Coffee consumption during pregnancy increases the risk of preeclampsia in rats by inhibiting 2-methoxyestradiol production.

Preeclampsia (PE) is a pregnancy-specific disease that causes maternal symptoms such as high blood pressure and adverse pregnancy outcomes. 2-methoxyestradiol (2-MeO-E2), an endogenous metabolite of 17ß-estradiol (E2) formed by Catechol-O-Methyltransferase (COMT), plays an important role in pregnancy. Our earlier studies have shown that polyphenols present in coffee can inhibit COMT activity, which may inhibit the formation of 2-MeO-E2 and contribute to PE. Therefore, the current study aims to investigate the possible effect and mechanism of coffee intake during pregnancy on PE in SD rats. Coffee is administered with or without cotreatment of 2-MeO-E2 to pregnant rats from the10th to the18th day of pregnancy. The results show that pregnant rats with coffee intake had prominent fetal growth restriction, hypertension and proteinuria, which can be ameliorated by co-treatment of 2-MeO-E2. In addition, coffee treatment leads to significantly decreased serum 2-MeO-E2. Therefore, the PE symptoms induced by coffee treatment is probably mediated by decreased 2-MeO-E2. Our findings provide new mechanistic insight into how coffee intake could lead to increased risk of PE, and demonstrate the effectiveness of 2-MeO-E2 supplementation as a potential therapeutic agent for PE.

Potential ecological risks of heavy metals and Cd accumulation characteristics of Suaeda salsa under different Cd input and water logging conditions in the Yellow River estuary, China.

To improve the remediation of heavy metal pollution by typical wetland vegetation and maintain the health of wetland ecosystems under the water-sediment regulation scheme (WSRS) application, we evaluated the potential ecological risk of heavy metals in surface sediment in the Yellow River estuary affected by the WSRS. The ranges of Cr, Cu, Zn, Cd, and Pb content in surface sediment were 52.44-100.80 mg·kg-1 dry weight (DW), 16.38-21.19 mg·kg-1 DW, 64.77-255.50 mg·kg-1 DW, 0.12-0.24 mg·kg-1 DW, and 5.40-8.63 mg·kg-1 DW, respectively, and potential ecological risk coefficients showed that Cd was associated with moderate potential risk. We further examined effects of Cd in a greenhouse experiment to explore the influence of short-term Cd input and water logging condition changes induced by WSRS on the Cd absorption characteristics of Suaeda salsa (L.) Pall in the Yellow River estuary. The results showed that total biomass decreased but Cd content in tissue of S. salsa increased with increasing Cd input and the accumulation factor reached maximum values at 100 µg·L-1 of Cd, indicating that S. salsa efficiently accumulated Cd. Water logging depth significantly affected S. salsa growth and Cd absorption with deeper water logging being detrimental to growth. The interaction effect of Cd input and water logging depth on Cd content and accumulation factor was significant. These results suggest that WSRS caused short-term heavy metal input and changes in water conditions affect wetland vegetation growth and heavy metal absorption in the downstream estuary.

Effects of adipocyte-conditioned cell culture media on S1P treatment of human triple-negative breast cancer cells.

Sphingosine-1-phosphate (S1P) is a potent sphingolipid metabolite that regulates a wide range of biological functions such as cell proliferation, cell apoptosis and angiogenesis. Its cellular level is elevated in breast cancer, which, in turn, would promote cancer cell proliferation, survival, growth and metastasis. However, the cellular concentration of S1P is normally in the low nanomolar range, and our previous studies showed that S1P selectively induced apoptosis of breast cancer cells at high concentrations (high nanomolar to low micromolar). Thus, local administration of high-concentration S1P alone or in combination of chemotherapy agents could be used to treat breast cancer. The breast mainly consists of mammary gland and connective tissue stroma (adipose), which are dynamically interacting each other. Thus, in the current study, we evaluated how normal adipocyte-conditioned cell culture media (AD-CM) and cancer-associated adipocyte-conditioned cell culture media (CAA-CM) would affect high-concentration S1P treatment of triple-negative breast cancer (TNBC) cells. Both AD-CM and CAA-CM may suppress the anti-proliferative effect and reduce nuclear alteration/apoptosis caused by high-concentration S1P. This implicates that adipose tissue is likely to be detrimental to local high-concentration S1P treatment of TNBC. Because the interstitial concentration of S1P is about 10 times higher than its cellular level, we undertook a secretome analysis to understand how S1P would affect the secreted protein profile of differentiated SGBS adipocytes. At 100 nM S1P treatment, we identified 36 upregulated and 21 downregulated secretome genes. Most of these genes are involved in multiple biological processes. Further studies are warranted to identify the most important secretome targets of S1P in adipocytes and illustrate the mechanism on how these target proteins affect S1P treatment of TNBC.

