UBL3 overexpression enhances EV-mediated Achilles protein secretion in conditioned media of MDA-MB-231 cells.

Cancer cells communicate within the tumor microenvironment (TME) through extracellular vesicles (EVs), which act as crucial messengers in intercellular communication, transporting biomolecules to facilitate cancer progression. Ubiquitin-like 3 (UBL3) facilitates protein sorting into small EVs as a post-translational modifier. However, the effect of UBL3 overexpression in EV-mediated protein secretion has not been investigated yet. This study aimed to investigate the effect of UBL3 overexpression in enhancing EV-mediated Achilles protein secretion in MDA-MB-231 (MM) cells by a dual-reporter system integrating Akaluc and Achilles tagged with Ubiquitin where self-cleaving P2A linker connects Akaluc and Achilles. MM cells stably expressing Ubiquitin-Akaluc-P2A-Achilles (Ubi-Aka/Achi) were generated. In our study, both the bioluminescence of Ubiquitin-Akaluc (Ubi-Aka) and the fluorescence of Achilles secretion were observed. The intensity of Ubi-Aka was thirty times lower, while the Achilles was four times lower than the intensity of corresponding cells. The ratio of Ubi-Aka and Achilles in conditioned media (CM) was 7.5. They were also detected within EVs using an EV uptake luciferase assay and fluorescence imaging. To investigate the effect of the UBL3 overexpression in CM, Ubi-Aka/Achi was transiently transfected into MM-UBL3-KO, MM, and MM-Flag-UBL3 cells. We found that the relative fluorescence expression of Achilles in CM of MM-UBL3-KO, MM, and MM-Flag-UBL3 cells was 30 %, 28 %, and 45 %, respectively. These findings demonstrated that UBL3 overexpression enhances EV-mediated Achilles protein secretion in CM of MM cells. Targeting UBL3 could lead to novel therapies for cancer metastasis by reducing the secretion of pro-metastatic proteins, thereby inhibiting disease progression.

Adhesion Mechanics and Detachment Dynamics of Vanishing Surface Layers on Hydrogels.

Cross-linked hydrogel surfaces exhibit reduced stiffness when polymerized against polymeric hydrophobic surfaces. As such, these layers play a critical role in contact mechanics, particularly exhibiting strong relative adhesion with colloidal probes when the contact area is small. This prevents the use of continuum models of adhesive soft contact. To connect mechanisms of stretch to the force response, depth-controlled nanoindentation experiments were conducted on polyacrylamide (pAAM) hydrogel samples using colloidal probe atomic force microscopy (AFM). The pAAM sample had a high water content of >90% and was molded against polyoxymethylene (POM) to create a more dilute surface layer with thickness ∼0.5 µm. Indentations to multiple depths between 50 nm and 1.25 µm were repeated 10 times each. First, the force drops during the unloading, and separation segments of each indentation were characterized. This described the detachment progression for increasing areas of contact, revealing that the pull-off force for a single chain was in the single-pN range. Second, the stretched polymer network was modeled as an array of parallel, linear springs. Assuming a constant areal chain density of α = 100 chains/µm2, the maximum force of adhesion was plotted versus the volume of chains stretched upward, and the average chain stiffness was calculated from a linear fit to be 22.8 × 10-6 N/m. A Weibull distribution analysis of detachment events revealed a dependence of chain stiffness on maximum indentation depth (dmax), with higher stiffness at shallower depths approaching kchain ≈ 20 × 10-6 N/m. These findings on adhesion mechanics between a vanishing hydrogel surface and probe can guide the development of multifunctional hydrogels for various biomedical applications.

Hazard-mapping and health risk analysis of iron and arsenic contamination in the groundwater of Sylhet district.

