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Introducing genetic material into hard-to-transfect mammalian cell lines and primary cells is often best achieved through retroviral infection. An ideal retroviral vector should offer a compact, selectable, and screenable marker while maximizing transgene delivery capacity. However, a previously published retroviral vector featuring an EGFP/Puromycin fusion protein failed to meet these criteria in our experiments. We encountered issues such as low infection efficiency, weak EGFP fluorescence, and selection against infected cells. To address these shortcomings, we developed a novel retroviral vector based on the Moloney murine leukemia virus. This vector includes a compact bifunctional EGFP and Puromycin resistance cassette connected by a 2A peptide. Our extensively tested vector demonstrated superior EGFP expression, efficient Puromycin selection, and no growth penalty in infected cells compared with the earlier design. These benefits were consistent across multiple mammalian cell types, underscoring the versatility of our vector. In summary, our enhanced retroviral vector offers a robust solution for efficient infection, reliable detection, and effective selection in mammalian cells. Its improved performance and compact design make it an ideal choice for a wide range of applications involving precise genetic manipulation and characterization in cell-based studies. [ABSTRACT FROM AUTHOR]

Bacterial peptidyl tRNA hydrolase (Pth) or Pth1 emerges as a pivotal enzyme involved in the maintenance of cellular homeostasis by catalyzing the release of peptidyl moieties from peptidyl-tRNA molecules and the maintenance of a free pool of specific tRNAs. This enzyme is vital for bacterial cells and an emerging drug target for various bacterial infections. Understanding the enzymatic mechanisms and structural intricacies of bacterial Pth is pivotal in designing novel therapeutics to combat antibiotic resistance. This review provides a comprehensive analysis of the multifaceted roles of Pth in bacterial physiology, shedding light on its significance as a potential drug target. This article delves into the diverse functions of Pth, encompassing its involvement in ribosome rescue, the maintenance of a free tRNA pool in bacterial systems, the regulation of translation fidelity, and stress response pathways within bacterial systems. Moreover, it also explores the druggability of bacterial Pth, emphasizing its promise as a target for antibacterial agents and highlighting the challenges associated with developing specific inhibitors against this enzyme. Structural elucidation represents a cornerstone in unraveling the catalytic mechanisms and substrate recognition of Pth. This review encapsulates the current structural insights of Pth garnered through various biophysical techniques, such as X-ray crystallography and NMR spectroscopy, providing a detailed understanding of the enzyme's architecture and conformational dynamics. Additionally, biophysical aspects, including its interaction with ligands, inhibitors, and substrates, are discussed, elucidating the molecular basis of bacterial Pth's function and its potential use in drug design strategies. Through this review article, we aim to put together all the available information on bacterial Pth and emphasize its potential in advancing innovative therapeutic interventions and combating bacterial infections. [ABSTRACT FROM AUTHOR]

GDF15, also known as MIC1, is a member of the TGF-beta superfamily. Previous studies reported elevated serum levels of GDF15 in patients with kidney disorder, and its association with kidney disease progression, while other studies identified GDF15 to have protective effects. To investigate the potential protective role of GDF15 on podocytes, we first performed in vitro studies using a Gdf15-deficient podocyte cell line. The lack of GDF15 intensified puromycin aminonucleoside (PAN)-triggered endoplasmic reticulum stress and induced cell death in cultivated podocytes. This was evidenced by elevated expressions of Xbp1 and ER-associated chaperones, alongside AnnexinV/PI staining and LDH release. Additionally, we subjected mice to nephrotoxic PAN treatment. Our observations revealed a noteworthy increase in both GDF15 expression and secretion subsequent to PAN administration. Gdf15 knockout mice displayed a moderate loss of WT1+ cells (podocytes) in the glomeruli compared to wild-type controls. However, this finding could not be substantiated through digital evaluation. The parameters of kidney function, including serum BUN, creatinine, and albumin–creatinine ratio (ACR), were increased in Gdf15 knockout mice as compared to wild-type mice upon PAN treatment. This was associated with an increase in the number of glomerular macrophages, neutrophils, inflammatory cytokines, and chemokines in Gdf15-deficient mice. In summary, our findings unveil a novel renoprotective effect of GDF15 during kidney injury and inflammation by promoting podocyte survival and regulating endoplasmic reticulum stress in podocytes, and, subsequently, the infiltration of inflammatory cells via paracrine effects on surrounding glomerular cells. [ABSTRACT FROM AUTHOR]

Prosthetic infections are associated with high morbidity, mortality, and relapse rates, making them still a serious problem for implantology. Staphylococcus aureus is one of the most common bacterial pathogens causing prosthetic infections. In response to the increasing rate of bacterial resistance to commonly used antibiotics, this work proposes a method for combating pathogenic microorganisms by modifying the surfaces of synthetic polymeric biomaterials using proteolytic enzyme inhibitors (serine protease inhibitors—4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and puromycin). While using techniques based on the immobilization of biologically active molecules, it is important to monitor the changes occurring on the surface of the modified biomaterial, where spectroscopic techniques (e.g., FTIR) are ideal. ATR-FTIR measurements demonstrated that the immobilization of both inhibitors caused large structural changes on the surface of the tested vascular prostheses (polyester or polytetrafluoroethylene) and showed that they were covalently bonded to the surfaces of the biomaterials. Next, the bactericidal and antibiofilm activities of the tested serine protease inhibitors were determined using the CLSM microscopic technique with fluorescent staining. During LIVE/DEAD analyses, a significant decrease in the formation of Staphylococcus aureus biofilm after exposure to selected concentrations of native inhibitors (0.02–0.06 mg/mL for puromycin and 0.2–1 mg/mL for 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) was demonstrated. [ABSTRACT FROM AUTHOR]

Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published [Hobson et al., Elife 9, e60048 (2020); and Enam et al., Elife 9, e60303 (2020)]. In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryoelectron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons. [ABSTRACT FROM AUTHOR]

Circadian-paced biological processes are key to physiology and required for metabolic, immunologic, and cardiovascular homeostasis. Core circadian clock components are transcription factors whose half-life is precisely regulated, thereby controlling the intrinsic cellular circadian clock. Genetic disruption of molecular clock components generally leads to marked pathological events phenotypically affecting behavior and multiple aspects of physiology. Using a transcriptional signature similarity approach, we identified anti-cancer protein synthesis inhibitors as potent modulators of the cardiomyocyte molecular clock. Eukaryotic protein translation inhibitors, ranging from translation initiation (rocaglates, 4-EGI1, etc.) to ribosomal elongation inhibitors (homoharringtonine, puromycin, etc.), were found to potently ablate protein abundance of REV-ERBα, a repressive nuclear receptor and component of the molecular clock. These inhibitory effects were observed both in vitro and in vivo and could be extended to PER2, another component of the molecular clock. Taken together, our observations suggest that the activity spectrum of protein synthesis inhibitors, whose clinical use is contemplated not only in cancers but also in viral infections, must be extended to circadian rhythm disruption, with potential beneficial or iatrogenic effects upon acute or prolonged administration. [ABSTRACT FROM AUTHOR]

