Characteristics of Hepatitis B Virus, Hepatitis C Virus, and Syphilis Coinfection in People With HIV/AIDS Contracted Through Different Sources: Retrospective Study.

BACKGROUND: The burden of hepatitis B virus (HBV), hepatitis C virus (HCV), and syphilis coinfections remains disproportionately high among people living with HIV/AIDS. Hubei province is located in central China, where there are distinct regional characteristics of the distribution of people living with HIV/AIDS acquired via diverse transmission routes and the AIDS epidemic itself. OBJECTIVE: We aimed to estimate the magnitude of HBV, HCV, or syphilis coinfections among people living with HIV/AIDS with blood-borne transmission, which includes former paid blood donors, contaminated blood recipients, and intravenous drug users, as well as among people with sex-borne HIV transmission (including heterosexual people and men who have sex with men) and people with mother-to-child HIV transmission. METHODS: From January 2010 to December 2020, people living with HIV/AIDS were tested for hepatitis B surface antigen (HBsAg), HCV antibodies, and syphilis-specific antibodies. The positive patients were further tested for HBV markers, HBV DNA, and HCV RNA, and received a rapid plasma reagin circle card test. All people living with HIV/AIDS were first divided into transmission groups (blood, sex, and mother-to-child); then, people with blood-borne HIV transmission were divided into former paid blood donors, contaminated blood recipients, and intravenous drug users, while people with sex-borne HIV transmission were divided into heterosexual people and men who have sex with men. RESULTS: Among 6623 people living with HIV/AIDS, rates of chronic HCV infection were 80.3% (590/735) in former paid blood donors, 73.3% (247/337) in intravenous drug users, 57.1% (444/777) in contaminated blood recipients, 19.4% (21/108) in people with mother-to-child HIV transmission, 8.1% (240/2975) in heterosexual people, and 1.2% (21/1691) in men who have sex with men. Chronic HBV infection rates were similar among all people with blood-borne HIV transmission. However, compared to heterosexual people, the chronic HBV infection rate was greater in men who have sex with men (213/1691, 12.6% vs 308/2975, 10.4%; χ21=5.469; P=.02), although HBV exposure was less common (827/1691, 48.9% vs 1662/2975, 55.9%; χ21=20.982; P<.001). Interestingly, the combination of HBsAg and hepatitis B e antigen (HBeAg) was found in 11 patients with sex-borne HIV transmission, but in 0 people with blood-borne HIV transmission (11/196, 5.6% vs 0/521, 0%; χ21=29.695, P<.001). In people with sex-borne HIV transmission, the proportions of patients with a syphilis titer ≥1:16 and neurosyphilis were 8.6% (105/1227) and 7.8% (37/473), respectively, whereas these values were 0 in people with blood-borne HIV transmission. CONCLUSIONS: In people living with HIV/AIDS, HCV transmission intensity was significantly associated with specific exposure modes of blood or sexual contact. The rate of chronic HBV infection among men who have sex with men was higher than in any other population. Attention should be paid to the high prevalence of neurosyphilis in people living with HIV/AIDS who contract HIV by sexual intercourse.

A self-regulated network for dynamically balancing multiple precursors in complex biosynthetic pathways.

Microbial synthesis has emerged as a promising and sustainable alternative to traditional chemical synthesis and plant extraction. However, the competition between synthetic pathways and central metabolic pathways for cellular resources may impair final production efficiency. Moreover, when the synthesis of target product requires multiple precursors from the same node, the conflicts of carbon flux have further negative impacts on yields. In this study, a self-regulated network was developed to relieve the competition of precursors in complex synthetic pathways. Using 4-hydroxycoumarin (4-HC) synthetic pathway as a proof of concept, we employed an intermediate as a trigger to dynamically rewire the metabolic flux of pyruvate and control the expression levels of genes in 4-HC synthetic pathway, achieving self-regulation of multiple precursors and enhanced titer. Transcriptomic analysis results additionally demonstrated that the gene transcriptional levels of both pyruvate kinase PykF and synthetic pathway enzyme SdgA dynamically changed according to the intermediate concentrations. Overall, our work established a self-regulated network to dynamically balance the metabolic flux of two precursors in 4-HC biosynthesis, providing insight into balancing biosynthetic pathways where multiple precursors compete and interfere with each other.

