Expression of phenylalanine ammonia lyases in Synechocystis sp. PCC 6803 and subsequent improvements of sustainable production of phenylpropanoids

Background Phenylpropanoids represent a diverse class of industrially important secondary metabolites, synthesized in plants from phenylalanine and tyrosine. Cyanobacteria have a great potential for sustainable production of phenylpropanoids directly from CO2, due to their photosynthetic lifestyle with a fast growth compared to plants and the ease of generating genetically engineered strains. This study focuses on photosynthetic production of the starting compounds of the phenylpropanoid pathway, trans-cinnamic acid and p-coumaric acid, in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Results A selected set of phenylalanine ammonia lyase (PAL) enzymes from different organisms was overexpressed in Synechocystis, and the productivities of the resulting strains compared. To further improve the titer of target compounds, we evaluated the use of stronger expression cassettes for increasing PAL protein levels, as well as knock-out of the laccase gene slr1573, as this was previously reported to prevent degradation of the target compounds in the cell. Finally, to investigate the effect of growth conditions on the production of trans-cinnamic and p-coumaric acids from Synechocystis, cultivation conditions promoting rapid, high density growth were tested. Comparing the different PALs, the highest specific titer was achieved for the strain AtC, expressing PAL from Arabidopsis thaliana. A subsequent increase of protein level did not improve the productivity. Production of target compounds in strains where the slr1573 laccase had been knocked out was found to be lower compared to strains with wild type background, and the Δslr1573 strains exhibited a strong phenotype of slower growth rate and lower pigment content. Application of a high-density cultivation system for the growth of production strains allowed reaching the highest total titers of trans-cinnamic and p-coumaric acids reported so far, at around 0.8 and 0.4 g L−1, respectively, after 4 days. Conclusions Production of trans-cinnamic acid, unlike that of p-coumaric acid, is not limited by the protein level of heterologously expressed PAL in Synechocystis. High density cultivation led to higher titres of both products, while knocking out slr1573 did not have a positive effect on production. This work contributes to capability of exploiting the primary metabolism of cyanobacteria for sustainable production of plant phenylpropanoids.

CerS1 but Not CerS5 Gene Silencing, Improves Insulin Sensitivity and Glucose Uptake in Skeletal Muscle

Skeletal muscle is perceived as a major tissue in glucose and lipid metabolism. High fat diet (HFD) lead to the accumulation of intramuscular lipids, including: long chain acyl-CoA, diacylglycerols, and ceramides. Ceramides are considered to be one of the most important lipid groups in the generation of skeletal muscle insulin resistance. So far, it has not been clearly established whether all ceramides adversely affect the functioning of the insulin pathway, or whether there are certain ceramide species that play a pivotal role in the induction of insulin resistance. Therefore, we designed a study in which the expression of CerS1 and CerS5 genes responsible for the synthesis of C18:0-Cer and C16:0-Cer, respectively, was locally silenced in the gastrocnemius muscle of HFD-fed mice through in vivo electroporation-mediated shRNA plasmids. Our study indicates that HFD feeding induced both, the systemic and skeletal muscle insulin resistance, which was accompanied by an increase in the intramuscular lipid levels, decreased activation of the insulin pathway and, consequently, a decrease in the skeletal muscle glucose uptake. CerS1 silencing leads to a reduction in C18:0-Cer content, with a subsequent increase in the activity of the insulin pathway, and an improvement in skeletal muscle glucose uptake. Such effects were not visible in case of CerS5 silencing, which indicates that the accumulation of C18:0-Cer plays a decisive role in the induction of skeletal muscle insulin resistance.

An increase in circulating levels of branched-chain amino acids during hemodialysis with regard to protein breakdown: three case reports

Background Hemodialysis (HD) is a protein catabolic event. However, the amino acid (AA) kinetics during HD sessions involved in protein breakdown have not been well investigated in patients with and without diabetes mellitus (DM). Case presentation Three patients (two patients with DM and one patient without DM) underwent fasting HD. Plasma levels of branched-chain AAs (BCAA; leucine, isoleucine, and valine), major non-essential AAs (alanine and glutamine, including glutamate), insulin, and ketone bodies were measured every hour during each HD session. After the start of the HD session, the plasma levels of insulin and all BCAAs dropped simultaneously. There was a significant subsequent increase in the plasma level of leucine and isoleucine levels, while valine levels remained constant. However, the recovery in levels of BCAAs during HD indicated a profound amount of BCAAs entering the blood from body tissues such as muscles. BCAAs may have surpassed their removal by HD. Ketone body levels increased continuously from the start of the sessions and reached high values in patients with DM. Synchronous changes in insulin depletion and an increase in the levels of ketone bodies may indicate disruption of energy metabolism. Conclusions This is the first report to demonstrate the time course of the changes in circulating levels of BCAAs and related metabolites in energy homeostasis during HD. An increase in BCAA levels during HD was found to be due to their transfer from the body tissue which suggested protein breakdown.