Background: Microbial secondary metabolites have shown promise as a source of novel antimicrobial agents. In this study, we aimed to isolate, characterize, and evaluate the antimicrobial activity of compound from a novel Streptomyces albofaciens strain MS38. The objective was to identify a potential bioactive compound with broad-spectrum antimicrobial properties. Methods: The isolated strain MS38 on starch casein agar was characterized using morphological, physiological, and molecular identification techniques. The compound was obtained from the fermented broth through extraction with n-butanol and further purification using silica gel column chromatography and high-performance liquid chromatography (HPLC). Structural elucidation was conducted using Ultraviolet (UV), Infrared (IR), nuclear magnetic resonance (NMR), and mass spectrometry (MS) techniques. The antimicrobial activity was evaluated using the agar well diffusion method and the microplate Alamar blue assay (MABA). Results: The isolated strain MS38 was identified as novel S. albofaciens based on morphological characteristics and confirmed by 16S sequences analysis and MALDI-TOF MS. The compound obtained from the fermented broth exhibited substantial antimicrobial activity against a variety of pathogenic bacteria and fungi. Structural analysis revealed a complex chemical structure with characteristic functional groups indicative of potential antimicrobial properties. The compound demonstrated strong activity against both Gram-positive (Staphylococcus Spp.) and Gram-negative (Klebsiella pneumoniae and Escherichia coli) bacteria, as well as fungi, including Candida albicans and Trichophyton rubrum. Conclusion: This study successfully isolated and characterized a bioactive compound from a novel S. albofaciens MS38. The compound exhibited significant antimicrobial activity against a range of pathogenic microorganisms. These findings underscore the importance of exploring microbial· biodiversity for the discovery of novel antimicrobial agents. This study contributes to the growing knowledge of microbial secondary metabolites with potential therapeutic value. [ABSTRACT FROM AUTHOR]

Objective(s): Exosomes are nanovesicular vehicles capable of transporting different cargoes. Based on their characteristics, exosomes are proposed as a class of vehicles for targeted delivery of therapeutics. We aimed to establish a HEK293T stable cell line capable of secreting GRP78-specific scFv-targeted exosomes. Methods: The pLEX-LAMP2b-GRP78 construct was developed by enzymatic replacement of DARPin in pLEX-LAMP2b-DARPin with a GRP78-specific scFv. pLEX-LAMP2b-GRP78 (or TurboGFP as control), psPAX2, and pMD2.G plasmids were co-transfected into HEK293T cells, and produced lentiviruses were harvested. Different multiplicities of infection (MOI; 10, 20, 30, 60, 120, and 240) were used for the transduction of HEK293T to select the most appropriate one as assessed by flow cytometry. Transduced HEK293T cells were subject to puromycin selection and the presence of the scFv was assessed in the established cell line at the DNA, transcript, and protein levels by PCR, RT-PCR, and Western blotting, respectively. Results: pLEX-LAMP2b-GRP78 was successfully developed. Co-transfection resulted in the expression of GFP by HEK293T in the control group 48 hours following transfection. The MOI of 60 was selected as 10% of cells were GFP+ 72 hours following transduction. Following puromycin selection, the presence of the integrated scFv DNA and transcript was confirmed. Moreover, Western blotting results confirmed the presence of the His-tagged scFv in the established cell line. Conclusions: HEK293T cells can be engineered for the production of targeted exosomes which could be applied for therapeutic purposes. Moreover, scFvs are potent targeting domains that could be leveraged for the development of targeted exosomes. [ABSTRACT FROM AUTHOR]

Introduction: Nephrotic syndrome (NS) is characterized by renal sodium and water retention. The mechanisms are not fully elucidated. Methods: The NS rat model was established by single intraperitoneal injection of 100 mg/kg puromycin aminonucleoside (PAN). The plasma electrolyte level and urinary sodium excretion were monitored dynamically. The changes of some sodium transporters, including epithelial Na+ channel (ENaC), Na+/H+ exchanger 3 (NHE3), Na+-K+-2Cl− cotransporter 2 (NKCC2) and Na+-Cl− cotransporter (NCC) in renal cortex at different time points and the level of peripheral circulation factors were detected. Results: The urinary sodium excretion of the model group increased significantly on the first day, then decreased compared with the control group, and there was no significant difference between the model group and the control group on the 12th day. The changes of peripheral circulation factors were not obvious. Some sodium transporters in renal cortex increased in varying degrees, while NKCC2 decreased significantly compared with the control group. Conclusions: The occurrence of NS edema may not be related to the angiotensin system. The decrease of urinary sodium excretion is independent of the development of albuminuria. During the 18 days of observation, it can be divided into three stages: sodium retention, sodium compensation, and simple water retention. The mechanism is related to the increased expression of α-ENaC, γ-ENaC, NHE3 and NCC in a certain period of time, the compensatory decrease of NKCC2 expression and the continuous increase of aquaporin 2 (AQP2) expression. [ABSTRACT FROM AUTHOR]

Puromycin derivatives containing an emissive thieno[3,4‐d]‐pyrimidine core, modified with azetidine and 3,3‐difluoroazetidine as Me2N surrogates, exhibit translation inhibition and bactericidal activity similar to the natural antibiotic. The analogues are capable of cellular puromycylation of nascent peptides, generating emissive products without any follow‐up chemistry. The 3,3‐difluoroazetidine‐containing analogue is shown to fluorescently label newly translated peptides and be visualized in both live and fixed HEK293T cells and rat hippocampal neurons. [ABSTRACT FROM AUTHOR]

Puromycin derivatives containing an emissive thieno[3,4‐d]‐pyrimidine core, modified with azetidine and 3,3‐difluoroazetidine as Me2N surrogates, exhibit translation inhibition and bactericidal activity similar to the natural antibiotic. The analogues are capable of cellular puromycylation of nascent peptides, generating emissive products without any follow‐up chemistry. The 3,3‐difluoroazetidine‐containing analogue is shown to fluorescently label newly translated peptides and be visualized in both live and fixed HEK293T cells and rat hippocampal neurons. [ABSTRACT FROM AUTHOR]