JCAD deficiency attenuates activation of hepatic stellate cells and cholestatic fibrosis.

BACKGROUND/AIMS: Cholestatic liver diseases including primary biliary cholangitis (PBC) are associated with active hepatic fibrogenesis, which ultimately progresses to cirrhosis. Activated hepatic stellate cells (HSCs) are the main fibrogenic effectors in response to cholangiocyte damage. JCAD regulates cell proliferation and malignant transformation in nonalcoholic steatoheaptitis-associated hepatocellular carcinoma (NASH-HCC). However, its participation in cholestatic fibrosis has not been explored yet. METHODS: Serial sections of liver tissue of PBC patients were stained with immunofluorescence. Hepatic fibrosis was induced by bile duct ligation (BDL) in wild-type (WT), global JCAD knockout mice (JCAD-KO) and HSC-specific JCAD knockout mice (HSC-JCAD-KO), and evaluated by histopathology and biochemical tests. In situ-activated HSCs isolated from BDL mice were used to determine effects of JCAD on HSC activation. RESULTS: In consistence with staining of liver sections from PBC patients, immunofluorescent staining revealed that JCAD expression was identified in smooth muscle α-actin (α-SMA)-positive fibroblast-like cells and was significantly up-regulated in WT mice with BDL. JCAD deficiency remarkably ameliorated BDL-induced hepatic injury and fibrosis, as documented by liver hydroxyproline content, when compared to WT mice with BDL. Histopathologically, collagen deposition was dramatically reduced in both JCAD-KO and HSC-JCAD-KO mice compared to WT mice, as visualized by Trichrome staining and semi-quantitative scores. Moreover, JCAD deprivation significantly attenuated in situ HSC activation and reduced expression of fibrotic genes after BDL. CONCLUSION: JCAD deficiency effectively suppressed hepatic fibrosis induced by BDL in mice, and the underlying mechanisms are largely through suppressed Hippo-YAP signaling activity in HSCs.

The expanded CRISPR toolbox for constructing microbial cell factories.

Microbial cell factories (MCFs) convert low-cost carbon sources into valuable compounds. The CRISPR/Cas9 system has revolutionized MCF construction as a remarkable genome editing tool with unprecedented programmability. Recently, the CRISPR toolbox has been significantly expanded through the exploration of new CRISPR systems, the engineering of Cas effectors, and the incorporation of other effectors, enabling multi-level regulation and gene editing free of double-strand breaks. This expanded CRISPR toolbox powerfully promotes MCF construction by facilitating pathway construction, enzyme engineering, flux redistribution, and metabolic burden control. In this article, we summarize different CRISPR tool designs and their applications in MCF construction for gene editing, transcriptional regulation, and enzyme modulation. Finally, we also discuss future perspectives for the development and application of the CRISPR toolbox.

Establishing Tunable Genetic Logic Gates with Versatile Dynamic Performance by Varying Regulatory Parameters.

Genetic logic gates can be employed in metabolic engineering and synthetic biology to regulate gene expression based on diverse inputs. Design of tunable genetic logic gates with versatile dynamic performance is essential for expanding the usability of these toolsets. Here, using the p-coumaric acid biosensor system as a proof-of-concept, we initially investigated the parameters influencing the buffer (BUF) genetic logic gates. Subsequently, integrating binding sequences from the p-coumaric acid biosensor system and tetR or lacI regulation systems into a constitutive promoter yielded AND genetic logic gates. Additionally, characterized antisense RNAs (asRNAs) or single guide RNAs (sgRNAs) with various repression efficiencies were combined with BUF gates to construct a suite of p-coumaric acid-triggered NOT genetic logic gates. Finally, the designed BUF and NOT gates were combined to construct bifunctional genetic circuits that were subjected to orthogonality evaluation. The genetic logic gates established in this study can serve as valuable tools in future applications of metabolic engineering and synthetic biology.

