An Integrated Approach to Protein Discovery and Detection From Complex Biofluids.

Ovarian cancer, a leading cause of cancer-related deaths among women, has been notoriously difficult to screen for and diagnose early, as early detection significantly improves survival. Researchers and clinicians seek routinely usable and noninvasive screening methods; however, available methods (i.e., biomarker screening) lack desirable sensitivity/specificity. The most fatal form, high-grade serous ovarian cancer, often originate in the fallopian tube; therefore, sampling from the vaginal environment provides more proximal sources for tumor detection. To address these shortcomings and leverage proximal sampling, we developed an untargeted mass spectrometry microprotein profiling method and identified cystatin A, which was validated in an animal model. To overcome the limits of detection inherent to mass spectrometry, we demonstrated that cystatin A is present at 100 pM concentrations using a label-free microtoroid resonator and translated our workflow to patient-derived clinical samples, highlighting the potential utility of early stage detection where biomarker levels would be low.

Cytomegalovirus pneumonia with intermittent pulmonary hemorrhage leading to asphyxia death: a case report and literature review.

Neonatal pulmonary hemorrhage is a late manifestation of various diseases. Premature delivery and low body weight are frequently observed as high-risk factors, characterized by acute onset, rapid progression, and high mortality rates. Pulmonary hemorrhage caused by cytomegalovirus infection in newborns with normal immune function is a rare occurrence. This case report focuses on a term neonate with normal birth weight who presented solely with nasal obstruction shortly after birth. However, 4 days after birth, the newborn experienced a sudden onset of blood gushing from both the mouth and nasal cavity. The patient was diagnosed with gastrointestinal bleeding, neonatal pneumonia and neonatal lung consolidation. And he was discharged after ten days of symptomatic treatment. However, upon returning home, the patient experienced a sudden onset of bleeding from the mouth and nose, leading to his untimely demise. Subsequent autopsy revealed the presence of pulmonary hemorrhage in newborn, which presented as interstitial pneumonia. The cause of pulmonary hemorrhage is cytomegalovirus infection. This case emphasizes the importance of pediatricians enhancing their skills in differentiating pulmonary hemorrhage, especially from cytomegalovirus pneumonia.

Advancing our understanding of bioreactors for industrial-sized cell culture: health care and cellular agriculture implications.

Bioreactors are advanced biomanufacturing tools that have been widely used to develop various applications in the fields of health care and cellular agriculture. In recent years, there has been a growing interest in the use of bioreactors to enhance the efficiency and scalability of these technologies. In cell therapy, bioreactors have been used to expand and differentiate cells into specialized cell types that can be used for transplantation or tissue regeneration. In cultured meat production, bioreactors offer a controlled and efficient means of producing meat without the need for animal farming. Bioreactors can support the growth of muscle cells by providing the necessary conditions for cell proliferation, differentiation, and maturation, including the provision of oxygen and nutrients. This review article aims to provide an overview of the current state of bioreactor technology in both cell therapy and cultured meat production. It will examine the various bioreactor types and their applications in these fields, highlighting their advantages and limitations. In addition, it will explore the future prospects and challenges of bioreactor technology in these emerging fields. Overall, this review will provide valuable insights for researchers and practitioners interested in using bioreactor technology to develop innovative solutions in the biomanufacturing of therapeutic cells and cultured meat.

Tubular cell transcriptional intermediary factor 1γ deficiency exacerbates kidney injury-induced tubular cell polyploidy and fibrosis.