Association of Pneumococcal Conjugate Vaccine Use With Hospitalized Pneumonia in Medicare Beneficiaries 65 Years or Older With and Without Medical Conditions, 2014 to 2017.

Importance: The association of 13-valent pneumococcal conjugate vaccine (PCV13) use with pneumonia hospitalization in older adults, especially those with underlying medical conditions, is not well described. Objective: To evaluate the association of PCV13 use with pneumonia, non-health care-associated (non-HA) pneumonia, and lobar pneumonia (LP) hospitalization among US Medicare beneficiaries 65 years or older. Design, Setting, and Participants: This cohort study with time-varying exposure assignment analyzed claims data from US Medicare beneficiaries 65 years or older enrolled in Parts A/B with a residence in the 50 US states or the District of Columbia by September 1, 2014. New Medicare Parts A/B beneficiaries within 6 months after their 65th birthday were continuously included in the cohort after September 1, 2014, and followed through December 31, 2017. Participants were censored if they died, changed enrollment status, or developed a study outcome. Most of the analyses were conducted from 2018 to 2019, and additional analyses were performed from 2021 to 2022. Exposures: Use of PCV13 vaccination 14 days or more before pneumonia hospitalization. Main Outcomes and Measures: Discrete-time survival models were used to estimate the incidence rate ratio (IRR) and number of pneumonia hospitalizations averted through PCV13 use. The adjusted IRR for the association of PCV13 vaccination with pneumonia hospitalization was used to estimate vaccine effectiveness (VE). Results: At the end of follow-up (December 2017), 24 121 625 beneficiaries (13 593 975 women [56.4%]; 418 005 [1.7%] Asian, 1 750 807 [4.8%] Black, 338 044 [1.4%] Hispanic, 111 508 [0.5%] Native American, and 20 700 948 [85.8%] White individuals) were in the cohort; 4 936 185 (20.5%) had received PCV13 only, and 10 646 220 (79.5%) had not received any pneumococcal vaccines. More than half of the beneficiaries in the cohort were younger than 75 years, White, and had either immunocompromising or chronic medical conditions. Coverage with PCV13 increased from 0.8% (September 2014) to 41.5% (December 2017). The VE for PCV13 was estimated at 6.7% (95% CI, 5.9%-7.5%) for pneumonia, 4.7% (95% CI, 3.9%-5.6%) for non-HA pneumonia, and 5.8% (95% CI, 2.6%-8.9%) for LP. From September 2014 through December 2017, an estimated 35 127 pneumonia (95% CI, 33 011-37 270), 24 643 non-HA pneumonia (95% CI, 22 761-26 552), and 1294 LP (95% CI, 797-1819) hospitalizations were averted through PCV13 use. Conclusions and Relevance: The study results suggest that PCV13 use was associated with reduced pneumonia hospitalization among Medicare beneficiaries 65 years or older, many of whom had underlying medical conditions. Increased PCV13 coverage and use of recently approved higher-valent pneumococcal conjugate vaccines may avert additional pneumonia hospitalizations in adults.

Influence of Potamogeton crispus harvesting on phosphorus composition of Lake Yimeng.

