Impact of SARS-CoV2 infection on gut microbiota dysbiosis.

The composition and function of the gut microbiota constantly influence health. Disruptions in this delicate balance, termed gut microbiota dysbiosis, have been implicated in various adverse health events. As the largest global epidemic since 1918, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had devastating consequences. While the primary impact of Corona Virus Disease 2019 (COVID-19) has been on the respiratory system, a growing body of research has unveiled the significant involvement of the gastrointestinal tract as well. Emerging evidence underscores notable alterations in the gut microbiome of COVID-19 patients. In addition, the gut microbiome is also characterized by an abundance of opportunistic pathogens, which is related to disease manifestations of COVID-19 patients. The intricate bidirectional interaction between the respiratory mucosa and the gut microbiota, known as the gut-lung axis, emerges as a crucial player in the pathological immune response triggered by SARS-CoV-2. Here, we discuss microbiota-based gut characteristics of COVID-19 patients and the long-term consequences of gut microbiota dysregulation. These insights could potentially transform the development of long-term interventions for COVID-19, offering hope for improved outcomes and enhanced patient recovery.

Epitranscriptome analysis of NAD-capped RNA by spike-in-based normalization and prediction of chronological age.

Nicotinamide adenine dinucleotide (NAD) can be used as an initiating nucleotide in RNA transcription to produce NAD-capped RNA (NAD-RNA). RNA modification by NAD that links metabolite with expressed transcript is a poorly studied epitranscriptomic modification. Current NAD-RNA profiling methods involve multi-steps of chemo-enzymatic labeling and affinity-based enrichment, thus presenting a critical analytical challenge to remove unwanted variations, particularly batch effects. Here, we propose a computational framework, enONE, to remove unwanted variations. We demonstrate that designed spike-in RNA, together with modular normalization procedures and evaluation metrics, can mitigate technical noise, empowering quantitative and comparative assessment of NAD-RNA across different datasets. Using enONE and a human aging cohort, we reveal age-associated features of NAD-capping and further develop an accurate RNA-based aging clock that combines signatures from both transcriptome and NAD-modified epitranscriptome. enONE facilitates the discovery of NAD-RNA responsive to physiological changes, laying an important foundation for functional investigations into this modification.

miR-22-3p ameliorates the symptoms of premature ovarian failure in mice by inhibiting CMKLR1 expression.

Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. This study was aimed at exploring the improving effects of miR-22-3p on the symptoms of POF in mice by inhibiting chemokine-like receptor 1 (CMKLR1) expression. Female mice were intraperitoneally injected with cyclophosphamide to construct POF mice models. Lentiviral vectors containing miR-22-3p, short hairpin RNA (sh)-CMKLR1, and overexpression (oe)-CMKLR1, respectively, or in combination, were injected into the ovaries of both sides of POF mice. miR-22-3p and CMKLR1 expression in ovarian tissues of mice was assessed, and the targeting relationship between miR-22-3p and CMKLR1 was predicted and verified. Serum estradiol (E2), anti-Mullerian hormone, and follicle-stimulating hormone levels were assessed. Ovarian weight was weighed, and pathological changes and the number of primordial follicles, primary follicles, secondary follicles, and atresia follicles were observed. Apoptosis of ovarian tissues was determined. In ovarian tissues of POF mice, miR-22-3p expression was decreased while CMKLR1 expression was increased. miR-22-3p up-regulation or CMKLR1 down-regulation restored sex hormone levels, improved ovarian weight and the number of primordial follicles, primary follicles, and secondary follicles, and reduced the number of atresia follicle and ovarian granulosa cell apoptosis in POF mice. miR-22-3p targeted CMKLR1, and overexpressing CMKLR1 reversed the ameliorative effects of miR-22-3p overexpression on POF mice. Our research highlights that overexpressed miR-22-3p down-regulates CMKLR1 to ameliorate the symptoms of POF in mice. Therefore, the miR-22-3p/CMKLR1 axis could improve the symptoms of POF.

Assessing Boron-Pleuromutilin AN11251 for the Development of Antibacterial Agents.

