Optical control of exopolysaccharide production in Sinorhizobium meliloti for studying biofilm formation and water retention

The rhizosphere contains many types of microbes interacting with plant roots, creating a complex symbiotic system. Microbial processes occurring in the rhizosphere are essential to the productivity of terrestrial ecosystems. In particular, exopolysaccharide produced by soil microbes allows dynamic regulation of soil moisture by modulating water transport. We have demonstrated that purified microbial exopolysaccharide (EPS) impacts soil water retention through enhancing the variability of water distributions in the soil microstructure. However, the impact of EPS on water transport in soil is not understood due to complex interaction of microbial EPS with soil microstructure and particle surface properties. To decipher the causal role of EPS in soil microstructures, we set out to develop engineered soil bacteria with spatially regulated EPS biosynthesis capabilities. Here we report genetic engineering of soil bacterium Sinorhizobium meliloti to enable in situ spatial control of EPS production. We show that the photo-sensitive transcription factor EL222, derived from Erythrobacter litoralis, allows robust control of gene expression in S. meliloti. Essential genes in the type II EPS (a major component of EPS from S. meliloti in the soil) production pathway were identified, and deletion strains were generated. Complementation of the essential gene using a synthetic promoter controlled by EL222 led to robust light-activated production of EPS. Optimization of the engineered genetic construct was performed by varying promoters, ribosome binding sites, and using alternative start codons. Using the engineered EPS production strain, we observed rapid settlement of EPS producing S. meliloti in liquid culture, and selective biofilm formation quantified by a crystal violet staining assay. This approach enables spatially regulated EPS production and biofilm formation. We will demonstrate control of gene expression in a synthetic soil microsystem that emulates aggregated sandy loam soil. We will also report our current progress on using these new strains of soil bacteria to study the impact of EPS production on water drying rate in the synthetic soil microsystem. We anticipate that the engineered genetic constructs will be broadly applicable for dissecting gene function in a defined population of microbes in the rhizosphere

Bacterial Extracellular Polymeric Substances Amplify Water Content Variability at the Pore Scale

The function of microbial communities in soil is inextricably linked with the complex physical, chemical, and biological structure of the soil itself. Pore-scale water content controls the hydraulic connectivity of microbial communities and microbes' access to aqueous and gaseous substrates. In turn, soil bacteria directly influence local moisture conditions through the secretion of extracellular polymeric substances (EPS). However, the effect of a soil's physical geometry on EPS-mediated water retention is not well understood. In this study, we systematically measured the rate and extent of water evaporation from pore structures as a function of both EPS concentration and pore size. Three different chamber types were employed: (i) glass capillary tubes (1.2 mm pore diameter) to represent a uniform macropore geometry; (ii) emulated soil micromodels (pore widths ~10 to >300 μm) to represent an aggregated sandy loam pore geometry; and (iii) microfluidic capillary arrays (uniform channels 20 μm wide) to represent a uniform micropore geometry. All chambers were initially saturated with dilute EPS solutions collected from stationary-phase Sinorhizobium meliloti cultures and then the infiltration of air was tracked over time. In the largest chambers, EPS concentration had no effect on the extent of evaporation or on the magnitude or variability of the evaporation rate. However, in the chambers with micropore-sized physical features, EPS concentration strongly influenced rate, extent, and variability of pore water evaporation. In micropores, higher EPS concentrations enhanced water retention and led to greater variability in pore-scale water distributions. In real soil, these phenomena could act together to promote the intermediate water contents associated with productive soil systems, and more variable pore-scale water distributions could increase microbial community diversity and the resiliency of soil systems

Comparative effectiveness of immediate antiretroviral therapy versus CD4-based initiation in HIV-positive individuals in high-income countries: observational cohort study

Background Recommendations have differed nationally and internationally with respect to the best time to start antiretroviral therapy (ART). We compared effectiveness of three strategies for initiation of ART in high-income countries for HIV-positive individuals who do not have AIDS: immediate initiation, initiation at a CD4 count less than 500 cells per mu L, and initiation at a CD4 count less than 350 cells per mu L. Methods We used data from the HIV-CAUSAL Collaboration of cohort studies in Europe and the USA. We included 55 826 individuals aged 18 years or older who were diagnosed with HIV-1 infection between January, 2000, and September, 2013, had not started ART, did not have AIDS, and had CD4 count and HIV-RNA viral load measurements within 6 months of HIV diagnosis. We estimated relative risks of death and of death or AIDS-defining illness, mean survival time, the proportion of individuals in need of ART, and the proportion of individuals with HIV-RNA viral load less than 50 copies per mL, as would have been recorded under each ART initiation strategy after 7 years of HIV diagnosis. We used the parametric g-formula to adjust for baseline and time-varying confounders. Findings Median CD4 count at diagnosis of HIV infection was 376 cells per mu L (IQR 222-551). Compared with immediate initiation, the estimated relative risk of death was 1.02 (95% CI 1.01-1.02) when ART was started at a CD4 count less than 500 cells per mu L, and 1.06 (1.04-1.08) with initiation at a CD4 count less than 350 cells per mu L. Corresponding estimates for death or AIDS-defining illness were 1.06 (1.06-1.07) and 1.20 (1.17-1.23), respectively. Compared with immediate initiation, the mean survival time at 7 years with a strategy of initiation at a CD4 count less than 500 cells per mu L was 2 days shorter (95% CI 1-2) and at a CD4 count less than 350 cells per mu L was 5 days shorter (4-6). 7 years after diagnosis of HIV, 100%, 98.7% (95% CI 98.6-98.7), and 92.6% (92.2-92.9) of individuals would have been in need of ART with immediate initiation, initiation at a CD4 count less than 500 cells per mu L, and initiation at a CD4 count less than 350 cells per mu L, respectively. Corresponding proportions of individuals with HIV-RNA viral load less than 50 copies per mL at 7 years were 87.3% (87.3-88.6), 87.4% (87.4-88.6), and 83.8% (83.6-84.9). Interpretation The benefits of immediate initiation of ART, such as prolonged survival and AIDS-free survival and increased virological suppression, were small in this high-income setting with relatively low CD4 count at HIV diagnosis. The estimated beneficial effect on AIDS is less than in recently reported randomised trials. Increasing rates of HIV testing might be as important as a policy of early initiation of ART