Harvesting is an important method used to control the overproduction of Potamogeton crispus in lakes. A three-year comparative field study was performed in a eutrophic lake (harvested area) and its connected lake (non-harvested area) to determine the effects of harvesting on the phosphorus (P) composition and environmental factors in the water and sediment. Results revealed that harvesting significantly reduced the dissolved total P and dissolved organic P (DOP) and increased the alkaline phosphatase activity and particulate P (PP) in the water. No significant differences were detected in the water total P (TP), soluble reactive P, chlorophyll-a, pH, and dissolved oxygen between the harvested and non-harvested areas. Sediment TP and organic P (OP) were significantly reduced in the harvested area. Harvesting changed the P composition in the water. In the non-harvested area, P was mainly formed by DOP (40%) in the water body, while in the harvested area, PP was the main water component (47%). Harvesting increased the proportion of inorganic P (IP) in the sediment and decreased the proportion of OP. In the water, the IP to TP ratio in the non-harvested and harvested areas were 58.26% and 63.51%, respectively. Our results showed that harvesting changed the P composition in the water and sediment. In the harvesting of submerged vegetation, our results can serve as a reference for the management of vegetation-rich lakes.

Relationship between the coprecipitation of phosphorus-on-calcite by submerged macrophytes and the phosphorus cycle in water.

To assess potential phosphorus removal, we utilized Potamogeton crispus to determine the effects of calcium addition on phosphorus removal. Plastic film was used to block material exchange between the overlying water and the sediment, and we compared the experimental results with long-term monitoring results of Yimeng Lake, which contained a dense population of P. crispus. The results revealed that the first 10-40 days constituted a period of rapid P decrease, as P. crispus could effectively remove the phosphorus in the water through coprecipitation of CaCO3-P. The treatment groups indicated that P. crispus released calcium into the overlying water, and after the addition of calcium ions, P. crispus showed increased phosphorus removal efficiency in the water. Total phosphorus (TP) and P/Ca content increased with increasing pH in the treatment groups, and the TP and pH declined as the calcium content increased in the treatment groups. Long-term field observations showed that the calcium-to-phosphorus ratio in the coprecipitates was dependent on the pH during the crystallization process. Thus, water calcium driven by P. crispus plays an important role in the phosphorus cycle of water, due to P. crispus assisted precipitation. This study revealed the effect of P. crispus on the water purification, the migration and transformation of Ca and P in sediment and overlying water under the condition of sediment calcium addition, so as to provide a theoretical basis for the ecological restoration of shallow lakes eutrophication.

Nutrients and Environmental Factors Cross Wavelet Analysis of River Yi in East China: A Multi-Scale Approach.

The accumulation of nutrients in rivers is a major cause of eutrophication, and the change in nutrient content is affected by a variety of factors. Taking the River Yi as an example, this study used wavelet analysis tools to examine the periodic changes in nutrients and environmental factors, as well as the relationship between nutrients and environmental factors. The results revealed that total phosphorus (TP), total nitrogen (TN), and ammonia nitrogen (NH4+-N) exhibit multiscale oscillation features, with the dominating periods of 16-17, 26, and 57-60 months. The continuous wavelet transform revealed periodic fluctuation laws on multiple scales between nutrients and several environmental factors. Wavelet transform coherence (WTC) was performed on nutrients and environmental factors, and the results showed that temperature and dissolved oxygen (DO) have a strong influence on nutrient concentration fluctuation. The WTC revealed a weak correlation between pH and TP. On a longer period, however, pH was positively correlated with TN. The flow was found to be positively correct with N and P, while N and P were found to be negatively correct with DO and electrical conductance (EC) at different scales. In most cases, TP was negatively correlated with 5-day biochemical oxygen demand (BOD5) and permanganate index (CODMn). The correlation between TN and CODMn and BOD5 was limited, and no clear dominant phase emerged. In a nutshell, wavelet analysis revealed that water temperature, pH, DO, flow, EC, CODMn, and BOD5 had a pronounced influence on nutrient concentration in the River Yi at different time scales. In the case of the combination of environmental factors, pH and DO play the largest role in determining nutrient concentration.

Cd absorption characteristics of Suaeda salsa under different sediment burial and exogenous Cd input conditions in the Yellow River estuary, China.

