Clinical identification and microbiota analysis of Chlamydia psittaci- and Chlamydia abortus- pneumonia by metagenomic next-generation sequencing.

Introduction: Recently, the incidence of chlamydial pneumonia caused by rare pathogens such as C. psittaci or C. abortus has shown a significant upward trend. The non-specific clinical manifestations and the limitations of traditional pathogen identification methods determine that chlamydial pneumonia is likely to be poorly diagnosed or even misdiagnosed, and may further result in delayed treatment or unnecessary antibiotic use. mNGS's non-preference and high sensitivity give us the opportunity to obtain more sensitive detection results than traditional methods for rare pathogens such as C. psittaci or C. abortus. Methods: In the present study, we investigated both the pathogenic profile characteristics and the lower respiratory tract microbiota of pneumonia patients with different chlamydial infection patterns using mNGS. Results: More co-infecting pathogens were found to be detectable in clinical samples from patients infected with C. psittaci compared to C. abortus, suggesting that patients infected with C. psittaci may have a higher risk of mixed infection, which in turn leads to more severe clinical symptoms and a longer disease course cycle. Further, we also used mNGS data to analyze for the first time the characteristic differences in the lower respiratory tract microbiota of patients with and without chlamydial pneumonia, the impact of the pattern of Chlamydia infection on the lower respiratory tract microbiota, and the clinical relevance of these characteristics. Significantly different profiles of lower respiratory tract microbiota and microecological diversity were found among different clinical subgroups, and in particular, mixed infections with C. psittaci and C. abortus resulted in lower lung microbiota diversity, suggesting that chlamydial infections shape the unique lung microbiota pathology, while mixed infections with different Chlamydia may have important effects on the composition and diversity of the lung microbiota. Discussion: The present study provides possible evidences supporting the close correlation between chlamydial infection, altered microbial diversity in patients' lungs and clinical parameters associated with infection or inflammation in patients, which also provides a new research direction to better understand the pathogenic mechanisms of pulmonary infections caused by Chlamydia.

The diagnosis of leptospirosis complicated by pulmonary tuberculosis complemented by metagenomic next-generation sequencing: A case report.

Leptospirosis is a zoonotic infection caused by the pathogenic Leptospira. Leptospirosis is transmitted mainly through contact with contaminated rivers, lakes, or animals carrying Leptospira. Human leptospirosis has a wide range of non-specific clinical manifestations ranging from fever, hypotension, and myalgia to multi-organ dysfunction, which severely hampers the timely clinical diagnosis and treatment of leptospirosis. Therefore, there is an urgent clinical need for an efficient strategy/method that can be used for the accurate diagnosis of leptospirosis, especially in critically ill patients. Here, we report a case of a 75-year-old male patient with clinical presentation of fever, cough, and diarrhea. Initial laboratory tests and a computed tomography (CT) scan of the chest suggested only tuberculosis. The patient was finally diagnosed with pulmonary tuberculosis (PTB) combined with leptospirosis by sputum Xpert MTB RIF, epidemiological investigations, and delayed serological testing. Furthermore, through metagenomic next-generation sequencing (mNGS) of clinical samples of cerebrospinal fluid (CSF), urine, plasma and sputum, the causative pathogens were identified as Mycobacterium tuberculosis complex and Leptospira spp. With specific treatment for both leptospirosis and tuberculosis, and associated supportive care (e.g., hemodialysis), the patient showed a good prognosis. This case report suggests that mNGS can generate a useful complement to conventional pathogenic diagnostic methods through more detailed etiological screening (i.e., at the level of species or species complex).

Human Rickettsia felis infections in Mainland China.

We identified four flea-borne spotted fever cases caused by Rickettsia felis in a retrospective survey of 182 patients with fever of unknown origin (FUO) in China between 2021 and 2022. The clinical signs and symptoms of the patients were similar to those of other rickettsioses, including fever, rash, and liver and kidney dysfunction. All four patients in the present study developed pneumonia or lung lesions after R. felis infection. The cases of R. felis infection, a neglected infectious disease, were sporadic in multiple provinces of the country. The high prevalence (2.14%, 4/187) of R. felis among patients with FUO highlights the risk posed by this pathogen to public health in China.