Tubulointerstitial fibrosis is considered the final convergent pathway of progressive chronic kidney diseases (CKD) regardless of etiology. However, mechanisms underlying kidney injury-induced fibrosis largely remain unknown. Recent studies have indicated that transcriptional intermediary factor 1γ (TIF1γ) inhibits the progression of fibrosis in other organs. Here, we found that TIF1γ was highly expressed in the cytoplasm and nucleus of the kidney proximal tubule. Interestingly, we found tubular TIF1γ expression was decreased in patients with CKD, including those with diabetes, hypertension, and IgA nephropathy, and in mouse models with experimental kidney fibrosis (unilateral ureteral obstruction [UUO], folic acid nephropathy [FAN], and aristolochic acid-induced nephrotoxicity). Tubule-specific knock out of TIF1γ in mice exacerbated UUO- and FAN-induced tubular cell polyploidy and subsequent fibrosis, whereas overexpression of kidney TIF1γ protected mice against kidney fibrosis. Mechanistically, in tubular epithelial cells, TIF1γ exerted an antifibrotic role via transforming growth factor-ß (TGF-ß)-dependent and -independent signaling. TIF1γ hindered TGF-ß signaling directly by inhibiting the formation and activity of the transcription factor Smad complex in tubular cells, and we discovered that TIF1γ suppressed epidermal growth factor receptor (EGFR) signaling upstream of TGF-ß signaling in tubular cells by ubiquitylating EGFR at its lysine 851/905 sites thereby promoting EGFR internalization and lysosomal degradation. Pharmacological inhibition of EGFR signaling attenuated exacerbated polyploidization and the fibrotic phenotype in mice with tubule deletion of TIF1γ. Thus, tubular TIF1γ plays an important role in kidney fibrosis by suppressing profibrotic EGFR and TGF-ß signaling. Hence, our findings suggest that maintaining homeostasis of tubular TIF1γ may be a new therapeutic option for treating tubulointerstitial fibrosis and subsequent CKD.

Regulation of the pleiotropic transcriptional regulator CodY on the conversion of branched-chain amino acids into branched-chain aldehydes in Lactococcus lactis.

IMPORTANCE: Branched-chain aldehydes are the primary compounds that contribute to the nutty flavor in cheddar cheese. Lactococcus lactis, which is often applied as primary starter culture, is a significant contributor to the nutty flavor of cheddar cheese due to its ability of conversion of BCAAs into branched-chain aldehydes. In the present study, we found that the regulatory role of CodY is crucial for the conversion. CodY acts as a pleiotropic transcriptional regulator via binding to various regulatory regions of key genes. The results presented valuable knowledge into the role of CodY on the regulation and biosynthetic pathway of branched-chain amino acids and the related aldehydes. Furthermore, it provided new insight for increasing the nutty flavor produced during the manufacture and ripening of cheese.

An intravitreal-injectable hydrogel depot doped borneol-decorated dual-drug-coloaded microemulsions for long-lasting retina delivery and synergistic therapy of wAMD.

Sustained retina drug delivery and rational drug combination are considered essential for enhancing the efficacy of therapy for wet age-related macular degeneration (wAMD) due to the conservative structure of the posterior ocular segment and the multi-factorial pathological mechanism. Designing a drug co-delivery system that can simultaneously achieve deep penetration and long-lasting retention in the vitreous is highly desired, yet remains a huge challenge. In this study, we fabricated Bor/RB-M@TRG as an intravitreal-injectable hydrogel depot for deep penetration into the posterior ocular segment and long-lasting distribution in the retinal pigment epithelium (RPE) layer. The Bor/RB-M@TRG consisted of borneol-decorated rhein and baicalein-coloaded microemulsions (Bor/RB-M, the therapy entity) and a temperature-responsive hydrogel matrix (the intravitreal depot). Bor/RB-M exhibited the strongest in vitro anti-angiogenic effects among all the groups studied, which is potentially associated with improved cellular uptake, as well as the synergism of rhein and baicalein, acting via anti-angiogenic and anti-oxidative stress pathways, respectively. Importantly, a single intravitreal (IVT) injection with Bor/RB-M@TRG displayed significant inhibition against the CNV of wAMD model mice, compared to all other groups. Particularly, coumarin-6-labeled Bor/RB-M@TRG (Bor/C6-M@TRG) could not only deeply penetrate into the retina but also stably accumulate in the RPE layer for at least 14 days. Our design integrates the advantages of borneol-decorated microemulsions and hydrogel depots, offering a promising new approach for clinically-translatable retinal drug delivery and synergistic anti-wAMD treatment.

GPS2 promotes erythroid differentiation by control of the stability of EKLF protein.