Suaeda salsa (L.) Pall., a typical halophyte plant in the Yellow River estuary, has high enrichment capacity for heavy metals. However, few studies have investigated the Cd absorption characteristics of S. salsa under different sediment burial and exogenous Cd input conditions, especially following the water-sediment regulation scheme (WSRS), which brought sediment burial and exogenous substances to the estuary. So, we established a greenhouse pot culture experiment with four sediment burial depths (0 cm, 3 cm, 6 cm, and 12 cm) and exogenous Cd input levels (0 mg·kg-1, 0.5 mg·kg-1, 1.0 mg·kg-1, and 1.5 mg·kg-1) and analyzed the leaf, stem, root, and total biomass; leaf, stem, and root Cd content; and storage, sediment Cd content, accumulation factor, root/leaf (R/L), root/stem (R/S), and stem/leaf (S/L) ratios to study the Cd absorption characteristics of S. salsa under the different sediment burial and exogenous Cd input. Results showed that high Cd content in roots, stems, and leaves was harmful to S. salsa growth, and then led to a decrease in biomass (characterized by stem, leaf, and total biomass). Suaeda salsa exhibited a survival strategy to deal with Cd toxicity, which involved the roots absorbing Cd from the sediment and storing it in stems and leaves (stem and leaf Cd content peaked at 0.5 mg·kg-1 Cd input) at low Cd input, whereas roots stored more Cd and reduced Cd transport to stems and leaves at high Cd input. Therefore, we observed the maximum value of leaf (500.63 ± 19.15 g·m-2), stem (648.22 ± 50.08 g·m-2), and total biomass (1246.92 ± 55.49 g·m-2) in the treatment with 1.5 mg·kg-1 Cd input and 3-cm sediment depth due to the Cd content in leaves and stems being relatively low. The accumulation factors of leaves, stems, and roots varied (0.39-0.99, 0.19-2.58, and 0.80-20.45, respectively), and most of the accumulation factors for roots and leaves and the R/L and R/S ratios were >1, which indicated that S. salsa had high enrichment levels of Cd, which mostly accumulated in the roots. Shallow or moderate burial depth was beneficial to S. salsa growth, but sediment burial was not beneficial to Cd absorption because the sum of leaf, stem, and root Cd storage was higher at 0-cm depth compared with the other depths. Variance analysis showed that the influence of Cd input on leaf, stem, root, and sediment Cd content and stem and root Cd storage was significant (P < 0.05), whereas sediment burial, interaction of sediment burial and Cd input on Cd content, storage, and biomass were not significant (P > 0.05). Therefore, we concluded that more attention should be paid to the control of sediment burial and heavy metal input, especially during the WSRS, in the Yellow River estuary.

Severe Hypoglycemia Risk With Long-Acting Insulin Analogs vs Neutral Protamine Hagedorn Insulin.

Importance: Previous studies have found that the risk of severe hypoglycemia does not differ between long-acting insulin analogs and neutral protamine Hagedorn (NPH) insulin in patients with type 2 diabetes. However, these studies did not focus on patients 65 years or older, who are at an increased risk for hypoglycemia, or did not include patients with concomitant prandial insulin use. Objective: To examine the risk of emergency department (ED) visits or hospitalizations for hypoglycemia among older community-residing patients with type 2 diabetes who initiated long-acting insulin or NPH insulin in real-world settings. Design, Setting, and Participants: This retrospective, new-user cohort study assessed Medicare beneficiaries 65 years or older who initiated insulin glargine (n = 407 018), insulin detemir (n = 141 588), or NPH insulin (n = 26 402) from January 1, 2007, to July 31, 2019. Exposures: Insulin glargine, insulin detemir, and NPH insulin. Main Outcomes and Measures: The primary outcome was time to first ED visit or hospitalization for hypoglycemia, defined using a modified validated algorithm. Propensity score-weighted Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% CIs. The risk of recurring hypoglycemia events was estimated using the Andersen-Gill model. Post hoc analyses were conducted investigating possible effect modification by age. Results: Of the 575 008 patients initiating use of insulin (mean [SD] age 74.9 [6.7] years; 53% female), 407 018 used glargine, 141 588 used detemir, and 26 402 used NPH insulin. The study included 7347 ED visits or hospitalizations for hypoglycemia (5194 for glargine, 1693 for detemir, and 460 for NPH insulin, with a median follow-up across the 3 cohorts of 0.37 years (interquartile range, 0.20-0.76 years). Initiation of glargine and detemir use was associated with a reduced risk of hypoglycemia compared with NPH insulin use (HR for glargine vs NPH insulin, 0.71; 95% CI, 0.63-0.80; HR, detemir vs NPH insulin, 0.72; 95% CI, 0.63-0.82). The HRs were similar for the recurrent event analysis. The protective association of long-acting insulin analogs varied by age and was not seen with concomitant prandial insulin use. Conclusions and Relevance: In this cohort study, initiation of long-acting analogs was associated with a lower risk of ED visits or hospitalizations for hypoglycemia compared with NPH insulin in older patients with type 2 diabetes in Medicare. However, this association was not seen with concomitant prandial insulin use.

