Research on the mechanism of TWSG1 in the malignant progression of glioma cells and tumor-associated macrophage infiltration.

Gliomas are malignant tumors of the central nervous system; current treatment methods have low efficacy. Twisted gastrulation BMP signaling modulator 1 (TWSG1) has been shown to play a role in gliomas but it is not known whether TWSG1 participates in glioma pathogenesis and macrophage immune regulation. This study identified a total of 24 differentially expressed genes with survival differences in gliomas using bioinformatics analysis. Among them, TWSG1 exhibited the strongest correlation with gliomas and was positively correlated with macrophage enrichment. The results showed that TWSG1 was highly expressed in various glioma cell lines, with the highest expression observed in the A172 cell line. Silencing TWSG1 significantly decreased the viability, migration, and invasion of A172 cells in vitro and tumor growth in a mouse xenograft model in vivo. It also reduced the expression of the matrix metalloproteinases MMP2 and MMP9 both in vivo and in vitro. Silencing TWSG1 significantly reduced the expression of M2 macrophage makers and upregulated the expression of M1 macrophage markers in A172 cells and tumor tissues. These data suggest that interference with TWSG1 suppressed the progression of A172 glioma cells and regulated immune infiltration.

Molecular Basis for Surface-Initiated Non-Thrombin-Generated Clot Formation Following Viral Infection.

In this paper, we propose a model that connects two standard inflammatory responses to viral infection, namely, elevation of fibrinogen and the lipid drop shower, to the initiation of non-thrombin-generated clot formation. In order to understand the molecular basis for the formation of non-thrombin-generated clots following viral infection, human epithelial and Madin-Darby Canine Kidney (MDCK, epithelial) cells were infected with H1N1, OC43, and adenovirus, and conditioned media was collected, which was later used to treat human umbilical vein endothelial cells and human lung microvascular endothelial cells. After direct infection or after exposure to conditioned media from infected cells, tissue surfaces of both epithelial and endothelial cells, exposed to 8 mg/mL fibrinogen, were observed to initiate fibrillogenesis in the absence of thrombin. No fibers were observed after direct viral exposure of the endothelium or when the epithelium cells were exposed to SARS-CoV-2 isolated spike proteins. Heating the conditioned media to 60 °C had no effect on fibrillogenesis, indicating that the effect was not enzymatic but rather associated with relatively thermally stable inflammatory factors released soon after viral infection. Spontaneous fibrillogenesis had previously been reported and interpreted as being due to the release of the alpha C domains due to strong interactions of the interior of the fibrinogen molecule in contact with hydrophobic material surfaces rather than cleavage of the fibrinopeptides. Contact angle goniometry and immunohistochemistry were used to demonstrate that the lipids produced within the epithelium and released in the conditioned media, probably after the death of infected epithelial cells, formed a hydrophobic residue responsible for fibrillogenesis. Hence, the standard inflammatory response constitutes the ideal conditions for surface-initiated clot formation.

Pathogenesis and virulence of Heartland virus.

Heartland virus (HRTV), an emerging tick-borne pathogenic bunyavirus, has been a concern since 2012, with an increasing incidence, expanding geographical distribution, and high pathogenicity in the United States. Infection from HRTV results in fever, thrombocytopenia, and leucopenia in humans, and in some cases, symptoms can progress to severe outcomes, including haemorrhagic disease, multi-organ failure, and even death. Currently, no vaccines or antiviral drugs are available for treatment of the HRTV disease. Moreover, little is known about HRTV-host interactions, viral replication mechanisms, pathogenesis and virulence, further hampering the development of vaccines and antiviral interventions. Here, we aimed to provide a brief review of HRTV epidemiology, molecular biology, pathogenesis and virulence on the basis of published article data to better understand this virus and provide clues for further study.

Asymmetric neurons are necessary for olfactory learning in the Drosophila brain.

