Biology subjects

Ma, X.

Publications and source records attributed to Ma, X..

At least 235 records · Page 13Linked to original sources

bioRxiv2023

Mechanisms of regulation of cryptic prophage-encoded gene products in Escherichia coli

The dicBF operon of Qin cryptic prophage in Escherichia coli K12 encodes the small RNA (sRNA) DicF and small protein DicB, which regulate host cell division and are toxic when overexpressed. While new functions of DicB and DicF have been identified in recent years, the mechanisms controlling the expression of the dicBF operon have remained unclear. Under standard laboratory growth conditions, transcription from dicBp, the major promoter of the dicBF operon, is repressed by DicA. Here, we discovered that transcription of the dicBF operon and processing of the polycistronic mRNA is regulated by multiple mechanisms. DicF sRNA accumulates during stationary phase and is processed from the polycistronic dicBF mRNA by the action of both RNase III and RNase E. DicA-mediated transcriptional repression of dicBp can be relieved by an antirepressor protein, Rem, encoded on the Qin prophage. Ectopic production of Rem results in cell filamentation due to strong induction of the dicBF operon and filamentation is mediated by DicF and DicB. Spontaneous derepression of dicBp occurs in a subpopulation of cells independent of the antirepressor. This phenomenon is reminiscent of the bistable switch of {lambda} phage with DicA and DicC performing functions similar to CI and Cro, respectively. Additional experiments demonstrate stress-dependent induction of the dicBF operon. Collectively, our results illustrate that toxic genes encoded on cryptic prophages are subject to layered mechanisms of control, some that are derived from the ancestral phage and some that are likely later adaptations. ImportanceCryptic or defective prophages have lost genes necessary to excise from the bacterial chromosome and produce phage progeny. In recent years, studies have found that cryptic prophage gene products influence diverse aspects of bacterial host cell physiology. However, to obtain a complete understanding of the relationship between cryptic prophages and the host bacterium, identification of the environmental, host or prophage-encoded factors that induce the expression of cryptic prophage genes is crucial. In this study, we examine the regulation of a cryptic prophage operon in Escherichia coli encoding a small RNA and a small protein that are involved in inhibiting bacterial cell division, altering host metabolism, and protecting the host bacterium from phage infections.

microbiology↗

bioRxiv2023

An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease

Main protease (MPro) of SARS-CoV-2, the viral pathogen of COVID-19, is a crucial nonstructural protein that plays a vital role in the replication and pathogenesis of the virus. Its protease function relies on three active site pockets to recognize P1, P2, and P4 amino acid residues in a substrate and a catalytic cysteine residue for catalysis. By converting the P1 C atom in an MPro substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the MPro catalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent MPro inhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC50 value in inhibiting SARS-CoV-2 from infecting ACE2+ A549 cells and a selectivity index of 875. The crystallography analyses of MPro bound with 6 inhibitors, including MPI89, revealed that inhibitors used their covalent warheads to covalently engage the catalytic cysteine and the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 represents one of the most potent MPro inhibitors developed so far, suggesting that further exploration of the azapeptide platform and the aza-2,2-dichloroacetyl warhead is needed for the development of potent inhibitors for the SARS-CoV-2 MPro as therapeutics for COVID-19.

biochemistry↗

bioRxiv2023

Dynein-Powered Cell Locomotion Guides Metastasis of Breast Cancer

Metastasis is a principal cause of death in cancer patients, which remains an unresolved fundamental and clinical problem. Conventionally, metastatic dissemination is linked to the actomyosin-driven cell locomotion. However, locomotion of cancer cells often does not strictly line up with the measured actomyosin forces. Here, we identify a complementary mechanism of metastatic locomotion powered by the dynein-generated forces. These forces that arise within a non-stretchable microtubule network drive persistent contact guidance of migrating cancer cells along the biomimetic collagen fibers. We also show that dynein-powered locomotion becomes indispensable during invasive 3D migration within a tissue-like luminal network between spatially confining hydrogel microspheres. Our results indicate that the complementary contractile system of dynein motors and microtubules is always necessary and in certain instances completely sufficient for dissemination of metastatic breast cancer cells. These findings advance fundamental understanding of cell locomotion mechanisms and expand the spectrum of clinical targets against metastasis.

