Human induced pluripotent stem cells (hiPSCs) derived from dermal fibroblasts having wild type (WT) SCN5A were engineered by CRISPR/Cas9-mediated genome editing to harbor a specific point mutation (C2204>T) in SCN5A, which results in a substitution of the WT alanine by valine at codon 735 (A735V). The established MUSli009-A-1 hiPSC line has a homozygous C2204>T mutation on exon 14 of SCN5A that was confirmed by DNA sequencing analysis. The cells exhibited normal karyotype, expressed pluripotent markers and retained its capability to differentiate into three germ layers. The cardiomyocytes derived from this line would be a useful model for investigating cardiac channelopathy.

This study evaluated the influence of prognostic factors and whole brain radiotherapy (WBRT) on overall survival (OS) of breast cancer (BC) patients with brain metastases (BM).

Cross-modal occipital responses appear to be essential for nonvisual processing in individuals with early blindness. However, it is not clear whether the recruitment of occipital regions depends on functional domain or sensory modality. The current study utilized a coordinate-based meta-analysis to identify the distinct brain regions involved in the functional domains of object, spatial/motion, and language processing and the common brain regions involved in both auditory and tactile modalities in individuals with early blindness. Following the PRISMA guidelines, a total of 55 studies were included in the meta-analysis. The specific analyses revealed the brain regions that are consistently recruited for each function, such as the dorsal fronto-parietal network for spatial function and ventral occipito-temporal network for object function. This is consistent with the literature, suggesting that the two visual streams are preserved in early blind individuals. The contrast analyses found specific activations in the left cuneus and lingual gyrus for language function. This finding is novel and suggests a reverse hierarchical organization of the visual cortex for early blind individuals. The conjunction analyses found common activations in the right middle temporal gyrus, right precuneus and a left parieto-occipital region. Clinically, this work contributes to visual rehabilitation in early blind individuals by revealing the function-dependent and sensory-independent networks during nonvisual processing.

Recently, modification of T cells with chimeric antigen receptor (CAR) has been an attractive approach for adoptive immunotherapy of cancers. Typically, CARs contain a single-chain variable domain fragment (scFv). Most often, scfvs are derived from a monoclonal antibody of murine origin and may be a trigger for host immune system that leads to the T-cell clearance. Nanobody is a specific antigen-binding fragment derived from camelid that has great homology to human VH and low immunogenic potential. Therefore, in this study, nanobody was employed instead of scFv in CAR construct.

It remains controversial whether and which fatty acids are different between prostate cancer (PCa) and benign prostatic tissues (BPT) in association with occurrence, progression and racial disparity between African American (AA) and Caucasian American (CA) populations.

Oxidative stress and cardiomyocyte apoptosis contributed to the progression of doxorubicin (Dox)-induced cardiotoxicity. Recent studies identified microRNA-22 (miR-22) as a cardiac- and skeletal muscle-enriched microRNA that functioned as a key regulator in stress-induced cardiac injury. The present study aimed to investigate the role and possible mechanism of miR-22 on Dox-induced oxidative stress and cardiomyocyte apoptosis. Mice were exposed to reduplicative injections of Dox (i.p., 4 mg/kg) weekly for consecutive 4 weeks to generate Dox-induced cardiotoxicity. Herein, we found that miR-22 level was significantly increased in murine hearts subjected to chronic Dox treatment. MiR-22 inhibition attenuated oxidative stress and cardiomyocyte apoptosis in vivo and in vitro, thereby preventing Dox-induced cardiac dysfunction. Mechanistically, we observed that miR-22 directly bound to the 3'-UTR of Sirt1 and caused SIRT1 downregulation. Conversely, miR-22 antagomir upregulated SIRT1 expression and SIRT1 inhibitor abolished the beneficial effects of miR-22 antagomir. In conclusion, miR-22 inhibition prevented oxidative stress and cardiomyocyte apoptosis via upregulating SIRT1 and miR-22 might be a new target for treating Dox-induced cardiotoxicity.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion of a CAG triplet repeat (encoding for a polyglutamine tract) within the first exon of the huntingtin gene. Expression of the mutant huntingtin (mHTT) protein can result in the production of N-terminal fragments with a robust propensity to form oligomers and aggregates, which may be causally associated with HD pathology. Several lines of evidence indicate that N17 phosphorylation or pseudophosphorylation at any of the residues T3, S13 or S16, alone or in combination, modulates mHTT aggregation, subcellular localization and toxicity. Consequently, increasing N17 phosphorylation has been proposed as a potential therapeutic approach. However, developing genetic/pharmacological tools to quantify these phosphorylation events is necessary in order to subsequently develop tool modulators, which is difficult given the transient and incompletely penetrant nature of such post-translational modifications. Here we describe the first ultrasensitive sandwich immunoassay that quantifies HTT phosphorylated at residue S13 and demonstrate its utility for specific analyte detection in preclinical models of HD.