Erythropoiesis is a complex multistage process that involves differentiation of early erythroid progenitors to enucleated mature red blood cells, in which lineage-specific transcription factors play essential roles. Erythroid Krüppel-like factor (EKLF/KLF1) is a pleiotropic erythroid transcription factor that is required for the proper maturation of the erythroid cells, whose expression and activation are tightly controlled in a temporal and differentiation stage-specific manner. Here, we uncover a novel role of G-protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor corepressor complex, in erythrocyte differentiation. Our study demonstrates that knockdown of GPS2 significantly suppresses erythroid differentiation of human CD34+ cells cultured in vitro and xenotransplanted in nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor γ-chain null mice. Moreover, global deletion of GPS2 in mice causes impaired erythropoiesis in the fetal liver and leads to severe anemia. Flow cytometric analysis and Wright-Giemsa staining show a defective differentiation at late stages of erythropoiesis in Gps2-/- embryos. Mechanistically, GPS2 interacts with EKLF and prevents proteasome-mediated degradation of EKLF, thereby increasing EKLF stability and transcriptional activity. Moreover, we identify the amino acids 191-230 region in EKLF protein, responsible for GPS2 binding, that is highly conserved in mammals and essential for EKLF protein stability. Collectively, our study uncovers a previously unknown role of GPS2 as a posttranslational regulator that enhances the stability of EKLF protein and thereby promotes erythroid differentiation.

NCAPG facilitates colorectal cancer cell proliferation, migration, invasion and epithelial-mesenchymal transition by activating the Wnt/ß-catenin signaling pathway.

BACKGROUND: The condensation complex gene non-SMC condensin I complex subunit G(NCAPG), a cell cycle-associated condensin, is over-expressed in various cancers. However, its biological function in colorectal cancer (CRC) has yet to be deciphered. In this study, we investigated the role of NCAPG in CRC progression. METHODS: Tissues and cells were used to measure NCAPG expression levels and their association with clinicopathological characteristics. NCAPG silencing and overexpression in CRC cells were used to measure its effect on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) progression. In addition, mRNA, and protein expression levels of key EMT biomarkers were measured. The underlying mechanism of NCAPG modulating CRC progression was further explored using western blotting, co-immunoprecipitation (CO-IP), and immunofluorescence (IF) assays. RESULTS: NCAPG was over-expressed in CRC tissues and cell lines. High expression levels were associated with differentiation levels, lymph metastasis, and vascular invasion in patients. NCAPG silencing suppressed, while NCAPG overexpression promoted the proliferative, migration, and invasive capacity of HCT116 and SW480 cells. Mechanistically, we discovered that NCAPG participated in regulating the EMT process and the Wnt/ß-catenin signaling pathway to facilitate CRC invasion and metastasis. Additional experiments demonstrated that NCAPG activated the Wnt/ß-catenin signaling pathway by binding to ß-catenin in CRC cells. CONCLUSION: NCAPG acts as an oncogene involved in the development and progression of CRC by binding to ß-catenin to activate the Wnt/ß-catenin signaling pathway.

Analysis of lumbar lateral instability on upright left and right bending radiographs in symptomatic patients with degenerative lumbar spondylolisthesis.

BACKGROUND: To evaluate lumbar mobility in various positions using upright left and right bending radiographs in patients with degenerative lumbar spondylolisthesis (DLS), as well as to assess the impact of lateral instability on patient-reported outcomes. METHODS: This study retrospectively reviewed a consecutive series of patients with DLS between January 2019 and October 2020. The enrolled patients were divided into two groups: the lateral instability group (group L) and non-lateral instability group (group NL). Translational and angular motion in both sagittal and coronal planes and patient-reported outcomes were compared between the two groups. RESULTS: There were 104 (59.8%) patients in group L and 70 (40.2%) patients in group NL, with an average age of 60.6 ± 7.8 years. Patients with a right bending posture in group L had a higher slip percentage (14.2 ± 7.4% vs 9.2 ± 3.2%, p = 0.01) and slip angle (6.3 ± 1.5° vs 2.2 ± 0.8°, p = 0.021). Compared with group NL, group L demonstrated significantly larger angular motion in the coronal plane (2.4 ± 1.3° vs 1.0 ± 0.7°, p = 0.008). Patients with lateral instability had worse preoperative back pain (6.1 ± 1.6 vs 2.7 ± 1.9, p = 0.01) and Oswestry Disability Index (ODI) scores (37.7 ± 5.5 vs 25.6 ± 2.6, p = 0.002). In terms of pain characteristics, group L was characterized by pain when getting out of a car, when rising from a chair, and when climbing stairs (all p values < 0.05). CONCLUSION: Lumbar lateral instability, that is, increased mobility in the coronal plane on lateral bending radiographs, translational and/or angular, correlates to more pronounced patient related symptoms in degenerative L4-5 spondylolisthesis. The existence of lumbar lateral instability leads to worse impacts on patient-reported outcomes when patients change their positions including getting out of a car, rising from a chair, and climbing stairs.

