Secreted R-spondin proteins (RSPOs1-4) function as adult stem cell growth factors by potentiating Wnt signaling. Simultaneous binding of distinct regions of the RSPO Fu1-Fu2 domain module to the extracellular domains (ECDs) of the LGR4 G protein-coupled receptor and the ZNRF3 transmembrane E3 ubiquitin ligase regulates Wnt receptor availability. Here, we examine the molecular basis for the differing signaling strengths of RSPOs1-4 using purified RSPO Fu1-Fu2, LGR4 ECD, and ZNRF3 ECD proteins in Wnt signaling and receptor binding assays, and we engineer novel high-potency RSPOs. RSPO2/3/4 had similar signaling potencies that were stronger than that of RSPO1, whereas RSPO1/2/3 had similar efficacies that were greater than that of RSPO4. The RSPOs bound LGR4 with affinity rank order RSPO4 > RSPO2/3 > RSPO1 and ZNRF3 with affinity rank order RSPO2/3 > > RSPO1 > RSPO4. An RSPO2-4 chimera combining RSPO2 ZNRF3 binding with RSPO4 LGR4 binding was a "Superspondin" that exhibited enhanced ternary complex formation and 10-fold stronger signaling potency than RSPO2 and efficacy equivalent to RSPO2. An RSPO4-1 chimera combining RSPO4 ZNRF3 binding with RSPO1 LGR4 binding was a "Poorspondin" that exhibited signaling potency similar to RSPO1 and efficacy equivalent to RSPO4. Conferring increased ZNRF3 binding upon RSPO4 with amino acid substitutions L56F, I58L, and I63M enhanced its signaling potency and efficacy. Our results reveal the molecular basis for RSPOs1-4 activity differences and suggest that signaling potency is determined by ternary complex formation ability, whereas efficacy depends on ZNRF3 recruitment. High-potency RSPOs may be of value for regenerative medicine and/or therapeutic applications.

R-loops, consisting of an RNA-DNA hybrid and displaced single-stranded DNA, are physiological structures that regulate various cellular processes occurring on chromatin. Intriguingly, changes in R-loop dynamics have also been associated with DNA damage accumulation and genome instability; however, the mechanisms underlying R-loop-induced DNA damage remain unknown. Here we demonstrate in human cells that R-loops induced by the absence of diverse RNA processing factors, including the RNA/DNA helicases Aquarius (AQR) and Senataxin (SETX), or by the inhibition of topoisomerase I, are actively processed into DNA double-strand breaks (DSBs) by the nucleotide excision repair endonucleases XPF and XPG. Surprisingly, DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB), but not the global genome repair protein XPC. These findings reveal an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability.

Unscheduled R loops can be a source of genome instability, a hallmark of cancer cells. Although targeted proteomic approaches and cellular analysis of specific mutants have uncovered factors potentially involved in R-loop homeostasis, we report a more open screening of factors whose depletion causes R loops based on the ability of activation-induced cytidine deaminase (AID) to target R loops. Immunofluorescence analysis of γH2AX caused by small interfering RNAs (siRNAs) covering 3,205 protein-coding genes identifies 59 potential candidates, from which 13 are analyzed further and show a significant increase of R loops. Such candidates are enriched in factors involved in chromatin, transcription, and RNA biogenesis and other processes. A more focused study shows that the DDX47 helicase is an R-loop resolvase, whereas the MeCP2 methyl-CpG-binding protein uncovers a link between DNA methylation and R loops. Thus, our results suggest that a plethora of gene dysfunctions can alter cell physiology via affecting R-loop homeostasis by different mechanisms.