This study investigates groundwater contamination by arsenic and iron and its health implications within the Sylhet district in Bangladesh. Utilizing geographic information system (GIS) and inverse distance weighting (IDW) methods, hazard maps have been developed to evaluate contamination risk across various upazilas. The findings show significant arsenic and iron pollution, particularly in the northwestern part of the district. In about 50% of the area, especially in Jaintiapur, Zakiganj, Companiganj, and Kanaighat where arsenic levels surpass 0.05 mg/L which is the standard limit of Bangladesh. Iron levels peak at 13.83 mg/L, severely impacting 45% of the region, especially in Gowainghat, northeastern Jaintiapur, Zakigonj, and Golabganj. The study employs USEPA health risk assessment methods to calculate the hazard quotient (HQ) and hazard index (HI) for both elements via oral and dermal exposure. Results indicate that children face greater noncarcinogenic and carcinogenic risks than adults, with oral HI showing significant risk in Balagonj and Bishwanath. Dermal adsorption pathways exhibit comparatively lower risks. Cancer risk assessments demonstrate high carcinogenic risks from oral arsenic intake in all areas. This comprehensive analysis highlights the urgent need for effective groundwater management and policy interventions in the Sylhet district to mitigate these health risks and ensure safe drinking water.

Alpha-Synuclein Interaction with UBL3 Is Upregulated by Microsomal Glutathione S-Transferase 3, Leading to Increased Extracellular Transport of the Alpha-Synuclein under Oxidative Stress.

Aberrant aggregation of misfolded alpha-synuclein (α-syn), a major pathological hallmark of related neurodegenerative diseases such as Parkinson's disease (PD), can translocate between cells. Ubiquitin-like 3 (UBL3) is a membrane-anchored ubiquitin-fold protein and post-translational modifier. UBL3 promotes protein sorting into small extracellular vesicles (sEVs) and thereby mediates intercellular communication. Our recent studies have shown that α-syn interacts with UBL3 and that this interaction is downregulated after silencing microsomal glutathione S-transferase 3 (MGST3). However, how MGST3 regulates the interaction of α-syn and UBL3 remains unclear. In the present study, we further explored this by overexpressing MGST3. In the split Gaussia luciferase complementation assay, we found that the interaction between α-syn and UBL3 was upregulated by MGST3. While Western blot and RT-qPCR analyses showed that silencing or overexpression of MGST3 did not significantly alter the expression of α-syn and UBL3, the immunocytochemical staining analysis indicated that MGST3 increased the co-localization of α-syn and UBL3. We suggested roles for the anti-oxidative stress function of MGST3 and found that the effect of MGST3 overexpression on the interaction between α-syn with UBL3 was significantly rescued under excess oxidative stress and promoted intracellular α-syn to extracellular transport. In conclusion, our results demonstrate that MGST3 upregulates the interaction between α-syn with UBL3 and promotes the interaction to translocate intracellular α-syn to the extracellular. Overall, our findings provide new insights and ideas for promoting the modulation of UBL3 as a therapeutic agent for the treatment of synucleinopathy-associated neurodegenerative diseases.

Ex vivo expansion of primary cells from limb tissue of Pleurodeles waltl.

Pleurodeles waltl is coming to light as a model animal, especially in regeneration studies, but deep studies on the molecular mechanisms have been limited due to the absence of primary tissue cells for wide usage. Therefore, we aimed to grow primary cells from limb tissue of P. waltl for in vitro experiments. Limb tissues were cut into small pieces and seeded as "explants" on culture dishes coated with fibronectin and gelatin. Compared to the control without coating, both fibronectin and gelatin supported quicker outgrowth of cells from explants and faster cell adhesion, and fibronectin showed significantly better performance than gelatin. Interestingly, the doubling time of cells on fibronectin- and gelatin-coated surfaces was almost the same (42.39 ± 2.79 h vs. 42.91 ± 3.69 h) and was not significantly different from that on non-coated plates (49.64 ± 3.63 h). The cryopreserved cells were successfully recovered and showed a multiplication capacity that was similar to that of fresh cells. Senescent cells were barely detected even after long-term sub-culture (>15 passages). Moreover, enhanced fluorescence of MitoSOX™ Red in cells under H2 O2 exposure confirmed the respondence to chemical stimuli. Collectively, our results show that we are able to grow enough good-quality cells from P. waltl limb tissue for in vitro experiments, and fibronectin coating provides the best biocompatible environment for cell outgrowth and attachment.