The safety and efficacy of PD-1 inhibitors in patients with advanced cancers and HIV/AIDS in China.

Purpose-Immunotherapy has revolutionized cancer therapy, becoming the standard of care for various malignancy treatments. Human immunodeficiency virus (HIV) patients, however, are an underserved group with limited access to clinical trials and cancer therapy. This study was to evaluate the safety and efficacy of programmed cell death 1 (PD - 1) inhibitors in patients with advanced cancer and HIV/acquired immunodeficiency syndrome (AIDS). Methods and Materials-We performed a prospective, open-label, nonrandomized, phase 1 single center study. Patients with advanced cancer and HIV/AIDS received the treatment of PD - 1 inhibitors (camrelizumab, 200 mg, administered intravenously every 3 weeks), along with combination antiretroviral therapy (cART) for HIV. Results-Sixteen participants (12 men and 4 women; median age, 46.5 (29 - 78) years) were enrolled; 1 had non - Hodgkin lymphoma (NHL), and 15 had non - AIDS - defining cancers. Safety was observed over 130 cycles of treatment with camrelizumab. Most treatment-emergent adverse events at least possibly attributed to camrelizumab were grade 1 or 2, including reactive cutaneous capillary endothelial proliferation (RCCEP) (9 participants), hearing loss (1 participant), hypophysitis (1 participant). 3 participants experienced hemorrhage due to poor performance status. HIV was controlled in all participants. Best tumor responses included 3 complete response, 5 partial response, 2 stable disease, and 6 progressive disease. The 2 years progression-free survival (PFS) was 67.0% (95% CI: -0.05, 0.00) and overall survival (OS) was 55.3% (95% CI: -0.05, 0.01) for the 16 patients who had received camrelizumab. Conclusions-This study demonstrates that camrelizumab treatment in patients with advanced cancers and HIV/AIDS was feasible and the clinical outcomes were acceptable.

Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria.

The RNA-guided Cas9 endonucleases have revolutionized gene editing and regulation, but their targeting scope is limited by the protospacer adjacent motif (PAM) requirement. The most extensively used SpCas9 from Streptococcus pyogenes recognizes the NGG PAM via an RxR PAM-binding motif within its PAM-interaction (PI) domain. To overcome the strict PAM requirement, we identified and characterized a Cas9 ortholog from Streptococcus equinus HC5 (SeHCas9) that shows high sequence identity with SpCas9 but harbors a different RxQ PAM-binding motif. Complete PAM profiling revealed that SeHCas9 recognized an NAG PAM and accommodated NKG and NAW PAMs. We investigated the PAM interaction mechanism by identifying the crucial role of R1336 within the RxQ motif in determining PAM specificity, as well as the essentiality of two conserved residues (R1152 and Q1229) across Cas9 orthologs bearing the RxQ motif for PAM recognition. Further protein engineering created two variants, SeHdCas9-Q1229R and SeHdCas9-RR, that showed robust repression across an NNG and NNN PAM range, respectively. Our work proposes a novel Cas9 PAM interaction mechanism and establishes PAM-free Cas9 variants for bacterial gene control with almost no targeting restriction.

Characterization of peripheral cytokine-secreting cells responses in HIV/TB co-infection.