Pleuromutilins are a group of antibiotics derived from the naturally occurring compound. The recent approval of lefamulin for both intravenous and oral doses in humans to treat community-acquired bacterial pneumonia has prompted investigations in modifying the structure to broaden the antibacterial spectrum, enhance the activity, and improve the pharmacokinetic properties. AN11251 is a C(14)-functionalized pleuromutilin with a boron-containing heterocycle substructure. It was demonstrated to be an anti-Wolbachia agent with therapeutic potential for Onchocerciasis and lymphatic filariasis. Here, the in vitro and in vivo PK parameters of AN11251 were measured including PPB, intrinsic clearance, half-life, systemic clearance, and volume of distribution. The results indicate that the benzoxaborole-modified pleuromutilin possesses good ADME and PK properties. AN11251 has potent activities against the Gram-positive bacterial pathogens tested, including various drug-resistant strains, and against the slow-growing mycobacterial species. Finally, we employed PK/PD modeling to predict the human dose for treatment of disease caused by Wolbachia, Gram-positive bacteria, or Mycobacterium tuberculosis, which might facilitate the further development of AN11251.

Human Kinase IGF1R/IR Inhibitor Linsitinib Controls the In Vitro and Intracellular Growth of Mycobacterium tuberculosis.

ATP provides energy in the biosynthesis of cellular metabolites as well as regulates protein functions through phosphorylation. Many ATP-dependent enzymes are antibacterial and anticancer targets including human kinases acted on by most of the successful drugs. In search of new chemotherapeutics for tuberculosis (TB), we screened repurposing compounds against the essential glutamine synthase (GlnA1) of Mycobacterium tuberculosis (Mtb) and identified linsitinib, a clinical-stage drug originally targeting kinase IGF1R/IR as a potent GlnA1 inhibitor. Linsitinib has direct antimycobacterial activity. Biochemical, molecular modeling, and target engagement analyses revealed the inhibition is ATP-competitive and specific in Mtb. Linsitinib also improves autophagy flux in both Mtb-infected and uninfected THP1 macrophages, as demonstrated by the decreased p-mTOR and p62 and the increased lipid-bound LC3B-II and autophagosome forming puncta. Linsitinib-mediated autophagy reduces intracellular growth of wild-type and isoniazid-resistant Mtb alone or in combination with bedaquiline. We have demonstrated that an IGF-IR/IR inhibitor can potentially be used to treat TB. Our study reinforces the concept of targeting ATP-dependent enzymes for novel anti-TB therapy.

Integrated Analysis Reveals the Gut Microbial Metabolite TMAO Promotes Inflammatory Hepatocellular Carcinoma by Upregulating POSTN.

Liver cancer has a high mortality rate. Chronic inflammation is one of the leading causes of hepatocellular carcinoma. Recent studies suggested high levels of trimethylamine N-oxide (TMAO) may correlate with increased risk of inflammatory-induced liver cancer. However, the mechanisms by which TMAO promotes liver cancer remain elusive. Here, we established a model of inflammatory-induced liver cancer by treating Hepa1-6 cells and Huh7 cells with TNF-α. TMAO synergistically increased the proliferation, migration and invasion of Hepa1-6 cells and Huh7 cells in the presence of TNF-α. We conducted bulk RNA-Seq of the TMAO-treated cell model of inflammatory Hepatocellular carcinoma (HCC) and evaluated the influence of the differentially expressed genes (DEGs) on clinical prognosis using Kaplan-Meier Plotter Database and Gene Expression Profiling Interactive Analysis (GEPIA) database. Univariate and multivariate Cox regression analyses of tumor microenvironment and DEGs were performed using Timer2.0. Upregulation of POSTN, LAYN and HTRA3 and downregulation of AANAT and AFM were positively related to poorer overall survival in human liver cancer. Moreover, higher expression of POSTN and HTRA3 positively correlated with infiltration of neutrophils, which can promote tumor progression. In vitro experiments showed TMAO activates ILK/AKT/mTOR signaling via POSTN, and knocking down POSTN significantly reduced ILK/AKT/mTOR signaling and the tumorigenicity of Hepa1-6 cells and Huh7 cells. Collectively, our results suggest the gut microbial metabolite TMAO and POSTN may represent potential therapeutic targets for liver cancer.

Investigate Natural Product Indolmycin and the Synthetically Improved Analogue Toward Antimycobacterial Agents.