Animals have complementary parallel memory systems that process signals from various sensory modalities. In the brain of the fruit fly Drosophila melanogaster, mushroom body (MB) circuitry is the primary associative neuropil, critical for all stages of olfactory memory. Here, our findings suggest that active signaling from specific asymmetric body (AB) neurons is also crucial for this process. These AB neurons respond to odors and electric shock separately and exhibit timing-sensitive neuronal activity in response to paired stimulation while leaving a decreased memory trace during retrieval. Our experiments also show that rutabaga-encoded adenylate cyclase, which mediates coincidence detection, is required for learning and short-term memory in both AB and MB. We observed additive effects when manipulating rutabaga co-expression in both structures. Together, these results implicate the AB in playing a critical role in associative olfactory learning and short-term memory.

Interactome profiling of Crimean-Congo hemorrhagic fever virus glycoproteins.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a biosafety level-4 pathogen requiring urgent research and development efforts. The glycoproteins of CCHFV, Gn and Gc, are considered to play multiple roles in the viral life cycle by interactions with host cells; however, these interactions remain largely unclear to date. Here, we analyzed the cellular interactomes of CCHFV glycoproteins and identified 45 host proteins as high-confidence Gn/Gc interactors. These host molecules are involved in multiple cellular biological processes potentially associated with the physiological actions of the viral glycoproteins. Then, we elucidated the role of a representative cellular protein, HAX1. HAX1 interacts with Gn by its C-terminus, while its N-terminal region leads to mitochondrial localization. By the strong interaction, HAX1 sequestrates Gn to mitochondria, thus depriving Gn of its normal Golgi localization that is required for functional glycoprotein-mediated progeny virion packaging. Consistently, the inhibitory activity of HAX1 against viral packaging and hence propagation was further elucidated in the contexts of pseudotyped and authentic CCHFV infections in cellular and animal models. Together, the findings provide a systematic CCHFV Gn/Gc-cell protein-protein interaction map, but also unravel a HAX1/mitochondrion-associated host antiviral mechanism, which may facilitate further studies on CCHFV biology and therapeutic approaches.

A subset of cholinergic mushroom body neurons blocks long-term memory formation in Drosophila.

Long-term memory (LTM) requires learning-induced synthesis of new proteins allocated to specific neurons and synapses in a neural circuit. Not all learned information, however, becomes permanent memory. How the brain gates relevant information into LTM remains unclear. In Drosophila adults, weak learning after a single training session in an olfactory aversive task typically does not induce protein-synthesis-dependent LTM. Instead, strong learning after multiple spaced training sessions is required. Here, we report that pre-synaptic active-zone protein synthesis and cholinergic signaling from the early α/ß subset of mushroom body (MB) neurons produce a downstream inhibitory effect on LTM formation. When we eliminated inhibitory signaling from these neurons, weak learning was then sufficient to form LTM. This bidirectional circuit mechanism modulates the transition between distinct memory phase functions in different subpopulations of MB neurons in the olfactory memory circuit.

SARS-CoV-2 NSP13 interacts with host IRF3, blocking antiviral immune responses.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses an unprecedented threat to human health since late 2019. Notably, the progression of the disease is associated with impaired antiviral interferon (IFN) responses. Although multiple viral proteins were identified as potential IFN antagonists, the underlying molecular mechanisms remain to be fully elucidated. In this study, we firstly demonstrate that SARS-CoV-2 NSP13 protein robustly antagonizes IFN response induced by the constitutively active form of transcription factor IRF3 (IRF3/5D). This induction of IFN response by IRF3/5D is independent of the upstream kinase, TBK1, a previously reported NSP13 target, thus indicating that NSP13 can act at the level of IRF3 to antagonize IFN production. Consistently, NSP13 exhibits a specific, TBK1-independent interaction with IRF3, which, moreover, is much stronger than that of NSP13 with TBK1. Furthermore, the NSP13-IRF3 interaction was shown to occur between the NSP13 1B domain and IRF3 IRF association domain (IAD). In agreement with the strong targeting of IRF3 by NSP13, we then found that NSP13 blocks IRF3-directed signal transduction and antiviral gene expression, counteracting IRF3-driven anti-SARS-CoV-2 activity. These data suggest that IRF3 is likely to be a major target of NSP13 in antagonizing antiviral IFN responses and provide new insights into the SARS-CoV-2-host interactions that lead to viral immune evasion.