Background: Currently the responses of peripheral cytokine-secreting cells in the natural course of human immunodeficiency virus (HIV) and tuberculosis (TB) co-infection haven't been fully elucidated. Methods: The function of peripheral proinflammatory, regulatory and cytotoxic cytokine-secreting cells were investigated by direct intracellular cytokine staining (ICS) and flow cytometry, additionally, the absolute numbers of different cytokine-secreting cells were measured among patients with HIV/TB co-infection (HT group), and compared them with the healthy controls (HC group), patients with TB (TB group) and patients with HIV infection (HIV group). After one week's anti-TB treatment, the changes of the percentages of cytokine-secreting cells were further evaluated in TB and HT groups. Results: Totally 26 individuals in the HC group, 51 in the TB group, 26 in the HIV group and 29 in the HT group were enrolled. The HT. HT group exhibited significantly lower absolute numbers of IFN-γ+CD4+, IFN-γ+CD8+, TNF-α+CD4+, IL17A+CD4+ T cells and TNF-α+CD14+ monocytes than the TB and HIV groups. Compared with the TB group, the percentages of CD8+ T cells secreting IFN-γ and perforin (p=0.010; p=0.043) were significantly lower among the HT group. Compared with the HIV group, the percentages of CD4+, CD8+ T cells and CD14+ monocytes secreting TNF-α (p=0.013; p=0.001; p<0.001) were significantly decreased, and the percentage of CD8+ T cells secreting IL-17A (p=0.015) was significantly increased among the HT group. Both the percentages of CD4+ T cells secreting TGF-ß (p<0.001; p=0.001), and CD4+ and CD8+ T cells secreting granzyme A (all p<0.001), were significantly higher among the HT group than among the TB group and HIV group. After one week's anti-TB treatment, an increased percentage of CD4+ T cells secreting TNF-α (p=0.003) was found in the TB group, and an increased percentage of CD8+ T cells secreting TNF-α (p=0.029) was found in the HT group. Conclusion: Significantly different functional profiles of peripheral proinflammatory, regulatory, and cytotoxic cytokine-secreting cells were observed in the natural course of HIV/TB co-infection compared to TB and HIV infection alone, even though the absolute numbers of those cells were significantly lower in HIV/TB co-infection. TNF-α-secreting CD8+ T cells may be a more sensitive marker for early evaluation of anti-TB treatment efficacy in patients with HIV/TB co-infection.

Identifying, Characterizing, and Engineering a Phenolic Acid-Responsive Transcriptional Factor from Bacillus amyloliquefaciens.

Transcriptional factors-based biosensors are commonly used in metabolic engineering for inducible control of gene expression and related applications such as high-throughput screening and dynamic pathway regulations. Mining for novel transcriptional factors is essential for expanding the usability of these toolsets. Here, we report the identification, characterization, and engineering of a phenolic acid responsive regulator PadR from Bacillus amyloliquefaciens (BaPadR). This BaPadR-based biosensor system showed a unique ligand preference and exhibited a high output strength comparable to that of commonly used inducible expression systems. Through engineering the DNA binding region of BaPadR, we further enhanced the dynamic range of the biosensor system. The DNA sequences that are responsible for BaPadR recognition were located by promoter truncation and hybrid promoter building. To further explore the tunability of the sensor system, base substitutions were performed on the BaPadR binding region of the phenolic acid decarboxylase promoter (PpadC) and the hybrid promoter. This novel biosensor system can serve as a valuable tool in future synthetic biology applications.

Adverse pregnancy outcomes associated with antiretroviral therapy initiated before pregnancy and during pregnancy: a retrospective study in Hubei province, China.