NAD+ Levels Are Augmented in Aortic Tissue of ApoE-/- Mice by Dietary Omega-3 Fatty Acids.

BACKGROUND: Maintaining bioenergetic homeostasis provides a means to reduce the risk of cardiovascular events during chronological aging. Nicotinamide adenine dinucleotide (NAD+) acts as a signaling molecule, and its levels were used to govern several biological pathways, for example, promoting angiogenesis by SIRT1 (sirtuin 1)-mediated inhibition of Notch signaling to rejuvenate capillary density of old-aged mice. NAD+ modulation shows promise in the vascular remodeling of endothelial cells. However, NAD+ distribution in atherosclerotic regions remains uncharacterized. Omega-3 polyunsaturated fatty acids consumption, such as docosahexaenoic acid and eicosapentaenoic acid, might increase the abundance of cofactors in blood vessels due to omega-3 polyunsaturated fatty acids metabolism. METHODS: Apolipoprotein E-deficient (ApoE-/-) mice were fed a Western diet, and the omega-3 polyunsaturated fatty acids-treated groups were supplemented with docosahexaenoic acid (1%, w/w) or eicosapentaenoic acid (1%, w/w) for 3 weeks. Desorption electrospray ionization mass spectrometry imaging was exploited to detect exogenous and endogenous NAD+ imaging. RESULTS: NAD+, NADH, NADP+, NADPH, FAD+, FADH, and nicotinic acid adenine dinucleotide of the aortic arches were detected higher in the omega-3 polyunsaturated fatty acids-treated mice than the nontreated control. Comparing the distribution in the outer and inner layers of the arterial walls, only NADPH was detected slightly higher in the outer part in eicosapentaenoic acid-treated mice. CONCLUSIONS: Supplementation of adding docosahexaenoic acid or eicosapentaenoic acid to the Western diet led to a higher NAD+, FAD+, and their metabolites in the aortic arch. Considering the pleiotropic roles of NAD+ in biology, this result serves as a beneficial therapeutic strategy in the animal model counter to pathological conditions.

Fewer polymer chains but higher adhesion: How gradient-stiffness hydrogel layers mediate adhesion through network stretch.

The presence of gradient softer outer layers, commonly observed in biological systems (such as cartilage and ocular tissues), as well as synthetic crosslinked hydrogels, profoundly influences their interactions with opposing surfaces. Our prior research demonstrated that gradient-stiffness hydrogel layers, characterized by increasing elasticity with depth, control contact mechanics, particularly in proximity to the layer thickness. We postulate that the distribution of polymers within these gradient layers imparts extraordinary stretch and adhesion characteristics due to network adaptability and stress-induced reorganization. To investigate this phenomenon, we utilized Atomic Force Microscopy nanoindentation to assess the depth-dependent adhesion behavior of polyacrylamide hydrogels with varying gradient layer thicknesses. Two gradient layer thicknesses were achieved by employing different molding materials: glass and polyoxymethylene (POM). Glass-molded hydrogels exhibited a thinner gradient layer alongside a stiffer bulk layer compared to their POM-molded counterparts. In indentation experiments, the POM-molded hydrogel had larger adhesion compared to glass-molded hydrogel. We find that indenting within the gradient layer engenders increased load-unload hysteresis due to heightened fluid transport in the sparse outer polymer network. Consequently, this led to augmented adhesion and work of separation at shallow depths. We suggest that the prominent stretching capability of the sparse outer polymer network during probe retraction contributes to enhanced adhesion. The Maugis-Dugdale adhesive model only fits well to indentations on the thin layer or indentations which engage significantly with the bulk. These results facilitate a comprehensive characterization of adhesion mechanics in gradient-stiffness hydrogels, which could foster their application across emerging contexts in health science and environmental domains.

In Silico Functional and Structural Analysis of Non-synonymous Single Nucleotide Polymorphisms (nsSNPs) in Human Paired Box 4 Gene.

