 and TPC2), ion channels previously thought to be gated by nicotinic acid adenine dinucleotide phosphate (NAADP), and the mammalian target of rapamycin (mTOR). The channel complex detects nutrient status, becomes constitutively open upon nutrient removal and mTOR translocation off the lysosomal membrane, and controls the lysosome's membrane potential, pH stability, and amino acid homeostasis. Mutant mice lacking lysoNa(ATP) have much reduced exercise endurance after fasting. Thus, TPCs make up an ion channel family that couples the cell's metabolic state to endolysosomal function and are crucial for physical endurance during food restriction.

I-motif sensor for the fluorometric detection of Monkeypox.

In this study, we developed an isothermal fluorometric diagnostic method for DNA virus-generating disorders such as Mpox. Our results showed that the release of a large number of protons during multiplex-LAMP markedly lowered the pH level, which transformed the retinoblastoma (Rb) linear ssDNA into i-motifs. Consequently, thiazole orange (TO; a fluorometric probe sensitive to the i-motif) boosted the signal-on fluorescence because of its ability to bind selectively to i-motifs. This multiplex-LAMP/i-motif-TO system enabled simultaneous detection aimed at numerous potential targets with remarkable sensitivity (1.47 pg per mL) and efficiency (30 minutes). Our method is expected to enable DNA-virus-related diseases to be efficiently and accurately assessed.

Microbial-transcriptome integrative analysis of heat stress effects on amino acid metabolism and lipid peroxidation in poultry jejunum.

Despite the significant threat of heat stress to livestock animals, only a few studies have considered the potential relationship between broiler chickens and their microbiota. Therefore, this study examined microbial modifications, transcriptional changes and host-microbiome interactions using a predicted metabolome data-based approach to understand the impact of heat stress on poultry. After the analysis, the host functional enrichment analysis revealed that pathways related to lipid and protein metabolism were elevated under heat stress conditions. In contrast, pathways related to the cell cycle were suppressed under normal environmental temperatures. In line with the transcriptome analysis, the microbial analysis results indicate that taxonomic changes affect lipid degradation. Heat stress engendered statistically significant difference in the abundance of 11 microorganisms, including Bacteroides and Peptostreptococcacea. Together, integrative approach analysis suggests that microbiota-induced metabolites affect host fatty acid peroxidation metabolism, which is correlated with the gene families of Acyl-CoA dehydrogenase long chain (ACADL), Acyl-CoA Oxidase (ACOX) and Acetyl-CoA Acyltransferase (ACAA). This integrated approach provides novel insights into heat stress problems and identifies potential biomarkers associated with heat stress.

Rolling circle transcription/G-quadruplex/QnMorpholine probe for highly selective and sensitive detection of alkaline phosphatase activity.

In this study, we combined a rolling circle transcription (RCT) system producing 22AG G-quadruplex RNA with a QnMorpholine (QNM) fluorescent probe for the selective and sensitive detection of alkaline phosphatase (ALP). ALP is involved in various biological functions, with monophosphate cleavage being one of its characteristic properties. Here, we developed a padlock RCT probing system in which a large amount of RCT 22AG RNA G-quadruplex was produced in the absence of ALP, providing a high fluorescence signal. In contrast, no RNA G-quadruplex was produced in the presence of ALP, with minimal fluorescence. This huge deviation in signal intensity allowed us to identify the presence or absence of ALP in a test sample. Under practical conditions, our system allowed the differentiation for ALP even when it was present at an extremely low concentration (0.0085 U/L), along with very high specificity. The simplicity and efficiency of this approach for ALP detection suggest its potential for use as a reliable diagnostic tool.

Triple ligation-based formation of a G-quadruplex for simultaneous detection of multiple miRNAs.

The simultaneous detection of multiple microRNAs (miRNA) is of great necessity but has not been extensively studied. This prompted our study, which involved the development of a triple ligation-based system for detecting three miRNAs at the same time. We designed a multi-ligation-padlock (MLP) probe that consists of three parts, each of which is complementary to two different miRNAs at the same time. In the presence of all three miRNAs, the probe becomes circularized, but in the absence of even one target, the probe remains linear. The first part of the MLP probe (MLP1) contains a T7 promoter part that can initiate RNA synthesis for any given target condition. However, it also includes a G-quadruplex complementary segment, which can only form a parallel RNA G-quadruplex through rolling circle transcription by the circularized template in the presence of all three targets. In this case, the application of our parallel G-quadruplex sensing fluorescent probe lutidine DESA (LutD) produces a strong signal. However, in the absence of any one of the targets, the RNA G-quadruplex cannot be formed and ultimately the LutD probe does not generate any signal. This difference in the signal intensity represents the presence or absence of all the target miRNAs. With our system, we were able to detect miRNA 21 at levels as low as 1.13 fM, miRNA 146a as low as 1.37 fM, and miRNA 25b as low as 1.51 fM within 45 minutes, confirming that our novel system can selectively and sensitively diagnose triple miRNAs.

