Dysregulation of Lipid Droplet Protein Expression in Adipose Tissues and Association with Metabolic Risk Factors in Adult Females with Obesity and Type 2 Diabetes.

BACKGROUND: Adipocyte dysregulation of lipid droplet (LD) metabolism caused by altered expression of LD proteins contributes to obesity-related metabolic diseases. OBJECTIVES: We aimed to investigate whether expression levels of PLIN1, CIDEA, and CIDEC were altered in adipose tissues of women with obesity and type 2 diabetes and whether their alterations were associated with metabolic risk factors. METHODS: Normal-weight (NW; 18.5 kg/m2 < BMI ≤ 25 kg/m2; n = 43), nondiabetic obese (OB; BMI > 30 kg/m2; n = 38), and diabetic obese (OB/DM; BMI > 30 kg/m2, fasting glucose ≥ 126 mg/dL, HbA1c ≥ 6.5%; n = 22) women were recruited. Metabolic parameters were measured, and expressions of PLIN1, CIDEA, CIDEC, and obesity-related genes were quantified in abdominal subcutaneous (SAT) and visceral adipose tissues (VAT). Effects of proinflammatory cytokines, endoplasmic reticulum (ER) stress inducers, and metabolic improvement agents on LD protein gene expressions were investigated in human adipocytes. RESULTS: PLIN1, CIDEA, and CIDEC expressions were lower in SAT and higher in VAT in OB subjects relative to NW subjects; however, they were suppressed in both fat depots in OB/DM subjects relative to OB (P < 0.05). Across the entire cohort, whereas VAT PLIN1 (r = 0.349) and CIDEC expressions (r = 0.282) were positively associated with BMI (P < 0.05), SAT PLIN1 (r = -0.390) and CIDEA expressions (r = -0.565) were inversely associated. After adjustment for BMI, some or all of the adipose LD protein gene expressions were negatively associated with fasting glucose (r = -0.259 or higher) and triglyceride levels (r = -0.284 or higher) and positively associated with UCP1 expression (r = 0.353 or higher) (P < 0.05). In adipocytes, LD protein gene expressions were 55-70% downregulated by increased proinflammatory cytokines and ER stress but 2-4-fold upregulated by the metabolic improvement agents exendin-4 and dapagliflozin (P < 0.05). CONCLUSIONS: The findings suggest that reduction of adipose LD protein expression is involved in the pathogenesis of metabolic disorders in women with obesity and type 2 diabetes and that increasing LD protein expression in adipocytes could control development of metabolic disorders.

Assessment of Pulmonary Ventilation Using 3D Ventilation Flow-Weighted and Ventilation-Weighted Maps From 3D Ultrashort Echo Time (UTE) MRI.

BACKGROUND: Three-dimensional (3D) ventilation flow-weighted (VFW) maps together with 3D ventilation-weighted (VW) maps may help to better assess pulmonary function. PURPOSE: To investigate the use of 3D VFW and VW maps for evaluating pulmonary ventilation function. STUDY TYPE: Prospective. POPULATION: Two patients (one male, 85 years old; one female, 64 years old) with chronic obstructive pulmonary disease (COPD) and nine healthy subjects (all male; 23-27 years). FIELD STRENGTH/SEQUENCE: 3-T, 3D radial UTE imaging. ASSESSMENT: 3D VFW and VW maps were calculated from 3D UTE MRI by voxel-wise subtraction of respiratory phase images. Their validation was tested in nine healthy volunteers using slow/deep and fast/shallow breathing conditions. Additional validation was performed by comparison with single photon emission computed tomography (SPECT) ventilation maps of one healthy participant. For comparison, gravity dependence of anterior-posterior regional ventilation was assessed by one-dimensional plot of the mean signal intensity for each coronal slice. Structural similarity index measure was also calculated. Finally, VW maps and VFW maps of two COPD patients were evaluated for emphysema lesions with reference to CT images. STATISTICAL TESTS: Wilcoxon sign-rank tests for regional Ventilation and Ventilation flow, analysis of variance, post-hoc t-tests and Bonferroni correction, coefficient of variation, Kullback-Liebler divergence. A P-value <0.05 was considered statistically significant. RESULTS: The validation of 3D VFW and VW maps was shown by statistically significant differences in ventilation flow and ventilation between the breathing conditions. Additionally, UTE-MRI and SPECT-based ventilation maps showed gravitational dependence in the anteroposterior direction. When applied to patients with COPD, the use of 3D VFW and VW maps was able to differentiate between two patients with different phenotypes. DATA CONCLUSION: The use of 3D VFW and VW maps can provide regional information on ventilation function and potentially contribute to assessment of COPD subtypes and disease progression. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 1.