Indolmycin (IND) is a microbial natural product that selectively inhibits bacterial tryptophanyl-tRNA synthetase (TrpRS). The tryptophan biosynthesis pathway was recently shown to be an important target for developing new antibacterial agents against Mycobacterium tuberculosis (Mtb). We investigated the antibacterial activity of IND against several mycobacterial model strains. A TrpRS biochemical assay was developed to analyze a library of synthetic IND analogues. The 4″-methylated IND compound, Y-13, showed improved anti-Mtb activity with a minimum inhibitory concentration (MIC) of 1.88 µM (∼0.5 µg/mL). The MIC increased significantly when overexpression of TrpRS was induced in the genetically engineered surrogate M. bovis BCG. The cocrystal structure of Mtb TrpRS complexed with IND and ATP has revealed that the amino acid pocket is in a state between the open form of apo protein and the closed complex with the reaction intermediate. In whole-cell-based experiments, we studied the combination effect of Y-13 paired with different antibacterial agents. We evaluated the killing kinetics, the frequency of resistance to INDs, and the mode of resistance of IND-resistant mycobacteria by genome sequencing. The synergistic interaction of Y-13 with the TrpE allosteric inhibitor, indole propionic acid, suggests that prospective IND analogues could shut down tryptophan biosynthesis and protein biosynthesis in pathogens, leading to a new class of antibiotics. Finally, we discuss a strategy to expand the genome mining of antibiotic-producing microbes specifically for antimycobacterial development.

[Sleep disturbance associated with Smith-Magenis syndrome].

Smith-Magenis syndrome (SMS) (OMIM #182290) is a rare genetic disorder with a prevalence of 1 in 25 000 live births. Approximately 90% of SMS patients have harbored a 3.7 Mb interstitial 17p11.2 deletion involving the RAI1 gene, while 10% of cases have carried pathogenic variants of the RAI1 gene. SMS is characterized by sleep disturbance, intellectual impairment, developmental delay, craniofacial and cardiovascular anomalies, obesity, self injury, aggressive and autistic-like behaviors. Most SMS patients have sleep disorders such as short total sleep time, frequent night waking, short sleep onset, and early morning waking. The sleep disturbance may aggravate with age and persist throughout life. Three mechanisms have been delineated. The first concern was the abnormal secretion of melatonin, with high levels during daytime and low levels at night. Evaluation of the integrity of the intrinsically photosensitive retinal ganglion cell (ipRGC)/melanopsin system has found that SMS patients showed dysfunction in the sustained component of the pupillary light responses to blue light. Synchronization of daily melatonin profile and its photoinhibition are dependent on the activation of melanopsin. Dysfunction of the retina-melanin system may be one of the causes of melatonin spectrum disorders. Secondly, dysregulation of circadian rhythm gene expression has also been noted in mice and SMS patients. Finally, there may be association between sleep deprivation symptoms and DNA methylation patterns, which has provided new insights for SMS-associated sleep disorders and symptoms alike. Treatment for SMS-related sleep disorders is administered primarily through medications like melatonin tablets, which can alleviate insomnia-related sleep difficulties, in particular externalizing behavior in children. Researchers are also actively exploring other treatments for SMS currently.

Analysis and Radiometric Calibration for Backscatter Intensity of Hyperspectral LiDAR Caused by Incident Angle Effect.

Hyperspectral LiDAR (HSL) is a new remote sensing detection method with high spatial and spectral information detection ability. In the process of laser scanning, the laser echo intensity is affected by many factors. Therefore, it is necessary to calibrate the backscatter intensity data of HSL. Laser incidence angle is one of the important factors that affect the backscatter intensity of the target. This paper studied the radiometric calibration method of incidence angle effect for HSL. The reflectance of natural surfaces can be simulated as a combination of specular reflection and diffuse reflection. The linear combination of the Lambertian model and Beckmann model provides a comprehensive theory that can be applied to various surface conditions, from glossy to rough surfaces. Therefore, an adaptive threshold radiometric calibration method (Lambertian-Beckmann model) is proposed to solve the problem caused by the incident angle effect. The relationship between backscatter intensity and incident angle of HSL is studied by combining theory with experiments, and the model successfully quantifies the difference between diffuse and specular reflectance coefficients. Compared with the Lambertian model, the proposed model has higher calibration accuracy, and the average improvement rate to the samples in this study was 22.67%. Compared with the results before calibration with the incidence angle of less than 70°, the average improvement rate of the Lambertian-Beckmann model was 62.26%. Moreover, we also found that the green leaves have an obvious specular reflection effect near 650-720 nm, which might be related to the inner microstructure of chlorophyll. The Lambertian-Beckmann model was more helpful to the calibration of leaves in the visible wavelength range. This is a meaningful and a breakthrough exploration for HSL.