Multiple gene detection using a selective fluorophore probe-RNA hybridization/graphene oxide quenching system.

Herein, we introduce a novel assay for multiple-gene recognition by ligation-double transcription mediated fluorometric profiling. We demonstrated the capability of the system to identify potential multi-gene classifiers for diagnostic use by employing a combination of a ligation-double transcription approach with a selective fluorophore probe-RNA hybridization/graphene oxide quenching system. The system is efficient and requires only 45 minutes for total experimentation and offers high sensitivity (369.6, 408, and 407.8 copies per mL for the O, E, and N genes of SARS-CoV-2, respectively) and specificity (selective to until two mismatches). Our system is expected to expedite the precise diagnosis of RNA-virus-related diseases with multiple gene classifiers. By focusing on distinct viral genes, our method allowed for the detection of various RNA viruses in a variety of sample pools.

Blocker-dUThiophene poly tailing-based method for assessing methyl transferase activity.

In this report, we present a method for the selective and sensitive detection of methyl transferase activity. The method uses a dsDNA probe that contains C3 spacers and is coupled with dUThioTP-TdT polymerase-based poly-tailing. The short dsDNA probe is designed with C3 spacers at both 3' ends to prevent any type of tailing reaction. However, the probe contains a methyl transferase recognition sequence that can methylate adenosines in the palindromic part of both strands. When a specific DpnI endonuclease is introduced, it selectively cleaves the dsDNA probe such that both strands are methylated, unblocking the probe into two separate dsDNA forms with exposed 3' OH groups. This makes the probe susceptible to tailing in the presence of a TdT tailing polymerase. The unblocked probe is then subjected to fluorescent dUThioTP-based tailing, which produces a strong fluorescent signal that indicates the presence of methyl transferase activity. In the absence of methyl transferase, the probe remains in the blocked state and does not undergo fluorescence. This method has a limit of detection of 0.049 U/mL with good selectivity and the potential for accurate MTase analysis.

22AG G-quadruplex RNA/QnMorpholine-mediated fluorimetric detection of miR-21.

Herein we report a simple ligation/transcription-mediated system, using a 22AG G-quadruplex RNA secondary structure and a fluorescence-inducing QnMorpholine probe, for the detection of miR-21. In the presence of the target miR-21, two oligonucleotide probes (promoter and reporter) were ligated, thereby transcribing the 22AG RNA sequence, a complement of the reporter probe. In contrast, in the absence of this target-induced ligation, the reporter complement could not be transcribed to produce the 22AG RNA sequence. Subsequent addition of the QnMorpholine probe resulted in binding with the 22AG G-quadruplex RNA, thereby generating high fluorescence; no fluorescence occurred in the absence of this secondary structure. Hence, the presence of miR-21 was evidenced by a target-induced high-intensity signal. This simple one-pot fluorimetric system, which could detect miR-21 of up to 3.08 femtomolar in less than 30 min, holds promise as a diagnostic tool for selective and sensitive miRNA detection.

Unnatural nucleotide-based rkDNA probe combined with graphene oxide for detection of alkaline phosphatase activity.

In this study we developed a fluorescent double-stranded DNA, incorporating an unnatural dUrk nucleotide, that we used as a probe for the detection of alkaline phosphatase (ALP) based on enzymatic cleavage of the non-fluorescent complementary strand. Primer extension performed using the unnatural nucleotide triphosphate dUrkTP and the natural deoxynucleotide triphosphates dATP, dCTP, and dGTP provided a simple fluorescent DNA strand that hybridized with the 5́-monophosphate non-fluorescent complementary strand. When applying the 5́-phosphate recognition and cleavage properties of lambda exonuclease (λ-exo), this probe could bind to graphene oxide (GO) and quench the fluorescence (in the absence of ALP) or not bind to GO and retain its fluorescence (in the presence of ALP). We obtained strongly fluorescent DNA strands through simple incorporation of multiple A sites in the complementary sequence, thereby increasing the number of dUrk residues during primer extension. This unnatural nucleotide-based rkDNA probing system exhibited high fluorescence differentiation for discriminating the status of ALP. This rkDNA-GO probing system appears to be a promising tool for monitoring the activity of disease-associated enzymes.

A fluorescent molecular rotor for the selective detection of the hybrid-conformation 22AG G-Quadruplex.