Simultaneous monitoring of each component on degradation of blended bioplastic using gas chromatography-mass spectrometry.

The increasing interest in bioplastics, with regard to future environmental issues, has rendered research on bioplastic biodegradation highly important. However, only a few tools directly monitor the degradation of bioplastics without measuring the levels of gaseous products, such as carbon dioxide. Classical nonquantitative methods, such as clear zone tests on solid plates, and less-sensitive weight-loss experiments in liquid media measured using a precision scale, are still employed to screen the microbial players associated with bioplastic degradation and monitor the biodegradation rates. However, the simultaneous monitoring of the degradation of each component of blended bioplastics has not been previously reported. In the present study, to provide information regarding the degradation rates and compositional changes of different bioplastics in a blend in a time-dependent manner, we simultaneously monitored and quantified the degradation of four bioplastics, polyhydroxybutyrate (PHB), polybutylene succinate (PBS), polycaprolactone (PCL), and poly(butylene adipate-co-terephthalate) (PBAT), by Bacillus sp. JY36 using gas chromatography-mass spectrometry (GC-MS) analysis after fatty acid methyl ester (FAME) derivatization. Our results demonstrate the feasibility of using the GC-MS-based method described here to obtain comprehensive data regarding blended bioplastics and their degradation. Moreover, our findings indicate that this method may support classical analytic tools for assessing bioplastic biodegradation.

Mental Health Crisis Screening in Youth with Autism Spectrum Disorder.

OBJECTIVE: While a growing body of evidence suggests youth with autism are at increased risk of experiencing a mental health crisis, no study has screened for crises in an outpatient setting. The current study fills this gap by examining a) the feasibility and utility of conducting routine crisis screenings; b) the psychometrics of a brief crisis screener (the Mental Health Crisis Assessment Scale-Revised; MCAS-R); and, c) the prevalence of and types of behaviors associated with crises. METHOD: This study was conducted at two different outpatient mental health clinics. Screenings were conducted using the MCAS-R, a 23-item parent report measure. A total of 406 youth with autism (76% Male; 72% White; M = 11.2y; SD = 3.5y), evenly divided across clinics, were screened. Seven clinicians conducted a clinical visit, which incorporated the results of the MCAS-R, to determine whether the child was in crisis. RESULTS: Eighty percent of youth were successfully screened, suggesting crisis screening is feasible. Most parents (73%) felt the MCAS-R helped communicate concerns with the clinician; few (<6%) felt the survey was too long or upsetting. All clinicians (100%) indicated that the MCAS-R was very helpful in facilitating communication and identifying/mitigating safety concerns; although, 33% reported screenings "sometimes" interrupted clinical flow. The MCAS-R strongly aligned with clinician ratings (88% correctly classified). Twenty percent of youth met the cutoff for crisis; aggression and self-injurious behaviors were the most common reasons for crises. CONCLUSION: This study suggests that outpatient crisis screening via the MCAS-R is feasible, accurate, and well received by parents and clinicians. ABBREVIATIONS: ASD: Autism Spectrum Disorder; MCAS-R: Mental Health Assessment Crisis Scale-Revised; DSM-5: Diagnostic and Statistical Manual, 5th Edition; ADOS-2: Autism Diagnostic Observation Schedule, Second Edition; ROC: Receiver Operating Curve.