A New Cellular Interactome of SARS-CoV-2 Nucleocapsid Protein and Its Biological Implications.

There is still much to uncover regarding the molecular details of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. As the most abundant protein, coronavirus nucleocapsid (N) protein encapsidates viral RNAs, serving as the structural component of ribonucleoprotein and virion, and participates in transcription, replication, and host regulations. Virus-host interaction might give clues to better understand how the virus affects or is affected by its host during infection and identify promising therapeutic candidates. Considering the critical roles of N, we here established a new cellular interactome of SARS-CoV-2 N by using a high-specific affinity purification (S-pulldown) assay coupled with quantitative mass spectrometry and immunoblotting validations, uncovering many N-interacting host proteins unreported previously. Bioinformatics analysis revealed that these host factors are mainly involved in translation regulations, viral transcription, RNA processes, stress responses, protein folding and modification, and inflammatory/immune signaling pathways, in line with the supposed actions of N in viral infection. Existing pharmacological cellular targets and the directing drugs were then mined, generating a drug-host protein network. Accordingly, we experimentally identified several small-molecule compounds as novel inhibitors against SARS-CoV-2 replication. Furthermore, a newly identified host factor, DDX1, was verified to interact and colocalize with N mainly by binding to the N-terminal domain of the viral protein. Importantly, loss/gain/reconstitution-of-function experiments showed that DDX1 acts as a potent anti-SARS-CoV-2 host factor, inhibiting the viral replication and protein expression. The N-targeting and anti-SARS-CoV-2 abilities of DDX1 are consistently independent of its ATPase/helicase activity. Further mechanism studies revealed that DDX1 impedes multiple activities of N, including the N-N interaction, N oligomerization, and N-viral RNA binding, thus likely inhibiting viral propagation. These data provide new clues to better depiction of the N-cell interactions and SARS-CoV-2 infection and may help inform the development of new therapeutic candidates.

A One-Size-Fits-All Approach to Pressure Ulcers: Whole-Buttock Fasciocutaneous Advancement Flap.

BACKGROUND: Buttock pressure injuries can be difficult to treat. There are many choices of flaps to reconstruct these wounds, but few are large, technically simple, and easily recycled. AIM AND OBJECTIVE: We are presenting our experience on surgical reconstruction of buttock pressure injuries using large whole-buttock fasciocutaneous flaps that are easily designed for ulcers regardless of location and size and are easily recycled for treatment of recurrences. MATERIAL AND METHODS: We conducted a retrospective review of all patients who received reconstruction with fasciocutaneous rotational flaps for buttock region pressure injuries from January 2013 to December 2018. The key steps of this one-size-fits-all flap include elevation of a large, oversized flap to achieve tension-free closure, avoiding fascial incisions over bony prominences, placing the V-Y type closure wound in the posteromedial thigh, and the use of closed incisional negative wound therapy postoperatively. RESULTS: Fifty patients underwent 54 flaps reconstruction for coverage of stage 4 gluteal pressure injuries between January 2013 and December 2018. Seventy-four percent healed without the need for further operation. The average size of the defect was 90 cm2 (maximum = 300 cm2). The average follow-up period was 31 months. Four of the 54 flaps were "recycled" flaps, 3 were performed for the coverage of recurrent ulcers and 1 flap was performed for treatment of a postoperative wound dehiscence. CONCLUSIONS: We recommend this simple, one-size-fits-all approach, whole-buttock fasciocutaneous flap when surgically treating gluteal pressure injuries for selected patients.

SFTS bunyavirus NSs protein sequestrates mTOR into inclusion bodies and deregulates mTOR-ULK1 signaling, provoking pro-viral autophagy.