Simple and Robust Microfabrication of Polymeric Piezoelectric Resonating MEMS Mass Sensors.

Resonating MEMS mass sensors are microdevices with broad applications in fields such as bioscience and biochemistry. Their advantageous surface-to-volume ratio makes their resonant frequency highly sensitive to variations in their mass induced by surface depositions. Recent global challenges, such as water quality monitoring or pandemic containment, have increased the need for low-cost (even disposable), rapidly fabricated microdevices as suitable detectors. Resonant MEMS mass sensors are among the best candidates. This paper introduces a simple and robust fabrication of polymeric piezoelectric resonating MEMS mass sensors. The microfabrication technology replaces the traditional layer-by-layer micromachining techniques with laser micromachining to gain extra simplicity. Membrane-based resonant sensors have been fabricated to test the technology. Their characterization results have proven that the technology is robust with good reproducibility (around 2% batch level variations in the resonant frequency). Initial tests for the MEMS mass sensors' sensitivity have indicated a sensitivity of 340 Hz/ng. The concept could be a starting point for developing low-cost MEMS sensing solutions for pandemic control, health examination, and pollution monitoring.

A small nucleolar RNA, SNORD126, promotes adipogenesis in cells and rats by activating the PI3K-AKT pathway.

Small nucleolar RNA (snoRNA) plays important role in various histogenesis. Whether snoRNA plays a role in adipogenesis is unknown. SNORD126 is a C/D box snoRNA. We previously demonstrated that SNORD126 promoted hepatocellular carcinoma cell growth by activating the phosphoinositide 3-kinase-protein kinase B (Akt) pathway through upregulating fibroblast growth factor receptor 2 expression. In the present study, we found that the expression of SNORD126 was downregulated in the obesity-related tissues in high-fat diet-fed rats. Overexpression of SNORD126 in 3T3-L1 cells promoted adipocytes differentiation. SNORD126 significantly increased the expression of CCAAT/enhancer-binding protein α, fatty acid-binding protein 4, peroxisome proliferative-activated receptor-γ, and the phosphorylation of Akt and p70S6K. Overexpression of SNORD126 in human adipose-derived stem cells stimulated adipogenesis and increased phosphorylation of Akt. Meanwhile, SNORD126 increased the messenger RNA and protein levels of cyclin D1 and cyclin-dependent kinase 2, which promoted mitotic clonal expansion progression during the early stage of 3T3-L1 cell differentiation. We further found that SNORD126 accelerated the growth of the groin fat pad and increased phosphorylation of Akt and p70S6K in rats. Overall, our results suggested that SNORD126 promoted adipocyte differentiation through increasing phosphorylation of Akt and p70S6K both in vitro and in vivo.

Fast readout method for multidimensional optical data storage using interferometry-aided reflectance spectroscopy.

The multiplex technique increases the capacity of optical data storage, but the current reading throughputs is limited by the single-bit reading. We propose a fast readout method of multidimensional optical data storage using interference-aided reflectance spectral measurement to readout multiple bits information simultaneously. The multidimensional data is recorded in the photoresist layer on the disc with dielectric multilayer substrate by laser direct writing. With the designed interference layer inside the disc, the relation of thickness of recording layer and the peak shift of the reflected spectra has been built up. With different writing depths representing different bit of data, 2 bits and 3 bits unit information have been recorded and successfully read out at one exposure. This fast readout method is not only suitable for optical data storage by engineering the optical path length for example Blu-ray disc but also for super resolution optical data storage.

EDAG mediates Hsp70 nuclear localization in erythroblasts and rescues dyserythropoiesis in myelodysplastic syndrome.