Aqueous Rechargeable Zn/ZnO Battery Based on Deposition/Dissolution Chemistry.

Recently, a novel electrochemical regulation associated with a deposition/dissolution reaction on an electrode surface has been proven to show superiority in large-scale energy storage systems (ESSs). Hence, in the search for high-performance electrodes showcasing these novel regulations, we utilized a low-cost ZnO microsphere electrode to construct aqueous rechargeable batteries (ARBs) that supplied a harvestable and storable charge through electrochemical deposition/dissolution via a reversible manganese oxidation reaction (MOR)/manganese reduction reaction (MRR), respectively, induced by the inherent formation/dissolution of zinc basic sulfate in a mild aqueous electrolyte solution containing 2 M ZnSO4 and 0.2 M MnSO4.

Key stakeholder perspectives on obstacles to an autism-specific Medicaid waiver service delivery model.

BACKGROUND: Children and youth with autism spectrum disorder have significant, multi-system needs that require supports, such as the autism waiver (AW) service delivery model. This study aimed to identify and describe characteristics of the AW, define obstacles and strengths in the provision of adequate services and provide recommendations for improving overall effectiveness. METHODS: This mixed-methods exploratory study used an electronic survey to gain information and perceptions of AW provider agency directors (n = 27) and service coordinators (n = 30). RESULTS: The key barriers reported were the shortage of qualified staff, inadequate staff training, complexity of cases or symptom severity of clients and lack of communication at multiple levels throughout the agency as well as with parents. CONCLUSIONS: Recommendations include reinforcing the workforce through higher salaries, greater training and communication interventions. These strategies may reduce staff turnover and shortage, lighten the caseload, reduce the waitlist period and improve the effectiveness and responsiveness of AW services.

Risk factors for heel pressure injury in cardiovascular intensive care unit patients.

This study analyzed the risk factors for heel pressure injury in cardiovascular intensive care unit patients with the aim of laying the groundwork for preventive nursing interventions. We conducted a retrospective case-control study of 92 patients who were admitted to the cardiovascular surgical or medical intensive care unit of a university hospital in South Korea between January and December 2017. Of these patients, 31 and 61 were included to the heel pressure injury group and the non-heel pressure injury group, respectively. Data on their demographic, disease-related, and intensive care unit treatment characteristics, as well as the degree of pressure injury, were collected from the hospital's electronic medical records using a standardized form. Cardiac surgery (P < .001), operation time (P = .001), use of a mechanical ventilator (P < .001), use of vasoconstrictors (P < .001), use of sedative drugs (P < .001), and extracorporeal membrane oxygenation treatment (P < .001) were identified as significant risk factors for heel pressure injury. A total of 22 patients (71%) from the heel pressure injury group developed deep tissue injury, and 16 patients (51.6%) who received extracorporeal membrane oxygenation treatment developed heel pressure injury.

Long-term and residual settlement behaviour of a multi-staged municipal solid waste landfill, Republic of Korea.

In this study, characteristics of the long-term landfill settlement are analysed and the starting date of residual settlement is deduced using the data of the landfill 1 of the Gimpo Metropolitan Landfill (GML) measured for 27 years. The landfill 1 is a multi-staged municipal solid waste landfill where dykes are constructed after landfilling for subsequent waste fills. The landfilling began in 1992 and finished in 2000, and the waste settlement measurement continued throughout this period. The older waste of the lower lift shows higher biodegradation and larger settlement than the waste of the upper lift, which acts as vertical load. Large settlement occurred in the lower lifts due to the collapse of waste voids caused by the overburden load of the upper lifts after biodegradation of waste in the lower lifts during long-term landfilling. In the conventional landfills, that is, mono-layer landfills, the time-dependent settlement generally occurs after the stress-dependent settlement. But, most of the time, this landfill showed mainly the time-dependent settlement. The duration, at which the biodegradation of organic matters is reduced and the residual settlement begins, was 8.8 years in average and the date range was from 7.6 to 11.7 years considering each disposed block in landfill 1. This date is obtained by calculating from the mid-point time of the landfilling duration.