Background: Antiretroviral therapy (ART) initiation before pregnancy was reported to have an increased risk of adverse pregnancy outcomes (APOs) than ART initiation during pregnancy. However, the risks of APOs associated with different ART regimens initiated before or during pregnancy remain unknown. Methods: Pregnant women living with HIV (PWLHIV) from Hubei Province, China, were retrospectively enrolled between January 1, 2004, and December 31, 2021. The trends of ART initiation time and application of different ART regimens were evaluated over time, separately. Using no ART exposure before and during pregnancy as control, the risks of APOs associated with protease inhibitor (PI) based regimens and non-nucleoside reverse transcriptase inhibitors (NNRTIs) based regimens initiated before pregnancy were analyzed; and the risks of APOs associated with PI-based regimens, NNRTIs based regimens and zidovudine (AZT) monotherapy initiated during pregnancy were analyzed. APOs, including low birthweight (LBW), stillbirth, preterm birth (PTB) and early miscarriage, were reviewed. Results: Among 781 PWLHIV including 1,010 pregnancies, 522 pregnancies (51.7%) were exposed to ART before or during pregnancy. Of them, the proportion of ART initiation before pregnancy per year increased from around 20% in the early period to more than 60% after 2019. Efavirenz (EFV)-nucleoside reverse transcriptase inhibitors (NRTIs) (32.2%), LPV/r-NRTIs (31.2%), and nevirapine (NVP)-NRTIs (27.4%) were the most commonly used regimens, and the proportion of LPV/r-NRTIs used per year has increased to around 50.0% in recent years. LPV/r-NRTIs was associated with higher risks of LBW whether initiated before pregnancy [adjusted OR (aOR) = 2.59, 95%CI 1.04-6.45, p = 0.041] or during pregnancy (aOR = 2.19, 95%CI 1.03-4.67, p = 0.041), compared with no exposure to ART before and during pregnancy. However, no matter initiated before or during pregnancy, LPV/r-NRTIs had no significantly increased risks of stillbirth, PTB and early miscarriage, and EFV /NVP-NRTIs and AZT monotherapy had no significantly increased risks of LBW, stillbirth, PTB and early miscarriage when compared with no exposure to ART before and during pregnancy. Conclusion: Our data suggests that LPV/r-NRTIs has been widely used among PWLHIV in recent years. However, the potential risk of LBW should be continuously monitored among PWLHIV whether LPV/r-NRTIs is initiated before or during pregnancy.

Application of Metabolite-Responsive Biosensors for Plant Natural Products Biosynthesis.

Plant natural products (PNPs) have shown various pharmaceutical activities, possessing great potential in global markets. Microbial cell factories (MCFs) provide an economical and sustainable alternative for the synthesis of valuable PNPs compared with traditional approaches. However, the heterologous synthetic pathways always lack native regulatory systems, bringing extra burden to PNPs production. To overcome the challenges, biosensors have been exploited and engineered as powerful tools for establishing artificial regulatory networks to control enzyme expression in response to environments. Here, we reviewed the recent progress involved in the application of biosensors that are responsive to PNPs and their precursors. Specifically, the key roles these biosensors played in PNP synthesis pathways, including isoprenoids, flavonoids, stilbenoids and alkaloids, were discussed in detail.

Charge order driven by multiple-Q spin fluctuations in heavily electron-doped iron selenide superconductors.

Intertwined spin and charge orders have been widely studied in high-temperature superconductors, since their fluctuations may facilitate electron pairing; however, they are rarely identified in heavily electron-doped iron selenides. Here, using scanning tunneling microscopy, we show that when the superconductivity of (Li0.84Fe0.16OH)Fe1-xSe is suppressed by introducing Fe-site defects, a short-ranged checkerboard charge order emerges, propagating along the Fe-Fe directions with an approximately 2aFe period. It persists throughout the whole phase space tuned by Fe-site defect density, from a defect-pinned local pattern in optimally doped samples to an extended order in samples with lower Tc or non-superconducting. Intriguingly, our simulations indicate that the charge order is likely driven by multiple-Q spin density waves originating from the spin fluctuations observed by inelastic neutron scattering. Our study proves the presence of a competing order in heavily electron-doped iron selenides, and demonstrates the potential of charge order as a tool to detect spin fluctuations.