Effect of invasive mechanical ventilation on the diversity of the pulmonary microbiota.

Pulmonary microbial diversity may be influenced by biotic or abiotic conditions (e.g., disease, smoking, invasive mechanical ventilation (MV), etc.). Specially, invasive MV may trigger structural and physiological changes in both tissue and microbiota of lung, due to gastric and oral microaspiration, altered body posture, high O2 inhalation-induced O2 toxicity in hypoxemic patients, impaired airway clearance and ventilator-induced lung injury (VILI), which in turn reduce the diversity of the pulmonary microbiota and may ultimately lead to poor prognosis. Furthermore, changes in (local) O2 concentration can reduce the diversity of the pulmonary microbiota by affecting the local immune microenvironment of lung. In conclusion, systematic literature studies have found that invasive MV reduces pulmonary microbiota diversity, and future rational regulation of pulmonary microbiota diversity by existing or novel clinical tools (e.g., lung probiotics, drugs) may improve the prognosis of invasive MV treatment and lead to more effective treatment of lung diseases with precision.

Stabilizing heterochromatin by DGCR8 alleviates senescence and osteoarthritis.

DiGeorge syndrome critical region 8 (DGCR8) is a critical component of the canonical microprocessor complex for microRNA biogenesis. However, the non-canonical functions of DGCR8 have not been studied. Here, we demonstrate that DGCR8 plays an important role in maintaining heterochromatin organization and attenuating aging. An N-terminal-truncated version of DGCR8 (DR8dex2) accelerated senescence in human mesenchymal stem cells (hMSCs) independent of its microRNA-processing activity. Further studies revealed that DGCR8 maintained heterochromatin organization by interacting with the nuclear envelope protein Lamin B1, and heterochromatin-associated proteins, KAP1 and HP1γ. Overexpression of any of these proteins, including DGCR8, reversed premature senescent phenotypes in DR8dex2 hMSCs. Finally, DGCR8 was downregulated in pathologically and naturally aged hMSCs, whereas DGCR8 overexpression alleviated hMSC aging and mouse osteoarthritis. Taken together, these analyses uncovered a novel, microRNA processing-independent role in maintaining heterochromatin organization and attenuating senescence by DGCR8, thus representing a new therapeutic target for alleviating human aging-related disorders.

Maintenance of Nucleolar Homeostasis by CBX4 Alleviates Senescence and Osteoarthritis.

CBX4, a component of polycomb repressive complex 1 (PRC1), plays important roles in the maintenance of cell identity and organ development through gene silencing. However, whether CBX4 regulates human stem cell homeostasis remains unclear. Here, we demonstrate that CBX4 counteracts human mesenchymal stem cell (hMSC) aging via the maintenance of nucleolar homeostasis. CBX4 protein is downregulated in aged hMSCs, whereas CBX4 knockout in hMSCs results in destabilized nucleolar heterochromatin, enhanced ribosome biogenesis, increased protein translation, and accelerated cellular senescence. CBX4 maintains nucleolar homeostasis by recruiting nucleolar protein fibrillarin (FBL) and heterochromatin protein KRAB-associated protein 1 (KAP1) at nucleolar rDNA, limiting the excessive expression of rRNAs. Overexpression of CBX4 alleviates physiological hMSC aging and attenuates the development of osteoarthritis in mice. Altogether, our findings reveal a critical role of CBX4 in counteracting cellular senescence by maintaining nucleolar homeostasis, providing a potential therapeutic target for aging-associated disorders.

Chemical screen identifies a geroprotective role of quercetin in premature aging.

Aging increases the risk of various diseases. The main goal of aging research is to find therapies that attenuate aging and alleviate aging-related diseases. In this study, we screened a natural product library for geroprotective compounds using Werner syndrome (WS) human mesenchymal stem cells (hMSCs), a premature aging model that we recently established. Ten candidate compounds were identified and quercetin was investigated in detail due to its leading effects. Mechanistic studies revealed that quercetin alleviated senescence via the enhancement of cell proliferation and restoration of heterochromatin architecture in WS hMSCs. RNA-sequencing analysis revealed the transcriptional commonalities and differences in the geroprotective effects by quercetin and Vitamin C. Besides WS hMSCs, quercetin also attenuated cellular senescence in Hutchinson-Gilford progeria syndrome (HGPS) and physiological-aging hMSCs. Taken together, our study identifies quercetin as a geroprotective agent against accelerated and natural aging in hMSCs, providing a potential therapeutic intervention for treating age-associated disorders.