During human erythroid maturation, Hsp70 translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. Failure of Hsp70 to localize to the nucleus was found in Myelodysplastic syndrome (MDS) erythroblasts and can induce dyserythropoiesis, with arrest of maturation and death of erythroblasts. However, the mechanism of the nuclear trafficking of Hsp70 in erythroblasts remains unknown. Here, we found the hematopoietic transcriptional regulator, EDAG, to be a novel binding partner of Hsp70 that forms a protein complex with Hsp70 and GATA-1 during human normal erythroid differentiation. EDAG overexpression blocked the cytoplasmic translocation of Hsp70 induced by EPO deprivation, inhibited GATA-1 degradation, thereby promoting erythroid maturation in an Hsp70-dependent manner. Furthermore, in myelodysplastic syndrome (MDS) patients with dyserythropoiesis, EDAG is dramatically down-regulated, and forced expression of EDAG has been found to restore the localization of Hsp70 in the nucleus and elevate the protein level of GATA-1 to a significant extent. In addition, EDAG rescued the dyserythropoiesis of MDS patients by increasing erythroid differentiation and decreasing cell apoptosis. This study demonstrates the molecular mechanism of Hsp70 nuclear sustaining during erythroid maturation and establishes that EDAG might be a suitable therapeutic target for dyserythropoiesis in MDS patients.

NLRP3 is dispensable for d-galactosamine/lipopolysaccharide-induced acute liver failure.

The nucleotide-binding domain and leucine-rich repeat-containing family pyrin domain containing 3 (NLRP3) inflammasome is involved in various acute and chronic liver diseases, however, it is not clear whether NLRP3 contributes to d-Galactosamine (D-GalN) plus lipopolysaccharide (LPS)-induced acute liver failure (ALF). This study aims to investigate the role of NLRP3 inflammasome in D-GalN/LPS-induced fatal hepatitis. We found that Nlrp3-/- and WT mice showed similar mortality against a lethal dose of D-GalN/LPS treatment. Serum ALT and AST levels, as well as liver necrosis area and hepatocyte apoptosis, were not significantly different between Nlrp3-/- and WT mice at 6 h after D-GalN/LPS injection. Moreover, the numbers of intrahepatic F4/80+ cells and Ly6G+ cells were comparable in two genotype mice following D-GalN/LPS treatment. Besides, Nlrp3-/- mice had reduced IL-1ß levels but similar TNF-α, IL-6, and MCP-1 levels compared with WT mice upon D-GalN/LPS administration. Our findings revealed that NLRP3 ablation does not protect mice from D-GalN/LPS-induced fatal hepatitis and has a marginal effect on intrahepatic inflammatory response upon D-GalN/LPS treatment. This suggests that NLRP3 inflammasome does not appear to be a major contributor to D-GalN/LPS-induced ALF.

Multiple miRNAs jointly regulate the biosynthesis of ecdysteroid in the holometabolous insects, Chilo suppressalis.

The accurate rise and fall of active hormones is important for insect development. The ecdysteroids must be cleared in a timely manner. However, the mechanism of suppressing the ecdysteroid biosynthesis at the right time remains unclear. Here, we sequenced a small RNA library of Chilo suppressalis and identified 300 miRNAs in this notorious rice insect pest. Microarray analysis yielded 54 differentially expressed miRNAs during metamorphosis development. Target prediction and in vitro dual-luciferase assays confirmed that seven miRNAs (two conserved and five novel miRNAs) jointly targeted three Halloween genes in the ecdysteroid biosynthesis pathway. Overexpression of these seven miRNAs reduced the titer of 20-hydroxyecdysone (20E), induced mortality, and retarded development, which could be rescued by treatment with 20E. Comparative analysis indicated that the miRNA regulation of metamorphosis development is a conserved process but that the miRNAs involved are highly divergent. In all, we present evidence that both conserved and lineage-specific miRNAs have crucial roles in regulating development in insects by controlling ecdysteroid biosynthesis, which is important for ensuring developmental convergence and evolutionary diversity.

Analysis of Drought Tolerance and Associated Traits in Upland Cotton at the Seedling Stage.