cell biology↗

bioRxiv2023

METTL3 is essential for small intestinal epithelial proliferation via regulation of growth factor signaling including KRAS

Intestinal epithelial transit amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite their critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit amplifying cell function. We report that the RNA methyltransferase, METTL3, is required for survival of transit amplifying cells in the murine small intestine. Transit amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Ribosome profiling and sequencing of methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of unique methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation confirmed a novel relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine, with important implications for both homeostatic tissue renewal and epithelial regeneration.

cell biology↗

bioRxiv2023

High throughput detection of variation in single-cell whole transcriptome through streamlined scFAST-seq

High-throughput single-cell full-length RNA sequencing is a powerful tool to explore the entire transcriptome, including non-polyadenylated transcripts. We developed a single cell Full-length RNA Sequence Transcriptome sequencing method (scFAST-seq), which combines semi-random primers with high reverse transcription efficiency, template switching and convenient rRNA removal methods, allowing the construction of full-length RNA libraries of up to 12,000 cells within 8 hours. Compared to regular 3 scRNA-seq, scFAST-seq has similar sensitivity to mRNA detection, sequencing cost and experimental workflow. Moreover, scFAST-seq has clear advantages in detecting non-polyadenylated transcripts, covering longer transcript length, and identifying more splice junctions. In addition, scFAST-seq can more accurately predict the direction of cell differentiation by calculating RNA velocity. Furthermore, we demonstrated that scFAST-seq combined with target region enrichment can simultaneously identify somatic mutations and cellular status of individual tumor cells, which is valuable information for precision medicine.

genomics↗

bioRxiv2023

Coupling of Slack and NaV1.6 sensitizes Slack to quinidine blockade and guides anti-seizure strategy development

Quinidine has been used as an anticonvulsant to treat patients with KCNT1-related epilepsy by targeting gain-of-function KCNT1 pathogenic mutant variants. However, the detailed mechanism underlying quinidines blockade against KCNT1 (Slack) remains elusive. Here, we report a functional and physical coupling of the voltage-gated sodium channel NaV1.6 and Slack. NaV1.6 binds to and highly sensitizes Slack to quinidine blockade. Homozygous knockout of NaV1.6 reduces the sensitivity of native sodium-activated potassium currents to quinidine blockade. NaV1.6-mediated sensitization requires the involvement of NaV1.6s N-and C-termini binding to Slacks C-terminus, and is enhanced by transient sodium influx through NaV1.6. Moreover, disrupting the Slack-NaV1.6 interaction by viral expression of Slacks C-terminus can protect against SlackG269S-induced seizures in mice. These insights about a Slack-NaV1.6 complex challenge the traditional view of "Slack as an isolated target" for anti-epileptic drug discovery efforts, and can guide the development of innovative therapeutic strategies for KCNT1-related epilepsy. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/532982v3_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1127858org.highwire.dtl.DTLVardef@19ad9f4org.highwire.dtl.DTLVardef@15b5542org.highwire.dtl.DTLVardef@13a86b4_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Calcium buffering tunes intrinsic excitability of spinal dorsal horn parvalbumin-expressing interneurons: A computational model

Parvalbumin-expressing interneurons (PVINs) play a crucial role within the dorsal horn of the spinal cord by preventing touch inputs from activating pain circuits. After nerve injury, their output is decreased via mechanisms that are not fully understood. In this study, we show that PVINs from nerve-injured mice change their firing pattern from tonic to adaptive. To examine the ionic mechanisms responsible for this decreased output, we employed a reparametrized Hodgkin-Huxley (HH) type model of PVINs, which predicted (1) the firing pattern transition is due to an increased contribution of small conductance calcium-activated potassium (SK) channels, enabled by (2) impairment in intracellular calcium buffering systems. Analyzing the dynamics of the HH-type model further demonstrated that a generalized Hopf bifurcation differentiates the two types of state transitions observed in the transient firing of PVINs. Importantly, this predicted mechanism holds true when we embed the PVINs model within the neuronal circuit model of the spinal dorsal horn. To experimentally validate this hypothesized mechanism, we used pharmacological modulators of SK channels and demonstrated that (1) tonic firing PVINs from naive mice become adaptive when exposed to an SK channel activator, and (2) adapting PVINs from nerve-injured mice return to tonic firing upon SK channel blockade. Our work provides important insights into the cellular mechanism underlying the decreased output of PVINs in the spinal dorsal horn after nerve injury and highlights potential pharmacological targets for new and effective treatment approaches to neuropathic pain. Significant StatementParvalbumin-expressing interneurons (PVINs) exert crucial inhibitory control over A {beta} fiber- mediated nociceptive pathways at the spinal dorsal horn. The loss of their inhibitory tone leads to neuropathic symptoms, like mechanical allodynia, via mechanisms that are not fully understood. This study identifies the reduced intrinsic excitability of PVINs as a potential cause for their decreased inhibitory output in nerve-injured condition. Combining computational and experimental approaches, we predict a calcium-dependent mechanism that modulates PVINs electrical activity following nerve injury: a depletion of cytosolic calcium buffer allows for the rapid accumulation of intracellular calcium through the active membranes, which in turn potentiates SK channels and impedes spike generation. Our results therefore pinpoint SK channels as interesting therapeutic targets for treating neuropathic symptoms.