SIRT6 deficiency results in developmental retardation in cynomolgus monkeys.

SIRT6 acts as a longevity protein in rodents1,2. However, its biological function in primates remains largely unknown. Here we generate a SIRT6-null cynomolgus monkey (Macaca fascicularis) model using a CRISPR-Cas9-based approach. SIRT6-deficient monkeys die hours after birth and exhibit severe prenatal developmental retardation. SIRT6 loss delays neuronal differentiation by transcriptionally activating the long non-coding RNA H19 (a developmental repressor), and we were able to recapitulate this process in a human neural progenitor cell differentiation system. SIRT6 deficiency results in histone hyperacetylation at the imprinting control region of H19, CTCF recruitment and upregulation of H19. Our results suggest that SIRT6 is involved in regulating development in non-human primates, and may provide mechanistic insight into human perinatal lethality syndrome.

ATF6 safeguards organelle homeostasis and cellular aging in human mesenchymal stem cells.

Loss of organelle homeostasis is a hallmark of aging. However, it remains elusive how this occurs at gene expression level. Here, we report that human mesenchymal stem cell (hMSC) aging is associated with dysfunction of double-membrane organelles and downregulation of transcription factor ATF6. CRISPR/Cas9-mediated inactivation of ATF6 in hMSCs, not in human embryonic stem cells and human adipocytes, results in premature cellular aging, characteristic of loss of endomembrane homeostasis. Transcriptomic analyses uncover cell type-specific constitutive and stress-induced ATF6-regulated genes implicated in various layers of organelles' homeostasis regulation. FOS was characterized as a constitutive ATF6 responsive gene, downregulation of which contributes to hMSC aging. Our study unravels the first ATF6-regulated gene expression network related to homeostatic regulation of membrane organelles, and provides novel mechanistic insights into aging-associated attrition of human stem cells.

Regulation of Stem Cell Aging by Metabolism and Epigenetics.

Stem cell aging and exhaustion are considered important drivers of organismal aging. Age-associated declines in stem cell function are characterized by metabolic and epigenetic changes. Understanding the mechanisms underlying these changes will likely reveal novel therapeutic targets for ameliorating age-associated phenotypes and for prolonging human healthspan. Recent studies have shown that metabolism plays an important role in regulating epigenetic modifications and that this regulation dramatically affects the aging process. This review focuses on current knowledge regarding the mechanisms of stem cell aging, and the links between cellular metabolism and epigenetic regulation. In addition, we discuss how these interactions sense and respond to environmental stress in order to maintain stem cell homeostasis, and how environmental stimuli regulate stem cell function. Additionally, we highlight recent advances in the development of therapeutic strategies to rejuvenate dysfunctional aged stem cells.

Visualization of aging-associated chromatin alterations with an engineered TALE system.

Visualization of specific genomic loci in live cells is a prerequisite for the investigation of dynamic changes in chromatin architecture during diverse biological processes, such as cellular aging. However, current precision genomic imaging methods are hampered by the lack of fluorescent probes with high specificity and signal-to-noise contrast. We find that conventional transcription activator-like effectors (TALEs) tend to form protein aggregates, thereby compromising their performance in imaging applications. Through screening, we found that fusing thioredoxin with TALEs prevented aggregate formation, unlocking the full power of TALE-based genomic imaging. Using thioredoxin-fused TALEs (TTALEs), we achieved high-quality imaging at various genomic loci and observed aging-associated (epi) genomic alterations at telomeres and centromeres in human and mouse premature aging models. Importantly, we identified attrition of ribosomal DNA repeats as a molecular marker for human aging. Our study establishes a simple and robust imaging method for precisely monitoring chromatin dynamics in vitro and in vivo.

Modeling xeroderma pigmentosum associated neurological pathologies with patients-derived iPSCs.