Recent whole-exome sequencing of malignancies have detected recurrent somatic mutations in U2 small nuclear ribonucleoprotein complex (snRNP) components of the spliceosome. These factors have also been identified as novel players in the DNA-damage response (DDR) in several genome-wide screens and proteomic analysis. Although accumulating evidence implies that the spliceosome has an important role in genome stability and is an emerging hallmark of cancer, its precise role in DNA repair still remains elusive. Here we identify two distinct mechanisms of how spliceosome U2 snRNP factors contribute to genome stability. We show that the spliceosome maintains protein levels of essential repair factors, thus contributing to homologous recombination repair. In addition, real-time laser microirradiation analysis identified rapid recruitment of the U2 snRNP factor SNRPA1 to DNA-damage sites. Functional analysis of SNRPA1 revealed a more immediate and direct role in preventing R-loop-induced DNA damage. Our present study implies a complex interrelation between transcription, mRNA splicing and the DDR. Cells require rapid spatio-temporal coordination of these chromatin transactions to cope with various forms of genotoxic stress.

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We aimed to investigate the associations between sociodemographic factors and instant messaging and social network site exposure among 9-year-old children.

Hidden variability is a fundamentally important issue in the context of gene expression studies. Collected tissue samples may have a wide variety of hidden effects that may alter their transcriptional landscape significantly. As a result their actual differential expression pattern can be potentially distorted, leading to inaccurate results from a genome-wide testing for the important transcripts.

The SARS-CoV-2 pandemic highlighted the need for broad-spectrum antivirals to increase our preparedness. Patients often require treatment by the time that blocking virus replication is less effective. Therefore, therapy should not only aim to inhibit the virus, but also to suppress pathogenic host responses, e.g., leading to microvascular changes and pulmonary damage. Clinical studies have previously linked SARS-CoV-2 infection to pathogenic intussusceptive angiogenesis in the lungs, involving the upregulation of angiogenic factors such as ANGPTL4. The β-blocker propranolol is used to suppress aberrant ANGPTL4 expression in the treatment of hemangiomas. Therefore, we investigated the effect of propranolol on SARS-CoV-2 infection and the expression of ANGPTL4. SARS-CoV-2 upregulated ANGPTL4 in endothelial and other cells, which could be suppressed with R-propranolol. The compound also inhibited the replication of SARS-CoV-2 in Vero-E6 cells and reduced the viral load by up to ~2 logs in various cell lines and primary human airway epithelial cultures. R-propranolol was as effective as S-propranolol but lacks the latter's undesired β-blocker activity. R-propranolol also inhibited SARS-CoV and MERS-CoV. It inhibited a post-entry step of the replication cycle, likely via host factors. The broad-spectrum antiviral effect and suppression of factors involved in pathogenic angiogenesis make R-propranolol an interesting molecule to further explore for the treatment of coronavirus infections.

Adaptation of bacteria to a changing environment is often accompanied by remodeling of the transcriptome. In the facultative phototroph Rhodobacter sphaeroides the alternative sigma factors RpoE, RpoHI and RpoHII play an important role in a variety of stress responses, including heat, oxidative stress and nutrient limitation. Photooxidative stress caused by the simultaneous presence of chlorophylls, light and oxygen is a special challenge for phototrophic organisms. Like alternative sigma factors, several non-coding sRNAs have important roles in the defense against photooxidative stress. RNAseq-based transcriptome data pointed to an influence of the stationary phase-induced StsR sRNA on levels of mRNAs and sRNAs with a role in the photooxidative stress response. Furthermore, StsR also affects expression of photosynthesis genes and of genes for regulators of photosynthesis genes. In vivo and in vitro interaction studies revealed that StsR, that is under control of the RpoHI and RpoHII sigma factors, targets rpoE mRNA and affects its abundance by altering its stability. RpoE regulates expression of the rpoHII gene and, consequently, expression of stsR. These data provide new insights into a complex regulatory network of protein regulators and sRNAs involved in defense against photooxidative stress and the regulation of photosynthesis genes.