neuroscience↗

bioRxiv2023

Seagrass genomes reveal a hexaploid ancestry facilitating adaptation to the marine environment

Seagrasses comprise the only submerged marine angiosperms, a feat of adaptation from three independent freshwater lineages within the Alismatales. These three parallel lineages offer the unique opportunity to study convergent versus lineage-specific adaptation to a fully marine lifestyle. Here, we present chromosome-level genome assemblies from a representative species of each of the seagrass lineages - Posidonia oceanica (Posidoniaceae), Cymodocea nodosa (Cymodoceaceae), and Thalassia testudinum (Hydrocharitaceae) - along with an improved assembly for Zostera marina (Zosteraceae). We also include a draft genome of Potamogeton acutifolius, a representative of Potamogetonaceae, the freshwater sister lineage to the Zosteraceae. Genome analysis reveals that all seagrasses share an ancient whole genome triplication (WGT) event, dating to the early evolution of the Alismatales. An additional whole genome duplication (WGD) event was uncovered for C. nodosa and P. acutifolius. Dating of ancient WGDs and more recent bursts of transposable elements correlate well with major geological and recent climatic events, supporting their role as rapid generators of genetic variation. Comparative analysis of selected gene families suggests that the transition from the submerged-freshwater to submerged-marine environment did not require revolutionary changes. Major gene losses related to, e.g., stomata, volatiles, defense, and lignification, are likely a consequence of the submerged lifestyle rather than the cause ( use it or lose it). Likewise, genes, often retained from the WGD and WGT, were co-opted for functions requiring the alignment of many small adaptations ( tweaking), e.g., osmoregulation, salinity, light capture, carbon acquisition, and temperature. Our ability to manage and conserve seagrass ecosystems depends on our understanding of the fundamental processes underpinning their resilience. These new genomes will accelerate functional studies and are expected to contribute to transformative solutions -- as continuing worldwide losses of the savannas of the sea are of major concern in times of climate change and loss of biodiversity.

evolutionary biology↗

bioRxiv2023

A New Behavioral Paradigm for Studying Frustrative Non-reward in Juvenile Mice

BackgroundIrritability, defined as proneness to anger, can reach a pathological extent. It is a defining symptom of Disruptive Mood Dysregulation Disorder (DMDD) and one of the most common reasons youth presents for psychiatric evaluation and care. Aberrant responses to frustrative non-reward (FNR, the response to omission of expected reward) are central to the pathophysiology of irritability. FNR is a translational construct to study irritability across species. The development of preclinical FNR models would advance mechanistic studies of the important and relatively understudied clinical phenomenon of irritability. MethodsWe used FNR as a conceptual framework to develop a novel mouse behavioral paradigm named Alternate Poking Reward Omission (APRO). After APRO, mice were examined with a battery of behavioral tests and processed for whole brain c-Fos imaging. FNR increases locomotion and aggression in mice regardless of sex. These behavioral changes resemble the symptoms observed in youth with severe irritability. There is no change in anxiety-like, depression-like, or non-aggressive social behaviors. FNR increases c-Fos+ neurons in 13 subregions of thalamus, iso-cortex and hippocampus including the prelimbic, ACC, hippocampus, dorsal thalamus, cuneiform nucleus, pons, and pallidum areas. FNR also shifts the brain network towards a more global processing mode. ConclusionOur novel FNR paradigm produces a frustration effect and alters brain processing in ways resembling the symptoms and brain network reconfiguration observed in youth with severe irritability. The novel behavioral paradigm and identified brain regions lay the groundwork for further mechanistic studies of frustration and irritability in rodents.