Comparative effectiveness of immediate antiretroviral therapy versus CD4-based initiation in HIV-positive individuals in high-income countries: observational cohort study.

BACKGROUND Recommendations have differed nationally and internationally with respect to the best time to start antiretroviral therapy (ART). We compared effectiveness of three strategies for initiation of ART in high-income countries for HIV-positive individuals who do not have AIDS: immediate initiation, initiation at a CD4 count less than 500 cells per μL, and initiation at a CD4 count less than 350 cells per μL. METHODS We used data from the HIV-CAUSAL Collaboration of cohort studies in Europe and the USA. We included 55 826 individuals aged 18 years or older who were diagnosed with HIV-1 infection between January, 2000, and September, 2013, had not started ART, did not have AIDS, and had CD4 count and HIV-RNA viral load measurements within 6 months of HIV diagnosis. We estimated relative risks of death and of death or AIDS-defining illness, mean survival time, the proportion of individuals in need of ART, and the proportion of individuals with HIV-RNA viral load less than 50 copies per mL, as would have been recorded under each ART initiation strategy after 7 years of HIV diagnosis. We used the parametric g-formula to adjust for baseline and time-varying confounders. FINDINGS Median CD4 count at diagnosis of HIV infection was 376 cells per μL (IQR 222-551). Compared with immediate initiation, the estimated relative risk of death was 1·02 (95% CI 1·01-1·02) when ART was started at a CD4 count less than 500 cells per μL, and 1·06 (1·04-1·08) with initiation at a CD4 count less than 350 cells per μL. Corresponding estimates for death or AIDS-defining illness were 1·06 (1·06-1·07) and 1·20 (1·17-1·23), respectively. Compared with immediate initiation, the mean survival time at 7 years with a strategy of initiation at a CD4 count less than 500 cells per μL was 2 days shorter (95% CI 1-2) and at a CD4 count less than 350 cells per μL was 5 days shorter (4-6). 7 years after diagnosis of HIV, 100%, 98·7% (95% CI 98·6-98·7), and 92·6% (92·2-92·9) of individuals would have been in need of ART with immediate initiation, initiation at a CD4 count less than 500 cells per μL, and initiation at a CD4 count less than 350 cells per μL, respectively. Corresponding proportions of individuals with HIV-RNA viral load less than 50 copies per mL at 7 years were 87·3% (87·3-88·6), 87·4% (87·4-88·6), and 83·8% (83·6-84·9). INTERPRETATION The benefits of immediate initiation of ART, such as prolonged survival and AIDS-free survival and increased virological suppression, were small in this high-income setting with relatively low CD4 count at HIV diagnosis. The estimated beneficial effect on AIDS is less than in recently reported randomised trials. Increasing rates of HIV testing might be as important as a policy of early initiation of ART. FUNDING National Institutes of Health