Autophagy is emerging as a critical player in host defense against diverse infections, in addition to its conserved function to maintain cellular homeostasis. Strikingly, some pathogens have evolved strategies to evade, subvert or exploit different steps of the autophagy pathway for their lifecycles. Here, we present a new viral mechanism of manipulating autophagy for its own benefit with severe fever with thrombocytopenia syndrome bunyavirus (SFTSV, an emerging high-pathogenic virus) as a model. SFTSV infection triggers autophagy, leading to complete autophagic flux. Mechanistically, we show that the nonstructural protein of SFTSV (NSs) interacts with mTOR, the pivotal regulator of autophagy, by targeting its kinase domain and captures mTOR into viral inclusion bodies (IBs) induced by NSs itself. Furthermore, NSsimpairs mTOR-mediated phosphorylation of unc-51-like kinase 1 (ULK1) at Ser757, disrupting the inhibitory effect of mTOR on ULK1 activity and thus contributing to autophagy induction. Pharmacologic treatment and Beclin-1 knockout experimental results establish that, in turn, autophagy enhances SFTSV infection and propagation. Moreover, the minigenome reporter system reveals that SFTSV ribonucleoprotein (the transcription and replication machinery) activity can be bolstered by autophagy. Additionally, we found that the NSs proteins of SFTSV-related bunyaviruses have a conserved function of targeting mTOR. Taken together, we unravel a viral strategy of inducing pro-viral autophagy by interacting with mTOR, sequestering mTOR into IBs and hence provoking the downstream ULK1 pathway, which presents a new paradigm for viral manipulation of autophagy and may help inform future development of specific antiviral therapies against SFTSV and related pathogens.

Transcriptome profiling highlights regulated biological processes and type III interferon antiviral responses upon Crimean-Congo hemorrhagic fever virus infection.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a biosafety level-4 (BSL-4) pathogen that causes Crimean-Congo hemorrhagic fever (CCHF) characterized by hemorrhagic manifestation, multiple organ failure and high mortality rate, posing great threat to public health. Despite the recently increasing research efforts on CCHFV, host cell responses associated with CCHFV infection remain to be further characterized. Here, to better understand the cellular response to CCHFV infection, we performed a transcriptomic analysis in human kidney HEK293 â€‹cells by high-throughput RNA sequencing (RNA-seq) technology. In total, 496 differentially expressed genes (DEGs), including 361 up-regulated and 135 down-regulated genes, were identified in CCHFV-infected cells. These regulated genes were mainly involved in host processes including defense response to virus, response to stress, regulation of viral process, immune response, metabolism, stimulus, apoptosis and protein catabolic process. Therein, a significant up-regulation of type III interferon (IFN) signaling pathway as well as endoplasmic reticulum (ER) stress response was especially remarkable. Subsequently, representative DEGs from these processes were well validated by RT-qPCR, confirming the RNA-seq results and the typical regulation of IFN responses and ER stress by CCHFV. Furthermore, we demonstrate that not only type I but also type III IFNs (even at low dosages) have substantial anti-CCHFV activities. Collectively, the data may provide new and comprehensive insights into the virus-host interactions and particularly highlights the potential role of type III IFNs in restricting CCHFV, which may help inform further mechanistic delineation of the viral infection and development of anti-CCHFV strategies.

Efficacy of hypochlorous acid (HOCl) fog in sanitizing surfaces against Enterococcus faecalis.

BACKGROUND: Fogging is an efficient method when disinfection of large areas is desired. METHODS: Two methods of ultrasonic fogging, pulsed and continuous, were compared on bacteria dried on either aluminum or polystyrene surfaces. We characterized commercial and home-made hypochlorous acid (HOCl) with respect to storage and means of production. RESULTS: We found that the initial chlorine concentration of the commercial solution was approximately 550 ppm, and when stored open under ambient conditions, the chlorine content decreased at a rate of 30% every 100 days. The HOCl produced using the home synthesizers had a maximum chlorine content of 257.6 ppm which decayed by 65% after 100 days. A second synthesizer produced a liquid with high chlorine content and pH, 750ppm and pH = 8.55. The anti-bacterial efficacy was probed using Enterococcus faecalis, a persistent source of infection in public and clinical spaces. Time course studies determined that E. faecalis could survive dry on surfaces for more than 12 weeks, but was easily eliminated in half the fogging time. CONCLUSIONS: The most effective mode of application was determined to be continuous fogging where a 6.59 log reduction was established in vertical geometry. The optimal pulsed fogging protocol produced a similar reduction, but required nearly 5 times as long. The home synthesized versions yielded much lower log bacterial reductions. No significant differences in outcome were determined between polymer or metal surfaces.