G-quadruplex (G4) DNA plays a vital role in myriad biological process and is linked to several human diseases, including Alzheimer's disease. Probing G4s with fluorescent probes can provide a better understanding their mechanisms of action and of their roles in Nature. In this study we developed a quinolinium-vinylaniline molecular rotor probe, featuring a diethylaminosalicylaldehyde unit that could discriminate the hybrid-22AG G4 sequence selectively amongst other G4 sequences. This probe underwent a significant red-shift upon binding to the target G4 (broad 575 nm â†’ sharp 630 nm) with enhanced fluorescence (up to 14-fold). We suspect that the vinylaniline unit of the molecular rotor, when bound to the hybrid-22 A G4, experienced restricted rotation, thereby undergoing enhanced intramolecular charge transfer. The presence of the diethylaminosalicylaldehyde moiety appeared to play a major role in the enhanced selectivity toward the 22AG G4.

Directly arylated oligonucleotides as fluorescent molecular rotors for probing DNA single-nucleotide polymorphisms.

We developed direct arylated oligonucleotide based molecular rotor (AOMR) to discriminate perfect matched DNA sequence from one base mismatched sequences. Quinolinium salts attached to vinyl aniline would be excellent fluorescent analogs with molecular rotor properties and are suitable for the detection of microenvironment change arising from dynamic motions with match-mismatch DNA base pairs. We applied direct N6 arylation of the adenosine located in natural oligonucleotide as a tool to incorporate the molecular rotor (Quinolinium salts attached vinyl aniline) and used it to discriminate perfect matched DNA sequence from one base mismatch sequences. The fluorescence and quantum yield of arylated oligonucleotide based molecular rotor (AOMR), particulary, RMAQn reveals 28.3 times higher discrimination factor with perfect matched sequence (RMAQn:T) (QY = 0.17) compare to single strand RMAQn (QY = 0.006) and one base mismatched sequence (RMAQn:G, RMAQn:A, and RMAQn:C) at λmax = 600 nm (orange emission), which would be useful for in vivo application. RMAQn:T duplex also showed high brightness (6068), 32.9 times higher than single strand RMAQn (192), as a result of restricted rotation of the Quinolinium salts attached vinyl aniline on adenosine moiety with perfect matched sequence compare to the mismatch sequences. Arylated oligonucleotide based molecular rotor (AOMR) proves to be an unprecedented sensitivity in detecting local dynamics of nucleic acids and also would be simple and cost-effective method to prepare SNP probe.

Point-of-care COVID-19 testing: colorimetric diagnosis using rapid and ultra-sensitive ramified rolling circle amplification.

In this paper, we report a molecular diagnostic system-combining a colorimetric probe (RHthio-CuSO4) for pyrophosphate sensing and isothermal gene amplification (ramified rolling circle amplification)-that operates with high selectivity and sensitivity for clinical point-of-care diagnosis of SARS-CoV-2. During the polymerase phase of the DNA amplification process, pyrophosphate was released from the nucleotide triphosphate as a side product, which was then sensed by our RHthio-CuSO4 probe with a visible color change. This simple colorimetric diagnostic system allowed highly sensitive (1.13 copies/reaction) detection of clinical SARS-CoV-2 within 1 h, while also displaying high selectivity, as evidenced by its discrimination of two respiratory viral genomes (human rhino virus and respiratory syncytial virus) from that of SARS-CoV-2. All of the reactions in this system were performed at a single temperature, with positive identification being made by the naked eye, without requiring any instrumentation. The high sensitivity and selectivity, short detection time (1 h), simple treatment (one-pot reaction), isothermal amplification, and colorimetric detection together satisfy the requirements for clinical point-of-care detection of SARS-CoV-2. Therefore, we believe that this combination of a colorimetric probe and isothermal amplification will be useful for point-of-care testing to prevent the propagation of COVID-19.

Dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for colorimetric and point-of-care determination of real SARS-CoV-2.

A probing system has been developed based on dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for the selective colorimetric detection of SARS-CoV-2. This approach can induce false-positive and -negative detection in real clinical samples; dLig-LAMP operates with improved selectivity. Unlike RT-LAMP, the selectivity of dLig-LAMP is determined in both the ligation and primer binding steps, not in the reverse transcription step. With this selective system in hand, we developed a colorimetric signaling system for point-of-care detection. We also developed a colorimetric probe for sensing pyrophosphate, which arises as a side product during the LAMP DNA amplification. Thus, dLig-LAMP appears to be an alternative method for improving the selectivity problems associated with reverse transcription. In addition, combining dLig-LAMP with colorimetric pyrophosphate probing allows point-of-care detection of SARS-CoV-2 within 1 h with high selectivity.