In human genome, members of Paired box (PAX) transcription factor family are highly sequence-specific DNA-binding proteins. Among PAX gene family members, PAX4 gene has significant role in growth, proliferation, differentiation, and insulin secretion of pancreatic ß-cells. Single nucleotide polymorphisms (SNPs) in PAX4 gene progress in the pathogenesis of various human diseases. Hence, the molecular mechanism of how these SNPs in PAX4 gene significantly progress diseases pathogenesis needs to be elucidated. For the reason, a series of bioinformatic analyzes were done to identify the SNPs of PAX4 gene that contribute in diseases pathogenesis. From the analyzes, 4145 SNPs (rsIDs) in PAX4 gene were obtained, where, 362 missense (8.73%), 169 synonymous (4.08%), and 2323 intron variants (56.04%). The rest SNPs were unspecified. Among the 362 missense variants, 118 nsSNPs were found as deleterious in SIFT analysis. Among those, 25 nsSNPs were most probably damaging and 23 were deleterious as observed in PolyPhen-2 and PROVEAN analyzes, respectively. Following all analyzes, 14 nsSNPs (rs149708455, rs115887120, rs147279315, rs35155575, rs370095957, rs373939873, rs145468905, rs121917718, rs2233580, rs3824004, rs372751660, rs369459316, rs375472849, rs372497946) were common and observed as deleterious, probably damaging, affective and diseases associated. Following structural analyzes, 11 nsSNPs guided proteins were found as most unstable and highly conserved. Among these, R20W, R39Q, R45Q, R60H, G65D, and A223D mutated proteins were highly harmful. Hence, the results from above-mentioned integrated comprehensive bioinformatic analyzes guide how different nsSNPs in PAX4 gene alter structural and functional characteristics of the protein that might progress diseases pathogenesis in human including type 2 diabetes.

Treatment Experiences for Patients Receiving Buprenorphine/Naloxone for Opioid Use Disorder: A Qualitative Study of Patients' Perceptions and Attitudes.

Background: Although buprenorphine/naloxone has been demonstrated to be an effective treatment for patients with opioid use disorder (OUD), treatment retention has been a challenge. This study extends what is presently a limited literature regarding patients' experiences with this medication and the implications for treatment retention. Methods: The study was conducted as a qualitative investigation of patients in treatment for OUD at the time of the study. Forty-three patients (27 men, 15 women, mean age 34.7) were recruited from three clinical settings, a community health center, an academically-based treatment site, and an independent substance abuse treatment facility. Most patients had returned to use in the past after attempts to become abstinent. Results: Patients generally reported positive experiences with this medication noting it helped to reduce opioid cravings quickly. As important considerations for treatment retention, patients emphasized a firm commitment to achieving abstinence when beginning treatment and a prescriber who is informed about and attentive to their emotional state. Diverging attitudes did exist regarding treatment duration as some patients were accepting of long-term treatment while others desired a relatively brief option. Among patients who had returned to use, potentially important issues emerged pertaining to the absence of patient outreach for missed medication appointments and inadequate discharge planning following stays at rehabilitation facilities. Conclusions: While results regarding the importance of patient motivation and strong patient-prescriber relationships have been noted in previous studies, other findings regarding opportunities to improve patient outreach and coordination of care have received relatively less attention and warrant further consideration.

Comparative evaluation of low-cost ceramic membrane and polymeric micro membrane in algal membrane photobioreactor for wastewater treatment.