neuroscience↗

bioRxiv2023

Leading and Following: Noise Differently Affects Semantic and Acoustic Processing during Naturalistic Speech Comprehension

Despite the distortion of speech signals caused by unavoidable noise in daily life, our ability to comprehend speech in noisy environments is relatively stable. However, the neural mechanisms underlying reliable speech-in-noise comprehension remain to be elucidated. The present study investigated the neural tracking of acoustic and semantic speech information during noisy naturalistic speech comprehension. Participants listened to narrative audio recordings mixed with spectrally matched stationary noise at three signal-to-ratio (SNR) levels (no noise, 3 dB, -3 dB), and 60-channel electroencephalography (EEG) signals were recorded. A temporal response function (TRF) method was employed to derive event-related-like responses to the continuous speech stream at both the acoustic and the semantic levels. Whereas the amplitude envelope of the naturalistic speech was taken as the acoustic feature, word entropy and word surprisal were extracted via the natural language processing method as two semantic features. Theta-band frontocentral TRF responses to the acoustic feature were observed at around 400 ms following speech fluctuation onset over all three SNR levels, and the response latencies were more delayed with increasing noise. Delta-band frontal TRF responses to the semantic feature of word entropy were observed at around 200 to 600 ms leading to speech fluctuation onset over all three SNR levels. The response latencies became more leading with increasing noise and were correlated with comprehension performance and perceived speech intelligibility. While the following responses to speech acoustics were consistent with previous studies, our study revealed the robustness of leading responses to speech semantics, which suggests a possible predictive mechanism at the semantic level for maintaining reliable speech comprehension in noisy environments. HighlightsO_LILeading responses were observed in the semantic-level neural tracking, with more leading latencies as noise increased. C_LIO_LIFollowing responses were observed in the acoustic-level neural tracking, with more delayed latencies as noise increased. C_LIO_LISemantic-level neural tracking is correlated with comprehension performance and perceived intelligibility. C_LIO_LIDistinct frequency bands were involved in speech semantic and acoustic processing. C_LI

neuroscience↗

bioRxiv2023

Probabilistic cell/domain-type assignment of spatial transcriptomics data with SpatialAnno

In the analysis of both single-cell RNA sequencing (scRNA-seq) and spatially resolved transcriptomics (SRT) data, classifying cells/spots into cell/domain types is an essential analytic step for many secondary analyses. Most of the existing annotation methods have been developed for scRNA-seq datasets without any consideration of spatial information. Here, we present SpatialAnno, an efficient and accurate annotation method for spatial transcriptomics datasets, with the capability to effectively leverage a large number of non-marker genes as well as "qualitative" information about marker genes without using a reference dataset. Uniquely, SpatialAnno estimates low-dimensional embeddings for a large number of non-marker genes via a factor model while promoting spatial smoothness among neighboring spots via a Potts model. Using both simulated and four real spatial transcriptomics datasets from the 10x Visium, ST, Slide-seqV1/2, and seqFISH platforms, we showcase the methods improved spatial annotation accuracy, including its robustness to the inclusion of marker genes for irrelevant cell/domain types and to various degrees of marker gene misspecification. SpatialAnno is computationally scalable and applicable to SRT datasets from different platforms. Furthermore, the estimated embeddings for cellular biological effects facilitate many downstream analyses.

bioinformatics↗

bioRxiv2023

Enhancer-promoter interactions are reconfigured through the formation of long-range multiway chromatin hubs as mouse ES cells exit pluripotency