The R-loops forming around DNA double-strand breaks (DSBs) within actively transcribed genes play a critical role in the DSB repair process. However, the mechanisms underlying R-loop formation at DSBs remain poorly understood, with diverse proposed models involving protein factors associated with RNA polymerase (RNAP) loading, pausing/backtracking or preexisting transcript RNA invasion. In this single-molecule study using Escherichia coli RNAP, we discovered that transcribing RNAP alone acts as a highly effective DSB sensor, responsible for generation of R-loops upon encountering downstream DSBs, without requiring any additional factors. The R-loop formation efficiency is greatly influenced by DNA end structures, ranging here from 2.8% to 73%, and notably higher on sticky ends with 3' or 5' single-stranded overhangs compared to blunt ends without any overhangs. The R-loops extend unidirectionally upstream from the DSB sites and can reach the transcription start site, interfering with ongoing-round transcription. Furthermore, the extended R-loops can persist and maintain their structures, effectively preventing the efficient initiation of subsequent transcription rounds. Our results are consistent with the bubble extension model rather than the 5'-end invasion model or the middle insertion model. These discoveries provide valuable insights into the initiation of DSB repair on transcription templates across bacteria, archaea and eukaryotes.

The current prevalence of tuberculosis (TB) in the People's Republic of China (P. R. China) demonstrates geographical heterogeneities, which show that the TB prevalence in the remote areas of Western China is more serious than that in the coastal plain of Eastern China. Although a lot of ecological studies have been applied in the exploration on the regional difference of disease risks, there is still a paucity of ecological studies on TB prevalence in P. R. China.

Characterizing the breeding sites of Culex pipiens complex is of major importance for the control of West Nile disease and other related diseases. However, little information is available about the characteristics and associated factors of the breeding sites of the Cx. pipiens complex in Lhasa, a representative high-altitude region in Southwestern China. In this study, a cross-sectional study concerning the breeding site characteristics and associated factors of the Cx. pipiens complex was carried out in Lhasa, Tibet from 2013-2016. Chi-square analysis and binary logistic regression analysis were applied to identify the key factors associated with the presence of Cx. pipiens complex larvae. Using a standard dipping method, 184 water bodies were examined and Cx. pipiens complex larvae were observed in 36 (19.57%) of them. There were significant differences in the composition of Cx. pipiens complex larvae among the breeding site stability (χ2 = 19.08, p = 0.00) and presence or absence of predators (χ2 = 6.986, p = 0.008). Binary logistic regression analysis indicated that breeding site stability and presence or absence of predators were significantly associated with the presence of Cx. pipiens complex larvae in Chengguan District, Lhasa. Relatively permanent water bodies such as water bodies along river fringes, ponds and puddles, and water bodies with no predators should be paid more attention for future Cx. pipiens complex larvae abatement campaigns in Lhasa, China.

We previously identified the heterogeneous ribonucleoprotein SAF-A/hnRNP U as a substrate for DNA-PK, a protein kinase involved in DNA damage response (DDR). Using laser micro-irradiation in human cells, we report here that SAF-A exhibits a two-phase dynamics at sites of DNA damage, with a rapid and transient recruitment followed by a prolonged exclusion. SAF-A recruitment corresponds to its binding to Poly(ADP-ribose) while its exclusion is dependent on the activity of ATM, ATR and DNA-PK and reflects the dissociation from chromatin of SAF-A associated with ongoing transcription. Having established that SAF-A RNA-binding domain recapitulates SAF-A dynamics, we show that this domain is part of a complex comprising several mRNA biogenesis proteins of which at least two, FUS/TLS and TAFII68/TAF15, exhibit similar biphasic dynamics at sites of damage. Using an original reporter for live imaging of DNA:RNA hybrids (R-loops), we show a transient transcription-dependent accumulation of R-loops at sites of DNA damage that is prolonged upon inhibition of RNA biogenesis factors exclusion. We propose that a new component of the DDR is an active anti-R-loop mechanism operating at damaged transcribed sites which includes the exclusion of mRNA biogenesis factors such as SAF-A, FUS and TAF15.