Establishing biosynthetic pathway for the production of p-hydroxyacetophenone and its glucoside in Escherichia coli.

p-Hydroxyacetophenone (p-HAP) and its glucoside picein are plant-derived natural products that have been extensively used in chemical, pharmaceutical and cosmetic industries owing to their antioxidant, antibacterial and antiseptic activities. However, the natural biosynthetic pathways for p-HAP and picein have yet been resolved so far, limiting their biosynthesis in microorganisms. In this study, we design and construct a biosynthetic pathway for de novo production of p-HAP and picein from glucose in E. coli. First, screening and characterizing pathway enzymes enable us to successfully establish functional biosynthetic pathway for p-HAP production. Then, the rate-limiting step in the pathway caused by a reversible alcohol dehydrogenase is completely eliminated by modulating intracellular redox cofactors. Subsequent host strain engineering via systematic increase of precursor supplies enables production enhancement of p-HAP with a titer of 1445.3 mg/L under fed-batch conditions. Finally, a novel p-HAP glucosyltransferase capable of generating picein from p-HAP is identified and characterized from a series of glycosyltransferases. On this basis, de novo biosynthesis of picein from glucose is achieved with a titer of 210.7 mg/L under fed-batch conditions. This work not only demonstrates a microbial platform for p-HAP and picein synthesis, but also represents a generalizable pathway design strategy to produce value-added compounds.

Characteristics of refined lymphocyte subsets changes in people living with HIV/AIDS during antiretroviral therapy period: An observation from Wuhan, China.

Background: To analyze the changing characteristics of continuous monitoring of refined lymphocyte subsets in people living with HIV/AIDS (PLWHA) during ART period. Methods: Refined lymphocyte subsets was continuously monitored using flow cytometry for 173 PLWHA, who were hospitalized in Zhongnan Hospital of Wuhan University from August 17, 2021 to September 14, 2022. The effect of ART status and duration of ART on changes of refined lymphocyte subsets were compared in different groups. Then, the levels of refined lymphocyte subsets in PLWHA treated for more than 10 years were compared to those of 1086 healthy individuals. Results: In addition to conventional CD4+ T lymphocytes and CD4+/CD8+ ratio, gradually increasing in numbers of CD3+CD4+CD45RO cells, CD3+CD4+CD45RA cells, CD45RA+CD3+CD4+CD25+CD127low and CD45RO+CD3+CD4+CD25+CD127low cells were found with the increase of ART duration. The number of CD4+CD28+ cells and CD8+CD28+ cells were 174/ul and 233/ul at 6 months post-ART, which gradually increased to 616/ul and 461/ul after ART initiation more than 10 years. Moreover, in ART ≤ 6 months, 6 months-3years, 3-10 years and >10 years groups, the percentage of CD3+CD8+HLA-DR+/CD8 were 79.66%, 69.73%, 60.19% and 57.90%, respectively, and the differences between groups showed statistical significance (F=5.727, P=0.001). For those PLWHA with ART more than 10 years, the levels of CD4+ T lymphocytes, CD3+CD4+CD45RO cells, CD3+CD4+CD45RA cells, CD4+CD28+ cells and CD8+CD28+ cells can increase to levels similar to those of healthy control. However, for those PLWHA with ART more than 10 years, CD4+/CD8+ ratio was 0.86 ± 0.47, which was lower than that of healthy control (0.86 ± 0.47 vs 1.32 ± 0.59, t=3.611, P=0.003); absolute counts and percentage of CD3+CD8+HLA-DR+ cells were 547/ul and 57.90%, which were higher than those of healthy control(547/ul vs 135/ul, t=3.612, P=0.003; 57.90% vs 22.38%, t=6.959, P<0.001). Conclusion: Persistent ART can gradually improve the immune status of PLWHA, which is manifested in the increase of lymphocytes, function recovery of lymphocytes and reduction of aberrant activation status of the immune system. After 10 years of standardized ART, most lymphocytes could return to levels of healthy persons, although it may take longer to complete recovery for CD4+/CD8+ ratio and CD3+CD8+HLA-DR+ cells.

Adverse pregnancy outcomes among pregnant women living with HIV in Hubei province, China: prevalence and risk factors.