Enhancers are genomic DNA sequences that bind transcription factors, chromatin regulators and non-coding transcripts to modulate the expression of target genes. They have been found to act from different locations relative to a gene and to modulate the activity of promoters up to [~]1 Mb away. Here we report the first 3D genome structures of single mouse ES cells as they are induced to exit pluripotency, transition through a formative stage and undergo neuroectodermal differentiation. In order to directly study how interactions between enhancers and promoters are reconfigured genome wide we have determined 3D structures of haploid cells using single cell Hi-C, where we can unambiguously map the contacts to particular chromosomes. We find that there is a remarkable reorganisation of 3D genome structure where inter-chromosomal intermingling increases dramatically in the formative state. This intermingling is associated with the formation of a large number of multiway hubs that bring together enhancers and promoters with similar chromatin states from typically 5-8 distant chromosomal sites that are often separated by many Mb from each other. Genes important for pluripotency exit establish contacts with emerging enhancers within multiway chromatin hubs as cells enter the formative state, consistent with these structural changes playing an important role in establishing new cell identities. Furthermore, we find that different multiway chromatin hubs, and thus different enhancer-promoter interactions, are formed in different individual cells. Our results suggest that studying genome structure in single cells will be important to identify key changes in enhancer-promoter interactions that occur as cells undergo developmental transitions.

developmental biology↗

bioRxiv2023

Syntaxin of plants 32 regulates pollen wall development and pollen tube cell wall integrity via controlling secretory pathway

Pollen tubes (PTs) elongate in a polar way to deliver sperm cells to the ovule. Pollen wall development and PT cell wall integrity (CWI) maintenance are critical for PT growth and double fertilization. Pollen wall development mainly relies on secretion of exine precursors in tapetum. RALF4/19-ANX/BUPS-MRI and RALF4/19-LRX-AUN are two distinct signaling pathways but converge to fine-tune CWI during PT growth. Here, we discovered that atsyp32+/-, AtSYP32 RNAi and AtSYP3132 RNAi lines were male sterile. The tapetum development in these lines were disturbed, and the pollen wall structure was impaired resulting in pollen grain and tube bursting and less PTs navigated to micropyles. Strikingly, there were numerous ectopic secretory vesicles retained in pollen cytoplasm, and the abundance or distribution of polysaccharides and AGPs altered significantly in PTs of the mutants and RNAi lines. AtSYP32 interacted with the vesicle transport regulators SEC31B, SEC22 and BET12, the PT CWI regulators RALF19 and LRX11, and the XyG xylosyltransferase XXT5, in the Golgi apparatus. Transcription of some genes related to pollen wall biosynthesis and PT CWI maintenance were seriously affected by AtSYP32 downregulation. Our findings illustrate that AtSYP32 plays essential roles in pollen wall development and PT CWI maintenance via controlling secretory pathway. IN A NUTSHELLO_ST_ABSBackgroundC_ST_ABSPollen wall is the most complex cell wall. Pollen wall development mainly relies on secretion of precursors of exine and pollen coat in tapetal cells. Pollen tubes (PTs) grow in a polar way to deliver sperm cells to the ovule. Maintenance of PT cell wall integrity (CWI) is critical for PT elongation and double fertilization. RALF4/19 ligands interact with BUPS-ANX receptors, signaling it in an autocrine manner to maintain CWI during PT elongation. RALF4/19-LRX-AUN pathway is distinct with RALF4/19-ANX/BUPS-MRI pathway but they converge to fine-tune CWI during PT growth. Biosynthesis of PT cell wall involves multiple subcellular compartments and vesicle transport pathways. Golgi apparatus acts as a hub in vesicle trafficking. Golgi-syntaxin AtSYP31 and AtSYP32 regulate pollen development by controlling intra-Golgi transport and Golgi morphology QuestionWhat is AtSYP32 role in pollen wall and tapetum development? Who are the AtSYP32 partners that regulate secretion of cell wall biosynthesis materials? FindingsWe found that no homozygote progeny was obtained from self-pollinated atsyp32+/- alleles due to pollen sterile. The tapetum development and degeneration in atsyp32+/- mutants was severely delayed, and the pollen wall and PT wall structure were impaired. Strikingly, there were numerous ectopic secretory vesicles retained in pollen cytoplasm in atsyp32+/- mutants, and the abundance or distribution of PT wall polysaccharides and AGPs altered obviously. AtSYP32 interacted with the vesicle transport regulators SEC31B, SEC22 and BET12, the PT CWI regulators RALF19 and LRX11, and XyG xylosyltransferase XXT5, in the Golgi. All these highlight that AtSYP32 regulates pollen wall development and maintenance of PT CWI via controlling secretory pathway. Next stepsThe biological significances and the molecular mechanisms of AtSYP32 interacting with XXT5, RALF19 and LRX11 are elusive but thought-provoking. We are going to clarify the mechanisms.