Engineering functional skin constructs: A quantitative comparison of three-dimensional bioprinting with traditional methods.

Tissue engineering has been successful in reproducing human skin equivalents while incorporating new approaches such as three-dimensional (3D) bioprinting. The latter method offers a plethora of advantages including increased production scale, ability to incorporate multiple cell types and printing on demand. However, the quality of printed skin equivalents compared to those developed manually has never been assessed. To leverage the benefits of this method, it is imperative that 3D-printed skin should be structurally and functionally similar to real human skin. Here, we developed four bilayered human skin epidermal-dermal equivalents: non-printed dermis and epidermis (NN), printed dermis and epidermis (PP), printed epidermis and non-printed dermis (PN), and non-printed epidermis and printed dermis (NP). The effects of printing induced shear stress [0.025 kPa (epidermis); 0.049 kPa (dermis)] were characterized both at the cellular and at the tissue level. At cellular level, no statistically significant differences in keratinocyte colony-forming efficiency (CFE) (p = 0.1641) were observed. In the case of fibroblasts, no significant differences in the cell alignment index (p < 0.1717) and their ability to contract collagen gel (p = 0.851) were detected. At the tissue levels, all the four skin equivalents were characterized using histological and immunohistochemical analysis with no significant differences found in either epidermal basal cell count, thickness of viable epidermis, and relative intensity of filaggrin and claudin-1. Our results demonstrated that 3D printing can achieve the same high-quality skin constructs as have been developed traditionally, thus opening new avenues for numerous high-throughput industrial and clinical applications.

Targeted DNA methylation from cell-free DNA using hybridization probe capture.

Cell-free (cf)DNA signatures are quickly becoming the target of choice for non-invasive screening, diagnosis, treatment and monitoring of human tumors. DNA methylation changes occur early in tumorigenesis and are widespread, making cfDNA methylation an attractive cancer biomarker. Already a proven technology for targeted genome sequencing, hybridization probe capture is emerging as a method for high-throughput targeted methylation profiling suitable to liquid biopsy samples. However, to date there are no reports describing the performance of this approach in terms of reproducibility, scalability, and accuracy. In the current study we performed hybridization probe capture using the myBaits® Custom Methyl-seq kit on 172 plasma samples and standards to evaluate its performance on cfDNA methylation analysis. The myBaits® assay showed high target recovery (>90%), demonstrated excellent reproducibility between captures (R 2 = 0.92 on average), and was unaffected by increasing the number of targets in a capture. Finally, myBaits® accurately replicated 'gold standard' beta values from WGBS (average R 2 = 0.79). The results of this study show that custom targeted methylation sequencing with myBaits® offers a cost-effective, reliable platform to profile DNA methylation at a set of discrete custom regions, with potential applicability to liquid biopsies for cancer monitoring.

Neuropeptide F inhibits dopamine neuron interference of long-term memory consolidation in Drosophila.