Human xylosyltransferases-I and -II (XT-I and XT-II) catalyze the initial and rate-limiting step in proteoglycan (PG)-biosynthesis. Because PG are major components of the extracellular matrix (ECM), an alternated XT expression is associated with the manifestation of ECM-related diseases. While Drosophila melanogaster and Caenorhabditis elegans only harbor one XT-isoform, all higher organisms contain two isoforms, which are expressed in a tissue-specific manner. The reason for the appearance of two isoenzymes remains unexplained and remarkable, as all other enzymes involved in the synthesis of the tetrasaccharid linker, which connects the PG core protein with attached glycosaminoglycans, only show one isoform. In human, mutations in the XYLT genes cause diseases affecting the homeostasis of the ECM, such as skeletal dysplasias. We investigated for the first time whether already XT-I-deficient human embryonic kidney (HEK293) cells can compensate for decreased expression levels of both XT-isoforms. A siRNA-mediated XYLT2 mRNA knockdown led to reduced cellular proliferation rates and a partially increased cellular senescence of treated HEK293 cells. These results were verified by conducting a stable CRISPR/Cas9-mediated XYLT2 knockout, which revealed that only cells expressing at least partially functional XT-II proteins remain proliferative. Our study, therefore, shows for the first time that cells lacking both XT-isoforms are not viable and clearly indicates the importance of the XT concerning the cellular metabolism.

Adipose tissue hypoxia occurs early in obesity and is associated with increased tissue macrophages and systemic inflammation that impacts muscle insulin responsiveness. We investigated how hypoxia interacted with adipocyte-macrophage crosstalk and inflammatory cytokine release, using co-culture and conditioned media (CM). Murine primary adipocytes from lean or obese mice were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions. RAW264.7 macrophages were incubated under normoxic or hypoxic conditions with or without adipocyte conditioned media. Macrophage and adipocyte-macrophage co-culture CM were also collected. We found hypoxia did not elicit direct cytokine release from macrophages. However, adipocyte CM or adipocyte co-culture, synergistically stimulated TNFα and MCP-1 release from macrophages that was not further impacted by hypoxia. Exposure of muscle cells to elevated cytokines led to reduced insulin and muscle stress/inflammatory signaling. We conclude hypoxia or obesity induces release of inflammatory TNFα and MCP-1 from mice primary adipocytes but the two environmental conditions do not synergize to worsen macrophage signal transduction or insulin responsiveness.

Francisella bacteria rely on a phylogenetically distinct type VI secretion system (T6SS) to escape host phagosomes and cause the fatal disease tularemia, but the structural and molecular mechanisms involved are unknown. Here we report the atomic structure of the Francisella T6SS central spike complex, obtained by cryo-electron microscopy. Our structural and functional studies demonstrate that, unlike the single-protein spike composition of other T6SS subtypes, Francisella T6SS's central spike is formed by two proteins, PdpA and VgrG, akin to T4-bacteriophage gp27 and gp5, respectively, and that PdpA has unique characteristics, including a putative cargo within its cavity and an N-terminal helical lid. Structure-guided mutagenesis demonstrates that the PdpA N-terminal lid and C-terminal spike are essential to Francisella T6SS function. PdpA is thus both an adaptor, connecting VgrG to the tube, and a likely carrier of secreted cargo. These findings are important to understanding Francisella pathogenicity and designing therapeutics to combat tularemia.