molecular biology↗

bioRxiv2023

UPF3A is a ubiquitously expressed NMD factor among mouse tissues

Nonsense-mediated mRNA decay (NMD), an important post-transcriptional regulatory mechanism in gene expression, is actively involved in a series of cellular and physiological processes, thus controlling cell fate and tissue homeostasis. Defects in NMD cause human diseases such as neurodevelopmental disorders, tumorigenesis and autoimmunity. UPF3 (Up- frameshift protein 3), first identified in the bakers yeast, is a core NMD factor. UPF3A and UPF3B, the two UPF3 paralogs emerging in vertebrates, have either activating or suppressing roles in NMD. Previous studies found that UPF3B protein is ubiquitously expressed in almost all mammalian organs, while UPF3A protein is hardly detectable in most of mammalian tissues, except in the testis. One hypothesis explaining this phenomena is the functional antognism between UPF3A and UPF3B in NMD. Thus, UPF3B competitively binds to UPF2 with higher affinity than UPF3A, which finally destabilizes UPF3A protein. In the present study, we quantitatively evaluated the expression of UPF3A and UPF3B in nine major tissues and reproductive organs of wild type male and female mice. Our study confirmed that UPF3A has the highest expression in male germlines. To our surprise, we found in most tissues, including brain and thymus, the protein level of UPF3A is comparable with that of UPF3B. In spleen and lung, UPF3A is higher than UPF3B. These findings are further supported by publicly available gene expression data. Thus, our study demonstrated that UPF3A protein is ubiquitously expressed in mouse tissues, and may play important roles in the homeostasis of multiple mammalian tissues.

cell biology↗

bioRxiv2023

In vivo photopharmacology with light-activated opioid drugs

Traditional methods for site-specific drug delivery in the brain are slow, invasive, and difficult to interface with recordings of neural activity. Here, we demonstrate the feasibility and experimental advantages of in vivo photopharmacology using "caged" opioid drugs that are activated in the brain with light after systemic administration in an inactive form. To enable bidirectional manipulations of endogenous opioid receptors in vivo, we developed PhOX and PhNX, photoactivatable variants of the mu opioid receptor agonist oxymorphone and the antagonist naloxone. Photoactivation of PhOX in multiple brain areas produced local changes in receptor occupancy, brain metabolic activity, neuronal calcium activity, neurochemical signaling, and multiple pain- and reward-related behaviors. Combining PhOX photoactivation with optical recording of extracellular dopamine revealed adaptations in the opioid sensitivity of mesolimbic dopamine circuitry during chronic morphine administration. This work establishes a general experimental framework for using in vivo photopharmacology to study the neural basis of drug action. HighlightsA photoactivatable opioid agonist (PhOX) and antagonist (PhNX) for in vivo photopharmacology. Systemic pro-drug delivery followed by local photoactivation in the brain. In vivo photopharmacology produces behavioral changes within seconds of photostimulation. In vivo photopharmacology enables all-optical pharmacology and physiology.

neuroscience↗

bioRxiv2023

Endothelial cell derived microvesicles modulate cerebrovascular and brain aging via miR-17-5p and serve as potential biomarkers for vascular aging

Vascular aging, characterized by brain endothelial cells (ECs) senescence and dysfunction, has been known to lead to various age-related cerebrovascular and neurodegenerative diseases. However, its underlying mechanisms remain elusive. ECs derived microvesicles (EMVs) and exosomes (EEXs) carry the characteristics of parent cells and transfer their contents to modulate the functions of recipient cells, holding the potential to evaluate or regulate vascular aging. Here, we found that young or aged ECs released EMVs were more effective than their released EEXs on alleviating or aggravating mice cerebrovascular and brain aging as indicated by SA-{beta}-gal staining, cerebral blood flow, blood brain barrier function, aging related markers and cognitive ability test. We further identified that these EMVs regulated cerebrovascular and brain aging by transferring miR-17-5p and could modulate ECs senescence and functions via miR-17-5p/PI3K/Akt pathway. Plasma EMVs and their contained miR-17-5p (EMV-miR-17-5p) were significantly increased or decreased in the elderly, and were closely correlated with vascular aging. Receiver Operating Characteristic (ROC) analysis showed that the area under the curve was 0.724 for EMVs, 0.77 for EMV-miR-17-5p and 0.815 for their combination for distinguishing vascular aging. Our results revealed the novel roles for EMVs that could more effectively modulate vascular and brain aging than EEXs by regulating ECs functions through miR-17-5p/PI3K/Akt pathway, and also suggested that EMVs and EMV-miR-17-5p represent promising biomarkers and therapeutic targets for vascular aging.