Novel antitubercular compounds are urgently needed to combat drug-resistant Mycobacterium tuberculosis (Mtb). Filamentous actinobacteria have historically been an excellent source of antitubercular drugs. Despite this, drug discovery from these microorganisms has fallen out of favour due to the continual rediscovery of known compounds. To increase the chance of discovering novel antibiotics, biodiverse and rare strains should be prioritised. Subsequently, active samples need to be dereplicated as early as possible to focus efforts on truly novel compounds. In this study, 42 South African filamentous actinobacteria were screened for antimycobacterial activity using the agar overlay method against the Mtb indicator Mycolicibacterium aurum under six different nutrient growth conditions. Known compounds were subsequently identified through extraction and high-resolution mass spectrometric analysis of the zones of growth inhibition produced by active strains. This allowed the dereplication of 15 hits from six strains that were found to be producing puromycin, actinomycin D and valinomycin. The remaining active strains were grown in liquid cultures, extracted and submitted for screening against Mtb in vitro. Actinomadura napierensis B60T was the most active sample and was selected for bioassay-guided purification. This resulted in the identification of tetromadurin, a known compound, but which we show for the first time to have potent antitubercular activity, with the MIC90s within the range of 73.7–151.6 nM against M. tuberculosis H37RvT in vitro under different test conditions. This shows that South African actinobacteria are a good source of novel antitubercular compounds and warrant further screening. It is also revealed that active hits can be dereplicated by HPLC-MS/MS analysis of the zones of growth inhibition produced by the agar overlay technique. [ABSTRACT FROM AUTHOR]

On-target integration of large cassettes via homology-directed repair (HDR) has several applications. However, the HDR-mediated targeted knock-in suffered from low efficiency. In this study, we made several large plasmids (12.1–13.4 kb) which included the CRISPR/Cas9 system along with a puromycin transgene as part of the large DNA donor (5.3–7.1 kb insertion cassettes) and used them to evaluate their targeted integration efficiency into a transgenic murine embryonic fibroblast (MEF) cell line carrying a single copy of a Venus transgene. We established a detection assay by which HDR events could be discriminated from the error-prone non-homologous end-joining (NHEJ) events. Improving the plasmid quality could considerably leverage the cell toxicity impediment of large plasmids. The use of the TILD (targeted integration with linearized dsDNA) cassettes did not improve the HDR rate compared to the circular plasmids. However, the direct inclusion of nocodazole into the electroporation solution significantly improved the HDR rate. Also, simultaneous delivery of RNase HII and the donor plasmids into the electroporated cells considerably improved the HDR events. In conclusion, the results of this study showed that using cell synchronization reagents in the electroporation medium can efficiently induce HDR rate in the mammalian genome. [ABSTRACT FROM AUTHOR]

Puromycin treatment can cause glomerular injury to the kidney, leading to proteinuria. However, the pathogenesis of acute kidney injury and subsequent regeneration after puromycin administration in animal models remain unclear. In this work, we examined the characteristics of kidney injury and subsequent regeneration following puromycin treatment in adult zebrafish. We intraperitoneally injected 100 μg of puromycin into zebrafish; sacrificed them at 1, 3, 5, 7, or 14 days post-injection (dpi); and examined the morphological, functional, and molecular changes in the kidney. Puromycin-treated zebrafish presented more rapid clearance of rhodamine dextran than control animals. Morphological changes were observed immediately after the puromycin injection (1–7 dpi) and had recovered by 14 dpi. The mRNA production of lhx1a, a renal progenitor marker, increased during recovery from kidney injury. Levels of NFκB, TNFα, Nampt, and p-ERK increased significantly during nephron injury and regeneration, and Sirt1, FOXO1, pax2, and wt1b showed an increasing tendency. However, TGF-β1 and smad5 production did not show any changes after puromycin treatment. This study provides evidence that puromycin-induced injury in adult zebrafish kidneys is a potential tool for evaluating the mechanism of nephron injury and subsequent regeneration. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) a key health problem chiefly in industrialized countries is characterize by memory loss in older persons. Treatments for AD include psychotropic mediators, disease-modifying managements, psychosocial interventions, and especially inhibition of cholinesterase (AChE) enzyme that will block the hydrolysis of a neurotransmitter acetylcholine (ACh). In the current study, it is reported for the first time that Anisomycine and Puromycin inhibit the activity of the AChE enzyme of krait venom. The results revealed that both Anisomycine (0.8–2.4 mM) and Puromycin (36.8–73.6 µM) exhibited AChE in a dose-dependent procedure. A kinetic study using the plot of Lineweaver Burk showed that both Anisomycine and Puromycin caused a competitive type of inhibition against AChE. The antibiotics (Anisomycin and Puromycin) compete with ACh for binding to the enzyme AChE active site. The Km of venom AChE for Anisomycin (0.8–1.6 mM) increased from 64 to 161% and the Vmax remains constant while for Puromycin (36.8–73.6 µM) the Km increased from 34 to 180% and did not affect the Vmax. The calculated IC50 for Anisomycine was found to be 1.4 mM while for Puromycine it was found to be 36.5 µM. In comparison, Puromycine has a more potent inhibitory effect than Anisomycine. It is concluded, that Anisomycin and Puromycin may possess the anti-AChE ability against Krait snake venom acetylcholinesterase. [ABSTRACT FROM AUTHOR]

The dimethylamino functionality has significant importance in industrially relevant molecules and methodologies to install these efficiently are highly desirable. We report herein a highly efficient, room‐temperature dimethylamination of chloroheteroarenes performed via the in‐situ generation of dimethylamine using N,N‐dimethylformamide (DMF) as precursor wiith a large substrate scope that includes various heteroarenes, purines as well as commercially relevant drugs such as altretamine, ampyzine and puromycin precursor. [ABSTRACT FROM AUTHOR]

Podocytes, alternatively called glomerular epithelial cells, are terminally differentiated cells that wrap around glomerular capillaries and function as a part of the glomerular filtration barrier in the kidney. Therefore, podocyte injury with morphological alteration and detachment from glomerular capillaries leads to severe proteinuria and subsequent renal failure through glomerulosclerosis. Previous RNA sequencing analysis of primary rat podocytes exposed to puromycin aminonucleoside (PAN), a well-known experimental model of injured podocytes, identified several transcripts as being aberrantly expressed. However, how the expression of these transcripts is regulated remains unclear. MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally inhibit the expression of their target transcripts. In this study, using small RNA sequencing analysis, miR-217-5p was identified as the most upregulated transcript in PAN-treated rat podocytes. MiR-217-5p overexpression in E11 podocyte cells led to shrunken cells with abnormal actin cytoskeletons. Consistent with these changes in cell morphology, gene ontology (GO) enrichment analysis showed that interactive GO terms related to cell morphogenesis were enriched with the predicted targets of miR-217-5p. Of the predicted targets highly downregulated by PAN, Myosin 1d (Myo1d) is a nonmuscle myosin predicted to be involved in actin filament organization and thought to play a role in podocyte morphogenesis and injury. We demonstrated that miR-217-5p targets Myo1d by luciferase assays, qRT–PCR, and Western blotting. Furthermore, we showed that miR-217-5p was present in urine from PAN- but not saline-administrated rats. Taken together, our data suggest that miR-217-5p may serve as a therapeutic target and a biomarker for podocyte injury. [ABSTRACT FROM AUTHOR]