We aimed to characterize the mechanisms involved in neuroprotection by R-PIA administered before pilocarpine-induced seizures. Caspase-1 and caspase-3 activities were assayed using fluorimetry, and cathepsin D, HSP-70, and AKT expression levels were assayed using Western Blot of hippocampal samples. R-PIA was injected before pilocarpine (PILO), and four groups were studied at 1 h 30 min and 7 days following initiation of status epilepticus (SE): PILO, R-PIA+PILO, SALINE, and R-PIA+SALINE. At 1 h 30 min, significantly higher activities of caspase-1 and -3 were observed in the PILO group than in the SALINE group. Caspase-1 and -3 activities were higher in the R-PIA+PILO group than in the PILO group. At 7 days following SE, caspase-1 and -3 activities were higher than in the initial post-seizure phase compared to the SALINE group. The pretreatment of rats receiving PILO significantly reduced caspase activities compared to the PILO group. Expression of HSP-70, AKT, and cathepsin D was significantly higher in the PILO group than in the SALINE. In the R-PIA+PILO group, the expression of AKT and HSP-70 was greater than in rats receiving only PILO, while cathepsin D presented decreased expression. Pretreatment with R-PIA in PILO-injected rats strongly inhibited caspase-1 and caspase-3 activities and cathepsin D expression. It also increased expression levels of the neuroprotective proteins HSP-70 and AKT, suggesting an important role in modulating the cellular survival cascade.

Background: The clinical course of relapsed or refractory (r/r) diffuse large B-cell lymphoma (DLBCL) is variable, with limited efficacy data of second-line treatment in a post-rituximab real-world context. Hence, we explored the predictors and constructed a nomogram for risk stratification in this population. Patients and methods: Among 296 r/r DLBCL patients pretreated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) at the Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College between 2006 and 2017, 231 were included for nomogram construction. After randomization, we constructed the prognostic nomogram in the primary cohort (n=161) based on a multivariate analysis and confirmed it in the validation cohort (n=70). Additionally, we explored predictive factors for second-line therapy using a ordinal regression analysis. Results: Four independent prognostic factors including rituximab in the second-line setting, initial Eastern Cooperative Oncology Group (ECOG) performance status (PS), response to front-line treatment, and invasion on progression/recurrence were used to construct the nomogram. The nomogram had a C index of 0.70 with AUC values of 0.73 and 0.71 for the primary and validation cohorts, respectively. Subsequently, three risk groups (low, intermediate, and high) were determined with median overall survival (OS) of 38.0, 25.0, and 7.0 months, respectively. Additionally, we conducted a multivariate ordinal regression analysis and identified prior response to front-line treatment (odds ratio=4.50, 95% CI: 1.84-11.27, p=0.001) and bulky disease at diagnosis (odds ratio=0.36, 95% CI: 0.182-0.702, p=0.003) were two independent factors for second-line treatment efficacy. Conclusions: The established predictors for treatment efficacy and the novel nomogram for survival in r/r DLBCL patients could potentially be applied for risk stratification and treatment guidance in the post-rituximab era.

We have recently described the presence of the erythropoietin receptor (EPO-R) on CD4(+) lymphocytes and demonstrated that its expression increases during their activation, reaching a level reported to be typical for erythroid progenitors. This observation suggests that EPO-R expression is modulated during lymphocyte activation, which may be important for the cells' function. Here we investigated whether the expression of GATA1, GATA3 and Sp1 transcription factors is correlated with the expression of EPO-R in human CD4(+) lymphocytes stimulated with monoclonal anti-CD3 antibody. The expression of GATA1, GATA3 and Sp1 transcription factors in CD4(+) cells was estimated before and after stimulation with anti-CD3 antibody by Western Blot and flow cytometry. The expression of EPO-R was measured using real-time PCR and flow cytometry. There was no change in the expression of GATA1 and GATA3 in CD4(+) lymphocytes after stimulation with anti-CD3 antibody. However, stimulation resulted in the significantly increased expression of the Sp1 factor. CD4(+) lymphocytes stimulated with anti-CD3 antibody exhibited an increase in both the expression level of EPOR gene and the number of EPO-R molecules on the cells' surface, the latter being significantly correlated with the increased expression of Sp1. Sp1 is noted to be the single transcription factor among the ones studied whose level changes as a result of CD4(+) lymphocyte stimulation. It seems that Sp1 may significantly affect the number of EPO-R molecules present on the surface of activated CD4(+) lymphocytes.