Recombinant Zoster Vaccine (Shingrix): Real-World Effectiveness in the First 2 Years Post-Licensure.

BACKGROUND: Shingrix (recombinant zoster vaccine) was licensed to prevent herpes zoster, dispensed as 2 doses given 2-6 months apart among adults aged ≥50 years. Clinical trials yielded efficacy of >90% for confirmed herpes zoster, but post-market performance has not been evaluated. Efficacy of a single dose and a delayed second dose and efficacy among persons with autoimmune or immunosuppressive conditions have not been studied. We aimed to assess post-market vaccine effectiveness of Shingrix. METHODS: We conducted a cohort study among Medicare Part D community-dwelling beneficiaries aged >65 years. Herpes zoster was identified using a medical office visit diagnosis with treatment, and postherpetic neuralgia was identified using a validated algorithm. We used inverse probability of treatment weighting to improve cohort balance and marginal structural models to estimate hazard ratios. RESULTS: We found a vaccine effectiveness of 70.1% (95% confidence interval [CI], 68.6-71.5) and 56.9% (95% CI, 55.0-58.8) for 2 and 1 doses, respectively. The 2-dose vaccine effectiveness was not significantly lower for beneficiaries aged >80 years, for second doses received at ≥180 days, or for individuals with autoimmune conditions. The vaccine was also effective among individuals with immunosuppressive conditions. Two-dose vaccine effectiveness against postherpetic neuralgia was 76.0% (95% CI, 68.4-81.8). CONCLUSIONS: This large real-world observational study of the effectiveness of Shingrix demonstrates the benefit of completing the 2-dose regimen. Second doses administered beyond the recommended 6 months did not impair effectiveness. Our effectiveness estimates were lower than the clinical trials estimates, likely due to differences in outcome specificity.

Effects of Sphingosine-1-Phosphate on Cell Viability, Differentiation, and Gene Expression of Adipocytes.

Sphingosine-1-phosphate (S1P) is a highly potent sphingolipid metabolite, which controls numerous physiological and pathological process via its extracellular and intracellular functions. The breast is mainly composed of epithelial cells (mammary gland) and adipocytes (stroma). Adipocytes play an important role in regulating the normal functions of the breast. Compared to the vast amount studies on breast epithelial cells, the functions of S1P in breast adipocytes are much less known. Thus, in the current study, we used human preadipocyte cell lines SGBS and mouse preadipocyte cell line 3T3-L1 as in vitro models to evaluate the effects of S1P on cell viability, differentiation, and gene expression in adipocytes. Our results showed that S1P increased cell viability in SGBS and 3T3-L1 preadipocytes but moderately reduced cell viability in differentiated SGBS and 3T3-L1 adipocytes. S1P was also shown to inhibit adipogenic differentiation of SGBS and 3T3-L1 at concentration higher than 1000 nM. Transcriptome analyses showed that S1P was more influential on gene expression in differentiated adipocytes. Furthermore, our network analysis in mature adipocytes showed that the upregulated DEGs (differentially expressed genes) were related to regulation of lipolysis, PPAR (peroxisome proliferator-activated receptor) signaling, alcoholism, and toll-like receptor signaling, whereas the downregulated DEGs were overrepresented in cytokine-cytokine receptor interaction, focal adhesion, starch and sucrose metabolism, and nuclear receptors pathways. Together previous studies on the functions of S1P in breast epithelial cells, the current study implicated that S1P may play a critical role in modulating the bidirectional regulation of adipocyte-extracellular matrix-epithelial cell axis and maintaining the normal physiological functions of the breast.

Similar Breast Cancer Risk in Women Older Than 65 Years Initiating Glargine, Detemir, and NPH Insulins.