Clinical evidence indicates that the melanocortin peptide ACTH is effective in inducing remission of nephrotic glomerulopathies like minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), including those resistant to steroids. This suggests that a steroid-independent melancortinergic mechanism may contribute. However, the type of melanocortin receptor (MCR) that conveys this beneficial effect as well as the underlying mechanisms remain controversial. Burgeoning evidence suggests that MC5R is expressed in glomeruli and may be involved in glomerular pathobiology. This study aims to test the effectiveness of a novel highly selective MC5R agonist (MC5R-A) in puromycin aminonucleoside (PAN) nephrosis. Upon PAN injury, rats developed evident proteinuria on day 5, denoting an established nephrotic glomerulopathy. Following vehicle treatment, proteinuria continued to persist on day 14 with prominent histologic signs of podocytopathy, marked by ultrastructural glomerular lesions, including extensive podocyte foot process effacement. Concomitantly, there was loss of podocyte homeostatic markers, such as synaptopodin and podocin, and de novo expression of the podocyte injury marker desmin. Treatment with MC5R-A attenuated urine protein excretion and mitigated the loss of podocyte marker proteins, resulting in improved podocyte ultrastructural changes. In vitro in cultured podocytes, MC5R-A prevented the PAN-induced disruption of actin cytoskeleton integrity and apoptosis. MC5R-A treatment in PAN-injured podocytes also reinstated inhibitory phosphorylation and thus averted hyperactivity of GSK3β, a convergent point of multiple podocytopathic pathways. Collectively, pharmacologic activation of MC5R by using the highly selective small-molecule agonist is likely a promising therapeutic strategy to improve proteinuria and glomerular injury in protenuric nephropathies. [ABSTRACT FROM AUTHOR]

Damage to podocytes is an important determinant of renal pathology. The puromycin aminonucleoside (PAN) mice nephropathy model is commonly used in the study of renal disease with podocyte injury. Hirudin has a broad nephroprotective effect and has been shown to treat renal interstitial fibrosis in previous studies. Mice were given PAN by gavage to prepare animal models, and MPC5 cells were incubated with PAN in vitro. Twenty‐four hours urine was collected for analysis of urinary protein levels. Renal pathological changes were observed by hematoxylin and eosin staining. Immunofluorescence detection of nephrin in kidney tissues and cells. Apoptosis was analyzed with over TUNEL. Cytoskeleton, endoplasmic reticulum stress (ERS), p38 MAPK signaling, and apoptosis‐related proteins were assessed by western blot analysis. The data suggested that hirudin attenuated reduced renal injury and increased urine protein in PAN mice. Hirudin also attenuated cytoskeletal protein (synaptopodin, nephrin, and podocin) disruption, ERS activation, and apoptosis in PAN mice and PAN‐induced podocytes. In addition, hirudin inhibited the expression of p38 MAPK signaling key proteins upregulated by PAN, thereby suppressing ERS. The p38 MAPK agonist was able to partially antagonize the inhibition of p38 MAPK signaling by hirudin in PAN‐induced podocytes, thereby reactivating the ERS inhibited by hirudin, promoting cytoskeletal protein degradation and increasing the level of apoptosis. In conclusion, hirudin could decrease podocyte injury by inhibiting p38 MAPK signaling‐mediated ERS, resulting in the protection of the kidney from PAN damage. These findings may provide an experimental basis for hirudin treatment of podocyte injury diseases. [ABSTRACT FROM AUTHOR]

The Caco-2 model is a common cell model for material intestinal absorption in vitro, which usually takes 21 days to establish. Although some studies have shown that adding puromycin (PM) can shorten the model establishment period to 7 days, this still requires a long modeling time. Therefore, exploring a shorter modeling method can reduce the experimental costs and promote the development and application of the model. Fucoidan is an acidic polysaccharide with various biological activities. Our study showed that the transepithelial electrical resistance (TEER) value could reach 600 Ω·cm2 on the fourth day after the addition of fucoidan and puromycin, which met the applicable standards of the model (>500 Ω). Moreover, the alkaline phosphatase (AKP) activity, fluorescein sodium transmittance, and cell morphology of this model all met the requirements of model establishment. Fucoidan did not affect the absorption of macromolecular proteins and drugs. The results indicate that fucoidan can be applied to establish the Caco-2 model and can shorten the model establishment period to 5 days. [ABSTRACT FROM AUTHOR]

Background: Spinal interneurons (INs) relay sensory and motor control information between the brain and body. When this relay circuitry is disrupted from injury or disease, it is devastating to patients due to the lack of native recovery in central nervous system (CNS) tissues. Obtaining a purified population of INs is necessary to better understand their role in normal function and as potential therapies in CNS. The ventral V0 (V0V) INs are excitatory neurons involved in locomotor circuits and are thus of interest for understanding normal and pathological spinal cord function. To achieve scalable amounts of V0V INs, they can be derived from pluripotent sources, such as mouse embryonic stem cells (mESCs), but the resultant culture is heterogenous, obscuring the specific role of V0V INs. This study generated a transgenic mESC line to enrich V0V INs from induced cultures to allow for a scalable, enriched population for future in vitro and in vivo studies. Methods: The transgenic Evx1-PAC mESC line was created by CRISPR-Cas9-mediated insertion of puromycin-N-acetyltransferase (PAC) into the locus of V0V IN marker Evx1. Evx1 and PAC mRNA expression were measured by qPCR. Viability staining helped establish the selection protocol for V0V INs derived from Evx1-PAC mESCs inductions. Immunostaining was used to examine composition of selected inductions. Cultures were maintained up to 30 days to examine maturation by expression of mature/synaptic markers, determined by immunostaining, and functional activity in co-cultures with selected motor neurons (MNs) and V2a INs on microelectrode arrays (MEAs). Results: V0V IN inductions were best selected with 4 µg/mL puromycin on day 10 to 11 and showed reduction of other IN populations and elimination of proliferative cells. Long-term selected cultures were highly neuronal, expressing neuronal nuclear marker NeuN, dendritic marker MAP2, pre-synaptic marker Bassoon, and glutamatergic marker VGLUT2, with some cholinergic VAChT-expressing cells. Functional studies on MEAs showed that co-cultures with MNs or MNs plus V2a INs created neuronal networks with synchronized bursting. Conclusions: Evx1-PAC mESCs can be used to purify V0V IN cultures for largely glutamatergic neurons that can be used in network formation studies or for rodent models requiring transplanted V0V INs. [ABSTRACT FROM AUTHOR]