Algal-bacterial membrane photobioreactor (AMPBR) is proven as a highly energy-efficient process for treating domestic wastewater. This study compared the application of polymeric micro-membrane (PMM) and a low-cost ceramic membrane (LCM) to the AMPBR process for treating domestic wastewater with low and high organic pollution levels. Experiments were conducted over 57 days using two PMM-AMPBRs and two LCM-AMPBRs, operating on a 12-h dark/light cycle in a continuous mode. Simulated wastewater containing varying levels of chemical oxygen demand (COD) was fed to reactors for a consistent hydraulic residence time (HRT) of 7 d and a flux rate of 100 L/m2/d. PMM and LCM-AMPBRs demonstrated efficient wastewater treatment capabilities, achieving COD removal rates exceeding 94% and 95% for high and low COD loadings, respectively. PMM-AMPBR achieved 54.1% TN removal at low COD loading, while LCM-AMPBR achieved 57.2%. These removal efficiencies decreased to 45.6% and 47.0% under high COD loading. Total Phosphorus (TP) removal reached 29-33% for PMM-AMPBRs and 21-24% for LCM-AMPBRs, irrespective of COD loading. LCM-AMPBRs showed significantly lower fouling frequency than PMM-AMPBRs. The biomass production rate decreased with increasing COD loading and achieved 40 mg/L/d at low COD loading for both AMPBRs. Net energy return (NER) values for both AMPBRs were close to 0.87, indicating them as energy-efficient processes. Considering the cost-effectiveness and comparable performance, LCM-AMPBR could be a viable alternative to PMM-AMPBR for wastewater treatment, particularly under low COD loading conditions.

Cardioprotective effects of arjunolic acid in LPS-stimulated H9C2 and C2C12 myotubes via the My88-dependent TLR4 signaling pathway.

CONTEXT: Arjunolic acid (AA) is a triterpenoid saponin found in Terminalia arjuna (Roxb.) Wight & Arn. (Combretaceae). It exerts cardiovascular protective effects as a phytomedicine. However, it is unclear how AA exerts the effects at the molecular level. OBJECTIVE: This study investigates the cardioprotective effects of arjunolic acid (AA) via MyD88-dependant TLR4 downstream signaling marker expression. MATERIALS AND METHODS: The MTT viability assay was used to assess the cytotoxicity of AA. LPS induced in vitro cardiovascular disease model was developed in H9C2 and C2C12 myotubes. The treatment groups were designed such as control (untreated), LPS control, positive control (LPS + pyrrolidine dithiocarbamate (PDTC)-25 µM), and treatment groups were co-treated with LPS and three concentrations of AA (50, 75, and 100 µM) for 24 h. The changes in the expression of TLR4 downstream signaling markers were evaluated through High Content Screening (HCS) and Western Blot (WB) analysis. RESULTS: After 24 h of co-treatment, the expression of TLR4, MyD88, MAPK, JNK, and NF-κB markers were upregulated significantly (2-6 times) in the LPS-treated groups compared to the untreated control in both HCS and WB experiments. Evidently, the HCS analysis revealed that MyD88, NF-κB, p38, and JNK were significantly downregulated in the H9C2 myotube in the AA treated groups. In HCS, the expression of NF-κB was downregulated in C2C12. Additionally, TLR4 expression was downregulated in both H9C2 and C2C12 myotubes in the WB experiment. DISCUSSION AND CONCLUSIONS: TLR4 marker expression in H9C2 and C2C12 myotubes was subsequently decreased by AA treatment, suggesting possible cardioprotective effects of AA.

Phylogenetic analysis and characterization of arsenic (As) transforming bacterial marker proteins following isolation of As-tolerant indigenous bacteria.

Marker proteins play a significant role in bacterial arsenic (As) transformation. Phylogenetic analysis and three-dimensional (3D) characteristics of As transforming bacterial marker proteins guide the evolutionary origin and As transforming potential of the species. Indeed, As-tolerant bacteria also show a significant level of As transformation. Hence, characterization of As transforming bacterial marker proteins, isolation of As transforming bacteria, and proper integration of the findings may guide to elucidate how bacteria transform As. Therefore, phylogenetic analysis and 3D characterization of As transforming bacterial marker protein following isolation of potential indigenous As-tolerant indigenous bacteria were done to explore the mechanism of bacterial As transformation. Phylogenetic analysis of ten As transforming marker proteins (arsA, arsB, arsC, arsD, arsR, aioA, arrA, aioB, acr1, and acr3) in 20 potential bacterial genomes (except 19 for the acr3) were studied. Some bacterial genomes featured up to five marker proteins, and therefore, 3D characteristics of the marker proteins were analyzed in those genomes having three-to-five marker proteins. In phylogeny, species in close clades represent their phylogenetic resemblances and may have similar functions. P. aeruginosa, E. coli, and K. pneumonia were found to be more effective due to having the highest number (five) of marker proteins. In 3D protein modeling, most of the marker proteins were found to be active. Among 19 indigenous bacterial isolates, multiple isolates showed tolerance up to 50 mM As(III) and 250 mM As(V), which may potentially transform a significant quantities of As. Hence, integration of the results of phylogenetic analysis, 3D protein characteristics, and As tolerance in the bacterial isolates could guide to explore the mechanism of how bacteria transform As at cellular and molecular levels.