Xeroderma pigmentosum (XP) is a group of genetic disorders caused by mutations of XP-associated genes, resulting in impairment of DNA repair. XP patients frequently exhibit neurological degeneration, but the underlying mechanism is unknown, in part due to lack of proper disease models. Here, we generated patient-specific induced pluripotent stem cells (iPSCs) harboring mutations in five different XP genes including XPA, XPB, XPC, XPG, and XPV. These iPSCs were further differentiated to neural cells, and their susceptibility to DNA damage stress was investigated. Mutation of XPA in either neural stem cells (NSCs) or neurons resulted in severe DNA damage repair defects, and these neural cells with mutant XPA were hyper-sensitive to DNA damage-induced apoptosis. Thus, XP-mutant neural cells represent valuable tools to clarify the molecular mechanisms of neurological abnormalities in the XP patients.

SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2.

SIRT6 belongs to the mammalian homologs of Sir2 histone NAD(+)-dependent deacylase family. In rodents, SIRT6 deficiency leads to aging-associated degeneration of mesodermal tissues. It remains unknown whether human SIRT6 has a direct role in maintaining the homeostasis of mesodermal tissues. To this end, we generated SIRT6 knockout human mesenchymal stem cells (hMSCs) by targeted gene editing. SIRT6-deficient hMSCs exhibited accelerated functional decay, a feature distinct from typical premature cellular senescence. Rather than compromised chromosomal stability, SIRT6-null hMSCs were predominately characterized by dysregulated redox metabolism and increased sensitivity to the oxidative stress. In addition, we found SIRT6 in a protein complex with both nuclear factor erythroid 2-related factor 2 (NRF2) and RNA polymerase II, which was required for the transactivation of NRF2-regulated antioxidant genes, including heme oxygenase 1 (HO-1). Overexpression of HO-1 in SIRT6-null hMSCs rescued premature cellular attrition. Our study uncovers a novel function of SIRT6 in maintaining hMSC homeostasis by serving as a NRF2 coactivator, which represents a new layer of regulation of oxidative stress-associated stem cell decay.

PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype.

PTEN is a tumour suppressor frequently mutated in many types of cancers. Here we show that targeted disruption of PTEN leads to neoplastic transformation of human neural stem cells (NSCs), but not mesenchymal stem cells. PTEN-deficient NSCs display neoplasm-associated metabolic and gene expression profiles and generate intracranial tumours in immunodeficient mice. PTEN is localized to the nucleus in NSCs, binds to the PAX7 promoter through association with cAMP responsive element binding protein 1 (CREB)/CREB binding protein (CBP) and inhibits PAX7 transcription. PTEN deficiency leads to the upregulation of PAX7, which in turn promotes oncogenic transformation of NSCs and instates 'aggressiveness' in human glioblastoma stem cells. In a large clinical database, we find increased PAX7 levels in PTEN-deficient glioblastoma. Furthermore, we identify that mitomycin C selectively triggers apoptosis in NSCs with PTEN deficiency. Together, we uncover a potential mechanism of how PTEN safeguards NSCs, and establish a cellular platform to identify factors involved in NSC transformation, potentially permitting personalized treatment of glioblastoma.

Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging.

Werner syndrome (WS) is a premature aging disorder caused by WRN protein deficiency. Here, we report on the generation of a human WS model in human embryonic stem cells (ESCs). Differentiation of WRN-null ESCs to mesenchymal stem cells (MSCs) recapitulates features of premature cellular aging, a global loss of H3K9me3, and changes in heterochromatin architecture. We show that WRN associates with heterochromatin proteins SUV39H1 and HP1α and nuclear lamina-heterochromatin anchoring protein LAP2ß. Targeted knock-in of catalytically inactive SUV39H1 in wild-type MSCs recapitulates accelerated cellular senescence, resembling WRN-deficient MSCs. Moreover, decrease in WRN and heterochromatin marks are detected in MSCs from older individuals. Our observations uncover a role for WRN in maintaining heterochromatin stability and highlight heterochromatin disorganization as a potential determinant of human aging.

CRISPR/Cas9 and TALE: beyond cut and paste.

Nuclease-based genome editing has proven to be a powerful and promising tool for disease modeling and gene therapy. Recent advances in CRISPR/Cas and TALE indicate that they could also be used as a targeted regulator of gene expression, as well as being utilized for illuminating specific chromosomal structures or genomic regions.