Mother-to-child transmission of Human Immunodeficiency Virus (HIV) has been greatly reduced with the advance of intervention technology. However, adverse pregnancy outcomes (APOs) are still common, and little is known about the driving forces of APOs among pregnant women living with HIV in China. Between January 2004 and December 2020, a total of 638 pregnancies among pregnant women living with HIV were enrolled in this study, 84 (13.2%) pregnancies with 87 APOs were reported. Preterm birth (3.8%), ectopic pregnancy (3.4%), spontaneous abortion (2.0%), and embryo arrest (1.7%) were the most common APOs in pregnant women living with HIV. Exposure to antiretroviral drugs (ARVs) during the first trimester (RR = 4.077, 95% CI: 0.521, 1.484, P<0.001) and the first CD4+ T lymphocyte count (CD4 count)≤ 350/µl (RR = 2.227, 95% CI: 0.063, 0.991, P = 0.026) were risk factors of APOs. The age≤ 30 years (RR = -2.513, 95% CI: -1.067, -0.132, P = 0.012) was associated with the decreasing of APOs. Encouraging people to initiate combination antiretroviral therapy and reach a high CD4 count level before pregnancy would be helpful to prevent APOs. Pregnant women exposed to ARVs in the first trimester needed more attention for APOs.

CmERF5-CmRAP2.3 transcriptional cascade positively regulates waterlogging tolerance in Chrysanthemum morifolium.

Waterlogging stress affects plant growth by limiting root respiration and reducing yield and economic value. Therefore, identifying genes involved in regulating waterlogging stress is vital. This study reports the ethylene-responsive VII transcription factor (CmRAP2.3) in the chrysanthemum. Subcellular localization and transactivation assay analyses revealed that CmRAP2.3 was localized in the nucleus and possessed transactivation activity. Overexpression of CmRAP2.3 in chrysanthemum was found to enhance waterlogging tolerance by decreasing reactive oxygen species (ROS) levels. Furthermore, we found that the transcription factor CmERF5 binds to GCC-like motifs in the CmRAP2.3 promoter region and activates CmRAP2.3 expression. Additionally, CmERF5 overexpression maintained a low ROS level and improved chrysanthemum waterlogging tolerance. Taken together, this study shows a molecular mechanism by which CmERF5 transcriptionally activates CmRAP2.3 to reduce waterlogging stress via the ROS pathway in the chrysanthemum.

Exploring and engineering PAM-diverse Streptococci Cas9 for PAM-directed bifunctional and titratable gene control in bacteria.

The RNA-guided Cas9s serve as powerful tools for programmable gene editing and regulation; their targeting scopes and efficacies, however, are always constrained by the PAM sequence stringency. Most Streptococci Cas9s, including the prototype SpCas9 from S. pyogenes, specifically recognize a canonical NGG PAM via a conserved RxR PAM-binding motif within the PAM-interaction (PI) domain. Here, SpCas9-based mining unveils three distinct and rarely presented PAM-binding motifs (QxxxR, QxQ and RxQ) among Streptococci Cas9 orthologs. With the catalytically-dead QxxxR-containing SedCas9 from S. equinus, we dissect its NAG PAM specificity and elucidate its underlying recognition mechanism via computational prediction and mutagenesis analysis. Replacing the SedCas9 PI domain with alternate PAM-binding motifs rewires its PAM specificity to NGG or NAA. Moreover, a semi-rational design with minimal mutation creates a SedCas9-NQ variant showing robust activity towards expanded NNG and NAA PAMs, based upon which we engineered a compact ω-SedCas9-NQ transcriptional regulator for PAM-directed bifunctional and titratable gene control. The ω-SedCas9-NQ mediated metabolic reprogramming of endogenous genes in Escherichia coli affords a 2.6-fold increase of 4-hydroxycoumarin production. This work reveals new Cas9 scaffolds with distinct PAM-binding motifs for PAM relaxation and creates a new PAM-diverse Cas9 variant for versatile gene control in bacteria.

Accuracy of FIB-4 and APRI scores compared to transient elastography for liver fibrosis in patients with HIV and HBV co-infection.