Soft Contact Mechanics with Gradient-Stiffness Surfaces.

The stiffness in the top surface of many biological entities like cornea or articular cartilage, as well as chemically cross-linked synthetic hydrogels, can be significantly lower or more compliant than the bulk. When such a heterogeneous surface comes into contact, the contacting load is distributed differently from typical contact models. The mechanical response under indentation loading of a surface with a gradient of stiffness is a complex, integrated response that necessarily includes the heterogeneity. In this work, we identify empirical contact models between a rigid indenter and gradient elastic surfaces by numerically simulating quasi-static indentation. Three key case studies revealed the specific ways in which (I) continuous gradients, (II) laminate-layer gradients, and (III) alternating gradients generate new contact mechanics at the shallow-depth limit. Validation of the simulation-generated models was done by micro- and nanoindentation experiments on polyacrylamide samples synthesized to have a softer gradient surface layer. The field of stress and stretch in the subsurface as visualized from the simulations also reveals that the gradient layers become confined, which pushes the stretch fields closer to the surface and radially outward. Thus, contact areas are larger than expected, and average contact pressures are lower than predicted by the Hertz model. The overall findings of this work are new contact models and the mechanisms by which they change. These models allow a more accurate interpretation of the plethora of indentation data on surface gradient soft matter (biological and synthetic) as well as a better prediction of the force response to gradient soft surfaces. This work provides examples of how gradient hydrogel surfaces control the subsurface stress distribution and loading response.

Accumulation of Sulfonic Acid Groups Anchored in Covalent Organic Frameworks as an Intrinsic Proton-Conducting Electrolyte.

Covalent organic frameworks (COFs) are a novel class of crystalline porous polymers, which possess high porosity, excellent stability, and regular nanochannels. 2D COFs provide a 1D nanochannel to form the proton transport channels. The abovementioned features afford a powerful potential platform for designing materials as proton transportation carriers. Herein, the authors incorporate sulfonic acid groups on the pore walls as proton sources for enhancing proton transport conductivity in the 1D channel. Interestingly, the sulfonic acid COFs (S-COFs) electrolytes being binder free exhibit excellent proton conductivity of ≈1.5 × 10-2 S cm-1 at 25 ℃ and 95% relative humidity (RH), which rank the excellent performance in standard proton-conducting electrolytes. The S-COFs electrolytes keep the high proton conduction over the 24 h. The activation energy is estimated to be as low as 0.17 eV, which is much lower than most reported COFs. This research opens a new window to evolve great potential of structural design for COFs as the high proton-conducting electrolytes.

Long-term patient outcomes following buprenorphine/naloxone treatment for opioid use disorder: a retrospective analysis in a commercially insured population.