Validation of PI-RADS v2 Scores at Various Non-University Radiology Practices

Purpose: The purpose of this study was to validate the second version of the Prostate Imaging Reporting and Data System (PI-RADSv2) scores in predicting positive in-bore MRI-guided targeted prostate biopsy results across different non-university related institutions. The study focuses on PI-RADS v2 scoring because during the study period, PI-RADS v2.1 had not been released. Materials and Methods: This was a retrospective review of 147 patients who underwent multiparametric magnetic resonance imaging (mpMRI) of the pelvis followed by in-bore MRI-guided targeted prostate biopsy from December 2014 to May 2018. All lesions on mpMRI were rated according to PI-RADS v2 criteria. PI-RADS v2 scores were then compared to MR-guided biopsy results and pre-biopsy PSA values. Results: Prostate Cancer (PCa) was detected in 54% (80/147) of patients, with more prostate cancer being detected with each subsequent increase in PI-RADS scores. Specifically, biopsy results in patients with PI-RADS 3, 4, and 5 lesions resulted in PCa in 25.6% (10/39), 58.1% (33/55), and 86.0% (37/43) respectively. Clinically significant PCa (Gleason score ≥7) was detected in 17.9% (7/39), 52.7% (29/55), and 72% (31/43) of cases for PI-RADS 3, 4, and 5 lesions respectively. When the PI-RADS scoring and biopsy results were compared across different institutions, there was no difference in the PI-RADS scoring of lesions or in the positive biopsy rates of the lesions. The sensitivity, specificity, PPV, and NPV for PI-RADS 3-4 lesions were also not statistically different across the institutions for detecting Gleason 7 or greater lesions. Conclusion: Our results agree with prior studies that higher PI-RADS scores are associated with the presence of clinically significant PCa and suggest prostate lesions with PI-RADS scores 3-5 have sufficient evidence to warrant targeted biopsy. The comparison of PI-RADS score across different types of non-university practices revealed no difference in scoring and biopsy outcome, suggesting that PI-RADS v2 can be easily applied outside of the university medical center setting. Clinical Relevance: PI-RADS v2 can be applied homogeneously in the non-university setting without significant difference in outcome.

FORMULATION DEVELOPMENT OF ORLISTAT NANOCRYSTALS: IN VITRO CHARACTERIZATION AND IN VIVO STUDIES

Poor solubility of orlistat limits its luminal concentration and hence needs to be administered in higher doses, leading to drug related side effects. The aim of the present research was to investigate nanocrystallization approach to increase the solubility of orlistat using melt extrusion and high-pressure homogenization (HPH) methods. The effect of factors like type and amount of polymer, homogenization pressure and time, and number of cycles on orlistat solubility was investigated. A ~10-fold increase in the solubility of orlistat was attained using OPo11N with a subsequent increase in the dissolution rate of the drug. Poloxamer 188-orlistat nanocrystals (OPo11N) as compared to pure orlistat led to a decrease in T90%(20 mins for OPo11N and 51 mins for marketed sample). In vivo studies in female Sprague Dawley (SD) rats showed that post one month of oral administration the total cholesterol and low-density lipoproteins of female SD rats remained unchanged compared to the control group. The triglycerides content and high-density lipoproteins levels were significantly increased with increase in the treatment time i.e. 12 weeks compared to the group treated with pure orlistat drug. In conclusion, the NC approach could serve as an effective formulation strategy for solubility enhancement of orlistat.

Impact of Air Pollution on Residents' Medical Expenses: A Study Based on the Survey Data of 122 Cities in China

Background: With the development of the social economy, air pollution has resulted in increased social costs. Medical costs and health issues due to air pollution are important aspects of environmental governance in various countries.Methods: This study uses daily air pollution monitoring data from 122 cities in China to empirically investigate the impact of air pollution on residents' medical expenses using the Heckman two-stage and instrumental variable methods, matching data from the 2018 China Health and Retirement Longitudinal Study (CHARLS) survey.Results: The study found that poor air quality, measured by the air quality index (AQI), significantly increased the probability of chronic lung disease, heart disease, and self-rated poor health. Additionally, the AQI (with an effect of 4.51%) significantly impacted health-seeking behavior and medical expenses. The medical expenditure effects of mild, moderate, severe, and serious pollution days were 3.27, 7.21, 8.62, and 42.66%, respectively.Conclusion: In the long run, residents' health in areas with a higher air pollution index, indicating poor air quality, is negatively impacted. The more extreme the pollution, the higher the probability of residents' medical treatment and the subsequent increase in medical expenses. Group and regional heterogeneity also play a role in the impact of air pollution on medical expenses. Compared with the existing literature, this study is based on individuals aged 15 years and above and produces reliable research conclusions.

Strength and dimensional stability of cement-bonded wood waste-sand bricks

Conservation of trees which belong to renewable natural resources in developing countries through judicious utilization in building industry has been seriously left unattended to over the years. The utilization of this unprocessed wealth materials which could serve as one of the alternative constituents in construction materials is now a global concern. Therefore, the needs to investigate the strength and water sorption property of wood waste as a partial replacing material in brick production for building construction arise. Sawdust, sand and cement were mixed together at three different mixing ratios of 1 : 1 : 1, 1 : 1 : 2 and 1 : 1 : 3. The brick samples were replicated three times and subjected to water absorption, density and compressive strength tests. The results show that the effect of the equal volume of sand and sawdust with a subsequent increase in the cement portion at each mixing ratio level gave compact bricks without sudden fracture, reduced unit weight of bricks and subsequent lower water sorption properties of the bricks. The bricks show potentials to be used for wall partitioning. The introduction of sawdust also serves as partial sand replacement in concrete brick making.