BACKGROUND: Due to economic shortages and concern about occupational exposure to HIV, liver biopsy and transient elastography (TE) are rarely available in patients with HIV/HBV co-infection in China, where HIV/HBV co-infection is prevalent. METHODS: The accuracy of FIB-4 and APRI for predicting liver fibrosis was compared with TE results in a series of 460 HIV/HBV co-infected patients. RESULTS: FIB-4 and APRI scores were strongly correlated to liver stiffness measurement scores by TE, and the correlation index was 81.4-96.3. An FIB-4 index >1.5 had a positive predictive value of 95.2% to consider fibrosis with a sensitivity of 85.7%. An APRI index >0.5 had a positive predictive value of 98.2% to consider fibrosis with a sensitivity of 76.0%. A FIB-4 value <1.5 or APRI <0.5 were concordant with TE results to exclude fibrosis in 94.4% and 96.8%, respectively. A FIB-4 value >1.5 or APRI >0.5 were concordant with fibrosis diagnosed by TE in 77.6-89.4% and 70.7-80.9%, respectively. CONCLUSIONS: In areas with limited resources, FIB-4 and APRI indexes were accurate, simple and inexpensive methods for assessing liver fibrosis in patients with HIV/HBV co-infection.

Biosensor-assisted titratable CRISPRi high-throughput (BATCH) screening for over-production phenotypes.

With rapid advances in the development of metabolic pathways and synthetic biology toolkits, a persisting challenge in microbial bioproduction is how to optimally rewire metabolic fluxes and accelerate the concomitant high-throughput phenotype screening. Here we developed a biosensor-assisted titratable CRISPRi high-throughput (BATCH) screening approach that combines a titratable mismatch CRISPR interference and a biosensor mediated screening for high-production phenotypes in Escherichia coli. We first developed a programmable mismatch CRISPRi that could afford multiple levels of interference efficacy with a one-pot sgRNA pool (a total of 16 variants for each target gene) harboring two consecutive random mismatches in the seed region of sgRNA spacers. The mismatch CRISPRi was demonstrated to enable almost a full range of gene knockdown when targeting different positions on genes. As a proof-of-principle demonstration of the BATCH screening system, we designed doubly mismatched sgRNA pools targeting 20 relevant genes in E. coli and optimized a PadR-based p-coumaric acid biosensor with broad dynamic range for the eGFP fluorescence guided high-production screening. Using sgRNA variants for the combinatorial knockdown of pfkA and ptsI, the p-coumaric acid titer was increased by 40.6% to o 1308.6 mg/l from glycerol in shake flasks. To further demonstrate the general applicability of the BATCH screening system, we recruited a HpdR-based butyrate biosensor that facilitated the screening of E. coli strains achieving 19.0% and 25.2% increase of butyrate titer in shake flasks with sgRNA variants targeting sucA and ldhA, respectively. This work reported the establishment of a plug-and-play approach that enables multilevel modulation of metabolic fluxes and high-throughput screening of high-production phenotypes.

Engineering Escherichia coli native metabolism for efficient biosynthesis of orotate.

Orotate (OA) is a precursor of pyrimidine nucleotides and is widely used in food, pharmaceutical, and cosmetic industries. Although various microorganisms have been used for OA production, the production efficiency needs to be further improved for industrial application. In this study, we engineered Escherichia coli native metabolism for efficient OA production. The entire pathway was divided into the downstream OA synthesis, the midstream aspartate/glutamine supply, and the upstream glycolysis modules. First, the downstream module was optimized by disrupting pyrE to block OA consumption and release the feedback inhibition, and tuning expression of the biosynthetic genes. Second, the midstream pathway was enhanced by increasing the supply of the precursors and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). More importantly, we observed that pyrE disruption may lead to metabolic disorder as indicated by the accumulation of large amount of acetate. This problem was solved by reducing the flux of glycolysis. With these efforts, the final strain produced 80.3 g/L OA with a yield of 0.56 g/g glucose in fed-batch fermentation, which are the highest titer and yield reported so far. This work paves the way for industrial production of OA and represents as a good example of modulating cell metabolism for efficient chemical production.