neuroscience↗

bioRxiv2023

Homeostatic control of an iron repressor in a GI tract resident

The transition metal iron plays a crucial role in living cells. However, high level of iron is potentially toxic through the production of reactive oxygen species (ROS), serving as a deterrent to the commensal fungus Candida albicans for colonization in the iron-rich gastrointestinal (GI) tract. We observe that the mutant lacking an iron-responsive transcription factor Hap43 is hyper-fit for colonization in murine gut. We demonstrate that high iron specifically triggers multiple post-translational modifications (PTMs) and proteasomal degradation of Hap43, a vital process guaranteeing the precision of intestinal ROS detoxification. Reduced levels of Hap43 lead to de-repression of antioxidant genes and therefore alleviate the deleterious ROS derived from iron metabolism. Our data reveal that Hap43 functions as a negative regulator for oxidative stress-adaptation of C. albicans to gut colonization and thereby provide a new insight into understanding the interplay between iron homeostasis and fungal commensalism. ImportanceIron homeostasis is critical for creatures. Candida albicans is one of the major commensals in the GI tract where is iron-replete environment. Transcriptional factor Hap43 was believed to repress iron utilizations genes in iron-depleted conditions for decades. However, the mystery in iron-replete conditions of Hap43 has never been uncovered. We discovered that reduced levels of Hap43 via phosphorylation-dependent nuclear export, followed by proteosome-mediated protein degradation, leads to de-repression of downstream antioxidant genes and promote its colonization in GI tract. We propose that C. albicans has a strict detoxification process to ensure its survival, which has important implications for understanding how the fungi survives in the mammalian host.

microbiology↗

bioRxiv2023

EVIDENCE FOR ANGIOTENSIN II AS A NATURALLY EXISTING SUPPRESSOR FOR THE NATRIURETIC PEPTIDE SYSTEM

BackgroundNatriuretic peptide system (NPS) and renin angiotensin aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date support this notion. ObjectivesThis study was designed to systematically investigate ANGII-NPS interaction in humans, in vivo, and in vitro for translational insights. MethodsCirculating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in rat model to determine influence of ANGII on ANP actions. Multiple engineered HEK293 cells and surface plasmon resonance (SPR) technology were leveraged for mechanistic exploration. ResultsIn humans, ANGII showed inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and interaction term between ANGII and natriuretic peptide increased predicting accuracy of base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed positive association between cGMP with ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at physiological dose attenuated blood pressure reduction and cGMP generation triggered by ANP infusion. In vitro, we showed that the suppression effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), which can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using SPR, we showed ANGII has low affinity for particulate guanylyl cyclase A (GC-A) receptor binding compared to ANP or BNP. ConclusionsOur study reveals ANGII as a natural suppressor for cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular disease. STRUCTURED GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/525806v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@6febe8org.highwire.dtl.DTLVardef@1e8e21corg.highwire.dtl.DTLVardef@54a98eorg.highwire.dtl.DTLVardef@47ade7_HPS_FORMAT_FIGEXP M_FIG C_FIG CLINICAL PERSPECTIVESAccumulating evidence continues to support the NPS as a promising therapeutic target via the function of the GC-A receptor and production of the second messenger cGMP for heart failure, hypertension, and other cardiovascular diseases. Improving our mechanistic understanding on GC-A/cGMP pathway regulation may further advance the development of novel NPS enhancing therapies. Here we report evidence from multiple avenues supporting a fundamental, yet previously underappreciated mechanism involving a negative action of ANGII in suppressing GC-A receptor-mediated cGMP production via an AT1 receptor-dependent manner. This study also provides a solid rationale for the superiority of combinatory neurohormonal therapies such as sacubitril/valsartan in treating cardiovascular disease, and further highlights a promising therapeutic avenue of dual targeting both the NPS and RAAS to maximize protection.

molecular biology↗