Naturally occurring smallpox has been eradicated but research stocks of variola virus (VARV), the causative agent of smallpox, still exist in secure laboratories. Clandestine stores of the virus or resurrection of VARV via synthetic biology are possible and have led to concerns that VARV could be used as a biological weapon. The US government has prepared for such an event by stockpiling smallpox vaccines and TPOXX®, SIGA Technologies' smallpox antiviral drug. While vaccination is effective as a pre-exposure prophylaxis, protection is limited when administered following exposure. Safety concerns preclude general use of the vaccine unless there is a smallpox outbreak. TPOXX is approved by the FDA for use after confirmed diagnosis of smallpox disease. Tecovirimat, the active pharmaceutical ingredient in TPOXX, targets a highly conserved orthopoxviral protein, inhibiting long-range dissemination of virus. Although indications for use of the vaccine and TPOXX do not overlap, concomitant use is possible, especially if the TPOXX indication is expanded to include post-exposure prophylaxis. It is therefore important to understand how vaccine and TPOXX may interact. In studies presented here, monkeys were vaccinated with the ACAM2000TM live attenuated smallpox vaccine and concomitantly treated with tecovirimat or placebo. Immune responses to the vaccine and protective efficacy versus a lethal monkeypox virus (MPXV) challenge were evaluated. In two studies, primary and anamnestic humoral immune responses were similar regardless of tecovirimat treatment while the third study showed reduction in vaccine elicited humoral immunity. Following lethal MPXV challenge, all (12 of 12) vaccinated/placebo treated animals survived, and 12 of 13 vaccinated/tecovirimat treated animals survived. Clinical signs of disease were elevated in tecovirimat treated animals compared to placebo treated animals. This suggests that TPOXX may affect the immunogenicity of ACAM2000 if administered concomitantly. These studies may inform on how vaccine and TPOXX are used during a smallpox outbreak.

Recent advances in gene therapy technologies have enabled the treatment of congenital disorders and cancers and facilitated the development of innovative methods, including induced pluripotent stem cell (iPSC) production and genome editing. We recently developed a novel non-transmissible and non-integrating measles virus (MV) vector capable of transferring multiple genes simultaneously into a wide range of cells through the CD46 and CD150 receptors. The MV vector expresses four genes for iPSC generation and the GFP gene for a period of time sufficient to establish iPSCs from human fibroblasts as well as peripheral blood T cells. The transgenes were expressed differentially depending on their gene order in the vector. Human hematopoietic stem/progenitor cells were directly and efficiently reprogrammed to naive-like cells that could proliferate and differentiate into primed iPSCs by the same method used to establish primed iPSCs from other cell types. The novel MV vector has several advantages for establishing iPSCs and potential future applications in gene therapy.

Liver is one of the most preferred destinations of distant metastasis in gastric cancer (GC). As effective treatment is still limited, the prognosis of GC patients bearing liver metastasis is poor. We filter out lysyl oxidase (LOX) to study its function in the tumor microenvironment (TME) and seek for potential therapeutic targets.

Actin waves are filamentous actin (F-actin)-rich structures that initiate in the somato-neuritic area and move toward neurite ends. The upstream cues that initiate actin waves are poorly understood. Here, using an optogenetic approach (Opto-cytTrkB), we found that local activation of the TrkB receptor around the neurite end initiates actin waves and triggers neurite elongation. During actin wave generation, locally activated TrkB signaling in the distal neurite was functionally connected with preferentially localized Rac1 and its signaling pathways in the proximal region. Moreover, TrkB activity changed the location of ankyrinG--the master organizer of the axonal initial segment-and initiated the stimulated neurite to acquire axonal characteristics. Taken together, these findings suggest that local Opto-cytTrkB activation switches the fate from minor to major axonal neurite during neuronal polarization by generating actin waves.