Long-term memory (LTM) formation requires consolidation processes to overcome interfering signals that erode memory formation. Olfactory memory in Drosophila involves convergent projection neuron (PN; odor) and dopaminergic neuron (DAN; reinforcement) input to the mushroom body (MB). How post-training DAN activity in the posterior lateral protocerebrum (PPL1) continues to regulate memory consolidation remains unknown. Here we address this question using targeted transgenes in behavior and electrophysiology experiments to show that (1) persistent post-training activity of PPL1-α2α'2 and PPL1-α3 DANs interferes with aversive LTM formation; (2) neuropeptide F (NPF) signaling blocks this interference in PPL1-α2α'2 and PPL1-α3 DANs after spaced training to enable LTM formation; and (3) training-induced NPF release and neurotransmission from two upstream dorsal-anterior-lateral (DAL2) neurons are required to form LTM. Thus, NPF signals from DAL2 neurons to specific PPL1 DANs disinhibit the memory circuit, ensuring that periodic events are remembered as consolidated LTM.

N-doped reduced graphene oxide for room-temperature NO gas sensors.

In this study, we use nitrogen-doped to improving the gas-sensing properties of reduced graphene oxide. Graphene oxide was prepared according to a modified Hummers' method and then nitrogen-doped reduced graphene oxide (N-rGO) was synthesized by a hydrothermal method using graphene oxide and NH4OH as precursors. The rGO is flat and smooth with a sheet-like morphology while the N-rGO exhibits folded morphology. This type of folding of the surface morphology can increase the gas sensitivity. The N-rGO and the rGO sensors showed n-type and p-type semiconducting behaviors in ambient conditions, respectively, and were responsive to low concentrations of NO gases (< 1000 ppb) at room temperature. The gas-sensing results showed that the N-rGO sensors could detect NO gas at concentrations as low as 400 ppb. The sensitivity of the N-rGO sensor to 1000 ppb NO (1.7) is much better than that of the rGO sensor (0.012). Compared with pure rGO, N-rGO exhibited a higher sensitivity and excellent reproducibility.

The impact of prenatal use of oral Clostridium butyricum on maternal group B Streptococcus colonization: A retrospective study.

OBJECTIVE: The aim of this study is to examine the effect of taking Clostridium butyricum (Miyarisan BM) orally for 4 weeks since the 32+0 weeks of gestation on preventing Group B Streptococcus colonization. MATERIALS AND METHODS: We retrospectively collected data on the pregnancy outcomes of 1602 women between October 2017 and August 2019. The control group received standard antenatal care, and the intervention group received standard antenatal care with a daily oral dose of probiotics since the 32+0 weeks of gestation. The daily dose was one pack of C. butyricum (Miyarisan BM) once or twice a day. A vaginal Group B Streptococcus swab was collected between 36+0 and 36+6 weeks of gestation. RESULTS: After applying the designated exclusion criteria, the total number of participants was 1576. The Group B Streptococcus colonization rate was significantly decreased in the intervention group (P = 0.0338; adjusted OR: 0.66 (0.45-0.97)). CONCLUSION: Probiotics can reduce the colonization rate of Group B Streptococcus in the vagina and rectum under three conditions: (1) intervention of adequate length, (2) sufficient probiotic dose, and (3) effective probiotics.

Combination of 3D Printing and ALD for Dentin Fabrication from Dental Pulp Stem Cell Culture.

A combination of fused deposition modeling printing with atomic layer deposition (ALD) of titania was designed to achieve templated biomineralization and terminal odontogenic differentiation of dental pulp stem cells on three-dimensional (3D) printed polylactic acid (PLA) scaffolds. In the absence of the ALD-deposited titania coating, we had previously shown that both plating efficiency and differentiation are adversely impacted when scaffolds are produced by 3D printing rather than traditional polymer molding. These differences were removed when both printed and molded structures were coated with ALD of titania, which improved the outcomes regardless of the manufacturing method. In this case, on all titania-coated substrates, the plating efficiency increased, copious mineral deposition was observed, and RT-PCR indicated a significant upregulation of osteocalcin, a gene associated with mineral deposition. The influence of additional coatings of collagen, gelatin, or fibronectin on the ALD titania-coated and uncoated PLA-printed and molded scaffolds was also investigated. Upregulation of the odontogenic late-stage differentiation sibling protein, dentin sialoprotein, was observed on the collagen ALD-titania-coated scaffolds and to a lesser extent on the gelatin ALD-titania-coated scaffolds.