Ursodeoxycholic acid in intrahepatic cholestasis of pregnancy

Background: Ursodeoxycholic acid is commonly used to treat intrahepatic cholestasis of pregnancy, yet its largest trial detected minimal benefit for a composite outcome (stillbirth, preterm birth, and neonatal unit admission). We aimed to examine whether ursodeoxycholic acid affects specific adverse perinatal outcomes. Methods: In this systematic review and individual participant data meta-analysis, we searched PubMed, Web of Science, Embase, MEDLINE, CINAHL, Global Health, MIDIRS, and Cochrane without language restrictions for relevant articles published between database inception, and Jan 1, 2020, using search terms referencing intrahepatic cholestasis of pregnancy, ursodeoxycholic acid, and perinatal outcomes. Eligible studies had 30 or more study participants and reported on at least one individual with intrahepatic cholestasis of pregnancy and bile acid concentrations of 40 μmol/L or more. We also included two unpublished cohort studies. Individual participant data were collected from the authors of selected studies. The primary outcome was the prevalence of stillbirth, for which we anticipated there would be insufficient data to achieve statistical power. Therefore, we included a composite of stillbirth and preterm birth as a main secondary outcome. A mixed-effects meta-analysis was done using multi-level modelling and adjusting for bile acid concentration, parity, and multifetal pregnancy. Individual participant data analyses were done for all studies and in different subgroups, which were produced by limiting analyses to randomised controlled trials only, singleton pregnancies only, or two-arm studies only. This study is registered with PROSPERO, CRD42019131495. Findings: The authors of the 85 studies fulfilling our inclusion criteria were contacted. Individual participant data from 6974 women in 34 studies were included in the meta-analysis, of whom 4726 (67·8%) took ursodeoxycholic acid. Stillbirth occurred in 35 (0·7%) of 5097 fetuses among women with intrahepatic cholestasis of pregnancy treated with ursodeoxycholic acid and in 12 (0·6%) of 2038 fetuses among women with intrahepatic cholestasis of pregnancy not treated with ursodeoxycholic acid (adjusted odds ratio [aOR] 1·04, 95% CI 0·35–3·07; p=0·95). Ursodeoxycholic acid treatment also had no effect on the prevalence of stillbirth when considering only randomised controlled trials (aOR 0·29, 95% CI 0·04–2·42; p=0·25). Ursodeoxycholic acid treatment had no effect on the prevalence of the composite outcome in all studies (aOR 1·28, 95% CI 0·86–1·91; p=0·22), but was associated with a reduced composite outcome when considering only randomised controlled trials (0·60, 0·39–0·91; p=0·016). Interpretation: Ursodeoxycholic acid treatment had no significant effect on the prevalence of stillbirth in women with intrahepatic cholestasis of pregnancy, but our analysis was probably limited by the low overall event rate. However, when considering only randomised controlled trials, ursodeoxycholic acid was associated with a reduction in stillbirth in combination with preterm birth, providing evidence for the clinical benefit of antenatal ursodeoxycholic acid treatment. Funding: Tommy's, the Wellcome Trust, ICP Support, and the National Institute for Health Research.</p

Ursodeoxycholic acid in intrahepatic cholestasis of pregnancy: a systematic review and individual participant data meta-analysis

Background: Ursodeoxycholic acid is commonly used to treat intrahepatic cholestasis of pregnancy, yet its largest trial detected minimal benefit for a composite outcome (stillbirth, preterm birth, and neonatal unit admission). We aimed to examine whether ursodeoxycholic acid affects specific adverse perinatal outcomes. Methods: In this systematic review and individual participant data meta-analysis, we searched PubMed, Web of Science, Embase, MEDLINE, CINAHL, Global Health, MIDIRS, and Cochrane without language restrictions for relevant articles published between database inception, and Jan 1, 2020, using search terms referencing intrahepatic cholestasis of pregnancy, ursodeoxycholic acid, and perinatal outcomes. Eligible studies had 30 or more study participants and reported on at least one individual with intrahepatic cholestasis of pregnancy and bile acid concentrations of 40 μmol/L or more. We also included two unpublished cohort studies. Individual participant data were collected from the authors of selected studies. The primary outcome was the prevalence of stillbirth, for which we anticipated there would be insufficient data to achieve statistical power. Therefore, we included a composite of stillbirth and preterm birth as a main secondary outcome. A mixed-effects meta-analysis was done using multi-level modelling and adjusting for bile acid concentration, parity, and multifetal pregnancy. Individual participant data analyses were done for all studies and in different subgroups, which were produced by limiting analyses to randomised controlled trials only, singleton pregnancies only, or two-arm studies only. This study is registered with PROSPERO, CRD42019131495. Findings: The authors of the 85 studies fulfilling our inclusion criteria were contacted. Individual participant data from 6974 women in 34 studies were included in the meta-analysis, of whom 4726 (67·8%) took ursodeoxycholic acid. Stillbirth occurred in 35 (0·7%) of 5097 fetuses among women with intrahepatic cholestasis of pregnancy treated with ursodeoxycholic acid and in 12 (0·6%) of 2038 fetuses among women with intrahepatic cholestasis of pregnancy not treated with ursodeoxycholic acid (adjusted odds ratio [aOR] 1·04, 95% CI 0·35–3·07; p=0·95). Ursodeoxycholic acid treatment also had no effect on the prevalence of stillbirth when considering only randomised controlled trials (aOR 0·29, 95% CI 0·04–2·42; p=0·25). Ursodeoxycholic acid treatment had no effect on the prevalence of the composite outcome in all studies (aOR 1·28, 95% CI 0·86–1·91; p=0·22), but was associated with a reduced composite outcome when considering only randomised controlled trials (0·60, 0·39–0·91; p=0·016). Interpretation: Ursodeoxycholic acid treatment had no significant effect on the prevalence of stillbirth in women with intrahepatic cholestasis of pregnancy, but our analysis was probably limited by the low overall event rate. However, when considering only randomised controlled trials, ursodeoxycholic acid was associated with a reduction in stillbirth in combination with preterm birth, providing evidence for the clinical benefit of antenatal ursodeoxycholic acid treatment. Funding: Tommy's, the Wellcome Trust, ICP Support, and the National Institute for Health Research