Background: While buprenorphine/naloxone (buprenorphine) has been demonstrated to be an effective medication for treating opioid use disorder (OUD), an important question exists about how long patients should remain in treatment.Objective: To examine the relationship between treatment duration and patient outcomes for individuals with OUD who have been prescribed buprenorphine.Methods: We conducted a retrospective, longitudinal study using the Massachusetts All Payer Claims Database, 2013 to 2017. The study comprised over 2,500 patients, approximately one-third of whom were female, who had been prescribed buprenorphine for OUD. The outcomes were hospitalizations and emergency room (ER) visits at 36 months following treatment initiation and 12 months following treatment discontinuation. Patients were classified into four groups based on treatment duration and medication adherence: poor adherence, duration <12 months; good adherence, duration <6 months; good adherence, duration 6 to 12 months, and good adherence, duration >12 months. We conducted analyses at the patient level of the relationship between duration and outcomes.Results: Better outcomes were observed for patients whose duration was greater than 12 months. Patients in the other groups had higher odds of hospitalization at 36 months following treatment initiation: poor adherence (2.71), <6 months (1.53), and 6 to 12 months (1.42). They also had higher odds of ER visits: poor adherence (1.69), <6 months (1.51), and 6 to 12 months (1.30). Similar results were observed following treatment discontinuation.Conclusions: OUD treatment with buprenorphine should be continued for at least 12 months to reduce hospitalizations and ED visits.

Mass spectrometry in the lipid study of cancer.

Introduction: Cancer is a heterogeneous disease that exploits various metabolic pathways to meet the demand for increased energy and structural components. Lipids are biomolecules that play essential roles as high energy sources, mediators, and structural components of biological membranes. Accumulating evidence has established that altered lipid metabolism is a hallmark of cancer.Areas covered: Mass spectrometry (MS) is a label-free analytical tool that can simultaneously identify and quantify hundreds of analytes. To date, comprehensive lipid studies exclusively rely on this technique. Here, we reviewed the use of MS in the study of lipids in various cancers and discuss its instrumental limitations and challenges.Expert opinion: MS and MS imaging have significantly contributed to revealing altered lipid metabolism in a variety of cancers. Currently, a single MS approach cannot profile the entire lipidome because of its lack of sensitivity and specificity for all lipid classes. For the metabolic pathway investigation, lipid study requires the integration of MS with other molecular approaches. Future developments regarding the high spatial resolution, mass resolution, and sensitivity of MS instruments are warranted.

Genetic engineering to improve essential and conditionally essential amino acids in maize: transporter engineering as a reference.

Ruminants and humans are unable to synthesize essential amino acids (EAAs) and conditionally essential amino acids (CEAAs) under normal conditions and need to acquire them from plant sources. Maize plays, as a major crop, a central role in global food security. However, maize is deficient in several EAAs and CEAAs. Genetic engineering has been successfully used to enrich the EAA content of maize to some extent, including the content of Lys, Trp, and Met. However, research on other EAAs is lacking. Genetic engineering provides several viable approaches for increasing the EAA content in maize, including transformation of a single gene, transformation of multiple genes in a single cassette, overexpression of putative amino acid transporters, engineering the amino acid biosynthesis pathway including silencing of feedback inhibition enzymes, and overexpression of major enzymes in this pathway. These challenging processes require a deep understanding of the biosynthetic and metabolic pathways of individual amino acids, and the interaction of individual amino acids with other metabolic pathways.

Desorption ionization using through-hole alumina membrane offers higher reproducibility than 2,5-dihydroxybenzoic acid, a widely used matrix in Fourier transform ion cyclotron resonance mass spectrometry imaging analysis.