OBJECTIVE: To assess whether initiation of insulin glargine (glargine), compared with initiation of NPH or insulin detemir (detemir), was associated with an increased risk of breast cancer in women with diabetes. RESEARCH DESIGN AND METHODS: This was a retrospective new-user cohort study of female Medicare beneficiaries aged ≥65 years initiating glargine (203,159), detemir (67,012), or NPH (47,388) from September 2006 to September 2015, with follow-up through May 2017. Weighted Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% CIs for incidence of breast cancer according to ever use, cumulative duration of use, cumulative dose of insulin, length of follow-up time, and a combination of dose and length of follow-up time. RESULTS: Ever use of glargine was not associated with an increased risk of breast cancer compared with NPH (HR 0.97; 95% CI 0.88-1.06) or detemir (HR 0.98; 95% CI 0.92-1.05). No increased risk was seen with glargine use compared with either NPH or detemir by duration of insulin use, length of follow-up, or cumulative dose of insulin. No increased risk of breast cancer was observed in medium- or high-dose glargine users compared with low-dose users. CONCLUSIONS: Overall, glargine use was not associated with an increased risk of breast cancer compared with NPH or detemir in female Medicare beneficiaries.

Zostavax vaccine effectiveness among US elderly using real-world evidence: Addressing unmeasured confounders by using multiple imputation after linking beneficiary surveys with Medicare claims.

PURPOSE: Medicare claims can provide real-world evidence (RWE) to support the Food and Drug Administration's ability to conduct postapproval studies to validate products' safety and effectiveness. However, Medicare claims do not contain comprehensive information on some important sources of bias. Thus, we piloted an approach using the Medicare Current Beneficiary Survey (MCBS), a nationally representative survey of the Medicare population, to (a) assess cohort balance with respect to unmeasured confounders in a herpes zoster vaccine (HZV) effectiveness claims-based study and (b) augment Medicare claims with MCBS data to include unmeasured covariates. METHODS: We reanalyzed data from our published HZV effectiveness Medicare analysis, using linkages to MCBS to obtain information on impaired mobility, education, and health-seeking behavior. We assessed survey variable balance between the matched cohorts and selected imbalanced variables for model adjustment, applying multiple imputation by chained equations (MICE) to impute these potential unmeasured confounders. RESULTS: The original HZV effectiveness study cohorts appeared well balanced with respect to variables we selected from the MCBS. Our imputed results showed slight shifts in HZV effectiveness point estimates with wider confidence intervals, but indicated no statistically significant differences from the original study estimates. CONCLUSIONS: Our innovative use of linked survey data to assess cohort balance and our imputation approach to augment Medicare claims with MCBS data to include unmeasured covariates provide potential solutions for addressing bias related to unmeasured confounding in large database studies, thus adding new tools for RWE studies.

[Effects of Sediment Burial and Exogenous Cd Input on Biomass Allocation and Antioxidative Enzyme Activities of Suaeda salsa in the Coastal Wetland of the Yellow River Delta].

The Yellow River Delta has been facing the threat of functional degradation during the recent years. The Water-Sediment Regulation Project not only supplements abundant freshwater, but also alters the sediment burial and heavy metal levels, which affects vegetation growth. Thus, we selected the pioneer species Suaeda salsa, to study the effects of different sediment burial depths (0, 3, 6, 12 cm) and exogenous Cd inputs (0, 0.5, 1.0, 1.5 mg·kg-1) on biomass allocation and activities of antioxidative enzymes in the coastal wetlands of the Yellow River delta. The results showed that a shallow or moderate burial depth had a stimulatory effect on chlorophyll content, while an excessive burial depth inhibited the growth of Suaeda salsa and chlorophyll content. With increasing Cd input, chlorophyll content and dry mass decreased. At a lower Cd input and moderate burial depth, activities of CAT and SOD increased, and at high levels, SOD activities decreased, while activities of CAT at a 12 cm burial depth and 1.0 mg·kg-1, 1.5 mg·kg-1 Cd input were higher than those for the control (62.66% and 58.56%). CAT activities reached high values (15.76 U·mg-1) at a high Cd input (1.5 mg·kg-1) and burial depth (12 cm). Analysis of variance showed that Cd input had a significant effect on protein content, and CAT and SOD activities, and sediment burial depth had a significant effect on the protein content and SOD activities. Interaction between Cd input and sediment burial depth had a significant effect on CAT and SOD activities (P<0.05). These results demonstrated that sediment burial depth and Cd input had a great influence on the growth of Suaeda salsa, and to some extent, Suaeda salsa could change its biomass allocation and antioxidative enzyme activities to adapt to severe environments.