Rediscovery of PF-3845 as a new chemical scaffold inhibiting phenylalanyl-tRNA synthetase in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) remains the deadliest pathogenic bacteria worldwide. The search for new antibiotics to treat drug-sensitive as well as drug-resistant tuberculosis has become a priority. The essential enzyme phenylalanyl-tRNA synthetase (PheRS) is an antibacterial drug target because of the large differences between bacterial and human PheRS counterparts. In a high-throughput screening of 2148 bioactive compounds, PF-3845, which is a known inhibitor of human fatty acid amide hydrolase, was identified inhibiting Mtb PheRS at Ki ∼ 0.73 ± 0.06 µM. The inhibition mechanism was studied with enzyme kinetics, protein structural modeling, and crystallography, in comparison to a PheRS inhibitor of the noted phenyl-thiazolylurea-sulfonamide class. The 2.3-Å crystal structure of Mtb PheRS in complex with PF-3845 revealed its novel binding mode, in which a trifluoromethyl-pyridinylphenyl group occupies the phenylalanine pocket, whereas a piperidine-piperazine urea group binds into the ATP pocket through an interaction network enforced by a sulfate ion. It represents the first non-nucleoside bisubstrate competitive inhibitor of bacterial PheRS. PF-3845 inhibits the in vitro growth of Mtb H37Rv at ∼24 µM, and the potency of PF-3845 increased against an engineered strain Mtb pheS-FDAS, suggesting on target activity in mycobacterial whole cells. PF-3845 does not inhibit human cytoplasmic or mitochondrial PheRS in biochemical assay, which can be explained from the crystal structures. Further medicinal chemistry efforts focused on the piperidine-piperazine urea moiety may result in the identification of a selective antibacterial lead compound.

Re-discovery of PF-3845 as a new chemical scaffold inhibiting phenylalanyl-tRNA synthetase in Mycobacterium tuberculosis.

Mycobacteria tuberculosis (Mtb) remains the deadliest pathogenic bacteria worldwide. The search for new antibiotics to treat drug-sensitive as well as drug-resistant tuberculosis has become a priority. The essential enzyme phenylalanyl-tRNA synthetase (PheRS) is an antibacterial drug target because of the large differences between bacterial and human PheRS counterparts. In a high-throughput screening of 2148 bioactive compounds, PF-3845, which is a known inhibitor of human fatty acid amide hydrolase (FAAH), was identified inhibiting Mtb PheRS at Ki ~0.73 ± 0.06 µM. The inhibition mechanism was studied with enzyme kinetics, protein structural modelling and crystallography, in comparison to a PheRS inhibitor of the noted phenyl-thiazolylurea-sulfonamide class. The 2.3-Å crystal structure of Mtb PheRS in complex with PF-3845 revealed its novel binding mode, in which a trifluoromethyl-pyridinylphenyl group occupies the Phe pocket while a piperidine-piperazine urea group binds into the ATP pocket through an interaction network enforced by a sulfate ion. It represents the first non-nucleoside bi-substrate competitive inhibitor of bacterial PheRS. PF-3845 inhibits the in vitro growth of Mtb H37Rv at ~24 µM, and the potency of PF-3845 increased against Mtb pheS-FDAS, suggesting on target activity in mycobacterial whole cells.  PF-3845 does not inhibit human cytoplasmic or mitochondrial PheRS in biochemical assay, which can be explained from the crystal structures. Further medicinal chemistry efforts focused on the piperidine-piperazine urea moiety may result in the identification of a selective antibacterial lead compound.

Derivatives of Natural Product Agrimophol as Disruptors of Intrabacterial pH Homeostasis in Mycobacterium tuberculosis.