Transgenic proteins can be routinely expressed in various mammalian cell types via different transgenic systems, but the efficiency of transgene expression is constrained by the complex interplay among factors such as the temporal consistency of expression and compatibility with specific cell types, including ocular cells. Here, we report a more efficient way to express an enhanced green fluorescent protein (EGFP) in human corneal fibroblasts, corneal epithelial cells, and conjunctival epithelial cells through a lentiviral expression system. The relative transducing unit criterion for EGFP-expressing pseudovirions was first determined in HEK-293T cells. Homogeneous populations of EGFP-positive and EGFP-negative cells could be isolated by cell sorting. The half-maximal inhibitory concentration (IC50) value for puromycin was calculated according to viability curves for each cell type. The results revealed that cell types differed with respect to EGFP expression efficiency after transduction with the same amount of EGFP-encoding pseudovirions. Using a cell sorter, the homogeneity of EGFP-positive cells reached >95%. In the initial sorting stage, however, the efficiency of EGFP expression in the sorted cells was noticeably reduced after two rounds of sequential culture, but repeated sorting for up to four rounds yielded homogeneous EGFP-positive human corneal fibroblasts that could be maintained in continuous culture in vitro. The sorted EGFP-positive cells retained their proper morphology and cell type-specific protein expression patterns. Puromycin resistance was found to depend on cell type, indicating that the IC50 for puromycin must be determined for each cell type to ensure the isolation of homogeneous EGFP-positive cells. Taken together, repeated cell sorting is an efficient means of obtaining homogeneous populations of ocular cells expressing a transgenic protein during continuous culture without the potential confounding effects of antibiotics. [ABSTRACT FROM AUTHOR]

Objective: Rapamycin is a potent inducer of autophagy in podocytes. However, we still understand very little about how autophagy is regulated under podocyte injury conditions. This study aimed to investigate the role of autophagy in podocyte injury and the regulatory mechanism of the PI3K/Akt/mTOR signaling pathway in this process. Methods: The podocytes were cultured in vitro, and the apoptosis rate of each group was determined by flow cytometry. The protein expression and distribution of LC3-II were examined by immunofluorescence. The phosphorylation levels of Akt, LC3-II, mTOR, 4EBP1, and P70S6K were measured using Western Blot. Transmission electron microscopy was used to examine the changes in autophagosomes in each group. Results: Compared with the control group, the puromycin group (PAN) increased podocyte apoptosis, decreased numbers of autophagosomes, and downregulated LC3-II protein expression. Compared with the PAN group, the podocyte apoptosis rate decreased in the Rapamycin group (RAPA), the number of autophagosomes increased, and LC3-II protein expression was upregulated. In addition, PAN evoked an increase in p-Akt expressions, RAPA treatment induced a reversal of PAN-induced p-Akt upregulation, and the phosphorylation levels of mTOR, 4EBP1, and P70S6K were downregulated. Conclusion: PAN can damage podocytes by inhibiting podocyte autophagic activity and promoting apoptosis. Rapamycin can ameliorate PAN-induced podocyte damage by activating autophagy. This effect may be related to rapamycin-mediated PI3K/AKT/mTOR signaling pathway and autophagy. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells. [ABSTRACT FROM AUTHOR]

Background: Cryopreservation of platelets (PLTs) could allow extension of their shelf‐life to years, compared to days for liquid stored platelets. Due to their greater hemostatic effect, reconstituted cryopreserved platelets (cryo‐PLTs) would be able to support bleeding emergencies. Since protein synthesis has been linked to PLT functions, such as clot formation and immune responses, the translational capacity of reconstituted cryo‐PLTs was assessed upon thawing and short‐term storage. Methods/materials: Platelets were frozen at −80°C with 5–6% DMSO. Upon thawing, they were reconstituted in plasma and then aliquoted (12 ml) into mini‐bags and assessed over 24 h of storage at RT. One series served as control; the second and third series were spiked with either 300 μM puromycin (Pm) or 227 nM biotin‐labeled Pm. Samples were tested for in vitro quality and PLT microvesicle enumeration by flow cytometry. Protein synthesis in cryo‐PLTs was assessed using a modified method based on puromycin‐associated nascent chain proteomics. Results: In vitro parameters of reconstituted and subsequently stored platelets were consistent with previously published results. Mass‐spectrometry analyses identified that 22 proteins were synthesized in PLTs and 13 of those were observed in platelet microvesicles (PMVs). Conclusion: Cryo‐PLTs can synthesize proteins upon reconstitution and storage. Discovery of a subset of these proteins in the PMV suggests a role in vesicle encapsulation, possibly in a selective manner. This observation provides novel insights into the capacity for protein synthesis in cryo‐PLTs and the potential regulation of protein packaging into PMV. [ABSTRACT FROM AUTHOR]

A novel aminopeptidase B (APB-AN) was identified from Aspergillus niger CGMCC 3.1454 for the first time and was cloned and expressed in Pichia pastoris. The mature enzyme of approximately 100 kDa was purified for characterization. The optimum pH and temperature of the recombinant APB-AN were determined to be 7.0 and 40 °C, respectively. The enzyme was stable below 40 °C and at pH values from 5.0 to 8.0. The Km and Vmax values were determined to be 0.61 mmol/L and 11.45 mmol/L/min, respectively, using Arg-pNA as the substrate. APB-AN was inhibited by Cu2+ and Fe2+ and activated by Co2+ and Na+. Most metal chelators (Ca2+, Mg2+ and Mn2+) and aminopeptidase inhibitors (bestatin and puromycin) suppressed its activity. APB-AN was found to be active towards 13 kinds of amino acid p-nitroanilide (pNA) substrates:Arg-pNA, Lys-pNA, Tyr- pNA, Trp-pNA, Phe-pNA, His-pNA, Ala-pNA, Met-pNA, Leu-pNA, Glu-pNA, Val-pNA, Pro-pNA and Ile-pNA, and the most preferred N-terminal amino acids were arginine and lysine. APB-AN also hydrolyzed 4 natural proteins: casein, bovine serum albumin, soy protein isolate and water-soluble wheat protein. It is expected that APB-AN has potential food processing applications. [ABSTRACT FROM AUTHOR]

A novel aminopeptidase B (APB-AN) was identified from Aspergillus niger CGMCC 3.1454 for the first time and was cloned and expressed in Pichia pastoris. The mature enzyme of approximately 100 kDa was purified for characterization. The optimum pH and temperature of the recombinant APB-AN were determined to be 7.0 and 40 °C, respectively. The enzyme was stable below 40 °C and at pH values from 5.0 to 8.0. The Km and Vmax values were determined to be 0.61 mmol/L and 11.45 mmol/L/min, respectively, using Arg-pNA as the substrate. APB-AN was inhibited by Cu2+ and Fe2+ and activated by Co2+ and Na+. Most metal chelators (Ca2+, Mg2+ and Mn2+) and aminopeptidase inhibitors (bestatin and puromycin) suppressed its activity. APB-AN was found to be active towards 13 kinds of amino acid p-nitroanilide (pNA) substrates:Arg-pNA, Lys-pNA, Tyr- pNA, Trp-pNA, Phe-pNA, His-pNA, Ala-pNA, Met-pNA, Leu-pNA, Glu-pNA, Val-pNA, Pro-pNA and Ile-pNA, and the most preferred N-terminal amino acids were arginine and lysine. APB-AN also hydrolyzed 4 natural proteins: casein, bovine serum albumin, soy protein isolate and water-soluble wheat protein. It is expected that APB-AN has potential food processing applications. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Modern Laboratory Medicine is the property of Journal of Modern Laboratory Medicine Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Progress in Modern Biomedicine is the property of Publishing House of Progress in Modern Biomedicine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Puromycin‐hydrolizing peptidases have been described as members of the prolyl oligopeptidase peptidase family. These enzymes are present across all domains of life but still little is known of the homologs found in the pathogenic bacterium Mycobacterium tuberculosis. The crystal structure of a M. tuberculosis puromycin hydrolase peptidase has been determined at 3 Angstrom resolution, revealing a conserved prolyl oligopeptidase fold, defined by α/β‐hydrolase and β‐propeller domains with two distinctive loops that occlude access of large substrates to the active site. The enzyme displayed amino peptidase activity with a substrate specificity preference for hydrophobic residues in the decreasing order of phenylalanine, leucine, alanine and proline. The enzyme's active site is lined by residues Glu564 for the coordination of the substrates amino terminal moiety and His561, Val608, Tyr78, Trp306, Phe563 and Ty567 for the accommodation of hydrophobic substrates. The availability of a crystal structure for puromycin hydrolase of M. tuberculosis shall facilitate the development of inhibitors with therapeutic applications. [ABSTRACT FROM AUTHOR]