Molecular mechanism of HIV-1 resistance to sifuvirtide, a clinical trial-approved membrane fusion inhibitor.

Host cell infection with HIV-1 requires fusion of viral and cell membranes. Sifuvirtide (SFT) is a peptide-based HIV-1 fusion inhibitor approved for phase III clinical trials in China. Here, we focused on characterizing HIV-1 variants highly resistant to SFT to gain insight into the molecular resistance mechanism. Three primary substitutions (V38A, A47I, and Q52R) located at the inhibitor-binding site of HIV-1's envelope protein (Env) and one secondary substitution (N126K) located at the C-terminal heptad repeat region of the viral protein gp41, which is part of the envelope, conferred high SFT resistance and cross-resistance to the anti-HIV-1 drug T20 and the template peptide C34. Interestingly, SFT's resistance profile could be dramatically improved with an M-T hook structure-modified SFT (MTSFT) and with short-peptide inhibitors that mainly target the gp41 pocket (2P23 and its lipid derivative LP-19). We found that the V38A and Q52R substitutions reduce the binding stabilities of SFT, C34, and MTSFT, but they had no effect on the binding of 2P23 and LP-19; in sharp contrast, the A47I substitution enhanced fusion inhibitor binding. Furthermore, the primary resistance substitutions impaired Env-mediated membrane fusion and cell entry and changed the conformation of the gp41 core structure. Importantly, whereas the V38A and Q52R substitutions disrupted the N-terminal helix of gp41, a single A47I substitution greatly enhanced its thermostability. Taken together, our results provide crucial structural insights into the mechanism of HIV-1 resistance to gp41-dependent fusion inhibitors, which may inform the development of additional anti-HIV drugs.

Mechanism of HIV-1 Resistance to an Electronically Constrained α-Helical Peptide Membrane Fusion Inhibitor.

SC29EK is an electronically constrained α-helical peptide HIV-1 fusion inhibitor that is highly effective against both wild-type and enfuvirtide (T20)-resistant viruses. In this study, we focused on investigating the mechanism of HIV-1 resistance to SC29EK by two approaches. First, SC29EK-escaping HIV-1 variants were selected and characterized. Three mutant viruses, which possessed two (N43K/E49A) or three (Q39R/N43K/N126K and N43K/E49A/N126K) amino acid substitutions in the N- and C-terminal repeat regions of gp41 were identified as conferring high resistance to SC29EK and cross-resistance to the first-generation (T20 and C34) and newly designed (sifuvirtide, MT-SC29EK, and 2P23) fusion inhibitors. The resistance mutations could reduce the binding stability of SC29EK, impair viral Env-mediated cell fusion and entry, and change the conformation of the gp41 core structure. Further, we determined the crystal structure of SC29EK in complex with a target mimic peptide, which revealed the critical intra- and interhelical interactions underlying the mode of action of SC29EK and the genetic pathway to HIV-1 resistance. Taken together, the present data provide new insights into the structure and function of gp41 and the structure-activity relationship (SAR) of viral fusion inhibitors.IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection, but it has relatively low anti-HIV activity and genetic barriers for resistance, thus calling for new drugs blocking the viral fusion process. As an electronically constrained α-helical peptide, SC29EK is highly potent against both wild-type and T20-resistant HIV-1 strains. Here, we report the characterization of HIV-1 variants resistant to SC29EK and the crystal structure of SC29EK. The key mutations mediating high resistance to SC29EK and cross-resistance to the first and new generations of fusion inhibitors as well as the underlying mechanisms were identified. The crystal structure of SC29EK bound to a target mimic peptide further revealed its action mode and genetic pathway to inducing resistance. Hence, our data have shed new lights on the mechanisms of HIV-1 fusion and its inhibition.