(1) Background: Upland cotton (Gossypium hirsutum L.) is the most important natural fiber worldwide, and it is extensively planted and plentifully used in the textile industry. Major cotton planting regions are frequently affected by abiotic stress, especially drought stress. Drought resistance is a complex, quantitative trait. A genome-wide association study (GWAS) constitutes an efficient method for dissecting the genetic architecture of complex traits. In this study, the drought resistance of a population of 316 upland cotton accessions was studied via GWAS. (2) Methods: GWAS methodology was employed to identify relationships between molecular markers or candidate genes and phenotypes of interest. (3) Results: A total of 8, 3, and 6 SNPs were associated with the euphylla wilting score (EWS), cotyledon wilting score (CWS), and leaf temperature (LT), respectively, based on a general linear model and a factored spectrally transformed linear mixed model. For these traits, 7 QTLs were found, of which 2 each were located on chromosomes A05, A11, and D03, and of which 1 was located on chromosome A01. Importantly, in the candidate regions WRKY70, GhCIPK6, SnRK2.6, and NET1A, which are involved in the response to abscisic acid (ABA), the mitogen-activated protein kinase (MAPK) signaling pathway and the calcium transduction pathway were identified in upland cotton at the seedling stage under drought stress according to annotation information and linkage disequilibrium (LD) block analysis. Moreover, RNA sequencing analysis showed that WRKY70, GhCIPK6, SnRK2.6, and NET1A were induced by drought stress, and the expression of these genes was significantly different between normal and drought stress conditions. (4) Conclusions: The present study should provide some genomic resources for drought resistance in upland cotton. Moreover, the germplasm of the different phenotypes, the detected SNPs and, the potential candidate genes will be helpful for molecular marker-assisted breeding studies about increased drought resistance in upland cotton.

Low dose irradiation facilitates hepatocellular carcinoma genesis involving HULC.

Irradiation exposure positive correlates with tumor formation, such as breast cancer and lung cancer. However, whether low dose irradiation induces hepatocarcinogenesis and the underlying mechanism remain poorly defined. In the present study, we reported that low dose irradiation facilitated the proliferation of hepatocyte through up-regulating HULC in vitro and in vivo. Low dose irradiation exposure elevated HULC expression level in hepatocyte. Deletion of heightened HULC erased the cells growth accelerated following low dose irradiation exposure. CDKN1, the neighbor gene of HULC, was down-regulated by overexpression of HULC following low dose irradiation exposure via complementary base pairing, resulting in promoting cell cycle process. Thus, our findings provide new insights into the mechanism of low dose irradiation-induced hepatocarcinogenesis through HULC/CDKN1 signaling, and shed light on the potential risk of low dose irradiation for the development of hepatocellular carcinoma in pre-clinical settings.

Toll-like receptor 5 signaling restrains T-cell/natural killer T-cell activation and protects against concanavalin A-induced hepatic injury.

Toll-like receptor-5 (TLR5) signaling regulates the immune privileged status of the liver and is involved in hepatic immune disorders. However, the role of TLR5 has not yet been investigated in experimental models of concanavalin A (Con A)-mediated liver injury. Here, we show that TLR5 is highly up-regulated in the hepatic mononuclear cells of mice during Con A-induced hepatitis. Increased mortality and liver histopathology of TLR5-deficient mice correlated with excessive production of proinflammatory cytokines, suggesting that TLR5 knockout mice were more susceptible to Con A-induced hepatitis. We also report that administration of CBLB502, an exogenous TLR5 agonist, substantially alleviated Con A-mediated hepatitis in wild-type mice as shown by increased survival rates, reduced aminotransferase and proinflammatory cytokine production, impaired lymphocyte infiltration, and ameliorated hepatocyte necrosis and/or apoptosis. Mechanistic studies revealed that CBLB502 acts as a negative regulator in limiting T-cell/natural killer T-cell activity and cytokine production in the Con A-hepatitis model. Bone marrow transplantation experiments showed that TLR5 in bone marrow-derived cells contributed to the hepatoprotective efficacy of CBLB502 against Con A-induced liver injury. Moreover, interleukin-6 elevation induced by CBLB502 is an important protective factor against Con A-induced liver injury. In addition, we demonstrate that CBLB502 suppresses α-galactosylceramide-induced natural killer T cell-dependent inflammatory liver injury. CONCLUSION: The TLR5 signaling pathway plays an important role in T cell-mediated hepatic injury and may be exploited for therapeutic treatment of inflammatory liver diseases. (Hepatology 2017;65:2059-2073).