Background: Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-propargyl-puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts are unsusceptible to puromycin. Results: We could increase susceptibility by using a Komagataella phaffii strain with an impaired ergosterol pathway (erg6Δ), but translation measurements are restricted to this strain background, which displayed growth deficits. Using surfactants, specifically Imipramine, instead, proved to be more advantageous and circumvents previous restrictions. Imipramine-supplemented OPP-labelling with subsequent flow cytometry analysis, enabled us to distinguish actively translating cells from negative controls, and to clearly quantify differences in translation activities in different strains and growth conditions. Specifically, we investigated K. phaffii at different growth rates, verified that methanol feeding alters translation activity, and analysed global translation in strains with genetically modified stress response pathways. Conclusions: We set up a simple protocol to measure global translation activity in yeast on a single cell basis. The use of surfactants poses a practical and non-invasive alternative to the commonly used ergosterol pathway impaired strains and thus impacts a wide range of applications where increased drug and dye uptake is needed. [ABSTRACT FROM AUTHOR]

We developed a cDNA TRAP display for the rapid selection of antibody-like proteins in various conditions. By modifying the original puromycin linker in the TRAP display, a monobody was covalently attached to the cDNA. As a proof-of-concept, we demonstrated a rapid model selection of an anti-EGFR1 monobody in a solution containing ribonuclease. [ABSTRACT FROM AUTHOR]

Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins. [ABSTRACT FROM AUTHOR]

Dermal microvascular endothelial cells (DMECs) play central roles in inflammation and angiogenesis and have become important cell models for studying various skin diseases. However, primary DMECs are difficult to culture and often contaminated by mesenchymal stem cells, fibroblasts, and other stromal cells. Surgically removed superfluous foreskin was first cut into pieces, digested with two types of enzymes, and dispersed into single cells. Cells obtained from the dermis were then subjected to Percoll density gradient centrifugation and cells located between densities 1.033 and 1.047 g/ml were further purified with endothelial growth medium containing decreasing concentrations of puromycin. Obtained HDMECs were identified by microscopy, flow cytometry, quantitative reverse‐transcription polymerase chain reaction, western blot analysis, and immunofluorescent staining. The expression of CD31 (PECAM‐1), CD34, VEGFR2, VWF (Von Willebrand Factor), VE‐Cadherin (CD144), and NOS was positive. HDMECs were found to have abilities of angiogenesis and uptake of acetylated low‐density lipoprotein. Growth curves and cell viability were analyzed, and a growth pattern consisting of the "latency phase‐logarithmic growth phase‐stagnation phase" was determined. In this study, a simple, rapid, effective, and low‐cost method is established to isolate HDMECs from the foreskin with a purity of over 91% and high viability. The method showed good repeatability and allowed a stable passage. This study provides technical support and theoretical guidance for studying the physiological characteristics of HDMECs, the pathogenesis of the skin associated, and other microvascular diseases. [ABSTRACT FROM AUTHOR]

Background: Thrombosis is a potentially life‐threatening nephrotic syndrome (NS) complication. We have previously demonstrated that hypercoagulopathy is proportional to NS severity in rat models and that pioglitazone (Pio) reduces proteinuria both independently and in combination with methylprednisolone (MP), a glucocorticoid (GC). However, the effect of these treatments on NS‐associated hypercoagulopathy remains unknown. We thus sought to determine the ability of Pio and GC to alleviate NS‐associated hypercoagulopathy. Methods: Puromycin aminonucleoside‐induced rat NS was treated with sham, Low‐ or High‐dose MP, Pio, or combination (Pio + Low‐MP) and plasma was collected at day 11. Plasma samples were collected from children with steroid‐sensitive NS (SSNS) and steroid‐resistant NS (SRNS) upon presentation and after 7 weeks of GC therapy. Plasma endogenous thrombin potential (ETP), antithrombin (AT) activity, and albumin (Alb) were measured using thrombin generation, amidolytic, and colorimetric assays, respectively. Results: In a rat model of NS, both High‐MP and Pio improved proteinuria and corrected hypoalbuminemia, ETP and AT activity (p <.05). Proteinuria (p =.005) and hypoalbuminemia (p <.001) were correlated with ETP. In childhood NS, while ETP was not different at presentation, GC therapy improved proteinuria, hypoalbuminemia, and ETP in children with SSNS (p <.001) but not SRNS (p =.330). Conclusions: Both Pio and GC diminish proteinuria and significantly alleviate hypercoagulopathy. Both Pio and MP improved hypercoagulopathy in rats, and successful GC therapy (SSNS) also improved hypercoagulopathy in childhood NS. These data suggest that even a partial reduction in proteinuria may reduce NS‐associated thrombotic risk. [ABSTRACT FROM AUTHOR]

Smad3 signaling and transgelin expression are often activated during puromycin aminonucleoside (PAN)‐induced podocyte injury. Here, we investigated whether the Smad3 inhibitor SIS3 can ameliorate damage to injured podocytes. A model of PAN‐induced podocyte injury was constructed using the MPC5 cell line. The effects of SIS3 on the expression of the podocyte cytoskeletal proteins transgelin, p15INK4B, phosphor‐smad3, phosphor‐JAK/stat3, the apoptotic marker cleaved caspase 3, and c‐myc were investigated using western blot. The distribution of F‐actin in PAN‐induced podocyte injury was observed under an immunofluorescence microscope. PAN‐induced podocyte injury altered the distribution of F‐actin and transgelin, and colocalization of these two proteins was observed. Transgelin expression and Smad3 phosphorylation were increased in the MPC5 cell line with prolonged PAN treatment. In addition, c‐myc expression, p15INK4B, and JAK phosphorylation were all increased after treatment with PAN. Treatment with the Smad3 inhibitor SIS3 reversed these phenomena and protected against PAN‐induced podocyte injury. Moreover, stimulating podocytes directly with TGFβ‐1 also led to enhanced expression of transgelin or phosphor‐JAK/stat3, and this could be inhibited by SIS3. In conclusion, transgelin expression was induced through the Smad3 signaling pathway during PAN‐induced podocyte injury, and the resulting abnormal distribution of F‐actin and the enhanced expression of transgelin could be reversed by blockade of this pathway. [ABSTRACT FROM AUTHOR]