Enfuvirtide (T20)-Based Lipopeptide Is a Potent HIV-1 Cell Fusion Inhibitor: Implications for Viral Entry and Inhibition.

The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and in vivo stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry, and infection. Unlike LP-11 and LP-19, which required a flexible linker between the peptide sequence and the lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying different binding modes with the extended N-terminal helices of gp41. Also, interestingly, LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor, but also revealed new insights into the mechanisms of viral inhibition.IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high doses and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report the design of LP-40, a T20-based lipopeptide inhibitor that has greatly improved anti-HIV activity and is a more potent inhibitor of cell-cell fusion than of cell-free virus infection. The binding modes of two classes of membrane-anchoring lipopeptides (LP-40 and LP-11) verify the current fusion model in which an extended prehairpin structure bridges the viral and cellular membranes, and their complementary effects suggest a vital strategy for combination therapy of HIV-1 infection. Moreover, our understanding of the mechanism of action of T20 and its derivatives benefits from the crystal structure of LP-40.

Development of potent and long-acting HIV-1 fusion inhibitors.

BACKGROUND: T20 (enfuvirtide) is the first approved HIV entry inhibitor and currently the only viral fusion inhibitor, but its low efficacy and genetic barrier to resistance significantly limit its application, calling for a next-generation drug. DESIGN: On the basis of the M-T hook structure, we recently developed a short-peptide named HP23, which mainly targets the deep pocket site of gp41 and possesses highly potent antiviral activity. To improve the pharmaceutical properties of a peptide-based inhibitor, we modified HP23 by different classes of lipids including fatty acid, cholesterol, and sphingolipids. To avoid the potential problem of oxidation, the methionine residue in the M-T hook sequence of HP23 was replaced with leucine. METHODS: Peptides were synthesized and their anti-HIV activity and biophysical properties were determined. RESULTS: A group of lipopeptides were generated with greatly improved anti-HIV activity. Promisingly, a fatty acid-conjugated lipopeptide named LP-11 showed potent and broad inhibitory activity against diverse primary HIV-1 isolates and clinically drug-resistant mutants, and it had dramatically increased ex-vivo antiviral activity and extended half-life. Also, LP-11 displayed highly enhanced α-helicity and thermal stability, and it was physically stable under high temperature and humidity. CONCLUSION: LP-11 has high potentials for clinical development and it can serve as an ideal tool for exploring the mechanisms of HIV-1 fusion and inhibition.

Thyroid storm following trauma: a pitfall in the emergency department.

We present a case of a patient with thyroid storm exhibiting symptoms similar to those of traumatic injury. This patient presented multiple traumatic wounds on his limbs and face, loss of consciousness, and tachycardia. Diagnosis was initially confounded by the similarity between the symptoms of traumatic hypovolemic shock and trauma-induced thyroid storm. This case report discusses the factors leading to the diagnostic delay and highlights the dangerous manifestations of thyroid storm.

[MRI characteristic of proximal femur bone marrow edema syndrome].

OBJECTIVE: To study the MRI features of proximal femur bone marrow edema syndrome for further improve the understanding of the disease. METHODS: MRI imaging of 10 patients with proximal femur bone marrow edema syndrome was retrospectively reviewed,including 6 males and 4 females with an average age of 41.5 years old ranging from 36 to 57. The courses of diseases ranged from 1 week to 3 months. Among them, 9 cases had clinical manifestations of sudden hip pain, 7 cases had limited ability of walking and hip movement;all patients had no obvious injury history, non of the female patients was pregnant. All patients were followed up from 3 to 12 months, the following-up were topped after MRI when the symptoms disappeared for 3 months. RESULTS: The MRI demonstrated diffuse bone marrow edema involving the femoral head, neck and the inter-trochanteric region, 13 hips of 10 patients with bone marrow edema included 6 cases in grade 1, 5 cases in grade 2,2 cases in grade 3; 9 hips with hip hydrarthrosis included 6 hips in grade I ,1 hip in grade II, 2 hips in grade III. After treatment for 3 to 12 months the hip symptoms of the patients disappeared and MRI images were normal. CONCLUSION: MRI is useful in defining the location and extent of proximal femur bone marrow edema syndrome.