This article reports the rational medicinal chemistry of a natural product, agrimophol (1), as a new disruptor of intrabacterial pH (pHIB) homeostasis in Mycobacterium tuberculosis (Mtb). Through the systematic investigation of the structure-activity relationship of 1, scaffold-hopping of the diphenylmethane scaffold, pharmacophore displacement strategies, and studies of the structure-metabolism relationship, a new derivative 5a was achieved. Compound 5a showed 100-fold increased potency in the ability to reduce pHIB to pH 6.0 and similarly improved mycobactericidal activity compared with 1 against both Mycobacterium bovis-BCG and Mtb. Compound 5a possessed improved metabolic stability in human liver microsomes and hepatocytes, lower cytotoxicity, higher selectivity index, and similar pKa value to natural 1. This study introduces a novel scaffold to an old drug, resulting in improved mycobactericidal activity through decreasing pHIB, and may contribute to the critical search for new agents to overcome drug resistance and persistence in the treatment of tuberculosis.

Rifampicin can induce antibiotic tolerance in mycobacteria via paradoxical changes in rpoB transcription.

Metrics commonly used to describe antibiotic efficacy rely on measurements performed on bacterial populations. However, certain cells in a bacterial population can continue to grow and divide, even at antibiotic concentrations that kill the majority of cells, in a phenomenon known as antibiotic tolerance. Here, we describe a form of semi-heritable tolerance to the key anti-mycobacterial agent rifampicin, which is known to inhibit transcription by targeting the ß subunit of the RNA polymerase (RpoB). We show that rifampicin exposure results in rpoB upregulation in a sub-population of cells, followed by growth. More specifically, rifampicin preferentially inhibits one of the two rpoB promoters (promoter I), allowing increased rpoB expression from a second promoter (promoter II), and thus triggering growth. Disruption of promoter architecture leads to differences in rifampicin susceptibility of the population, confirming the contribution of rifampicin-induced rpoB expression to tolerance.

Kasugamycin potentiates rifampicin and limits emergence of resistance in Mycobacterium tuberculosis by specifically decreasing mycobacterial mistranslation.

Most bacteria use an indirect pathway to generate aminoacylated glutamine and/or asparagine tRNAs. Clinical isolates of Mycobacterium tuberculosis with increased rates of error in gene translation (mistranslation) involving the indirect tRNA-aminoacylation pathway have increased tolerance to the first-line antibiotic rifampicin. Here, we identify that the aminoglycoside kasugamycin can specifically decrease mistranslation due to the indirect tRNA pathway. Kasugamycin but not the aminoglycoside streptomycin, can limit emergence of rifampicin resistance in vitro and increases mycobacterial susceptibility to rifampicin both in vitro and in a murine model of infection. Moreover, despite parenteral administration of kasugamycin being unable to achieve the in vitro minimum inhibitory concentration, kasugamycin alone was able to significantly restrict growth of Mycobacterium tuberculosis in mice. These data suggest that pharmacologically reducing mistranslation may be a novel mechanism for targeting bacterial adaptation.

Translational misreading in Mycobacterium smegmatis increases in stationary phase.

The study of errors in gene translation has largely been confined to a small number of model organisms. We have examined all possible misreading errors at a defined codon in Mycobacterium smegmatis. Using a dual-luciferase gain of function reporter system that employs a mutated essential lysine in firefly luciferase, we accurately quantified mistranslation errors. Overall, accuracy of gene translation was comparable with Escherichia coli at <1/2000 errors/codon during exponential growth. Stationary phase was associated with a dramatic increase in misincorporation errors by Lys-tRNACUU(Lys) at a subset of three codons, each with a single base changed from the AAG lysine codon. The maximum error rate detected was 0.2% with codon AUG. Treatment with streptomycin increased misreading errors at several codons associated in particular with U·U, G·U and C·U codon·anti-codon mismatches, but oxidative stress did not change translational fidelity. Our study is the first comprehensive examination of misreading errors for a defined codon in mycobacteria.

Mycobacterial mistranslation is necessary and sufficient for rifampicin phenotypic resistance.

Errors are inherent in all biological systems. Errors in protein translation are particularly frequent giving rise to a collection of protein quasi-species, the diversity of which will vary according to the error rate. As mistranslation rates rise, these new proteins could produce new phenotypes, although none have been identified to date. Here, we find that mycobacteria substitute glutamate for glutamine and aspartate for asparagine at high rates under specific growth conditions. Increasing the substitution rate results in remarkable phenotypic resistance to rifampicin, whereas decreasing mistranslation produces increased susceptibility to the antibiotic. These phenotypic changes are reflected in differential susceptibility of RNA polymerase to the drug. We propose that altering translational fidelity represents a unique form of environmental adaptation.