5-Aminolevulinic acid (5-ALA) is an unnatural amino acid and has been approved as a biodegradable, non-toxic pesticide and herbicide with applications in sustainable agriculture. 5-ALA can also be applied for cancer targeting via tumor localization and photodynamic therapy. Herein, we developed a feasible quantification, regulation and production method of 5-ALA in Escherichia coli is based on the chimera of 5-ALA synthetase from Rhodobacter sphaeroides (RshemA) and super-fold green fluorescent protein (sfGFP) under the control of dual promoters/double plasmids. 5-ALA production based on quantification with the reporter sfGFP was unsuccessfully for the RshemA-sfGFP fusion protein owing to a steric hindrance effect, but was effective using dual constitutive promoters (i.e., J23100 and PLacI) for RshemA and sfGFP independently. Moreover, a simple quantification method based on the linear relationship between 5-ALA concentration and the change in sfGFP intensity was calculated with the Hill equation according to the results of dual plasmids which composed of RshemA-threonine/homoserine exporter (RhtA) and the sensing plasmid pSU-T7-sfGFP. Compared with the conventional detection method for 5-ALA using Ehrlich's reagent, our proposed method is advantages in effectiveness, real-time detection, and outstanding sensitivity. Finally, the highest yield of 5-ALA was obtained in E. coli D2TT strain, reaching 2.46 g/L of 5-ALA produced in a 2.5-L baffle flask fermentation. Hence, this approach shows strong potential for improving 5-ALA production with appropriate regulation and detection based on the fluorescent signal.

Spinal cord injury (SCI) can have profound effects on the autonomic and cardiovascular systems, notably with injuries above high-thoracic levels that result in the development of autonomic dysreflexia (AD) characterized by volatile hypertension in response to exaggerated sympathetic reflexes triggered by afferent stimulation below the injury level. Pathophysiological changes associated with the development of AD include sprouting of both nociceptive afferents and ascending propriospinal 'relay' neurons below the injury, as well as dynamic changes in synaptic inputs onto sympathetic preganglionic neurons. However, it remains uncertain whether synapse formation between sprouted c-fibers and propriospinal neurons contributes to the development of exaggerated sympathetic reflexes produced during AD. We previously reported that once daily treatment with the anti-epileptic and neuropathic pain medication, gabapentin (GBP), at low dosage (50 mg/kg) mitigates experimentally induced AD soon after injections, likely by impeding glutamatergic signaling. Since much higher doses of GBP are reported to block the formation of excitatory synapses, we hypothesized that continuous, high dosage GBP treatment after SCI might prevent the formation of aforementioned aberrant synapses and, accordingly, reduce the incidence and severity of AD. Adult female rats implanted with aortic telemetry probes for hemodynamic monitoring underwent T4-transection SCI and immediately received 100 mg/kg (i.p.) of GBP and then every six hours (400 mg/kg/day) for 4-weeks after injury. We assessed daily body weight, mean arterial pressure, heart rate, frequency of spontaneous AD, and hemodynamic changes during colorectal distension (CRD) to establish whether high dose GBP treatment prophylactically mitigates both AD and associated aberrant synaptic plasticity. This regimen significantly reduced both the absolute blood pressure reached during experimentally induced AD and the time required to return to baseline afterwards. Conversely, GBP prevented return to pre-injury body weights and paradoxically increased the frequency of spontaneously occurring AD. While there were significant decreases in the densities of excitatory and inhibitory pre-synaptic markers in the lumbosacral dorsal horn following injury alone, they were unaltered by continuous GBP treatment. This indicates distinct mechanisms of action for acute GBP to mitigate induced AD whereas chronic GBP increases non-induced AD frequencies. While high dose prophylactic GBP is not recommended to treat AD, acute low dose GBP may hold therapeutic value to mitigate evoked AD, notably during iatrogenic procedures under controlled clinical conditions.

The cost of SNP genotyping to screen different breeds and to estimate the exact proportion of ancestry level is quite high, which can be compensated through deriving a small panel of ancestry informative markers (AIMs). Hence, we carried out the present study to provide an insight into ancestry level inferred from a panel of informative markers in the crossbred Vrindavani population developed at ICAR-IVRI, India. We have performed a new method i.e., discriminant analysis of principal components (DAPC) for the first time on the dataset of Vrindavani cattle. To confirm our method, we had performed DAPC on two other well-known crossbred cattle, i.e., Frieswal and Beefmaster. Three sets of panels (500, 1000 and 2000 markers) were tested for clustering of individuals. Among all the panels, we found the panel (1000 markers) with DAPC based contribution method was of the smallest size and comparatively of the highest accuracy.