RATIONALE: DIUTHAME (desorption ionization using through-hole alumina membrane), a recently developed matrix-free ionization-assisting substrate, was examined for reproducibility in terms of mass accuracy and intensity using standard lipid and mouse brain sections. The impregnation property of DIUTHAME significantly improved the reproducibility of mass accuracy and intensity compared with 2,5-dihydroxybenzoic acid (DHB). METHODS: Frozen tissue sections were mounted on indium tin oxide-coated glass slides. DIUTHAME and DHB were applied to individual sections. Subsequently, a solution of a phosphatidylcholine standard, PC(18:2/18:2), was poured onto the DIUTHAME and matrix. Finally, the samples were subjected to laser desorption ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry. The reproducibility was tested by calculating the mean ± standard deviation values of mass errors and intensities of individual ion species. RESULTS: Analysis of the PC(18:2/18:2) standard showed significantly (p < 0.01) lower mass error for DIUTHAME-MS than for MALDI-MS. Endogenous PC(36:4) analysis in mouse brain section also showed significantly (p < 0.05) lower mass errors for DIUTHAME-MS. Furthermore, we investigated the mass error of some abundant lipid ions in brain sections and observed similar results. DIUTHAME-MS displayed lower signal intensity in standard PC analysis. Interestingly, it offered higher signal intensities for all the endogenous lipid ions. Lower fluctuations of both mass accuracies and signal intensities were observed in DIUTHAME-MS. CONCLUSIONS: Our results demonstrated that DIUTHAME-MS offers higher reproducibility for mass accuracies and intensities than MALDI-MS in both standard lipid and mouse brain tissue analyses. It can potentially be used instead of conventional MALDI-MS and mass spectrometry imaging analyses to achieve highly reproducible data for mass accuracy and intensity.

CD24hiCD38hi and CD24hiCD27+ Human Regulatory B Cells Display Common and Distinct Functional Characteristics.

Although IL-10-producing regulatory B cells (Bregs) play important roles in immune regulation, their surface phenotypes and functional characteristics have not been fully investigated. In this study, we report that the frequency of IL-10-producing Bregs in human peripheral blood, spleens, and tonsils is similar, but they display heterogenous surface phenotypes. Nonetheless, CD24hiCD38hi transitional B cells (TBs) and CD24hiCD27+ B cells (human equivalent of murine B10 cells) are the major IL-10-producing B cells. They both suppress CD4+ T cell proliferation as well as IFN-γ/IL-17 expression. However, CD24hiCD27+ B cells were more efficient than TBs at suppressing CD4+ T cell proliferation and IFN-γ/IL-17 expression, whereas they both coexpress IL-10 and TNF-α. TGF-ß1 and granzyme B expression were also enriched within CD24hiCD27+ B cells, when compared with TBs. Additionally, CD24hiCD27+ B cells expressed increased levels of surface integrins (CD11a, CD11b, α1, α4, and ß1) and CD39 (an ecto-ATPase), suggesting that the in vivo mechanisms of action of the two Breg subsets are not the same. Lastly, we also report that liver allograft recipients with plasma cell hepatitis had significant decreases of both Breg subsets.

Deciphering the molecular pathways of apoptosis using purified fractions from leaf extract of Basella alba through studying the regulation of apoptosis related genes.

Apoptosis plays a pivotal role in the exclusion of abnormal cells without any ruin of surrounding healthy cells. Generally, it occurs through an orderly and autonomously process which is controlled by proper function of various genes. Therefore, the current experiments detect the expression level/pattern of those genes to confirm the involvement of extrinsic and intrinsic pathway using Basella alba leaf (BAL). Several fractions after gel filtration chromatography of BAL extract have been pooled to evaluates its apoptosis induction potentiality on Ehrlich's Ascites Carcinoma (EAC) cells through conducting a number of bio-assays such as cell growth inhibition assay, fluorescence and optical microscopy, DNA fragmentation assay and gene expression analysis etc. The pooled fractions of BAL showed 12-56% inhibitory effect on EAC cell line at the concentration range of 25-400 µg/ml that was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. They also exhibited excellent cell growth inhibition at in vivo and in vitro condition when treated with 10, 20 and 40 mg/kg day. After administration of six consequent days, significant morphological features of apoptosis were observed in EAC cells under both fluorescence and optical microscope which was further supported by DNA fragmentation assay. The polymerase chain reaction amplification of bax, bcl-2 (B-cell lymphoma 2), p53, tumor necrosis factor-α, Fas, NF-kß (Nuclear factor-Kappa-B), PARP-1 (Poly (ADP-ribose) polymerase), Cyt-c cas-8, cas-9 and cas-3 revealed that the experimental sample able to induce apoptosis in both extrinsic and intrinsic pathways through altering the gene expression. The current findings suggest that sample from BAL occupy wonderful competence to induce cell apoptosis and become an ideal resource for cancer treatment.