Endoplasmic reticulum stress increases LECT2 expression via ATF4.

Non-alcoholic fatty liver disease (NAFLD) is frequently associated with obesity, insulin resistance, and endoplasmic reticulum (ER) stress. Elevated circulating levels of the hepatokine leukocyte cell-derived chemotaxin-2 (LECT2) have also been noted in NAFLD; however, the mechanism underlying this association is unclear. To investigate a possible link between ER stress/unfolded protein response (UPR) signaling and LECT2 secretion, HepG2 cells were incubated with ER stress inducers with or without an ER stress-reducing chemical chaperone. Additionally, UPR pathway genes were knocked down and overexpressed, and a ChIP assay was performed. In diet-induced obese mice, hepatic expression of LECT2 and activating transcription factor 4 (ATF4) was measured. In HepG2 cells, LECT2 expression was increased by ER stressors, an effect blocked by the chemical chaperone. Among UPR pathway proteins, only knockdown of ATF4 suppressed ER stress-induced LECT2 expression, while overexpression of ATF4 enhanced LECT2 expression. The ChIP assay revealed that ATF4 binds to three putative binding sites on the LECT2 promoter and binding is promoted by an ER stress inducer. In steatotic livers of obese mice, LECT2 and ATF4 expression was concomitantly elevated. Our data indicate that activation of ER stress/UPR signaling induces LECT2 expression in steatotic liver; specifically, ATF4 appears to mediate upregulation of LECT2 transcription.

Critical Role of Functional Groups Containing N, S, and O on Graphene Surface for Stable and Fast Charging Li-S Batteries.

Lithium-sulfur (Li-S) batteries are considered one of the most promising energy storage technologies, possibly replacing the state-of-the-art lithium-ion (Li-ion) batteries owing to their high energy density, low cost, and eco-compatibility. However, the migration of high-order lithium polysulfides (LiPs) to the lithium surface and the sluggish electrochemical kinetics pose challenges to their commercialization. The interactions between the cathode and LiPs can be enhanced by the doping of the carbon host with heteroatoms, however with relatively low doping content (<10%) in the bulk of the carbon, which can hardly interact with LiPs at the host surface. In this study, the grafting of versatile functional groups with designable properties (e.g., catalytic effects) directly on the surface of the carbon host is proposed to enhance interactions with LiPs. As model systems, benzene groups containing N/O and S/O atoms are vertically grafted and uniformly distributed on the surface of expanded reduced graphene oxide, fostering a stable interface between the cathode and LiPs. The combination of experiments and density functional theory calculations demonstrate improvements in chemical interactions between graphene and LiPs, with an enhancement in the electrochemical kinetics, power, and energy densities.

An equal-TE ultrafast 3D gradient-echo imaging method with high tolerance to magnetic susceptibility artifacts: Application to BOLD functional MRI.

PURPOSE: To develop an ultrafast 3D gradient echo-based MRI method with constant TE and high tolerance to B0 inhomogeneity, dubbed ERASE (equal-TE rapid acquisition with sequential excitation), and to introduce its use in BOLD functional MRI (fMRI). THEORY AND METHODS: Essential features of ERASE, including spin behavior, were characterized, and a comparison study was conducted with conventional EPI. To demonstrate high tolerance to B0 inhomogeneity, in vivo imaging of the mouse brain with a fiber-optic implant was performed at 9.4 T, and human brain imaging (including the orbitofrontal cortex) was performed at 3 T and 7 T. To evaluate the performance of ERASE in BOLD-fMRI, the characteristics of SNR and temporal SNR were analyzed for in vivo rat brains at 9.4 T in comparison with multislice gradient-echo EPI. Percent signal changes and t-scores are also presented. RESULTS: For both mouse brain and human brain imaging, ERASE exhibited a high tolerance to magnetic susceptibility artifacts, showing much lower distortion and signal dropout, especially in the regions involving large magnetic susceptibility effects. For BOLD-fMRI, ERASE provided higher temporal SNR and t-scores than EPI, but exhibited similar percent signal changes in in vivo rat brains at 9.4 T. CONCLUSION: When compared with conventional EPI, ERASE is much less sensitive, not only to EPI-related artifacts such as Nyquist ghosting, but also to B0 inhomogeneity including magnetic susceptibility effects. It is promising for use in BOLD-fMRI, providing higher temporal SNR and t-scores with constant TE when compared with EPI, although further optimization is needed for human fMRI.