Fish otolith geochemistry, environmental conditions and human occupation at Lake Mungo, Australia

Fish otoliths from the Willandra Lakes Region World Heritage Area (south-western New South Wales, Australia) have been analysed for oxygen isotopes and trace elements using in situ techniques, and dated by radiocarbon. The study focused on the lunettes of Lake Mungo, an overflow lake that only filled during flooding events and emptied by evaporation, and Lake Mulurulu, which was part of the running Willandra Creek system. Samples were collected from two different contexts: from hearths directly associated with human activity, and isolated surface finds. AMS radiocarbon dating constrains the human activity documented by five different hearths to a time span of less than 240 years around 19,350 cal. BP. These hearths were constructed in aeolian sediments with alternating clay and sand layers, indicative of fluctuating lake levels and occasional drying out. The geochemistry of the otoliths confirms this scenario, with shifts in Sr/Ca and Ba/Ca marking the entry of the fish into Lake Mungo several years before their death, and a subsequent increase in the δ18O by ∼4‰ indicating increasing evaporation of the lake. During sustained lake-full conditions there are considerably fewer traces of human presence. It seems that the evaporating Lake Mungo attracted people to harvest fish that might have become sluggish through oxygen starvation in an increasingly saline water body (easy prey hypothesis). In contrast, surface finds have a much wider range in radiocarbon age as a result of reworking, and do not necessarily indicate evaporative conditions, as shown by comparison with otoliths from upstream Lake Mulurulu.

Limited water access is associated with food insecurity and diarrheal episodes among children suffering from moderate acute malnutrition in Far-North Cameroon

To gain further understanding of the interlinkages between poor water access, household food insecurity, and undernutrition among children, this study used a cross-sectional design with 474 female caretakers of children suffering from moderate acute malnutrition (MAM) to explore the relationship between limited access to water and diarrheal diseases among children, aged <5 years, experiencing MAM. The mean age of the caretakers was 28.50±6.88 years and that of their MAM children (sex ratio=0.7) was 17.79±9.59 months. The participants reported spending an average of 19.29±15.69 min for one trip to fetch water. A negative correlation was found between mean time spent fetching drinking water and hygiene and handwashing score (r=−0.141, p=0.003). Furthermore, the more severe the food insecurity status of a household, the farther the family member likely had to go to fetch for drinking water [F(2, 444)=8.64, p≤0.001]. Results from binary logistic regression showed that children from households practicing open defecation (p=0.008) and/or having inadequate hygiene practices (p=0.004) had increased odds of developing diarrhea. Therefore, ameliorating water access in households with MAM children could contribute to improvements in hygiene and sanitation attitudes with a subsequent increase in the effectiveness of nutrition interventions aiming at reducing acute malnutrition among children.

Large spontaneous HBV DNA fluctuations and potential usefulness of a single point measurement of combined HBV DNA and quantitative HBsAg for the exclusion of HBeAg negative chronic hepatitis B: A prospective Tunisian cohort study

Background/Aims: We aimed to describe spontaneous short-term hepatitis B Virus (HBV) DNA fluctuations and to assess the usefulness of quantitative HBsAg (qHBsAg) in Tunisian patients with HBeAg-negative chronic HBV infection.Methods: We included 174 treatment-naïve patients with chronic HBeAg-negative HBV. A one-year prospective follow-up was carried out with serial determinations of HBV DNA, alanine aminotransferase (ALT) levels and qHBsAg. Patients were classified into three groups: inactive carriers (G1), patients with HBeAg negative CHB (G2) and patients with “indeterminant state” (G3). For this latter group, a liver biopsy was indicated.Results: Genotype D was the only detected. During the follow-up, 21.6% and 19.5% of patients with low initial (<2000 IU/mL) and intermediate viral load (2000-20000 IU/mL), experienced a subsequent increase in their HBV DNA levels above 2000 and 20000 IU/mL, respectively. Significant variations of HBV DNA levels (≥0.5 log10 IU/mL) were observed in 61.1% of patients at 6 months-interval. Among the 174 patients, 89 (51.1%) belonged to G1, 33 (19%) to G2 and 52 (29.9%) to G3. Fourteen patients have undergone liver biopsy, among whom 7 (50%) showed moderate to severe liver disease. Combination of HBV DNA <2000 IU/mL and qHBsAg <832 IU/mL excluded CHB in 98.4% of cases.Conclusions: This study highlights the large short-term HBV DNA fluctuations in Tunisian patients with HBeAg negative chronic HBV of genotype D. HBV DNA < 2000 IU/mL along with qHBsAg < 832 IU/mL excluded CHB in 98.4% of cases. Significant proportion of patients with “indeterminant state” within genotype D would have HBeAg negative CHB.