Abundant with organelle-like membranous structures, the tumor microenvironment is composed of cancer cells that secrete exosomes. Studies have shown that these secreted exosomes transport RNA and active molecules to other cells to reshape the tumor microenvironment and promote tumor growth. In fact, we found that exosomes derived from melanoma cells drive pre-malignant transition in primary melanocytes. However, there is little available in the scientific literature on how exosomes modulate melanocytes in the microenvironment to optimize conditions for tumor progression and metastasis. We therefore focused this current study on identifying these conditions genetically. Through RNA sequencing, we analyzed gene expression levels of melanocytes driven by exosomes derived from melanoma and lung cancer cells compared with those without exosome controls. Significant differences were found in gene expression patterns of melanocytes driven by exosomes derived from melanoma and lung cancer cells. In the melanocytes responding to exosomes derived from melanoma cells, genes of lipopolysaccharide and regulation of leukocyte chemotaxis were predominant. In the melanocytes responding to exosomes derived from lung cancer cells, genes of DNA replication and mitotic nuclear division played an important role. These results provide further mechanistic understanding of tumor progression promoted by tumor-derived exosomes. This will also help identify potential therapeutic targets for melanoma progression.

Nitrogen mustard (NM) is a cytotoxic vesicant known to cause acute lung injury which progresses to fibrosis. Herein, we developed a murine model of NM-induced pulmonary toxicity with the goal of assessing inflammatory mechanisms of injury. C57BL/6J mice were euthanized 1-28 d following intratracheal exposure to NM (0.08 mg/kg) or PBS control. NM caused progressive alveolar epithelial thickening, perivascular inflammation, bronchiolar epithelial hyperplasia, interstitial fibroplasia and fibrosis, peaking 14 d post exposure. Enlarged foamy macrophages were also observed in the lung 14 d post NM, along with increased numbers of microparticles in bronchoalveolar lavage fluid (BAL). Following NM exposure, rapid and prolonged increases in BAL cells, protein, total phospholipids and surfactant protein (SP)-D were also detected. Flow cytometric analysis showed that CD11b+Ly6G-F4/80+Ly6Chi proinflammatory macrophages accumulated in the lung after NM, peaking at 3 d. This was associated with macrophage expression of HMGB1 and TNFα in histologic sections. CD11b+Ly6G-F4/80+Ly6Clo anti-inflammatory/pro-fibrotic macrophages also increased in the lung after NM peaking at 14 d, a time coordinate with increases in TGFβ expression and fibrosis. NM exposure also resulted in alterations in pulmonary mechanics including increases in tissue elastance and decreases in compliance and static compliance, most prominently at 14 d. These findings demonstrate that NM induces structural and inflammatory changes in the lung that correlate with aberrations in pulmonary function. This mouse model will be useful for mechanistic studies of mustard lung injury and for assessing potential countermeasures.

Tumor associated macrophages (TAMs) have a crucial role in cancer progression, metastasis and drug response. Piroxicam and sulindac sulfide are non-steroidal anti-inflammatory drugs (NSAID) that decrease the incidence and progression of several types of cancer. However, their role in suppressing the interactions between TAMs and cancer cells remain unclear. Herein, we studied the impact of human monocytes conditioned media (CM) on cellular proliferation of ER-dependent MCF-7 and ER-independent MDA-MB-231 cells, and the effects of piroxicam and sulindac sulfide on the expression levels of RAS, COX-2, IL-6, IL-1β and PAR-4 (qRT-PCR), BCL-2 and BAX (western blot), Caspase-3, VEGF-a and PGE2 (ELISA), MMP-2 and -9 (zymography) in the stimulated cells. Our results showed that CM caused a significant increase in cells survival through significant increase in RAS expression which resulted in upregulation of COX-2, PGE2, BCL-2, IL-6, IL-1β, VEGF-A and MMP-9 and down regulation of PAR-4. Treatment with one of the NSAIDs used in this study produced a time and concentration dependent growth inhibition of stimulated cells by inhibiting RAS expression. Suppression of RAS was accompanied by downregulation of its downstream signaling of IL-1β, IL-6, COX-2 and PGE2, activation of apoptotic machinery through upregulation of PAR-4 and caspase-3, as well as, inhibition of BCL-2, VEGF-A, MMP-2 and MMP-9. In conclusion, our data support the role of piroxicam and sulindac sulfide in suppressing inflammation-driven breast cancer progression and identifies promising novel target in RAS and PAR-4 signaling.