Improvement of cadaverine production in whole cell system with baker's yeast for cofactor regeneration.

Cadaverine, 1,5-diaminopentane, is one of the most promising chemicals for biobased-polyamide production and it has been successfully produced up to molar concentration. Pyridoxal 5'-phosphate (PLP) is a critical cofactor for inducible lysine decarboxylase (CadA) and is required up to micromolar concentration level. Previously the regeneration of PLP in cadaverine bioconversion has been studied and salvage pathway pyridoxal kinase (PdxY) was successfully introduced; however, this system also required a continuous supply of adenosine 5'-triphosphate (ATP) for PLP regeneration from pyridoxal (PL) which add in cost. Herein, to improve the process further a method of ATP regeneration was established by applying baker's yeast with jhAY strain harboring CadA and PdxY, and demonstrated that providing a moderate amount of adenosine 5'-triphosphate (ATP) with the simple addition of baker's yeast could increase cadaverine production dramatically. After optimization of reaction conditions, such as PL, adenosine 5'-diphosphate, MgCl2, and phosphate buffer, we able to achieve high production (1740 mM, 87% yield) from 2 M L-lysine. Moreover, this approach could give averaged 80.4% of cadaverine yield after three times reactions with baker's yeast and jhAY strain. It is expected that baker's yeast could be applied to other reactions requiring an ATP regeneration system.

Nano/Microstructured Silicon-Carbon Hybrid Composite Particles Fabricated with Corn Starch Biowaste as Anode Materials for Li-Ion Batteries.

Silicon has a great potential as an alternative to graphite which is currently used commercially as an anode material in lithium-ion batteries (LIBs) because of its exceptional capacity and reasonable working potential. Herein, a low-cost and scalable approach is proposed for the production of high-performance silicon-carbon (Si-C) hybrid composite anodes for high-energy LIBs. The Si-C composite material is synthesized using a scalable microemulsion method by selecting silicon nanoparticles, using low-cost corn starch as a biomass precursor and finally conducting heat treatment under C3H6 gas. This produces a unique nano/microstructured Si-C hybrid composite comprised of silicon nanoparticles embedded in micron-sized amorphous carbon balls derived from corn starch that is capsuled by thin graphitic carbon layer. Such a dual carbon matrix tightly surrounds the silicon nanoparticles that provides high electronic conductivity and significantly decreases the absolute stress/strain of the material during multiple lithiation-delithiation processes. The Si-C hybrid composite anode demonstrates a high capacity of 1800 mAh g-1, outstanding cycling stability with capacity retention of 80% over 500 cycles, and fast charge-discharge capability of 12 min. Moreover, the Si-C composite anode exhibits good acceptability in practical LIBs assembled with commercial Li[Ni0.6Co0.2Mn0.2]O2 and Li[Ni0.80Co0.15Al0.05]O2 cathodes.

Engineering Sodium-Ion Solvation Structure to Stabilize Sodium Anodes: Universal Strategy for Fast-Charging and Safer Sodium-Ion Batteries.

Sodium-ion batteries are promising alternatives for lithium-ion batteries due to their lower cost caused by global sodium availability. However, the low Coulombic efficiency (CE) of the sodium metal plating/stripping process represents a serious issue for the Na anode, which hinders achieving a higher energy density. Herein, we report that the Na+ solvation structure, particularly the type and location of the anions, plays a critical role in determining the Na anode performance. We show that the low CE results from anion-mediated corrosion, which can be tackled readily through tuning the anion interaction at the electrolyte/anode interface. Our strategy thus enables fast-charging Na-ion and Na-S batteries with a remarkable cycle life. The presented insights differ from the prevailing interpretation that the failure mechanism mostly results from sodium dendrite growth and/or solid electrolyte interphase formation. Our anionic model introduces a new guideline for improving the electrolytes for metal-ion batteries with a greater energy density.

Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion.

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.

DJ-1 Can Replace FGF-2 for Long-Term Culture of Human Pluripotent Stem Cells in Defined Media and Feeder-Free Condition.

Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF-2 mandates media change every day, which makes hPSC culture costly and cumbersome. Human DJ-1 (hDJ-1) can bind to and stimulate FGF receptor-1. In this study, for the first time, we have replaced hFGF-2 with hDJ-1 in the essential eight media and maintained the human embryonic stem cells (hESCs), H9, in the defined media at feeder-free condition. After more than ten passages, H9 in both groups still successfully maintained the typical hESC morphology and high protein levels of pluripotency markers, SSEA4, Tra1-60, Oct4, Nanog, and ALP. DNA microarray revealed that more than 97% of the 21,448 tested genes, including the pluripotency markers, Sox2, Nanog, Klf4, Lin28A, Lin28B, and Myc, have similar mRNA levels between the two groups. Karyotyping revealed no chromosome abnormalities in both groups. They also differentiated sufficiently into three germ layers by forming in vitro EBs and in vivo teratomas. There were some variations in the RT-qPCR assay of several pluripotency markers. The proliferation rates and the mitochondria of both groups were also different. Taken together, we conclude that hDJ-1 can replace hFGF-2 in maintaining the self-renewal and the pluripotency of hESCs in feeder-free conditions.

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b'a' Domain.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b'a' domain of protein disulfide isomerase (PDIb'a') greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb'a'-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.

Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor.

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a') tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.

Secondary transmission of SARS-CoV-2 through wastewater: Concerns and tactics for treatment to effectively control the pandemic.

The SARS-CoV-2 virus has spread globally and has severely impacted public health and the economy. Hand hygiene, social distancing, and the usage of personal protective equipment are considered the most vital tools in controlling the primary transmission of the virus. Converging evidence indicated the presence of SARS-CoV-2 in wastewater and its persistence over several days, which may create secondary transmission of the virus via waterborne and wastewater pathways. Although, researchers have started focusing on this mode of virus transmission, limited knowledge and societal unawareness of the transmission through wastewater may lead to significant increases in the number of positive cases. To emphasize the severe issue of virus transmission through wastewater and create societal awareness, we present a state of the art critical review on transmission of SARS-CoV-2 in wastewater and the potential remedial strategies to effectively control the viral spread and safeguard society. For low-income countries with high population densities, it is suggested to identify the virus in large scale municipal wastewater plants before following up with one-to-one testing for effective control of the secondary transmission. Ultrafiltration is an effective method for wastewater treatment and usually more than 4 logs of virus removal are achieved while safeguarding good protein permeability. Decentralized wastewater treatment facilities using solar-assisted disinfestation methods are most economical and can be effectively used in hospitals, isolation wards, and medical centers for reducing the risk of transmission from high local concentration sites, especially in tropical countries with abundant solar energy. Disinfection with chlorine, sodium hypochlorite, benzalkonium chloride, and peracetic acid have shown potential in terms of virucidal properties. Biological wastewater treatment using micro-algae will be highly effective in removal of virus and can be incorporated into membrane bio-reaction to achieve excellent virus removal rate. Though promising results have been shown by initial research for inactivation of SARS-CoV-2 in wastewater using physical, chemical and biological based treatment methods, there is a pressing need for extensive investigation of COVID-19 specific disinfectants with appropriate concentrations, their environmental implications, and regular monitoring of transmission. Effective wastewater treatment methods with high virus removal capacity and low treatment costs should be selected to control the virus spread and safeguard society from this deadly virus.

Translational Detection of Nonproteinogenic Amino Acids Using an Engineered Complementary Cell-Free Protein Synthesis Assay.

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.