Background: The lumen of the endoplasmic reticulum (ER) acts as a cellular Ca2+ store and a site for oxidative protein folding, which is controlled by the reduced glutathione (GSH) and glutathione-disulfide (GSSG) redox pair. Although depletion of luminal Ca2+ from the ER provokes a rapid and reversible shift towards a more reducing poise in the ER, the underlying molecular basis remains unclear. Results: We found that Ca2+ mobilization-dependent ER luminal reduction was sensitive to inhibition of GSH synthesis or dilution of cytosolic GSH by selective permeabilization of the plasma membrane. A glutathionecentered mechanism was further indicated by increased ER luminal glutathione levels in response to Ca2+ efflux. Inducible reduction of the ER lumen by GSH flux was independent of the Ca2+-binding chaperone calreticulin, which has previously been implicated in this process. However, opening the translocon channel by puromycin or addition of cyclosporine A mimicked the GSH-related effect of Ca2+ mobilization. While the action of puromycin was ascribable to Ca2+ leakage from the ER, the mechanism of cyclosporine A-induced GSH flux was independent of calcineurin and cyclophilins A and B and remained unclear. Conclusions: Our data strongly suggest that ER influx of cytosolic GSH, rather than inhibition of local oxidoreductases, is responsible for the reductive shift upon Ca2+ mobilization. We postulate the existence of a Ca2+- and cyclosporine A-sensitive GSH transporter in the ER membrane. These findings have important implications for ER redox homeostasis under normal physiology and ER stress. [ABSTRACT FROM AUTHOR]

Podocytes are specialized cells with a limited capacity for cell division that do not regenerate in response to injury and loss. Insults that compromise the integrity of podocytes promote proteinuria and progressive renal disease. The aim of this study was to evaluate the potential renoprotective and regenerative effects of mesenchymal stromal cells (mSC) in a severe form of the podocyte injury model induced by intraperitoneal administration of puromycin, aggravated by unilateral nephrectomy. Bone derived mSC were isolated and characterized according to flow cytometry analyses and to their capacity to differentiate into mesenchymal lineages. Wistar rats were divided into three groups: Control, PAN, and PAN+ mSC, consisting of PAN rats treated with 2 × 105 mSC. PAN rats developed heavy proteinuria, hypertension, glomerulosclerosis and significant effacement of the foot process. After 60 days, PAN rats treated with mSC presented a significant amelioration of all these abnormalities. In addition, mSC treatment recovered WT1 expression, improved nephrin, podocin, synaptopodin, podocalyxin, and VEGF expression, and downregulated proinflammatory Th1 cytokines in the kidney with a shift towards regulatory Th2 cytokines. In conclusion, mSC administration induced protection of podocytes in this experimental PAN model, providing new perspectives for the treatment of renal diseases associated with podocyte damage. [ABSTRACT FROM AUTHOR]

GCN2 (general control nonrepressed 2) is a serine/threonine-protein kinase that regulates translation in response to stressors such as amino acid and purin deprivation, cold shock, wounding, cadmium, and UV-C exposure. Activated GCN2 phosphorylates the α-subunit of the eukaryotic initiation factor 2 (eIF2) leading to a drastic inhibition of protein synthesis and shifting translation to specific mRNAs. To investigate the role of GCN2 in responses to UV-B radiation its activity was analyzed through eIF2α phosphorylation assays in mutants of the specific UV-B and stress signaling pathways of Arabidopsis thaliana. EIF2α phosphorylation was detectable 30 min after UV-B exposure, independent of the UV-B photoreceptor UV RESISTANCE LOCUS8 and its downstream signaling components. GCN2 dependent phosphorylation of eIF2α was also detectable in mutants of the stress related MAP kinases, MPK3 and MPK6 and their negative regulator map kinase phosphatase1 (MKP 1). Transcription of downstream components of the UV-B signaling pathway, the Chalcone synthase (CHS) was constitutively higher in gcn2-1 compared to wildtype and further increased upon UV-B while GLUTATHIONE PEROXIDASE7 (GPX7) behaved similarly to wildtype. The UVR8 independent FAD-LINKED OXIDOREDUCTASE (FADox) had a lower basal expression in gcn2-1 which was increased upon UV-B. Since high fluence rates of UV-B induce DNA damage the expression of the RAS ASSOCIATED WITH DIABETES PROTEIN51 (RAD51) was quantified before and after UV-B. While the basal expression was similar to wildtype it was significantly less induced upon UV-B in the gcn2-1 mutant. This expression pattern correlates with the finding that gcn2 mutants develop less cyclobutane pyrimidine dimers after UV-B exposure. Quantification of translation with the puromycination assay revealed that gcn2 mutants have an increased rate of translation which was also higher upon UV-B. Growth of gcn2 mutants to chronic UV-B exposure supports GCN2 's role as a negative regulator of UV-B responses. The elevated resistance of gcn2 mutants towards repeated UV-B exposure points to a critical role of GCN2 in the regulation of translation upon UV-B. [ABSTRACT FROM AUTHOR]

Coiled-coil domain containing 67 (CCDC67) gene is a tumor suppressor gene that exhibits a significant inhibitory effect on a variety of tumors. Our previous study demonstrated that the upregulation of CCDC67 gene in TPC-1 cells inhibited cell proliferation, migration and invasion, and promoted apoptosis in vitro. However, due to the lack of a suitable cell tool, these results were not validated in vivo. In the present study, a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated and identified. Firstly, cDNA clones of the CCDC67 gene were obtained by reverse transcription using a custom-designed primer. The results of subsequent electrophoresis analysis and sequencing revealed that the cDNA clones of CCDC67 gene were obtained successfully, with a length of 1,862 bp. The lentiviral vectors, containing the CCDC67, luciferase reporter and puromycin acetyltransferase genes, were co-transfected with two plasmids that encode lentiviral structural proteins and envelope proteins into 293T cells. Following ultracentrifugation, the titer of lentivirus was determined by ELISA to be 5.0×108 TU/ml. The constructed lentiviral vector was used to transfect TPC-1 thyroid cancer cells, and stabilization was achieved by puromycin screening. The expression of CCDC67 gene, luciferase activity and tumorigenic ability of the generated cell line were detected. Reverse transcription-qPCR results demonstrated that the expression levels of CCDC67 gene in TPC-1 cells following transfection were increased 194,46.782-fold compared with those in the negative control group (P<0.01). A higher fluorescence intensity was detected in the generated cell line, while no detectable fluorescence was observed in untransfected TPC-1 cells. The tumorigenic ability of TPC-1-Luc-Puromycin-CCDC67 cells was verified by bioluminescence imaging and histopathological analysis using a pulmonary metastasis model. These results demonstrated that a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated successfully. The TPC-1-Luc-Puromycin-CCDC67 cell line may be a helpful tool for further research on CCDC67 in vivo. [ABSTRACT FROM AUTHOR]

Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable markers that each allow for selection of multiple "unlinked" transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called "inteins" can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors. Using a lentiviral system, we create and validate 2-split Hygromycin, Puromycin, Neomycin and Blasticidin resistance genes as well as mScarlet fluorescent proteins. By combining split points, we create 3- and 6-split Hygromycin resistance genes, demonstrating that higher-degree split markers can be generated by a "chaining" design. We adapt the split marker system for selecting biallelically engineered cells after CRISPR gene editing. Future engineering of split markers may allow selection of a higher number of genetic modifications in target cells. Selectable markers are widely used in cell engineering but there is only a limited variety to choose from. Here the authors split markers using inteins, allowing up to six transgene integration events to be selected for with one marker. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Xi'an Jiaotong University (Medical Sciences) is the property of Xian Jiaotong University and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Glomerular diseases are the leading cause of chronic kidney disease, and mesangial cells (MCs) have been demonstrated to be involved in the pathogenesis. Puromycin aminonucleoside (PAN) is a nephrotoxic drug that induces glomerular injury with elusive mechanisms. The present study was undertaken to investigate the role of PAN in MC apoptosis, as well as the underlying mechanism. Here we found that PAN induced MC apoptosis accompanied by declined cell viability and enhanced inflammatory response. The apoptosis was further evidenced by increments of apoptosis regulator BAX (BAX) and caspase-3 expression. In line with the apoptotic response in MCs following PAN treatment, we also found a remarkable induction of estrogen-related receptor-α (ERRα), an orphan nuclear receptor, at both mRNA and protein levels. Interestingly, ERRα silencing by an siRNA approach resulted in an attenuation of the apoptosis and inflammatory response caused by PAN. More importantly, overexpression of ERRα in MCs significantly triggered MC apoptosis in line with increased BAX and caspase-3 expression. In PAN-treated MCs, ERRα overexpression further aggravated PAN-induced apoptosis. In agreement with the in vitro study, we also observed increased ERRα expression in line with enhanced apoptotic response in renal cortex from PAN-treated rats. These data suggest a detrimental effect of ERRα on PAN-induced MC apoptosis and inflammatory response, which could help us to better understand the pathogenic mechanism of MC injury in PAN nephropathy. [ABSTRACT FROM AUTHOR]

Cathepsin L, a lysosomal cysteine proteinase, may have a key role in various biological and disease processes by intracellular and extracellular degradation of proteins. We examined the levels of cathepsin L and its intrinsic inhibitors in glomeruli of rats with puromycin aminonucleoside (PAN) nephrosis. In contrast to the weak levels of cathepsin L in normal glomeruli, on days 4 and 8, strong immunostaining was detected in almost all podocytes when proteinuria and pathological changes of the podocytes developed. Cathepsin L was reduced after day 28, but remained in a focal and segmental manner. Cystatin β, an intracellular inhibitor, was not detected in podocytes. However, cystatin C, an extracellular inhibitor, was detected in podocytes after day 4, coincident with cathepsin L. Cystatin C levels were gradually reduced but sustained in many podocytes on day 28, while cystatin C was not detected in podocytes sustained cathepsin L. These results demonstrated that cathepsin L levels are not always accompanied by the levels of its inhibitors in podocytes of PAN nephrosis, suggesting a potential role of cathepsin L in podocyte injury, which is a critical process for the development and progression of tuft adhesion and sclerosis. [ABSTRACT FROM AUTHOR]

The translational capability of ribosomes deprived of specific non-fundamental ribosomal proteins may be altered. Physiological mechanisms are scanty and it is unclear whether free ribosomal proteins can crosstalk with the signaling machinery. RACK1 (Receptor for Activated C Kinase 1) is a highly conserved scaffold protein, located on the 40S subunit near the mRNA exit channel. RACK1 is involved in a variety of intracellular contexts, both on and off the ribosomes, acting as a receptor for proteins in signaling like PKCs. Here we show that binding of RACK1 to ribosomes is essential for full translation of capped mRNAs and efficient recruitment of eIF4E (eukaryotic Initiation Factor 4E). In vitro, supplementing the ribosome machinery with wild-type RACK1 restores the translational capability, whereas addition of a RACK1 mutant unable to bind ribosomes does not. Out of the ribosome, RACK1 has a reduced half-life. By accumulating in living cells, free RACK1 exerts an inhibitory phenotype by impairing cell cycle progression and repressing global translation. Here we present RACK1 binding to ribosomes as a crucial way to regulate translation, possibly by interacting with known signaling-involved partners on or out of the ribosome. [ABSTRACT FROM AUTHOR]

Drug-induced kidney injury (DIKI) remains a significant concern during drug development. Whereas FDA-endorsed urinary protein biomarkers encounter limitations including the lack of translatability, there is a considerable interest surrounding the application of microRNAs (miRNAs) in the renal biomarker space. Current knowledge about the value of these novel biomarkers for subacute preclinical rodent studies is still sparse. In this work, Wistar rats were treated with three nephrotoxic compounds—cisplatin (CIS, proximal tubule, 2.5 mg/kg, intraperitoneal [i.p.]), puromycin (PUR, glomerulus, 20/10 mg/kg, i.p.) and N-phenylanthranylic acid (NPAA, collecting ducts, 500 mg/kg, per os)—for up to 28 days to evaluate the performance of a panel of 68 urinary miRNAs as potential nephron segment-specific biomarkers. Out of these 68 kidney injury associated-miRNAs, our selection strategy ultimately revealed rno-miR-34c-5p significantly dysregulated after CIS single administration, and rno-miR-335 and rno-miR-155-5p significantly dysregulated after PUR treatment. In contrast, NPAA daily administration strongly altered the expression profile of 28 miRNAs, with rno-miR-210-3p displaying the most robust changes. A thorough evaluation showed that these miRNA candidates could complement urinary protein biomarkers to detect CIS- or PUR-induced kidney injury in a subacute setting, with a mechanistic (based on rno-miR-34c-5p) and/or a kidney injury detection potential. Our results also provide the first evidence that urinary miRNAs could enhance the detection of collecting duct damage. Overall, these data improve our understanding of the utility of urinary miRNAs as DIKI biomarkers in a subacute DIKI preclinical setting and support the value of using urinary biomarker panels comprising proteins and miRNAs. [ABSTRACT FROM AUTHOR]