The prevalence of allergic diseases in children is markedly increasing to epidemic proportions. The aim of this study is to describe the presence and examine associated parental and child characteristics of allergic sensitization and physician-diagnosed allergy in Dutch children at age 10 years. This study among 5471 children was performed in a population-based prospective cohort from fetal life onwards. Allergic sensitization was measured by skin prick tests. Physician-diagnosed allergy and parental and child characteristics were collected by questionnaires. In children aged 10 years, inhalant and food allergic sensitization was present in 32.2% and 7.1%, and physician-diagnosed inhalant and food allergy in 12.4% and 2.3%. Maternal and paternal history of allergy, eczema or asthma was associated with increased risks of physician-diagnosed inhalant allergy (aOR (95% CI) 1.44 (1.23-1.70) and 1.59 (1.30-1.94), respectively), but not with food allergy. Asthma and eczema ever at age 10 years were associated with increased risks of physician-diagnosed inhalant allergy (4.60 (3.55-5.96) and 2.42 (1.94-3.03), respectively). Eczema ever at age 10 years was associated with an increased risk of physician-diagnosed food allergy (5.78, 3.04-9.52), with the highest risk of cashew (7.36, 3.20-16.94) and peanut (5.58, 3.08-10.10) food allergy.Conclusions: We found strong effects of parental history of allergy, eczema or asthma on the presence of physician-diagnosed inhalant allergy in children at age 10 years. Eczema ever at age 10 years was a strong risk factor for the development of physician-diagnosed inhalant and food allergy. What is Known: • The prevalence of allergic diseases in children has markedly increased. • Early-life influences are critically important in the development of allergic diseases. What is New: • Maternal and paternal history of allergy, eczema or asthma is associated with increased risks of physician-diagnosed inhalant allergy but not with food allergy. • Eczema ever at age 10 years is associated with an increased risk of physician-diagnosed food allergy, with the highest risk for cashew and peanut food allergy.

To assess the prevalence of HIV infection and characteristically risk of factors which associated with HIV infection among MSM in Harbin, China.

Primary gastric diffuse large B-cell lymphoma (GDLBCL) is a heterogeneous disease in clinicopathological features and prognosis. Programmed death ligand-1 (PD-L1) and microRNA-34a (miR-34a) play crucial roles in GDLBCL progress. The purpose of this research is to explore the clinical significance of PD-L1 and miR-34a expression in GDLBCL.

R-loops are three-stranded nucleic acid structures formed from the hybridization of RNA and DNA. While the pathological consequences of R-loops have been well-studied to date, the locations, classes, and dynamics of physiological R-loops remain poorly understood. R-loop mapping studies provide insight into R-loop dynamics, but their findings are challenging to generalize. This is due to the narrow biological scope of individual studies, the limitations of each mapping modality, and, in some cases, poor data quality. In this study, we reprocessed 810 R-loop mapping datasets from a wide array of biological conditions and mapping modalities. From this data resource, we developed an accurate R-loop data quality control method, and we reveal the extent of poor-quality data within previously published studies. We then identified a set of high-confidence R-loop mapping samples and used them to define consensus R-loop sites called 'R-loop regions' (RL regions). In the process, we identified a stark divergence between RL regions detected by S9.6 and dRNH-based mapping methods, particularly with respect to R-loop size, location, and colocalization with RNA binding factors. Taken together, this work provides a much-needed method to assess R-loop data quality and offers novel context regarding the differences between dRNH- and S9.6-based R-loop mapping approaches.