Correction: Polymerase-mediated synthesis of p-vinylaniline-coupled fluorescent DNA for the sensing of nucleolin protein-c-myc G-quadruplex interactions.

Correction for 'Polymerase-mediated synthesis of p-vinylaniline-coupled fluorescent DNA for the sensing of nucleolin protein-c-myc G-quadruplex interactions' by Guralamatta Siddappa Ravi Kumara et al., Org. Biomol. Chem., 2021, DOI: 10.1039/D1OB00863C.

Polymerase-mediated synthesis of p-vinylaniline-coupled fluorescent DNA for the sensing of nucleolin protein-c-myc G-quadruplex interactions.

In this paper we report the synthesis of two deoxyuridine derivatives (dUCN2, dUPy)-featuring p-vinylaniline-based fluorophores linked through a propargyl unit at the 5' position-that function as molecular rotors. This probing system proved to be useful for the sensing of gene regulation arising from interactions between this G-quadruplex and nucleolin.

Loop-mediated fluorescent probes for selective discrimination of parallel and antiparallel G-Quadruplexes.

Herein we report simple pyridinium (1-3) and quinolinium (4) salts for the selective recognition of G-quadruplexes (G4s). Among them, the probe 1, interestingly, selectively discriminated parallel (c-KIT-1, c-KIT-2, c-MYC) G4s from anti-parallel/hybrid (22AG, HRAS-1, BOM-17, TBA) G4s at pH 7.2, through a switch on response in the far-red window. Significant changes in the absorption (broad 575 nm â†’ sharp 505 nm) and emission of probe 1 at 620 nm, attributed to selective interaction with parallel G4s, resulted in complete disaggregation-induced monomer emission. Symmetrical push/pull molecular confinements across the styryl units in probe 1 enhanced the intramolecular charge transfer (ICT) by restricting the free rotation of CC units in the presence of sterically less hindered and highly accessible G4 surface/bottom tetrads in the parallel G4s, which is relatively lower extent in antiparallel/hybrid G4s. We confirm that the disaggregation of probe 1 was very effective in the presence of parallel G4-forming ODNs, due to the presence of highly available free surface area, resulting in additional π-stacking interactions. The selective sensing capabilities of probe 1 were analyzed using UV-Vis spectroscopy, fluorescence spectroscopy, molecular dynamics (MD)-based simulation studies, and 1H NMR spectroscopy. This study should afford insights for the future design of selective compounds targeting parallel G4s.

Highly fluorescent morpholine naphthalimide deoxyuridine nucleotide for the detection of miRNA 24-3P through rolling circle amplification.

In this study we synthesized the nucleotide dUrkTP, a highly fluorescent naphthalimide deoxyuridine triphosphate that undergoes aggregation-induced emission (AIE). We incorporated and extended dUrkTP during the primer extension of DNA mediated by DNA polymerase, and also in the rolling circle amplification of DNA mediated by Phi29 polymerase. Accordingly, we could use this fluorescent nucleotide for the detection of microRNA 24-3P, a biomarker of porcine reproductive and respiratory syndrome virus. The direct labeling system obtained during rolling circle DNA amplification exhibited increased fluorescence, due to AIE of the dUrkTP residue upon gel formation, thereby allowing the detection of miRNA 24-3P. This direct labeling system facilitated the simple and inexpensive detection of miRNA 24-3P with high sensitivity (limit of detection: 3.58 fM) and selectivity.

rkDNA-graphene oxide as a simple probe for the rapid detection of miRNA21.

In this study we developed a novel diagnostic tool for the detection of miRNA21, based on the fluorescent nucleotide morpholine naphthalimide deoxyuridine (dUrkTP). We incorporated dUrkTP into DNA through primer extension to obtain rkDNA displaying high fluorescence. We then used lambda exonuclease, a specific nuclease for 3́-monophosphate-containing DNA, to separate rkDNA from its complementary sequence. The fluorescence of the free rkDNA was quenched dramatically upon interacting with graphene oxide (GO). Our rkDNA-GO fluorescence probing system exhibited high sensitivity and selectivity for the detection of miRNA21. This inexpensive probing system, employing simple primer extension and exonuclease degradation, required only 30 min to detect its target miRNA. This strategy appears suitable for the detection of diverse types of miRNA.

Preferred display white prediction model based on mixed chromatic adaptation between "prototypical display white" and surround lighting color.

The correlated color temperature (CCT) of the monitor white needs to be controlled for the preferred image reproduction according to the surround lighting changes. The preferred display white prediction model according to the surround lighting color is proposed both for the emissive transparent display and opaque displays. To develop the model, the preferred CCT of the monitor white of a simulated emissive transparent display and an opaque display were investigated under four different surround lighting CCTs by conducting psychophysical experiments with twenty subjects.